Maochun Qin

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

BackgroundChanges in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease.ResultsIn this study Me thylated D NA I mmunoP recipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify T issue and D evelopmental S tage specific D ifferentially M ethylated DNA R egions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array).ConclusionsOur data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer

Significance Genetic alterations are frequently observed in bladder cancer. In this study, we demonstrate that bladder tumors can be classified into two different types based on the spectrum of genetic diversity they confer. In one class of tumors, we observed tumor protein p53 mutations and a large number of single-nucleotide and structural variants. Another characteristic of this group was chromosome shattering, known as chromothripsis, and mutational heterogeneity. The other two bladder tumors did not show these profound genetic aberrations, but we found a novel translocation and amplification of the gene glutamate receptor ionotropic N-methyl D-aspertate, a potentially druggable target. Advancements in bladder cancer treatment have been slow. Understanding the genetic landscape of bladder cancer may therefore help to identify new therapeutic targets and bolster management of this disease. Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as “stitchers,” to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication–licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.

FUSIM: a software tool for simulating fusion transcripts

BackgroundGene fusions are the result of chromosomal aberrations and encode chimeric RNA (fusion transcripts) that play an important role in cancer genesis. Recent advances in high throughput transcriptome sequencing have given rise to computational methods for new fusion discovery. The ability to simulate fusion transcripts is essential for testing and improving those tools.ResultsTo facilitate this need, we developed FUSIM (FUsion SIMulator), a software tool for simulating fusion transcripts. The simulation of events known to create fusion genes and their resulting chimeric proteins is supported, including inter-chromosome translocation, trans-splicing, complex chromosomal rearrangements, and transcriptional read through events.ConclusionsFUSIM provides the ability to assemble a dataset of fusion transcripts useful for testing and benchmarking applications in fusion gene discovery.

Nomogram to predict the likelihood of upgrade of atypical ductal hyperplasia diagnosed on a core needle biopsy in mammographically detected lesions.

To estimate the likelihood of the upgrade for atypical ductal hyperplasia (ADH) diagnosed on a core needle biopsy of a mammographically detected lesion.

OSAT: a tool for sample-to-batch allocations in genomics experiments

BackgroundBatch effect is one type of variability that is not of primary interest but ubiquitous in sizable genomic experiments. To minimize the impact of batch effects, an ideal experiment design should ensure the even distribution of biological groups and confounding factors across batches. However, due to the practical complications, the availability of the final collection of samples in genomics study might be unbalanced and incomplete, which, without appropriate attention in sample-to-batch allocation, could lead to drastic batch effects. Therefore, it is necessary to develop effective and handy tool to assign collected samples across batches in an appropriate way in order to minimize the impact of batch effects.ResultsWe describe OSAT (Optimal Sample Assignment Tool), a bioconductor package designed for automated sample-to-batch allocations in genomics experiments.ConclusionsOSAT is developed to facilitate the allocation of collected samples to different batches in genomics study. Through optimizing the even distribution of samples in groups of biological interest into different batches, it can reduce the confounding or correlation between batches and the biological variables of interest. It can also optimize the homogeneous distribution of confounding factors across batches. It can handle challenging instances where incomplete and unbalanced sample collections are involved as well as ideally balanced designs.

Robust detection of immune transcripts in FFPE samples using targeted RNA sequencing

Current criteria for identifying cancer patients suitable for immunotherapy with immune checkpoint blockers (ICBs) are subjective and prone to misinterpretation, as they mainly rely on the visual assessment of CD274 (best known as PD-L1) expression levels by immunohistochemistry (IHC). To address this issue, we developed a RNA sequencing (RNAseq)-based approach that specifically measures the abundance of immune transcripts in formalin-fixed paraffin embedded (FFPE) specimens. Besides exhibiting superior sensitivity as compared to whole transcriptome RNAseq, our assay requires little starting material, implying that it is compatible with RNA degradation normally caused by formalin. Here, we demonstrate that a targeted RNAseq panel reliably profiles mRNA expression levels in FFPE samples from a cohort of ovarian carcinoma patients. The expression profile of immune transcripts as measured by targeted RNAseq in FFPE versus freshly frozen (FF) samples from the same tumor was highly concordant, in spite of the RNA quality issues associated with formalin fixation. Moreover, the results of targeted RNAseq on FFPE specimens exhibited a robust correlation with mRNA expression levels as measured on the same samples by quantitative RT-PCR, as well as with protein abundance as determined by IHC. These findings demonstrate that RNAseq profiling on archival FFPE tissues can be used reliably in studies assessing the efficacy of cancer immunotherapy.

SCNVSim: somatic copy number variation and structure variation simulator

BackgroundSomatically acquired structure variations (SVs) and copy number variations (CNVs) can induce genetic changes that are directly related to tumor genesis. Somatic SV/CNV detection using next-generation sequencing (NGS) data still faces major challenges introduced by tumor sample characteristics, such as ploidy, heterogeneity, and purity. A simulated cancer genome with known SVs and CNVs can serve as a benchmark for evaluating the performance of existing somatic SV/CNV detection tools and developing new methods.ResultsSCNVSim is a tool for simulating somatic CNVs and structure variations SVs. Other than multiple types of SV and CNV events, the tool is capable of simulating important features related to tumor samples including aneuploidy, heterogeneity and purity.ConclusionsSCNVSim generates the genomes of a cancer cell population with detailed information of copy number status, loss of heterozygosity (LOH), and event break points, which is essential for developing and evaluating somatic CNV and SV detection methods in cancer genomics studies.

A Signature of Genomic Instability Resulting from Deficient Replication Licensing

Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea). In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats.

Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

Whole-genome sequencing of a malignant granular cell tumor with metabolic response to pazopanib

Granular cell tumors are an uncommon soft tissue neoplasm. Malignant granular cell tumors comprise <2% of all granular cell tumors, are associated with aggressive behavior and poor clinical outcome, and are poorly understood in terms of tumor etiology and systematic treatment. Because of its rarity, the genetic basis of malignant granular cell tumor remains unknown. We performed whole-genome sequencing of one malignant granular cell tumor with metabolic response to pazopanib. This tumor exhibited a very low mutation rate and an overall stable genome with local complex rearrangements. The mutation signature was dominated by C>T transitions, particularly when immediately preceded by a 5′ G. A loss-of-function mutation was detected in a newly recognized tumor suppressor candidate, BRD7. No mutations were found in known targets of pazopanib. However, we identified a receptor tyrosine kinase pathway mutation in GFRA2 that warrants further evaluation. To the best of our knowledge, this is only the second reported case of a malignant granular cell tumor exhibiting a response to pazopanib, and the first whole-genome sequencing of this uncommon tumor type. The findings provide insight into the genetic basis of malignant granular cell tumors and identify potential targets for further investigation.