Novel gene fusion discovery in Spitz tumours and its relevance in diagnostics.

In addition to morphologic analysis, molecular diagnostic work up of Spitz tumours is often of great value for their accurate diagnosis/classification. Nowadays, next-generation sequencing (NGS) is the predominant screening method in molecular diagnostics. Up to 80% of these melanocytic neoplasms comprise gene fusions as genetic anomalies for which the driver codes for a protein harbouring a kinase domain. However, because of the variety of fusion partners the use of PCR-based targeted enrichment NGS methods is not recommended. We describe a series of four Spitz tumour samples in which distinct gene fusions were detected by hybridisation-based capture NGS (TPM3::ALK, LIMA1::ROS1, LRRFIP2::ROS1 and MYO5A::RET). Two of these fusions are not previously described. All 4 fusions were confirmed by reverse transcription-PCR. These findings demonstrate the need for molecular analysis that can detect unknown fusions in Spitz neoplasms for optimal diagnosis.

Lung proliferation is dependent on the duration not the timepoint of tracheal occlusion in nitrofen rats with diaphragmatic hernia.

OBJECTIVE: Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with congenital diaphragmatic hernia (CDH). Limited data are available regarding the effect of duration versus timepoint of TO. Our objective was to document the impact of TO on lung development in the near-term period in rats with nitrofen-induced CDH. METHOD: Nitrofen was administered on embryonic day (ED)9 and fetal TO was performed on ED18.5, 19, or 20 (term = ED22). Sham-operated and untouched littermates served as controls. Lungs were harvested in 0.5-day steps and only fetuses with a left-sided CDH were included in further analyses. Healthy fetuses provided a reference for normal near-term lung development. RESULTS: Duration of TO in the nitrofen rat model for CDH predicts lung growth in terms of lung-body-weight ratio as well as an increased mRNA level of the proliferation marker Ki67. Longer TO also induced a more complex airway architecture. The timepoint of TO was not predictive of lung growth. CONCLUSION: In the nitrofen rat model of CDH, a longer period of TO leads to enhanced lung growth and more refined airway architecture.

Liquid Biopsy in Early-Stage Lung Cancer: Current and Future Clinical Applications.

Lung cancer remains the leading cause of cancer death worldwide, with the majority of cases diagnosed in an advanced stage. Early-stage disease non-small cell lung cancer (NSCLC) has a better outcome, nevertheless the 5-year survival rates drop from 60% for stage IIA to 36% for stage IIIA disease. Early detection and optimized perioperative systemic treatment are frontrunner strategies to reduce this burden. The rapid advancements in molecular diagnostics as well as the growing availability of targeted therapies call for the most efficient detection of actionable biomarkers. Liquid biopsies have already proven their added value in the management of advanced NSCLC but can also optimize patient care in early-stage NSCLC. In addition to having known diagnostic benefits of speed, accessibility, and enhanced biomarker detection compared to tissue biopsy, liquid biopsy could be implemented for screening, diagnostic, and prognostic purposes. Furthermore, liquid biopsy can optimize therapeutic management by overcoming the issue of tumor heterogeneity, monitoring tumor burden, and detecting minimal residual disease (MRD), i.e., the presence of tumor-specific ctDNA, post-operatively. The latter is strongly prognostic and is likely to become a guidance in the postsurgical management. In this review, we present the current evidence on the clinical utility of liquid biopsy in early-stage lung cancer, discuss a selection of key trials, and suggest future applications.

HLA-I diversity and tumor mutational burden by comprehensive next-generation sequencing as predictive biomarkers for the treatment of non-small cell lung cancer with PD-(L)1 inhibitors.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) improved outcomes in non-small cell lung cancer (NSCLC) patients. We report the predictive utility of human leukocyte antigen class I (HLA-I) diversity and tumor mutational burden (TMB) by comprehensive next-generation sequencing. METHODS: 126 patients were included. TMB high was defined as ≥ 10 nonsynonymous mutations/Mb. Patients exhibit high HLA-I diversity if at least one locus was in the upper 15th percentile for DNA alignment scores. RESULTS: No difference in response rate (RR; 44.4% versus 30.9%; p = 0.1741) or 6-month survival rate (SR; 75.6% versus 77.8%; p = 0.7765) was noted between HLA-I high diversity and low diversity patients. HLA-I high diversity patients did significantly more often exhibit durable clinical benefit (DCB), defined as response or stable disease lasting minimally 6 months (64.4% [29/45] versus 43.2% [35/81]; p = 0.0223). TMB high patients exhibited higher RR (49.1% versus 25.4%; p = 0.0084) and SR 6 months after start ICI (85.5% versus 70.4%; p = 0.0468) than TMB low patients. The proportion of patients with DCB, did not differ significantly between TMB high and low subgroups (60.0% [33/55] versus 42.3% [30/71]; p = 0.0755). Patients with combined dual high TMB and HLA-I diversity had higher RR (63.2% versus 22.2%; p = 0.0033), but SR at 6 months did not differ significantly (84.2% versus 64,4%; p = 0.1536). A significantly higher rate of patients experienced DCB in dual high compared to the dual low group (73.7% [14/19] versus 35.6% [16/45]; p = 0.0052). Triple positive patients (high TMB and HLA-I diversity and PD-L1 positive) had higher RR (63.6% versus 0.0%; p = 0.0047) and SR at 6 months (100% versus 66.7%; p = 0.0378) compared to triple-negative patients. CONCLUSION: HLA-I diversity was able to predict durable clinical benefit in ICI treated NSCLC patients, but failed to confirm as a predictor of response or survival. TMB confirmed as a predictive biomarker.

Diagnostic Validation of a Comprehensive Targeted Panel for Broad Mutational and Biomarker Analysis in Solid Tumors.

The use of targeted Next Generation Sequencing (NGS) for the diagnostic screening of somatic variants in solid tumor samples has proven its high clinical value. Because of the large number of ongoing clinical trials for a multitude of variants in a growing number of genes, as well as the detection of proven and emerging pan-cancer biomarkers including microsatellite instability (MSI) and tumor mutation burden (TMB), the currently employed diagnostic gene panels will become vastly insufficient in the near future. Here, we describe the validation and implementation of the hybrid capture-based comprehensive TruSight Oncology (TSO500) assay that is able to detect single-nucleotide variants (SNVs) and subtle deletions and insertions (indels) in 523 tumor-associated genes, copy-number variants (CNVs) of 69 genes, fusions with 55 cancer driver genes, and MSI and TMB. Extensive validation of the TSO500 assay was performed on DNA or RNA from 170 clinical samples with neoplastic content down to 10%, using multiple tissue and specimen types. Starting with 80 ng DNA and 40 ng RNA extracted from formalin-fixed and paraffine-embedded (FFPE) samples revealed a precision and accuracy >99% for all variant types. The analytical sensitivity and specificity were at least 99% for SNVs, indels, CNVs, MSI, and gene rearrangements. For TMB, only values around the threshold could yield a deviating outcome. The limit-of-detection for SNVs and indels was well below the set threshold of 5% variant allele frequency (VAF). This validated comprehensive genomic profiling assay was then used to screen 624 diagnostic samples, and its success rate for adoption in a clinical diagnostic setting of broad solid tumor screening was assessed on this cohort.

The Dynamics of Nucleotide Variants in the Progression from Low-Intermediate Myeloma Precursor Conditions to Multiple Myeloma: Studying Serial Samples with a Targeted Sequencing Approach.

Multiple myeloma (MM), or Kahler's disease, is an incurable plasma cell (PC) cancer in the bone marrow (BM). This malignancy is preceded by one or more asymptomatic precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). The molecular mechanisms and exact cause of this progression are still not completely understood. In this study, the mutational profile underlying the progression from low-intermediate risk myeloma precursor conditions to MM was studied in serial BM smears. A custom capture-based sequencing platform was developed, including 81 myeloma-related genes. The clonal evolution of single nucleotide variants and short insertions and deletions was studied in serial BM smears from 21 progressed precursor patients with a median time of progression of six years. From the 21 patients, four patients had no variation in one of the 81 studied genes. Interestingly, in 16 of the 17 other patients, at least one variant present in MM was also detected in its precursor BM, even years before progression. Here, the variants were present in the pre-stage at a median of 62 months before progression to MM. Studying these paired BM samples contributes to the knowledge of the evolutionary genetic landscape and provides additional insight into the mutational behavior of mutant clones over time throughout progression.

Whole-genome sequencing reveals progressive versus stable myeloma precursor conditions as two distinct entities.

Multiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n = 15) are characterized by later initiation in the patient's life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

Clinical Validation of Targeted Solid Tumor Profiling.

Large-scale tumor profiling studies have generated massive amounts of data that have been instrumental for the detection of recurrent driver mutations in many tumor types. These driver mutations as well as the concurrent passenger mutations are now being used for a more accurate diagnosis of the tumor and prognosis for the patient. Moreover, therapeutic inhibitors toward specific mutations are already on the market and many clinical trials are ongoing to approve novel therapeutic drugs. The broad-range identification of these somatic mutations is key to this tailored personalized medicine approach, which preferentially has to be performed by a multigene multihotspot method such as massive parallel sequencing, also called next generation sequencing (NGS). The implementation of NGS in molecular diagnostics of tumor profiling however, requires a firm validation to minimize the occurrence of false positives and false negatives, thereby yielding highly accurate and robust clinical data.Here, we describe the different performance characteristics as well as quality metrics that should be analyzed for the robust diagnostic validation of tumor profiling in order to meet the requirements of international standards specific for medical laboratories, such as the ISO15189:2012 standard. These metrics include assays that assess the precision, limit of detection, accuracy, sensitivity, specificity, and robustness of the entire workflow from DNA enrichment up to the final report.

Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes: An Initiative of the Belgian ComPerMed Expert Panel.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software.

Archival May-Grünwald-Giemsa-Stained Bone Marrow Smears Are an Eligible Source for Molecular DNA Research.

Biobanking is increasingly important in studying complex heterogeneous diseases. Therefore, it is essential to ensure the sample quality after long-term storage for reliable downstream analyses. The Clinical Biobank of the Jessa Hospital and the University Biobank Limburg (UBiLim) hold a continuously growing collection of hematological samples, including May-Grünwald-Giemsa (MGG)- and Perls' Prussian Blue (PPB)-stained bone marrow (BM) smears, stored at room temperature (RT) for up to 20 years. In this study, we investigated the effect of short- and long-term storage on the quality of DNA and RNA extracted from these BM smears to assess their fitness-for-purpose in downstream molecular applications, including agarose gel electrophoresis, bio-analyzer analysis, quantitative polymerase chain reaction (qPCR), and targeted next-generation sequencing (NGS). The RNA quality was very low for all samples, independent of storage time or staining method. The DNA from PPB-stained BM smears was already degraded after 1 year of storage and correspondingly could not be used for reliable downstream molecular analysis. In contrast, DNA extracted from MGG-stained BM smears stored for up to 10 years was able to generate high-quality data in qPCR and targeted NGS analyses. Longer storage periods (>15 years) of these samples revealed a high degree of degradation and a significant amount of DNA transitions and transversions. In conclusion, the DNA extracted from archival MGG-stained BM smears with a storage time up to at least 10 years was qualitatively good and fit for downstream analysis, including targeted NGS. This indicates that these samples are an eligible source for molecular DNA research and for studying complex diseases.

Mapping the landscape of tandem repeat variability by targeted long read single molecule sequencing in familial X-linked intellectual disability.

BACKGROUND: The etiology of more than half of all patients with X-linked intellectual disability remains elusive, despite array-based comparative genomic hybridization, whole exome or genome sequencing. Since short read massive parallel sequencing approaches do not allow the detection of larger tandem repeat expansions, we hypothesized that such expansions could be a hidden cause of X-linked intellectual disability. METHODS: We selectively captured over 1800 tandem repeats on the X chromosome and characterized them by long read single molecule sequencing in 3 families with idiopathic X-linked intellectual disability. RESULTS: In male DNA samples, full tandem repeat length sequences were obtained for 88-93% of the targets and up to 99.6% of the repeats with a moderate guanine-cytosine content. Read length and analysis pipeline allow to detect cases of > 900 bp tandem repeat expansion. In one family, one repeat expansion co-occurs with down-regulation of the neighboring MIR222 gene. This gene has previously been implicated in intellectual disability and is apparently linked to FMR1 and NEFH overexpression associated with neurological disorders. CONCLUSIONS: This study demonstrates the power of single molecule sequencing to measure tandem repeat lengths and detect expansions, and suggests that tandem repeat mutations may be a hidden cause of X-linked intellectual disability.

Publisher Correction: Impaired oxidative stress response characterizes HUWE1-promoted X-linked intellectual disability.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Prognostic Biomarkers in the Progression From MGUS to Multiple Myeloma: A Systematic Review.

Multiple myeloma (MM), characterized by malignant plasma cells in the bone marrow, is consistently preceded by asymptomatic premalignant stage monoclonal gammopathy of undetermined significance (MGUS). These MGUS patients have an annual risk of 1% to progress to MM. Clinical, imaging, and genomic (genetic and epigenetic) factors were identified, whose presence increased the risk of progression from MGUS to MM. In this systematic review we summarize the currently identified clinical, imaging, and genomic biomarkers suggested to increase the progression risk or shown to be differentially expressed/present between both cohorts of patients. Despite the wide range of proposed markers, there are still no reliable biomarkers to individually predict which MGUS patient will progress to MM and which will not. Research on biomarkers in the progression from MGUS to MM will give more insight in the unknown pathogenesis of this hematological malignancy. This would improve research by elucidating new pathways and potential therapeutic targets as well as clinical management by closer follow-up and earlier treatment of high-risk MGUS patients.

Impaired oxidative stress response characterizes HUWE1-promoted X-linked intellectual disability.

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID.

Identification of Intellectual Disability Genes in Female Patients with a Skewed X-Inactivation Pattern.

Intellectual disability (ID) is a heterogeneous disorder with an unknown molecular etiology in many cases. Previously, X-linked ID (XLID) studies focused on males because of the hemizygous state of their X chromosome. Carrier females are generally unaffected because of the presence of a second normal allele, or inactivation of the mutant X chromosome in most of their cells (skewing). However, in female ID patients, we hypothesized that the presence of skewing of X-inactivation would be an indicator for an X chromosomal ID cause. We analyzed the X-inactivation patterns of 288 females with ID, and found that 22 (7.6%) had extreme skewing (>90%), which is significantly higher than observed in the general population (3.6%; P = 0.029). Whole-exome sequencing of 19 females with extreme skewing revealed causal variants in six females in the XLID genes DDX3X, NHS, WDR45, MECP2, and SMC1A. Interestingly, variants in genes escaping X-inactivation presumably cause both XLID and skewing of X-inactivation in three of these patients. Moreover, variants likely accounting for skewing only were detected in MED12, HDAC8, and TAF9B. All tested candidate causative variants were de novo events. Hence, extreme skewing is a good indicator for the presence of X-linked variants in female patients.

Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors.

The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.

Increased STAG2 dosage defines a novel cohesinopathy with intellectual disability and behavioral problems.

Next generation genomic technologies have made a significant contribution to the understanding of the genetic architecture of human neurodevelopmental disorders. Copy number variants (CNVs) play an important role in the genetics of intellectual disability (ID). For many CNVs, and copy number gains in particular, the responsible dosage-sensitive gene(s) have been hard to identify. We have collected 18 different interstitial microduplications and 1 microtriplication of Xq25. There were 15 affected individuals from 6 different families and 13 singleton cases, 28 affected males in total. The critical overlapping region involved the STAG2 gene, which codes for a subunit of the cohesin complex that regulates cohesion of sister chromatids and gene transcription. We demonstrate that STAG2 is the dosage-sensitive gene within these CNVs, as gains of STAG2 mRNA and protein dysregulate disease-relevant neuronal gene networks in cells derived from affected individuals. We also show that STAG2 gains result in increased expression of OPHN1, a known X-chromosome ID gene. Overall, we define a novel cohesinopathy due to copy number gain of Xq25 and STAG2 in particular.

Nxf7 deficiency impairs social exploration and spatio-cognitive abilities as well as hippocampal synaptic plasticity in mice.

Nuclear RNA export factors (NXF) are conserved in all metazoans and are deemed essential for shuttling RNA across the nuclear envelope and other post-transcriptional processes (such as mRNA metabolism, storage and stability). Disruption of human NXF5 has been implicated in intellectual and psychosocial disabilities. In the present report, we use recently described Nxf7 knockout (KO) mice as an experimental model to analyze in detail the behavioral consequences of clinical NXF5 deficiency. We examined male Nxf7 KO mice using an extended cognitive and behavioral test battery, and recorded extracellular field potentials in the hippocampal CA1 region. We observed various cognitive and behavioral changes including alterations in social exploration, impaired spatial learning and spatio-cognitive abilities. We also defined a new experimental paradigm to discriminate search strategies in Morris water maze and showed significant differences between Nxf7 KO and control animals. Furthermore, while we observed no difference in a nose poke suppression in an conditioned emotional response (CER) protocol, Nxf7 KO mice were impaired in discriminating between differentially reinforced cues in an auditory fear conditioning protocol. This distinct neurocognitive phenotype was accompanied by impaired hippocampal Long-term potentiation (LTP), while long-term depression (LTD) was not affected by Nxf7 deficiency. Our data demonstrate that disruption of murine Nxf7 leads to behavioral phenotypes that may relate to the intellectual and social deficits in patients with NXF5 deficiency.

Microdeletion of the escape genes KDM5C and IQSEC2 in a girl with severe intellectual disability and autistic features.

Intellectual disability (ID) is a very heterogeneous disorder with over 100 ID genes located on the X chromosome alone. Of these, KDM5C and IQSEC2 are located adjacent to each other at the Xp11.22 locus. While mutations in either of these genes are associated with severe ID in males, female carriers are mostly unaffected. Here, we report on a female patient with severe ID and autistic features carrying a de novo 0.4 Mb deletion containing six coding genes including KDM5C and IQSEC2. X-inactivation analysis revealed skewing in a lymphocyte-derived cell line from this patient with preferential inactivation of the mutant X chromosome. As the brain-expressed KDM5C and IQSEC2 genes escape X-inactivation, deletion of these alleles could still be detrimental despite skewing of X-inactivation. Indeed, mutations in either of both genes have been reported in a few female ID patients. Expression analysis in the patients' cell line revealed decreased KDM5C mRNA levels compared to female controls. IQSEC2 levels could not be compared due to very low expression in blood. Overall, our data suggest that heterozygous loss-of-function of the escape genes KDM5C and/or IQSEC2 can contribute to severe ID in female patients and should be taken into account in diagnostics.

Novel microduplications at Xp11.22 including HUWE1: clinical and molecular insights into these genomic rearrangements associated with intellectual disability.

Recently, we defined a minimal overlapping region for causal Xp11.22 copy number gains in males with intellectual disability (ID), and identified HECT, UBA and WWE domain-containing protein-1 (HUWE1) as the primary dosage-sensitive gene, whose overexpression leads to ID. In the present study, we used this minimal interval to search for HUWE1 copy number variations by quantitative polymerase chain reaction in a large cohort of Brazilian males with idiopathic ID. We detected two unrelated sporadic individuals with syndromic ID carrying unique overlapping duplications encompassing HUWE1. Breakpoint junction analysis showed a simple tandem duplication in the first patient, which has probably arisen by microhomology-mediated break-induced repair mechanism. In the second patient, the rearrangement is complex having an insertion of an intrachromosomal sequence at its junction. This kind of rearrangement has not been reported in Xp11.22 duplications and might have emerged by a replication- or recombination-based mechanism. Furthermore, the presence of infantile seizures in the second family suggests a potential role of increased KDM5C expression on epilepsy. Our findings highlight the importance of microduplications at Xp11.22 to ID, even in sporadic cases, and reveal new clinical and molecular insight into HUWE1 copy number gains.