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BackgroundAcute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer, and patients with relapsed ALL have a poor prognosis. Detection of ALL blasts remaining at the end of treatment, or minimal residual disease (MRD), and spread of ALL into the central nervous system (CNS) have prognostic importance in ALL. Current methods to detect MRD and CNS disease in ALL rely on the presence of ALL blasts in patient samples. Cell-free DNA, or small fragments of DNA released by cancer cells into patient biofluids, has emerged as a robust and sensitive biomarker to assess cancer burden, although cfDNA analysis has not previously been applied to ALL.MethodsWe present a simple and rapid workflow based on NanoporeMinION sequencing of PCR amplified B cell-specific rearrangement of the (IGH) locus in cfDNA from B-ALL patient samples. A cohort of 5 pediatric B-ALL patient samples was chosen for the study based on the MRD and CNS disease status.ResultsQuantitation of IGH-variable sequences in cfDNA allowed us to detect clonal heterogeneity and track the response of individual B-ALL clones throughout treatment. cfDNA was detected in patient biofluids with clinical diagnoses of MRD and CNS disease, and leukemic clones could be detected even when diagnostic cell-count thresholds for MRD were not met. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient, even when blasts are not physically present in the biofluid sample.ConclusionsThe Nanopore IGH detection workflow to monitor cell-free DNA is a simple, rapid, and inexpensive assay that may ultimately serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

10034 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relatively high relapse rate, which is associated with poor prognosis. Currently, minimal residual disease (MRD) at the end of induction and consolidation therapy is the best predictor of patient relapse, however obtaining bone marrow aspirate is invasive and not always accurate. Another major concern in ALL is the presence of central nervous system (CNS) disease, which is often present long before clinical diagnosis can be made by flow cytometry. To circumvent these clinical challenges, we developed a new assay quantifying cell-free, circulating tumor (cfDNA) as a biomarker of disease progression, which can be correlated with MRD status as a predictor of relapse. cfDNA is frequently used to monitor progression of solid tumors, but pediatric leukemias lack common mutations that can be used to distinguish leukemic cfDNA from normal cfDNA. Methods: We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We developed a novel workflow for identifying VDJ rearrangements in leukemia cells and tracking their presence in cfDNA. We collected bone marrow, blood, and CSF samples from newly diagnosed patients, and cfDNA was isolated from blood and CSF samples throughout treatment. Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements of leukemic clones in genomic DNA. The MinION assay relies on patient-specific sequencing. We are also in the process of developing a universal assay that utilizes recurrent methylation changes in ALL to identify leukemic cfDNA in patient samples. Results: The MinION workflow was used to follow leukemic cfDNA throughout the course of treatment, and accurately identified MRD and CNS disease in patients. This workflow performed equivalent or better at detecting leukemic clones compared to MiSeq and droplet digital polymerase chain reaction (ddPCR), and is faster and less expensive than traditional Illumina sequencing. Methylation analysis of 865 ALL and 79 healthy samples yielded 55 regions and 19 specific methylation sites that were uniquely present in ALL samples. We are validating these sites by ddPCR to establish a panel of biomarkers to track ALL over time via cfDNA. Conclusions: The end goal of our study is provide a more sensitive and less invasive method for tracking MRD and CNS disease than current approaches. Results will ultimately be correlated with patient response to therapy, the presence of relapse or CNS disease, and overall outcomes determined by standard clinical diagnostic procedures.

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification.

e18018Background: GDT produces higher response rates and improved survival compared to empiric therapy in some series of patients (pts) with refractory cancers. Limited data exists on the utility o...

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Stage 4 NB is characterized with its clinical heterogynous outcome. The special category, stage 4S subgroup (2-5% of all NB) has a cure rate of above 90%. On the other hand, MYCN amplification (25-30% of all NB) is associated with poor outcome of 26% event free survival rate. Alternative splicing events have crucial roles in tumorigenesis and progression, and are nearly universal in human genes. However, biomarkers and drug designs typically focus on the main transcript of coding genes. High-throughput RNA sequencing (RNA-seq) experiments allow us to quantify all genes and their isoforms across samples. The purposes of this study are to systematically exam and compare the splicing programs in stage 4 NB subgroups, to evaluate the effects of splicing program, to identify key factors controlling the splicing program, and also to report isoform candidates that play important roles in dramatic different outcomes of advanced-stage NB subtypes, which represents potential diagnostic, prognostics and therapeutic targets for advanced-stage NB. In this study, we analyzed RNA-seq data of 29 stage 4 NB tumors and discovered an alternative splicing signature for MYCN amplified stage 4 tumors compared to MYCN non-amplified 4S tumors. In summary, 460 genes were alternatively spliced between MYCN non-amplified 4S and MYCN amplified stage 4 NB tumors. Pathway analysis with Ingenuity IPA demonstrated that alternatively spliced genes held enriched roles in cancer hallmarks biological functions including cell death, cell proliferation, apoptosis, cell movement and immune response. In particular, tissue-specific splicing factors such as RBFOX, CELF, and hnRNP families were differentially expressed between tumor subgroups. Regulatory motif sequences of these splicing factors were enriched in adjacent introns of differentially spliced exons. In addition, we used SHEP neuroblastoma Tet21N system and siRNA on IMR32 NB cell line to either induce or knockdown MYCN expression. RNA-seq analysis of MYCN induction and knockdown NB cell lines showed evidence of MYCN regulation of some of the splicing factors. This study systematically examined and compared the splicing programs in stage 4 NB subgroups. Moreover, it demonstrated that in addition to MYCN's well characterized role in transcriptional regulation, it regulates splicing events by controlling the expression of splicing factors. Citation Format: Shile Zhang, Tom Badgett, Jun Wei, Young K. Song, Dominik Bogen, Peter Johansson, Catherine Tolman, Jianbin He, Xinyu Wen, Zhihui Liu, Carol Thiele, Javed Khan. Massively parallel transcriptome sequencing in neuroblastoma reveals specific mRNA splicing patterns controlled by RNA binding splicing factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3161. doi:10.1158/1538-7445.AM2013-3161

Genetic alterations are thought to enable cancers to proliferate and survive more effectively or to resist cytotoxic therapies. However, the molecular basis of tumorigenesis and progression is not fully understood. In order to understand the genetic aberrations underlying tumor progression in refractory neuroblastoma, we performed next-generation sequencing on the whole exomes of three sequential tumor samples taken from a high-risk stage 4 neuroblastoma patient at diagnosis, after cytotoxic therapies, and at death, paired with a germline DNA sample from the same patient. Each genomic DNA sample was first partitioned using SureSelect whole human exome kits targeting ∼38Mb of genomic regions, then subjected to sequencing. Each exome sequencing experiment yielded approximately 240 million mappable reads representing 12 billion base pairs. We used DiBayes to identify single nucleotide variants (SNVs). After filtering away the low-quality SNVs, we identified approximately 18,000 high-quality SNVs in each sample, of which ∼7500 are within the protein coding regions. Using the frequencies of the SNVs derived from the sequencing experiments, we discovered a shared LOH signature at the chromosome level among all three tumors indicating they originate from a common cancer progenitor. When we examined the germline DNA, we found about 97% of SNVs for each tumor sample were also found in the germline DNA, and each tumor had more than 300 unique somatic SNVs. These data showed that despite of the shared LOH pattern, tumor genomes are highly unstable at the single nucleotide level. Furthermore, we also found that the three tumor samples shared 64 SNVs which may be potential driver mutations. Twenty of those common 64 SNVs, located on 7 genes, are predicted to be damaging mutations by SIFT algorithm. These mutations are currently being validated using Sanger sequencing, and further RNA-seq in the same tumor samples to investigate for pathway disruption that may lead to chemo-refractory disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2011-4833

Introduction: Neuroblastoma (NB) is one of the small round blue cell tumors of childhood arising in the peripheral nervous system. Fifty percent of patients present with metastatic or high risk disease and despite aggressive multimodal therapy approximately 60% percent of these patients eventually succumb to NB. Currently only a handful of molecular alterations are known to influence prognosis, but no clear mechanism of pathogenesis has been demonstrated. Recently, targeted sequencing efforts identified recurrent single nucleotide variants (SNVs) in the ALK gene in 6.1-15% of NB. In an effort to better understand the biology driving this tumor and to identify new targets for therapy, we sequenced the transcriptomes of 20 stage four tumors, including ten MYCN amplified and ten MYCN non-amplified samples, using massively parallel sequencing technology. Methods: In our analysis pipeline, 50 base nucleotide reads are filtered against a database of rRNA, tRNA, repetitive regions, and adapter sequences. The remaining reads were aligned to the reference human genome (hg 18) and a database of spice junctions. Reads that align were analyzed for: 1) base coverage, 2) transcript expression levels, 3) calling SNVs and 4) determination of which of the SNVs were damaging by Sorting Intolerant From Tolerant (SIFT) analysis Results: Initial analysis of the first six samples, yielded an average of 86.6 million uniquely mapped reads per sample. On average we detected the expression of 6,000 genes to a depth of 10x. The RNA seq expression profile correlated well with expression array data from the same sample (r=0.62), while the sequencing data identified an additional 3,000 genes that were not detected by microarray. Our sequencing method maintains “strandedness” allowing the identification of multiple novel antisense non-coding transcripts. Using the SAMtool, an average of 1,255 nonsynonymous SNVs predicted per sample. Of these nonsynonymous SNVs, 69-160 per sample were predicted by the SIFT algorithm to be damaging. Interestingly, 3 different genes had damaging nonsynonymous SNVs in at least 30% of the samples. Conclusion: Next generation sequencing of transcriptome is a powerful and more sensitive method than microarrays for expression profiling and allows for the identification of novel transcripts including non-coding RNAs. Here we report the most extensive profiling of the neuroblastoma transcriptome to date using this technology. We identified several hundred protein disrupting SNVs, and of these 3 were commonly altered. Ongoing analysis is underway to validate our results. The identification of recurrent genetic alterations in NB will assist in developing a better understanding of the mechanisms of pathogenesis of this pediatric cancer and lead to new therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1137.

Accumulation of genetic and epigenetic alterations is thought to enable cancers to proliferate and survive more effectively than their neighboring cells, or for aggressive tumors to resist cytotoxic therapies. However, the molecular basis of tumorigenesis and chemoresistance is not fully understood. With the advent of next-generation sequencing technologies, we can determine the whole transcriptome or genome sequence of a tumor and thereby detect all the changes that may result in a particular phenotype such as resistance to cytotoxic therapies. To understand the genetic aberrations underlying the chemoresistance in neuroblastoma, we attempted to catalogue all genetic aberrations in two tumor samples before and after cytotoxic therapies using RNA-seq (including whole transcriptome and miRNA) and genomic DNA sequencing from the same neuroblastoma patient who was refractory to standard chemotherapies. First, we performed whole genome sequencing on patient's constitutional DNA to establish a genetic background. Multiple mate-paired libraries were constructed and subjected to next-generation sequencing. We acquired 53 Gb high-quality mappable data, equivalent to 16X coverage of this patient's genome. Next, whole transcriptome sequencing of the two tumors yielded total 345 million 50bp mappable reads representing 14,128 and 14,484 genes (20875 and 21330 transcripts) respectively. The average coverage for each transcript was >14, while 5297 genes for one sample and 6912 genes for the other had an average coverage of >10. With such deep sequencing, single nucleotide variant (SNV) analysis on the transcriptome sequencing data identified 9103 and 10556 exonic SNVs of total 154871 and 191033 SNVs respectively in two samples. Among the exonic SNVs, 3965 and 4470 SNVs were in the coding regions and about half of them were nonsynonymous. Using SIFT analysis, we predicted 353 and 443 SNVs to be damaging before and after chemotherapies. Of them, 165 SNVs were shared among two samples. Therefore, these shared SNVs and newly acquired SNVs after chemotherapies may contain the genetic alterations that are resistant to the standard neuroblastoma therapies in this patient. In addition, 188 SNVs were present in the original tumor but not in the refractory sample indicating genomic instability of tumors. In parallel, we performed microRNA sequencing on the two samples. While most of 987 mature microRNAs were detected at similar levels after normalized against total reads, 64 microRNAs were significantly differentially expressed before and after chemotherapies using stringent selection criteria (total read counts in two samples>500, >5 fold change, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2212.