195 research outputs found
Sequencing human-gibbon breakpoints of synteny reveals mosaic new insertions at rearrangement sites
No full textThe gibbon genome exhibits extensive karyotypic diversity with an increased rate of chromosomal rearrangements during evolution. In an effort to understand the mechanistic origin and implications of these rearrangement events, we sequenced 24 synteny breakpoint regions in the white-cheeked gibbon (Nomascus leucogenys, NLE) in the form of high-quality BAC insert sequences (4.2 Mbp). While there is a significant deficit of breakpoints in genes, we identified seven human gene structures involved in signaling pathways (DEPDC4, GNG10), phospholipid metabolism (ENPP5, PLSCR2), beta-oxidation (ECH1), cellular structure and transport (HEATR4), and transcription (ZNF461), that have been disrupted in the NLE gibbon lineage. Notably, only three of these genes show the expected evolutionary signatures of pseudogenization. Sequence analysis of the breakpoints suggested both nonclassical nonhomologous end-joining (NHEJ) and replication-based mechanisms of rearrangement. A substantial number (11/24) of human-NLE gibbon breakpoints showed new insertions of gibbon-specific repeats and mosaic structures formed from disparate sequences including segmental duplications, LINE, SINE, and LTR elements. Analysis of these sites provides a model for a replication-dependent repair mechanism for double-strand breaks (DSBs) at rearrangement sites and insights into the structure and formation of primate segmental duplications at sites of genomic rearrangements during evolution
Molecular refinement of gibbon genome rearrangements
No full textThe gibbon karyotype is known to be extensively rearranged when compared to the human and to the ancestral primate karyotype. By combining a bioinformatics (paired-end sequence analysis) approach and a molecular cytogenetics approach, we have refined the synteny block arrangement of the white-cheeked gibbon (Nomascus leucogenys, NLE) with respect to the human genome. We provide the first detailed clone framework map of the gibbon genome and refine the location of 86 evolutionary breakpoints to <1 Mb resolution. An additional 12 breakpoints, mapping primarily to centromeric and telomeric regions, were mapped to approximately 5 Mb resolution. Our combined FISH and BES analysis indicates that we have effectively subcloned 49 of these breakpoints within NLE gibbon BAC clones, mapped to a median resolution of 79.7 kb. Interestingly, many of the intervals associated with translocations were gene-rich, including some genes associated with normal skeletal development. Comparisons of NLE breakpoints with those of other gibbon species reveal variability in the position, suggesting that chromosomal rearrangement has been a longstanding property of this particular ape lineage. Our data emphasize the synergistic effect of combining computational genomics and cytogenetics and provide a framework for ultimate sequence and assembly of the gibbon genome
Next-Generation Sequencing: Application in Liver Cancer—Past, Present and Future?
No full textHepatocellular Carcinoma (HCC) is the third most deadly malignancy worldwide characterized by phenotypic and molecular heterogeneity. In the past two decades, advances in genomic analyses have formed a comprehensive understanding of different underlying pathobiological layers resulting in hepatocarcinogenesis. More recently, improvements of sophisticated next-generation sequencing (NGS) technologies have enabled complete and cost-efficient analyses of cancer genomes at a single nucleotide resolution and advanced into valuable tools in translational medicine. Although the use of NGS in human liver cancer is still in its infancy, great promise rests in the systematic integration of different molecular analyses obtained by these methodologies, i.e., genomics, transcriptomics and epigenomics. This strategy is likely to be helpful in identifying relevant and recurrent pathophysiological hallmarks thereby elucidating our limited understanding of liver cancer. Beside tumor heterogeneity, progress in translational oncology is challenged by the amount of biological information and considerable “noise” in the data obtained from different NGS platforms. Nevertheless, the following review aims to provide an overview of the current status of next-generation approaches in liver cancer, and outline the prospects of these technologies in diagnosis, patient classification, and prediction of outcome. Further, the potential of NGS to identify novel applications for concept clinical trials and to accelerate the development of new cancer therapies will be summarized
Writing the book you'll teach
No full textAs the field of LIS education changes--re-engineering standard practices to become viable in a rapidly changing educational environment--LIS textbooks must change with the times. Students need to learn both the practicalities of librarianship and the history, ethics, and pedagogies of their chosen career. It's increasingly important for textbook authors to be innovative thinkers while still being deeply grounded in the area of their expertise. Textbooks are changing along with the field--professors require that books include the importance of diversity, equity, inclusion, and access. Students may read a big foundations textbook or a number of smaller texts more specific to a particular track. Student demographics and interests are changing with the times, and textbooks need to keep up. So who writes these textbooks? You and your colleagues do. In this informative and entertaining discussion designed to introduce you to textbook authorship and publication, you'll learn from a senior acquisitions editor and published authors how to propose, write, and market a textbook. Senior acquisitions editor Jessica Gribble will offer information about coming up with and refining an idea, writing a proposal, signing a contract, working with an editor, and the challenges and joys of writing the book. Coming up with and refining an idea: Professional development books do one of two things, and they're two sides of the same coin. Either a librarian has found a way to meet a challenge, or they've had an exciting new idea for a program or service. Ideas for textbooks are different; in some ways they're easier to come by, and in others they're far more difficult. Many textbooks come about because a professor has started teaching a new course and has cobbled together some reading materials, but wishes there were a textbook. That professor is a great potential author. Of course, we'll do some research together to learn if this class is being taught at other schools and if other professors would use a textbook. It can be more difficult to publish a textbook as an alternative to books that are already established in their courses, but sometimes it's warranted. This is when we (the potential author and publisher) need to determine what's missing or outdated in the current texts, what will make the new textbook different, and how we'll let professors know about it. Writing a proposal: I'll share our proposal document and some things to think about when proposing a textbook. What courses will use the book? How many schools are teaching that course? What will the table of contents look like? If the textbook is a revision, which parts will be updated? How will the author take pains to keep the textbook relevant in the 5 or so years between editions? Are there any new theories or practices that need to be included? How does it compare to the competition? How long should the book be? How long does it take to write a book? Signing a contract: Although most contracts are standard, what should potential authors understand? Areas for conversation include royalties, index preparation, the often-confusing "protection of the work from competing publications" clause, and registration of copyright vs. publication rights. Some important things aren't included in the contract, like title and cover decisions. Working with an editor (from the editor's perspective): I'm available to work with authors in many ways, depending on how they work best. I'll talk about my role as a therapist of sorts (guilt/nudging/check-ins) and tell a funny story about an author encounter at a conference. I'll describe what authors can expect from an acquisitions editor and what will happen later in the process, when the book is in production. Writing the book: Authors Marcia A. Mardis and Laura Saunders will share their experiences. Both have authored and edited books, and both have worked alone and with co-authors or co-editors. They'll share the nitty-gritty of what it's like to write a textbook, what makes it different from an article or professional development book, how to overcome writer's block and fatigue, and how to use your published textbook as part of your professional development moving forward. They'll talk about time management, collaboration tools, responding to changing community needs, new technologies, learning approaches, professional standards, strategies for engaging readers (students and faculty), ideas for formative feedback, managing multiple authors and drafts, and dealing with the "too much to cover" problem. Marketing the book: I'll give a brief overview of our general marketing efforts, including catalogs, e-mail marketing, and conferences. In our question-and-answer session, we'll encourage all participants to share their experiences with textbooks, both good and bad, and to ask questions about the publication process and the writing process. Our conversation will be open to discussion of the future of textbooks, open educational resources, and textbook affordability initiatives
Abstract P1-06-06: Evidence for tumor heterogeneity and clonal evolution during invasive progression of breast cancer
No full textAbstract
Purpose: Intratumoral heterogeneity is well recognized to be an important driver of treatment resistance and metastasis. We undertook this N of one study to measure the degree of heterogeneity in a single large preinvasive lesion with an invasive component to determine the relationship between tumor heterogeneity, spatial distribution, clonal evolution, and invasive progression.
Methods: We identified a patient with extensive DCIS measuring 7.5 cm, associated with 1.5 cm of an invasive component. We segregated the tumor sample into 32 unique blocks with precise geospatial localization; invasive cancer was identified in 3 of 32 blocks. NGS libraries were made from FFPE derived DNA (20-40ng) for full exome sequencing and hybridization to a 4.8 million element SNP array. All data were analyzed and a phylogentic tree was constructed.
Results: The sequence data was analyzed with Platypus[1] and 3922 somatic mutation sites were found in total. These sites were concatenated into one sequence for each sample. Then a Neighbor-Joining tree was built with a Jukes-Cantor model and 1000 bootstrap replicates using FastME 2.0[2], to assess the reliability of the tree (Figure 1). Phylogenetic analysis revealed that invasive cancer evolved twice, independently. Dense sampling allowed reconstruction of the temporal order of mutations that accumulated in the cell lineage of the invasive cancers. Furthermore, the phylogeny revealed that distant regions may be closely genetically related, the oldest parts of the tumor were in the interior of the tumor, and that the invasive tumors evolved near the oldest parts of the tumor, rather than at the expanding front.
Conclusions: Extensive sampling and sequencing of a single tumor yields important insights about tumor heterogeneity and tumor progression for DCIS to invasive cancer. Foci of invasion were geospatially associated with preinvasive regions of progressively higher mutational load.
1. Rimmer A, Phan H, Mathieson I, Iqbal Z, Twigg SR, Wilkie AO, McVean G, Lunter G, Consortium W: Integrating mapping-, assembly-and haplotype-based approaches for calling variants in clinical sequencing applications. Nature genetics 2014, 46(8):912-918.
2. Lefort V, Desper R, Gascuel O: FastME 2.0: a comprehensive, accurate, and fast distance-based phylogeny inference program. Molecular biology and evolution 2015:msv150.
Citation Format: Ding Y, Marks JR, King LM, Hall AH, Mardis ER, Rodrigo AG, Maley CC, Hwang E-S. Evidence for tumor heterogeneity and clonal evolution during invasive progression of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-06.</jats:p
Abstract P1-05-30: Genomic and microenvironmental intra-tumor heterogeneity in DCIS
No full textAbstract
Intra-tumor heterogeneity drives neoplastic progression by supplying the fuel for natural selection among neoplastic cells. It also complicates screening and treatment of neoplasms. We hypothesize that the degree of intra-tumor heterogeneity in DCIS should predict which tumors are likely to become invasive and metastatic. We initiated a pilot project to test this hypothesis by comparing 9 cases of pure DCIS to 9 cases of DCIS with adjacent invasive disease. For each case, we sequenced the exome from two spatially distinct regions of DCIS as well as normal tissue taken from a lymph node with no tumor involvement. This required the development of new methods to extract high quality sequencing data from small amounts of DNA extracted from FFPE samples. We calculated the genetic divergence between the two tumor regions, defined as percent of the sequenced regions of the genome showing differences between the two samples that had sufficient sequencing coverage and quality scores for confident scoring. We also employed automated imaging analysis to score microenvironmental differences between the two tumor regions. These microenvironmental measures are based on ecological methods for measuring organismal interactions and habitats. We will present initial data on differences in phenotypic and genotypic intra-tumor heterogeneity comparing pure DCIS to DCIS associated with invasive breast cancer. Our methods can be readily translated to large tissue banks of FFPE samples from DCIS.
Citation Format: Fortunato A, King L, Mallo D, Kovacheva V, Yuan Y, Boddy A, Graham T, Aktipis A, Mardis ER, Hall A, Marks JR, Hwang S, Maley CC. Genomic and microenvironmental intra-tumor heterogeneity in DCIS [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-30.</jats:p
Abstract P2-05-01: Whole genome sequencing reveals enrichment of mutations in mucin gene family in breast cancer diagnosed during pregnancy
No full textAbstract
Background
Pregnancy is known to modulate breast cancer (BC) risk. Different reproductive behaviors have been shown to impact not only the risk of developing BC but also the phenotypes of these tumors. Breast cancer diagnosed during pregnancy (BCP) is a rare disease but could serve as a good model to understand how pregnancy modulates BC biology. In this project, we aim to interrogate the effect of pregnancy on the biology of BC by performing whole genome sequencing (WGS) using a unique series of BC patients diagnosed during pregnancy (BCP).
Method
Whole genome sequencing was performed for 35 BCP and 20 non-pregnant controls matched for age and stage with available clinic-pathological data. DNA extracted from primary tumor and matched adjacent normal FFPE tissues was assessed using WGS on Illumina HiSeqXTen platform targeting 60x and 30x coverage for tumor and normal DNA respectively. Briefly, 2x150bp paired end sequence data were generated, cleaned, trimmed and aligned to the reference genome (hg19) using bwa-mem. Somatic mutations were detected using Strelka and annotated using SnpEff. Mutational signatures were extracted using deconstructSigs. Differences on mutational profiles between BCP and case controls were assessed using Wilcoxon test for continuous variables and Fisher exact test for categorical variables.
Result
No difference in clinic-pathological features was observed between BCP and control patients. A median of 10084 and 13829 SNVs and of 26 and 21 indels were identified in the BCP and controls respectively, no significant difference between the two groups being observed (p = 0.703 and p = 0.851). Of interest, a significantly higher number of mutations was found in the BCP as compared to the control group when considering only mutations associated with a deleterious effect (median: 20 vs. 12, p = 0.027). As expected, TP53 and PIK3CA were the most frequently mutated genes both in BCP and control cases without any significant difference between the groups (34.3% vs. 22.2%, p = 0.53 and 20.0% vs. 16.7%, p = 1, respectively). Interestingly, there was a significant enrichment of non-silent mutations in the mucin genes family (MUC2, MUC4, MUC12, MUC16, MUC17, MUC20) in the BCP group: 45.7% of BCP vs. 11.1% of control cases had at least one such mutation (p = 0.015). A similar significant result (45.7% vs. 23.1%, p = 0.034) was found when comparing BCP with BC control cases from the TCGA dataset (selected to have similar age, ER and PR distribution, N = 56). When comparing the distribution of the twelve BC mutational signatures, a borderline significant enrichment with a signature depicting mismatch-repair deficiency (signature 20) was observed in the BCP patients (p = 0.059).
Conclusion
This is the first study reporting the mutational landscape of breast cancer diagnosed during pregnancy using WGS. We found that BCP are associated with a higher number of putative driver mutations including mutations in mucin genes, shown to be implicated in tumorigenesis. Furthermore, BCP were enriched with a mismatch-repair deficiency signature. These results could open new avenues for the development of targeted therapeutic approaches for patients diagnosed with breast cancer during pregnancy.
Citation Format: Nguyen B, Venet D, Desmedt C, Pruneri G, Peccatori F, Mardis ER, Azim HA, Rothé F, Sotiriou C. Whole genome sequencing reveals enrichment of mutations in mucin gene family in breast cancer diagnosed during pregnancy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-01.</jats:p
Abstract GS2-01: Discovery and characterization of an estrogen bound LncRNA in late-Stage breast cancer
No full textAbstract
Breast cancer is the second most common newly diagnosed cancer and the second leading cause of cancer death among women in the United States. Despite the proven benefits of adjuvant endocrine therapy in women with hormone receptor positive breast cancer, relapses still occur over 5 years after initial treatment with endocrine therapy, referred to as late-stage relapse. Currently, the mechanisms of driving late-stage relapse are poorly characterized. To date, breast cancer research has primarily focused on protein-coding genes thereby missing the emerging class of long intergenic non-coding RNAs (lncRNAs) that may serve as critical regulators of relapse. Furthermore, the current understanding of lncRNA function is still in its infancy representing a critical gap for translating lncRNA discoveries into real-world applications to benefit patient care. Several well-described examples indicate that lncRNAs may be master epigenetic regulators in cancer biology through their interactions with proteins regulating target gene expression. Therefore, we hypothesize that lncRNAs may interact with estrogen receptor alpha 1 (ESR1) to regulate genes promoting late-stage relapse. To address this, we performed a transcriptome analysis of tumors from a unique cohort of 24 patients that had late-stage relapse to discover a novel set of lncRNAs, most of which have not yet been characterized. Next, we used RNA Immunoprecipitation coupled with transcriptome sequencing (RIP-Seq) to identify transcripts bound to ESR1 in T47D cells. We discovered 217 lncRNAs bound to ESR1 of which 50 were up-regulated in late-stage breast cancer. We chose to focus the most up-regulated lncRNA in late-stage relapse. Since it is an unannotated lncRNA we will refer to it as 'LAte-Stage relapse ESR1-Bound lncRNA 1', or LASER-1. To further understand the interplay between LASER-1 and ESR1, cells endogenously expressing LASER-1 were subjected to partial digestion to preserve lncRNA and protein interactions. Protected RNA fragments were subsequently immunoprecipitated with ESR1 and quantified by qPCR to reveal specific ESR1 interaction sites within LASER-1. Next, we observed increased expression of LASER-1 in ER+ breast cancer cell lines. Notably, LASER-1 expression was elevated in MCF7 long-term estrogen deprived (MCF7 LTED) cells -- that have amplified ESR1 -- relative to parental MCF7 cells. To demonstrate that LASER-1 promotes oncogenic phenotypes we transiently silenced LASER-1 in two cell lines with high endogenous expression of LASER-1 (including MCF LTED) and observed a decrease in cellular proliferation and invasion. Subsequent gene expression analysis after silencing LASER-1 altered mRNA and protein levels of critical cell cycle genes (i.e., p27). Overall, this is the first study to discover ESR1 bound lncRNAs that may be contributing to late-stage relapse in breast cancer. In the short-term, our ongoing research may lead to significant breakthroughs establishing the importance of LASER-1 as a master regulator in late-stage relapse. In the longer-term, we envision this research may lead to the development of novel therapeutics targeting LASER-1 with the potential for rapid clinical translation.
Citation Format: Maher CA, Silva-Fisher J, Eteleeb A, Tang C, Perou C, Reis-Filho JS, Mardis ER, Ellis MJ. Discovery and characterization of an estrogen bound LncRNA in late-Stage breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-01.</jats:p
Proliferation and estrogen signaling can distinguish patients at risk for early versus late relapse among estrogen receptor positive breast cancers
No full textIntroduction: We examined if a combination of proliferation markers and estrogen receptor (ER) activity could predict early versus late relapses in ER-positive breast cancer and inform the choice and length of adjuvant endocrine therapy.
Methods: Baseline affymetrix gene-expression profiles from ER-positive patients who received no systemic therapy (n = 559), adjuvant tamoxifen for 5 years (cohort-1: n = 683, cohort-2: n = 282) and from 58 patients treated with neoadjuvant letrozole for 3 months (gene-expression available at baseline, 14 and 90 days) were analyzed. A proliferation score based on the expression of mitotic kinases (MKS) and an ER-related score (ERS) adopted from Oncotype DX® were calculated. The same analysis was performed using the Genomic Grade Index as proliferation marker and the luminal gene score from the PAM50 classifier as measure of estrogen-related genes. Median values were used to define low and high marker groups and four combinations were created. Relapses were grouped into time cohorts of 0-2.5, 0-5, 5-10 years.
Results: In the overall 10 years period, the proportional hazards assumption was violated for several biomarker groups indicating time-dependent effects. In tamoxifen-treated patients Low-MKS/Low-ERS cancers had continuously increasing risk of relapse that was higher after 5 years than Low-MKS/High-ERS cancers [0 to 10 year, HR 3.36; p = 0.013]. High-MKS/High-ERS cancers had low risk of early relapse [0-2.5 years HR 0.13; p = 0.0006], but high risk of late relapse which was higher than in the High-MKS/Low-ERS group [after 5 years HR 3.86; p = 0.007]. The High-MKS/Low-ERS subset had most of the early relapses [0 to 2.5 years, HR 6.53; p < 0.0001] especially in node negative tumors and showed minimal response to neoadjuvant letrozole. These findings were qualitatively confirmed in a smaller independent cohort of tamoxifen-treated patients. Using different biomarkers provided similar results.
Conclusions: Early relapses are highest in highly proliferative/low-ERS cancers, in particular in node negative tumors. Relapses occurring after 5 years of adjuvant tamoxifen are highest among the highly-proliferative/high-ERS tumors although their risk of recurrence is modest in the first 5 years on tamoxifen. These tumors could be the best candidates for extended endocrine therapy
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