Molecular basis of ETV6-mediated predisposition to childhood acute lymphoblastic leukemia.

There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with wild-type ETV6 with potentially dominant-negative effects. Whole-transcriptome and whole-genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy-number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Targeted Next-Generation Sequencing Reveals Exceptionally High Rates of Molecular Driver Mutations in Never-Smokers With Lung Adenocarcinoma.

INTRODUCTION: Historically, high rates of actionable driver mutations have been reported in never-smokers with lung adenocarcinoma (ADC). In the era of modern, comprehensive cancer mutation sequencing, this relationship necessitates a more detailed analysis. METHODS: All Mount Sinai patients between January 1, 2015, and June 1, 2020, with a diagnosis of ADC of any stage with known smoking status who received genomic testing were included. Most patients were analyzed using the Sema4 hotspot panel or the Oncomine Comprehensive Assay version 3 next-generation sequencing (NGS) panel conducted at Sema4. Patients were considered fully genotyped if they were comprehensively analyzed for alterations in EGFR, KRAS, MET, ALK, RET, ROS1, BRAF, NTRK1-3, and ERBB2, otherwise they were considered partially genotyped. RESULTS: Two hundred and thirty-six never-smokers and 671 smokers met the above criteria. Of the never-smokers, 201 (85%) had a driver mutation with 167 (71%) considered actionable (ie, those with US Food and Drug Administration-approved agents). Among smokers, 439 (65%) had an identified driver mutation with 258 (38%) actionable (P < .0001). When comprehensively sequenced, 95% (70/74) of never-smokers had a driver mutation with 78% (58/74) actionable; whereas, for smokers, 75% (135/180) had a driver with only 47% (74/180) actionable (P < .0001). Within mutations groups, EGFR G719X and KRAS G12Cs were more common to smokers. For stage IV patients harboring EGFR-mutant tumors treated with EGFR-directed therapies, never-smokers had significantly improved OS compared to smokers (hazard ratio = 2.71; P = .025). In multivariable analysis, Asian ancestry and female sex remained significant predictors of (1) OS in stage IV patients and (2) likelihood of harboring a receptor of fusion-based driver. CONCLUSION: Comprehensive NGS revealed driver alterations in 95% of never-smokers, with the majority having an associated therapy available. All efforts should be exhausted to identify or rule out the presence of an actionable driver mutation in all metastatic lung ADC.

Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE): a cloud-based platform for curating and classifying germline variants.

Variant interpretation in the era of massively parallel sequencing is challenging. Although many resources and guidelines are available to assist with this task, few integrated end-to-end tools exist. Here, we present the Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE), a web- and cloud-based platform for annotation, identification, and classification of variations in known or putative disease genes. Starting from a set of variants in variant call format (VCF), variants are annotated, ranked by putative pathogenicity, and presented for formal classification using a decision-support interface based on published guidelines from the American College of Medical Genetics and Genomics (ACMG). The system can accept files containing millions of variants and handle single-nucleotide variants (SNVs), simple insertions/deletions (indels), multiple-nucleotide variants (MNVs), and complex substitutions. PeCanPIE has been applied to classify variant pathogenicity in cancer predisposition genes in two large-scale investigations involving >4000 pediatric cancer patients and serves as a repository for the expert-reviewed results. PeCanPIE was originally developed for pediatric cancer but can be easily extended for use for nonpediatric cancers and noncancer genetic diseases. Although PeCanPIE's web-based interface was designed to be accessible to non-bioinformaticians, its back-end pipelines may also be run independently on the cloud, facilitating direct integration and broader adoption. PeCanPIE is publicly available and free for research use.

Impact of body mass index on the efficacy of aromatase inhibitors in patients with metastatic breast cancer.

PURPOSE: Higher levels of estrogen in obese patients may lead to incomplete inhibition by aromatase inhibitors (AIs). The aim of this study was to determine the impact of body mass index (BMI) on efficacy of AIs in patients with metastatic hormone receptor (HR)-positive breast cancer (BC). METHODS: We performed a retrospective chart review of all female patients with metastatic HR-positive BC on an AI in first- or second-line settings and seen at our academic institution between 2001 and 2020. The primary endpoint was progression-free survival (PFS), defined as the time from start of AI to disease progression or death from any cause. RESULTS: We identified 219 patients who had received an AI in the first- or second-line settings for metastatic HR-positive BC and with documented information on BMI. Of the 219 patients, 56% (123) had a low BMI (defined as < 27 kg/m2) and 44% (96) had a high BMI (≥ 27 kg/m2). The median PFS was 21.9 months (95% CI 14.5 to 28.4) in the low BMI group versus 20.2 months (95% CI 14.3 to 27.5) in the high BMI group (p = 0.73). CONCLUSION: While BMI influences efficacy of AIs in the adjuvant setting, our results suggest that in the metastatic setting, BMI may not impact the efficacy of AIs. This discrepancy could be due to other differences in disease characteristics that make complete aromatase inhibition more important in the adjuvant setting when disease burden is the lowest.

Identifying modules of cooperating cancer drivers.

Identifying cooperating modules of driver alterations can provide insights into cancer etiology and advance the development of effective personalized treatments. We present Cancer Rule Set Optimization (CRSO) for inferring the combinations of alterations that cooperate to drive tumor formation in individual patients. Application to 19 TCGA cancer types revealed a mean of 11 core driver combinations per cancer, comprising 2-6 alterations per combination and accounting for a mean of 70% of samples per cancer type. CRSO is distinct from methods based on statistical co-occurrence, which we demonstrate is a suboptimal criterion for investigating driver cooperation. CRSO identified well-studied driver combinations that were not detected by other approaches and nominated novel combinations that correlate with clinical outcomes in multiple cancer types. Novel synergies were identified in NRAS-mutant melanomas that may be therapeutically relevant. Core driver combinations involving NFE2L2 mutations were identified in four cancer types, supporting the therapeutic potential of NRF2 pathway inhibition. CRSO is available at https://github.com/mikekleinsgit/CRSO/.

Analysis of Real-World Data to Investigate the Impact of Race and Ethnicity on Response to Programmed Cell Death-1 and Programmed Cell Death-Ligand 1 Inhibitors in Advanced Non-Small Cell Lung Cancers.

BACKGROUND: Racial disparities among clinical trial participants present a challenge to assess whether trial results can be generalized into patients representing diverse races and ethnicities. The objective of this study was to evaluate the impact of race and ethnicity on treatment response in patients with advanced non-small cell lung cancer (aNSCLC) treated with programmed cell death-1 (PD-1) or programmed cell death-ligand 1 (PD-L1) inhibitors through analysis of real-world data (RWD). MATERIALS AND METHODS: A retrospective cohort study of 11,138 patients with lung cancer treated at hospitals within the Mount Sinai Health System was performed. Patients with confirmed aNSCLC who received anti-PD-1/PD-L1 treatment were analyzed for clinical outcomes. Our cohort included 249 patients with aNSCLC who began nivolumab, pembrolizumab, or atezolizumab treatment between November 2014 and December 2018. Time-to-treatment discontinuation (TTD) and overall survival (OS) were the analyzed clinical endpoints. RESULTS: After a median follow-up of 14.8 months, median TTD was 7.8 months (95% confidence interval, 5.4-not estimable [NE]) in 75 African American patients versus 4.6 (2.4-7.2) in 110 White patients (hazard ratio [HR], 0.63). Median OS was not reached (18.4-NE) in African American patients versus 11.6 months (9.7-NE) in White patients (HR, 0.58). Multivariable Cox regression conducted with potential confounders confirmed longer TTD (adjusted HR, 0.65) and OS (adjusted HR, 0.60) in African American versus White patients. Similar real-world response rate (42.6% vs. 43.5%) and disease control rate (59.6% vs. 56.5%) were observed in the African American and White patient populations. Further investigation revealed the African American patient group had lower incidence (14.7%) of putative hyperprogressive diseases (HPD) upon anti-PD-1/PD-L1 treatment than the White patient group (24.5%). CONCLUSION: Analysis of RWD showed longer TTD and OS in African American patients with aNSCLC treated with anti-PD-1/PD-L1 inhibitors. Lower incidence of putative HPD is a possible reason for the favorable outcomes in this patient population. IMPLICATIONS FOR PRACTICE: There is a significant underrepresentation of minority patients in randomized clinical trials, and this study demonstrates that real-world data can be used to investigate the impact of race and ethnicity on treatment response. In retrospective analysis of patients with advanced non-small cell lung cancer treated with programmed cell death-1 or programmed cell death-ligand 1 inhibitors, African American patients had significantly longer time-to-treatment discontinuation and longer overall survival. Analysis of real-world data can yield clinical insights and establish a more complete picture of medical interventions in routine clinical practice.

RNAIndel: discovering somatic coding indels from tumor RNA-Seq data.

MOTIVATION: Reliable identification of expressed somatic insertions/deletions (indels) is an unmet need due to artifacts generated in PCR-based RNA-Seq library preparation and the lack of normal RNA-Seq data, presenting analytical challenges for discovery of somatic indels in tumor transcriptome. RESULTS: We present RNAIndel, a tool for predicting somatic, germline and artifact indels from tumor RNA-Seq data. RNAIndel leverages features derived from indel sequence context and biological effect in a machine-learning framework. Except for tumor samples with microsatellite instability, RNAIndel robustly predicts 88-100% of somatic indels in five diverse test datasets of pediatric and adult cancers, even recovering subclonal (VAF range 0.01-0.15) driver indels missed by targeted deep-sequencing, outperforming the current best-practice for RNA-Seq variant calling which had 57% sensitivity but with 14 times more false positives. AVAILABILITY AND IMPLEMENTATION: RNAIndel is freely available at https://github.com/stjude/RNAIndel. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Incorporating heterogeneous sampling probabilities in continuous phylogeographic inference - Application to H5N1 spread in the Mekong region.

MOTIVATION: The potentially low precision associated with the geographic origin of sampled sequences represents an important limitation for spatially explicit (i.e. continuous) phylogeographic inference of fast-evolving pathogens such as RNA viruses. A substantial proportion of publicly available sequences is geo-referenced at broad spatial scale such as the administrative unit of origin, rather than more precise locations (e.g. geographic coordinates). Most frequently, such sequences are either discarded prior to continuous phylogeographic inference or arbitrarily assigned to the geographic coordinates of the centroid of their administrative area of origin for lack of a better alternative. RESULTS: We here implement and describe a new approach that allows to incorporate heterogeneous prior sampling probabilities over a geographic area. External data, such as outbreak locations, are used to specify these prior sampling probabilities over a collection of sub-polygons. We apply this new method to the analysis of highly pathogenic avian influenza H5N1 clade data in the Mekong region. Our method allows to properly include, in continuous phylogeographic analyses, H5N1 sequences that are only associated with large administrative areas of origin and assign them with more accurate locations. Finally, we use continuous phylogeographic reconstructions to analyse the dispersal dynamics of different H5N1 clades and investigate the impact of environmental factors on lineage dispersal velocities. AVAILABILITY AND IMPLEMENTATION: Our new method allowing heterogeneous sampling priors for continuous phylogeographic inference is implemented in the open-source multi-platform software package BEAST 1.10. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A composite biomarker of neutrophil-lymphocyte ratio and hemoglobin level correlates with clinical response to PD-1 and PD-L1 inhibitors in advanced non-small cell lung cancers.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have been incorporated into various clinical oncology guidelines for systemic treatment of advanced non-small cell lung cancers (aNSCLC). However, less than 50% (and 20%) of the patients responded to the therapy as a first (or second) line of therapy. PD-L1 immunohistochemistry (IHC) is an extensively studied biomarker of response to ICI, but results from this test have equivocal predictive power. In order to identify other biomarkers that support clinical decision-making around whether to treat with ICIs or not, we performed a retrospective study of patients with aNSCLC who underwent ICI-based therapy in the Mount Sinai Health System between 2014 and 2019. METHODS: We analyzed data from standard laboratory tests performed in patients as a part of the routine clinical workup during treatment, including complete blood counts (CBC) and a comprehensive metabolic panel (CMP), to correlate test results with clinical response and survival. RESULTS: Of 11,138 NSCLC patients identified, 249 had been treated with ICIs. We found associations between high neutrophil-to-lymphocyte ratio (NLR ≥ 5) and poor survival in ICI-treated NSCLC. We further observed that sustained high NLR after initiation of treatment had a more profound impact on survival than baseline NLR, regardless of PD-L1 status. Hazard ratios when comparing patients with NLR ≥ 5 vs. NLR < 5 are 1.7 (p = 0.02), 3.4 (p = 4.2 × 10- 8), and 3.9 (p = 1.4 × 10- 6) at baseline, 2-8 weeks, and 8-14 weeks after treatment start, respectively. Mild anemia, defined as hemoglobin (HGB) less than 12 g/dL was correlated with survival independently of NLR. Finally, we developed a composite NLR and HGB biomarker. Patients with pretreatment NLR ≥ 5 and HGB < 12 g/dL had a median overall survival (OS) of 8.0 months (95% CI 4.5-11.5) compared to the rest of the cohort with a median OS not reached (95% CI 15.9-NE, p = 1.8 × 10- 5), and a hazard ratio of 2.6 (95% CI 1.7-4.1, p = 3.5 × 10- 5). CONCLUSIONS: We developed a novel composite biomarker for ICI-based therapy in NSCLC based on routine CBC tests, which may provide meaningful clinical utility to guide treatment decision. The results suggest that treatment of anemia to elevate HGB before initiation of ICI therapy may improve patient outcomes or the use of alternative non-chemotherapy containing regimens.

The genomic profiling and MAMLD1 expression in human and canines with Cushing's disease.

BACKGROUND: Cushing's disease (CD) is defined as hypercortisolemia caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (corticotroph PA) that afflicts humans and dogs. In order to map common aberrant genomic features of CD between humans and dogs, we performed genomic sequencing and immunostaining on corticotroph PA. METHODS: For inclusion, humans and dog were diagnosed with CD. Whole exome sequencing (WES) was conducted on 6 human corticotroph PA. Transcriptome RNA-Seq was performed on 6 human and 7 dog corticotroph PA. Immunohistochemistry (IHC) was complete on 31 human corticotroph PA. Corticotroph PA were compared with normal tissue and between species analysis were also performed. RESULTS: Eight genes (MAMLD1, MNX1, RASEF, TBX19, BIRC5, TK1, GLDC, FAM131B) were significantly (P < 0.05) overexpressed across human and canine corticotroph PA. IHC revealed MAMLD1 to be positively (3+) expressed in the nucleus of ACTH-secreting tumor cells of human corticotroph PA (22/31, 70.9%), but absent in healthy human pituitary glands. CONCLUSIONS: In this small exploratory cohort, we provide the first preliminary insights into profiling the genomic characterizations of human and dog corticotroph PA with respect to MAMLD1 overexpression, a finding of potential direct impact to CD microadenoma diagnosis. Our study also offers a rationale for potential use of the canine model in development of precision therapeutics.

Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation.

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC).

The detection of acquired copy-number abnormalities (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) in neoplastic disorders by chromosomal microarray analysis (CMA) has significantly increased over the past few years with respect to both the number of laboratories utilizing this technology and the broader number of tumor types being assayed. This highlights the importance of standardizing the interpretation and reporting of acquired variants among laboratories. To address this need, a clinical laboratory-focused workgroup was established to draft recommendations for the interpretation and reporting of acquired CNAs and CN-LOH in neoplastic disorders. This project is a collaboration between the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). The recommendations put forth by the workgroup are based on literature review, empirical data, and expert consensus of the workgroup members. A four-tier evidence-based categorization system for acquired CNAs and CN-LOH was developed, which is based on the level of available evidence regarding their diagnostic, prognostic, and therapeutic relevance: tier 1, variants with strong clinical significance; tier 2, variants with some clinical significance; tier 3, clonal variants with no documented neoplastic disease association; and tier 4, benign or likely benign variants. These recommendations also provide a list of standardized definitions of terms used in the reporting of CMA findings, as well as a framework for the clinical reporting of acquired CNAs and CN-LOH, and recommendations for how to deal with suspected clinically significant germline variants.

Dedifferentiation in SDH-Deficient Gastrointestinal Stromal Tumor: A Report With Histologic, Immunophenotypic, and Molecular Characterization.

One-third of gastrointestinal stromal tumors (GISTs) that lack KIT or PDGFRA mutations show succinate dehydrogenase (SDH) mutations or promoter hypermethylation. Most SDH-deficient GISTs occur in the pediatric, adolescent, or young adult setting and have unique features including predilection for the stomach, multinodular plexiform architecture, epithelioid cytology, prominence of lymphovascular invasion, and predilection for nodal metastasis. Dedifferentiation in GIST is a rare histologic change which may occur de novo or secondary to imatinib therapy and is characterized by abrupt transition of well-differentiated (WD) GIST to a subclonal anaplastic process that shows loss of immunohistochemical marks (CD117, DOG1). We describe the case of a previously healthy 18-year-old man who presented with a large gastric wall mass that contained 2 distinct morphologic populations. The first was WD and characterized by sweeping fascicles of bland spindled cells. This population abruptly transitioned to dedifferentiated (DD) foci composed of large sheets of discohesive cells that displayed a spectrum of rhabdoid and epithelioid morphologies with marked pleomorphism and mitotic activity. Immunohistochemically, the tumor showed variable staining in the 2 components with diffuse DOG-1 and CD117 positivity in the WD component and complete absence in the DD foci. SDH-B staining was lost in both components. Whole exome and transcriptome analysis was performed on tissue from both components and both showed an SDHB mutation (c.286G>A) as well as unique mutational burden and copy number profiles. Herein, we describe the first case of a DD SDH-deficient GIST with morphologic, immunophenotypic, and molecular characterization.

Next-generation sequencing of duplication CNVs reveals that most are tandem and some create fusion genes at breakpoints.

Interpreting the genomic and phenotypic consequences of copy-number variation (CNV) is essential to understanding the etiology of genetic disorders. Whereas deletion CNVs lead obviously to haploinsufficiency, duplications might cause disease through triplosensitivity, gene disruption, or gene fusion at breakpoints. The mutational spectrum of duplications has been studied at certain loci, and in some cases these copy-number gains are complex chromosome rearrangements involving triplications and/or inversions. However, the organization of clinically relevant duplications throughout the genome has yet to be investigated on a large scale. Here we fine-mapped 184 germline duplications (14.7 kb-25.3 Mb; median 532 kb) ascertained from individuals referred for diagnostic cytogenetics testing. We performed next-generation sequencing (NGS) and whole-genome sequencing (WGS) to sequence 130 breakpoints from 112 subjects with 119 CNVs and found that most (83%) were tandem duplications in direct orientation. The remainder were triplications embedded within duplications (8.4%), adjacent duplications (4.2%), insertional translocations (2.5%), or other complex rearrangements (1.7%). Moreover, we predicted six in-frame fusion genes at sequenced duplication breakpoints; four gene fusions were formed by tandem duplications, one by two interconnected duplications, and one by duplication inserted at another locus. These unique fusion genes could be related to clinical phenotypes and warrant further study. Although most duplications are positioned head-to-tail adjacent to the original locus, those that are inverted, triplicated, or inserted can disrupt or fuse genes in a manner that might not be predicted by conventional copy-number assays. Therefore, interpreting the genetic consequences of duplication CNVs requires breakpoint-level analysis.

Gene integrated set profile analysis: a context-based approach for inferring biological endpoints.

The identification of genes with specific patterns of change (e.g. down-regulated and methylated) as phenotype drivers or samples with similar profiles for a given gene set as drivers of clinical outcome, requires the integration of several genomic data types for which an 'integrate by intersection' (IBI) approach is often applied. In this approach, results from separate analyses of each data type are intersected, which has the limitation of a smaller intersection with more data types. We introduce a new method, GISPA (Gene Integrated Set Profile Analysis) for integrated genomic analysis and its variation, SISPA (Sample Integrated Set Profile Analysis) for defining respective genes and samples with the context of similar, a priori specified molecular profiles. With GISPA, the user defines a molecular profile that is compared among several classes and obtains ranked gene sets that satisfy the profile as drivers of each class. With SISPA, the user defines a gene set that satisfies a profile and obtains sample groups of profile activity. Our results from applying GISPA to human multiple myeloma (MM) cell lines contained genes of known profiles and importance, along with several novel targets, and their further SISPA application to MM coMMpass trial data showed clinical relevance.

Avian influenza H5N1 viral and bird migration networks in Asia.

The spatial spread of the highly pathogenic avian influenza virus H5N1 and its long-term persistence in Asia have resulted in avian influenza panzootics and enormous economic losses in the poultry sector. However, an understanding of the regional long-distance transmission and seasonal patterns of the virus is still lacking. In this study, we present a phylogeographic approach to reconstruct the viral migration network. We show that within each wild fowl migratory flyway, the timing of H5N1 outbreaks and viral migrations are closely associated, but little viral transmission was observed between the flyways. The bird migration network is shown to better reflect the observed viral gene sequence data than other networks and contributes to seasonal H5N1 epidemics in local regions and its large-scale transmission along flyways. These findings have potentially far-reaching consequences, improving our understanding of how bird migration drives the periodic reemergence of H5N1 in Asia.

Clear cell sarcoma of kidney involving a horseshoe kidney and harboring EGFR internal tandem duplication.

Clear cell sarcoma of kidney (CCSK) is a rare renal malignancy, previously unreported in horseshoe kidney (HSK). B-cell lymphoma 6 corepressor (BCOR) gene internal tandem duplication (ITD) was identified as a recurrent somatic alteration in approximately 85% of CCSKs. This and the YWHAE-NUTM2B/E fusion, the second most common recurrent molecular alteration in CCSK (10%), are considered to be mutually exclusive. However, there is a subset of CCSKs that do not harbor either the BCOR-ITD or YWHAE-NUTM2 translocation and lack known molecular alterations. Herein, we report the first case of CCSK arising in HSK and harboring epidermal growth factor receptor ITD.

Aberrant chromosome morphology in human cells defective for Holliday junction resolution.

In somatic cells, Holliday junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom's syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday junctions. Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom's syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability. MUS81-EME1 (refs 4-7), SLX1-SLX4 (refs 8-11) and GEN1 (refs 12, 13) also process Holliday junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom's syndrome cells to analyse human cells compromised for the known Holliday junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom's syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom's syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom's syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom's syndrome.

Large inverted duplications in the human genome form via a fold-back mechanism.

Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. For half of the rearrangements in our study, we sequenced at least one breakpoint junction. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted sequences are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a "fold-back" intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the fold-back loop. This process can lead to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes.

Interactive analysis of large cancer copy number studies with Copy Number Explorer.

UNLABELLED: Copy number abnormalities (CNAs) such as somatically-acquired chromosomal deletions and duplications drive the development of cancer. As individual tumor genomes can contain tens or even hundreds of large and/or focal CNAs, a major difficulty is differentiating between important, recurrent pathogenic changes and benign changes unrelated to the subject's phenotype. Here we present Copy Number Explorer, an interactive tool for mining large copy number datasets. Copy Number Explorer facilitates rapid visual and statistical identification of recurrent regions of gain or loss, identifies the genes most likely to drive CNA formation using the cghMCR method and identifies recurrently broken genes that may be disrupted or fused. The software also allows users to identify recurrent CNA regions that may be associated with differential survival. AVAILABILITY AND IMPLEMENTATION: Copy Number Explorer is available under the GNU public license (GPL-3). Source code is available at: https://sourceforge.net/projects/copynumberexplorer/ CONTACT: scott.newman@emory.edu.