Most large structural variants in cancer genomes can be detected without long reads.

Short-read sequencing is the workhorse of cancer genomics yet is thought to miss many structural variants (SVs), particularly large chromosomal alterations. To characterize missing SVs in short-read whole genomes, we analyzed 'loose ends'-local violations of mass balance between adjacent DNA segments. In the landscape of loose ends across 1,330 high-purity cancer whole genomes, most large (>10-kb) clonal SVs were fully resolved by short reads in the 87% of the human genome where copy number could be reliably measured. Some loose ends represent neotelomeres, which we propose as a hallmark of the alternative lengthening of telomeres phenotype. These pan-cancer findings were confirmed by long-molecule profiles of 38 breast cancer and melanoma cases. Our results indicate that aberrant homologous recombination is unlikely to drive the majority of large cancer SVs. Furthermore, analysis of mass balance in short-read whole genome data provides a surprisingly complete picture of cancer chromosomal structure.

Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

Somatic whole genome dynamics of precancer in Barrett's esophagus reveals features associated with disease progression.

While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett's esophagus compared to 40 Barrett's patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett's tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett's versus those who progress to cancer is acquisition and expansion of TP53-/- cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett's esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies.

Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

Integrated mutational landscape analysis of uterine leiomyosarcomas.

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.

Structural variant evolution after telomere crisis.

Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis.

Whole-genome characterization of lung adenocarcinomas lacking the RTK/RAS/RAF pathway.

RTK/RAS/RAF pathway alterations (RPAs) are a hallmark of lung adenocarcinoma (LUAD). In this study, we use whole-genome sequencing (WGS) of 85 cases found to be RPA(-) by previous studies from The Cancer Genome Atlas (TCGA) to characterize the minority of LUADs lacking apparent alterations in this pathway. We show that WGS analysis uncovers RPA(+) in 28 (33%) of the 85 samples. Among the remaining 57 cases, we observe focal deletions targeting the promoter or transcription start site of STK11 (n = 7) or KEAP1 (n = 3), and promoter mutations associated with the increased expression of ILF2 (n = 6). We also identify complex structural variations associated with high-level copy number amplifications. Moreover, an enrichment of focal deletions is found in TP53 mutant cases. Our results indicate that RPA(-) cases demonstrate tumor suppressor deletions and genome instability, but lack unique or recurrent genetic lesions compensating for the lack of RPAs. Larger WGS studies of RPA(-) cases are required to understand this important LUAD subset.

Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs.

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

The evolutionary history of 2,658 cancers.

Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.

Divergent Coupling of 2-Carbonyl-anilines and Diazo-cyclopentanones: Asymmetric Total Synthesis of (+)-Leucomidine A.

The direct coupling of 2-carbonyl-anilines and diazo-cyclopentanones, promoted by a rhodium catalyst and diphenyl phosphate, is reported for the divergent generation of both carbazolones and indolones. The strategy allows for the successful transfer of the substituents/functionality and the chirality of the coupling partners into the functionalized heterocyclic products, thus serving as the strategic basis for natural product synthesis as demonstrated by the concise asymmetric total synthesis of (+)-leucomidine A.

SvABA: genome-wide detection of structural variants and indels by local assembly.

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA's performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs and substantially improves detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (<1000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in ∼4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized (50-300 bp) SVs.

Total Synthesis of (±)-Grandilodine B.

The first total synthesis of the opened-type Kopsia alkaloid grandilodine B is reported. Four stereocenters of this alkaloid, three of them quaternary, are stereoselectively generated by a Diels-Alder reaction, a diastereoselective cyanation of tertiary alcohol, and a facial-selective nitrone 1,3-dipolar cycloaddition.

Changes in soil bacterial community structure as a result of incorporation of Brassica plants compared with continuous planting eggplant and chemical disinfection in greenhouses.

Greenhouse eggplant monocropping in China has contributed to the aggravation of soil-borne diseases, reductions in crop quality and yield, and the degradation of physical and chemical soil properties. Crop rotation is one effective way of alleviating the problems of continuous cropping worldwide; however, few studies have reported changes in soil bacterial community structures and physical and chemical soil properties after Brassica vegetables had been rotated with eggplant in greenhouses. In this experiment, mustard-eggplant (BFN) and oilseed rape-eggplant (BFC) rotations were studied to identify changes in the physicochemical properties and bacterial community structure in soil that was previously subject to monocropping. Samples were taken after two types of Brassica plants incorporated into soil for 15 days to compare with continually planted eggplant (control, CN) and chemical disinfection of soil (CF) in greenhouses. MiSeq pyrosequencing was used to analyze soil bacterial diversity and structure in the four different treatments. A total of 55,129 reads were identified, and rarefaction analysis showed that the soil treatments were equally sampled. The bacterial richness of the BFC treatment and the diversity of the BFN treatment were significantly higher than those of the other treatments. Further comparison showed that the bacterial community structures of BFC and BFN treatments were also different from CN and CF treatments. The relative abundance of several dominant bacterial genera in the BFC and BFN treatments (such as Flavobacteria, Stenotrophomonas, Massilia and Cellvibrio, which played different roles in improving soil fertility and advancing plant growth) was distinctly higher than the CN or CF treatments. Additionally, the total organic matter and Olsen-P content of the BFC and BFN treatments were significantly greater than the CN treatment. We conclude that Brassica vegetables-eggplant crop rotations could provide a more effective means of solving the problems of greenhouse eggplant monocultures.

The Role of ΔFosB on the Pro-survival Effect of PTHrP in Goat Mammary Epithelial Cells.

The mechanism of regulation mammary epithelial cell number in ruminant is not fully understood, but is thought to be dependent on the balance of cell proliferation and cell apoptosis. Parathyroid hormone-related protein (PTHrP) could express in mammary epithelial cells and breast cancer cells, and has been reported to regulate cell survival. Here, we showed that PTHrP induced cell proliferation and increased the expression of CyclinD1 and proliferating cell nuclear antigen (PCNA) in goat mammary epithelial cells (GMEC). PTHrP increased the mRNA levels of anti-apoptosis genes Bcl-2 and Bcl-xl, and protected GMEC from apoptosis. We also found ΔFosB, an alternative splicing of finkel-biskis-jinkins murine osteosarcoma B (fosB), inhibited GMEC apoptosis, and induced cell proliferation with increased Bcl-2/Bax and Bcl-xl/Bax ratios. Interestingly, ΔFosB could further promote the pro-survival effect of PTHrP, and the Bcl-2/Bax and Bcl-xl/Bax ratios showed higher levels. We conclude that the pro-survival role of PTHrP in GMEC may be regulated by ΔFosB.

Regulatory effects of ΔFosB on proliferation and apoptosis of MCF-7 breast cancer cells.

Matrix metalloproteinase-9 (MMP-9) plays a vital role in tumor angiogenesis, cell migration, and invasiveness because it can degrade almost all basement membrane and extracellular matrix components. MMP-9 has been reported in many cancers including breast cancer, lung cancer, and colon cancer. ΔFosB in mammary epithelial cells has been shown to regulate cell proliferation, differentiation, and death. We found that ΔFosB increased the expression of MMP-9 in MCF-7 breast cancer cells. ΔFosB overexpression in MCF-7 cells increased cellular viability and decreased cell apoptosis. SB-3CT, an inhibitor of MMP-9, promoted apoptosis, inhibited cell proliferation, induced cell cycle arrest, and downregulated the expression of antiapoptotic genes Bcl-2 and Bcl-xl in MCF-7 cells. ΔFosB increased the number of MCF-7 cells in G2/M and S phases, upregulated the expression of Bcl-2 and Bcl-xl, and protected MCF-7 cells from apoptosis induced by MMP-9 inhibition. We also found that ΔFosB overexpression in MCF-7 cells inhibited Ca(2+)-induced apoptosis and promoted cell proliferation. Therefore, ΔFosB may be a potential target in breast cancer cell apoptosis by regulating the expression of MMP-9.

Rapid Assembly of Functionalized Hydrodibenzofurans via Semipinacol Rearrangements.

A distinct strategy via unprecedented semipinacol rearrangements for the synthesis of functionalized hydrodibenzofurans is reported. The versatile reactivity of benzofuran-3-one as a building block enabled the convergent coupling of simple starting materials and, thus, allowed for the facile variation of R group and the construction of hydrodibenzofurans with fused rings.

Parathyroid hormone-related protein overexpression protects goat mammary gland epithelial cells from calcium-sensing receptor activation-induced apoptosis.

Normal mammary gland epithelial cells and breast cancer cells express the calcium-sensing receptor (CaSR), which is the master regulator of systemic calcium metabolism. During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. However, very little information is available on the role of CaSR in goat mammary gland epithelial cells (GMECs) apoptosis. In this investigation, the full-length cDNA of CaSR from Xinong Saanen dairy goats was cloned, which contains an open-reading frame of 3,258 bp encoding 1,085 amino acids with a predicted molecular weight of 121.0 kDa and an isoelectric point of 5.65. The amino acid sequence is highly homologous with sheep, and the goat CaSR gene is mapped to chromosome 1. Quantitative real-time PCR suggested that CaSR was predominantly expressed in the heart, kidney and mammary gland. Then, we found the stimulation of CaSR with its activator gadolinium chloride (GdCl3) contributed to increase CaSR mRNA levels in GMECs and simultaneously promoted cell apoptosis, and these effects were abrogated partially by NPS2390 which is an inhibitor of CaSR. We also demonstrated that Ca(2+) increased CaSR mRNA levels and induced GMECs apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from calcium-induced apoptosis, and promoted cell proliferation. In conclusion, these results suggest that PTHrP overexpression protects GMECs from CaSR activation-induced apoptosis.

Efficient assembly of polysubstituted pyrroles via a (3 + 2) cycloaddition/skeletal rearrangement/redox isomerization cascade reaction.

An unprecedented cascade strategy, used in conjunction with a redox isomerization, for the synthesis of 3-allyl pyrroles is reported. In a single step, readily accessible simple starting materials are transformed into highly substituted pyrroles with high efficiency. The products obtained contain allyl substituents, which can be readily elaborated to other useful functional groups. The reaction proceeds through an unusual (3 + 2) cycloaddition/skeletal rearrangement/redox isomerization pathway.