Epigenetic control of chromosome-associated lncRNA genes essential for replication and stability.

ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilize a set of distinctive ASAR characteristics that allow for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci result in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Differential Allelic Expression among Long Non-Coding RNAs.

Long non-coding RNAs (lncRNA) comprise a diverse group of non-protein-coding RNAs >200 bp in length that are involved in various normal cellular processes and disease states, and can affect coding gene expression through mechanisms in cis or in trans. Since the discovery of the first functional lncRNAs transcribed by RNA Polymerase II, H19 and Xist, many others have been identified and noted for their unusual transcriptional pattern, whereby expression from one chromosome homolog is strongly favored over the other, also known as mono-allelic or differential allelic expression. lncRNAs with differential allelic expression have been observed to play critical roles in developmental gene regulation, chromosome structure, and disease. Here, we will focus on known examples of differential allelic expression of lncRNAs and highlight recent research describing functional lncRNAs expressed from both imprinted and random mono-allelic expression domains.

The Feasibility of Patient-Specific Circulating Tumor DNA Monitoring throughout Multi-Modality Therapy for Locally Advanced Esophageal and Rectal Cancer: A Potential Biomarker for Early Detection of Subclinical Disease.

As non-operative management (NOM) of esophageal and rectal cancer is becoming more prevalent, blood-biomarkers such as circulating tumor DNA (ctDNA) may provide clinical information in addition to endoscopy and imaging to aid in treatment decisions following chemotherapy and radiation therapy. In this feasibility study, we prospectively collected plasma samples from locally advanced esophageal (n = 3) and rectal cancer (n = 2) patients undergoing multimodal neoadjuvant therapy to assess the feasibility of serial ctDNA monitoring throughout neoadjuvant therapy. Using the Dual-Index Degenerate Adaptor-Sequencing (DIDA-Seq) error-correction method, we serially interrogated plasma cell-free DNA at 28-41 tumor-specific genomic loci throughout therapy and in surveillance with an average limit of detection of 0.016% mutant allele frequency. In both rectal cancer patients, ctDNA levels were persistently elevated following total neoadjuvant therapy with eventual detection of clinical recurrence prior to salvage surgery. Among the esophageal cancer patients, ctDNA levels closely correlated with tumor burden throughout and following neoadjuvant therapy, which was associated with a pathologic complete response in one patient. In this feasibility study, patient- and tumor-specific ctDNA levels correlated with clinical outcomes throughout multi-modality therapy suggesting that serial monitoring of patient ctDNA has the potential to serve as a highly sensitive and specific biomarker to risk-stratify esophageal and rectal cancer patients eligible for NOM. Further prospective investigation is warranted.

Reciprocal monoallelic expression of ASAR lncRNA genes controls replication timing of human chromosome 6.

DNA replication occurs on mammalian chromosomes in a cell-type distinctive temporal order known as the replication timing program. We previously found that disruption of the noncanonical lncRNA genes ASAR6 and ASAR15 results in delayed replication timing and delayed mitotic chromosome condensation of human chromosomes 6 and 15, respectively. ASAR6 and ASAR15 display random monoallelic expression and display asynchronous replication between alleles that is coordinated with other random monoallelic genes on their respective chromosomes. Disruption of the expressed allele, but not the silent allele, of ASAR6 leads to delayed replication, activation of the previously silent alleles of linked monoallelic genes, and structural instability of human chromosome 6. In this report, we describe a second lncRNA gene (ASAR6-141) on human chromosome 6 that when disrupted results in delayed replication timing in cisASAR6-141 is subject to random monoallelic expression and asynchronous replication and is expressed from the opposite chromosome 6 homolog as ASAR6 ASAR6-141 RNA, like ASAR6 and ASAR15 RNAs, contains a high L1 content and remains associated with the chromosome territory where it is transcribed. Three classes of cis-acting elements control proper chromosome function in mammals: origins of replication, centromeres, and telomeres, which are responsible for replication, segregation, and stability of all chromosomes. Our work supports a fourth type of essential chromosomal element, the "Inactivation/Stability Center," which expresses ASAR lncRNAs responsible for proper replication timing, monoallelic expression, and structural stability of each chromosome.

Multiregion exome sequencing of ovarian immature teratomas reveals 2N near-diploid genomes, paucity of somatic mutations, and extensive allelic imbalances shared across mature, immature, and disseminated components.

Immature teratoma is a subtype of malignant germ cell tumor of the ovary that occurs most commonly in the first three decades of life, frequently with bilateral ovarian disease. Despite being the second most common malignant germ cell tumor of the ovary, little is known about its genetic underpinnings. Here we performed multiregion whole-exome sequencing to interrogate the genetic zygosity, clonal relationship, DNA copy number, and mutational status of 52 pathologically distinct tumor components from ten females with ovarian immature teratomas, with bilateral tumors present in five cases and peritoneal dissemination in seven cases. We found that ovarian immature teratomas are genetically characterized by 2N near-diploid genomes with extensive loss of heterozygosity and an absence of genes harboring recurrent somatic mutations or known oncogenic variants. All components within a single ovarian tumor (immature teratoma, mature teratoma with different histologic patterns of differentiation, and yolk sac tumor) were found to harbor an identical pattern of loss of heterozygosity across the genome, indicating a shared clonal origin. In contrast, the four analyzed bilateral teratomas showed distinct patterns of zygosity changes in the right versus left sided tumors, indicating independent clonal origins. All disseminated teratoma components within the peritoneum (including gliomatosis peritonei) shared a clonal pattern of loss of heterozygosity with either the right or left primary ovarian tumor. The observed genomic loss of heterozygosity patterns indicate that diverse meiotic errors contribute to the formation of ovarian immature teratomas, with 11 out of the 15 genetically distinct clones determined to result from nondisjunction errors during meiosis I or II. Overall, these findings suggest that copy-neutral loss of heterozygosity resulting from meiotic abnormalities may be sufficient to generate ovarian immature teratomas from germ cells.

Patient-specific factors influence somatic variation patterns in von Hippel-Lindau disease renal tumours.

Cancer development is presumed to be an evolutionary process that is influenced by genetic background and environment. In laboratory animals, genetics and environment are variables that can largely be held constant. In humans, it is possible to compare independent tumours that have developed in the same patient, effectively constraining genetic and environmental variation and leaving only stochastic processes. Patients affected with von Hippel-Lindau disease are at risk of developing multiple independent clear cell renal carcinomas. Here we perform whole-genome sequencing on 40 tumours from six von Hippel-Lindau patients. We confirm that the tumours are clonally independent, having distinct somatic single-nucleotide variants. Although tumours from the same patient show many differences, within-patient patterns are discernible. Single-nucleotide substitution type rates are significantly different between patients and show biases in trinucleotide mutation context. We also observe biases in chromosome copy number aberrations. These results show that genetic background and/or environment can influence the types of mutations that occur.