Large Chromosomal Aberrations Research Articles

The kinetics of blast clearance are associated with copy number alterations in childhood B-cell acute lymphoblastic leukemia

We analyzed the pattern of whole-genome copy number alterations (CNAs) and their association with the kinetics of blast clearance during the induction treatment among 195 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) who displayed intermediate or high levels of minimal residual disease (MRD). Using unsupervised hierarchical clustering of CNAs > 5 Mbp, we dissected three clusters of leukemic samples with distinct kinetics of blast clearance [A – early slow responders (n=105), B – patients with persistent leukemia (n=24), C – fast responders with the low but detectable disease at the end of induction (n=66)] that corresponded with the patients’ clinical features, the microdeletion profile,the presence of gene fusions and patients survival. Low incidence of large CNAs and chromosomal numerical aberrations occurred in cluster A which included ALL samples showing recurrent microdeletions within the genes encoding transcription factors (i.e., IKZF1, PAX5, ETV6, and ERG), DNA repair genes (XRCC3 and TOX), or harboring chromothriptic pattern of CNAs. Low hyperdiploid karyotype with trisomy 8 or hypodiploidy was predominantly observed in cluster B. Whereas cluster C included almost exclusively high-hyperdiploid ALL samples with concomitant mutations in RAS pathway genes. The pattern of CNAs influences the kinetics of leukemic cell clearance and selected aberrations affecting DNA repair genes may contribute to BCP-ALL chemoresistance.

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum.

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma.

Common Variable Immunodeficiency and Neurodevelopmental Delay Due to a 13Mb Deletion on Chromosome 4 Including the NFKB1 Gene: A Case Report

Syndromic immunodeficiencies are a heterogeneous group of inborn errors of immunity that can affect the development of non-immune organs and systems. The genetic basis of these immunodeficiencies is highly diverse, ranging from monogenic defects to large chromosomal aberrations. Antibody deficiency is the most prevalent immunological abnormality in patients with syndromic immunodeficiencies caused by chromosomal rearrangements, and usually manifests as a common variable immunodeficiency (CVID)-like phenotype. Here we describe a patient with a complex phenotype, including neurodevelopmental delay, dysmorphic features, malformations, and CVID (hypogammaglobulinemia, reduced pre-switch and switch memory B cells, and impaired vaccine response). Microarray-based comparative genomic hybridization (aCGH) revealed a 13-Mb deletion on chromosome 4q22.2-q24 involving 53 genes, some of which were related to the developmental manifestations in our patient. Although initially none of the affected genes could be linked to his CVID phenotype, subsequent reanalysis identified NFKB1 haploinsufficiency as the cause. This study underscores the value of periodic reanalysis of unsolved genetic studies performed with high-throughput technologies (eg, next-generation sequencing and aCGH). This is important because of the ongoing incorporation of new data establishing the relationship between genes and diseases. In the present case, NFKB1 had not been associated with human disease at the time aCGH was performed. Eight years later, reanalysis of the genes included in the chromosome 4 deletion enabled us to identify NFKB1 haploinsufficiency as the genetic cause of our patient’s CVID. In the future, other genes included in the deletion may be linked to human disease, allowing us to better define the molecular basis of our patient’s complex clinical phenotype.

LYmphoid NeXt-Generation Sequencing (LYNX) Panel: A Comprehensive Capture-Based Sequencing Tool for the Analysis of Prognostic and Predictive Markers in Lymphoid Malignancies

B-cell neoplasms represent a clinically heterogeneous group of hematologic malignancies with considerably diverse genomic architecture recently endorsed by next-generation sequencing (NGS) studies. Because multiple genetic defects have a potential or confirmed clinical impact, a tendency toward more comprehensive testing of diagnostic, prognostic, and predictive markers is desired. This study introduces the design, validation, and implementation of an integrative, custom-designed, capture-based NGS panel titled LYmphoid NeXt-generation sequencing (LYNX) for the analysis of standard and novel molecular markers in the most common lymphoid neoplasms (chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma). A single LYNX test provides the following: i) accurate detection of mutations in all coding exons and splice sites of 70 lymphoma-related genes with a sensitivity of 5% variant allele frequency, ii) reliable identification of large genome-wide (≥6 Mb) and recurrent chromosomal aberrations (≥300 kb) in at least 20% of the clonal cell fraction, iii) the assessment of immunoglobulin and T-cell receptor gene rearrangements, and iv) lymphoma-specific translocation detection. Dedicated bioinformatic pipelines were designed to detect all markers mentioned above. The LYNX panel represents a comprehensive, up-to-date tool suitable for routine testing of lymphoid neoplasms with research and clinical applicability. It allows a wide adoption of capture-based targeted NGS in clinical practice and personalized management of patients with lymphoproliferative diseases.

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.

Patient-friendly integrated first trimester screening by NIPT and fetal anomaly scan

Many major structural fetal anomalies can be diagnosed by first trimester fetal anomaly scan. NIPT can accurately detect aneuploidies and large chromosomal aberrations in cfDNA in maternal blood plasma. This study shows how a patient-friendly first trimester screening for both chromosomal and structural fetal anomalies in only two outpatient visits can be provided. Genotype-first approach assures not only the earliest diagnosis of trisomy 21 (the most prevalent chromosome aberration), but also completion of the screening at 12–14 weeks. To ensure proper management and avoid unnecessary anxiety abnormal NIPT different from trisomy 21, 18 and 13 should be referred for genetic counseling.

Single-Cell Multi-Omics Reveals Distinct Paths to Survival of Admixed BTKC481 Mutant Vs. Wild-Type Cells in Clinically Progressing Chronic Lymphocytic Leukemia

Mutations in the kinase binding domain of BTK at position C481 are associated with resistance to BTK inhibitor (BTKi) therapy in chronic lymphocytic leukemia (CLL). Nearly half of patients manifesting clinical progression with these alterations exhibit a subclonal burden of resistance. Intriguingly, measured BTKC481 variant allelic fractions (VAF) are commonly lower than 10% [Ahn et al, Blood 2017]. This raises the important question of how BTKC481-mutated (MUT) and wildtype (WT) cells differ in their response to therapeutic challenge, and how low-burden MUT subclones facilitate escape from therapy. While the admixture of MUT and WT cells within the same individual presents an opportunity to directly study the downstream effects of subclonal resistance mutations, these cells cannot be separated through sorting. To overcome this limitation, we utilized Genotyping of Transcriptomes (GoT)-a strategy to jointly capture genotyping of a locus of interest together with whole transcriptomes at the single cell level (Fig. 1A). Importantly, GoT eliminates patient-specific and technical confounders, enabling direct linkage of BTK genotypes to transcriptional phenotypes. We applied GoT to 64,099 CD19+ cells across a cohort of seven patients with clinically progressive CLL found to have low-burden BTKC481 subclones (Fig. 1B). Samples were obtained at the time of progression and were screened for mutations in PLCG2. We genotyped 33.3% of cells, consistent with 34.0% of cells expressing BTK in whole transcriptome data. Clustering of the gene expression profiles showed that MUT and WT clones did not segregate by genotype in most (6/7) patients (Fig. 1C), implying a large degree of transcriptional similarity between MUT and WT cells, and further highlighting the need for multi-omics single-cell sequencing to directly link genotype to phenotype in this context. We note that the single exception with distinct genotypic clustering (CLL06, Fig. 1D) was driven by co-occurring large chromosomal aberrations within the MUT cells, including del(8p), which is associated with BTKi resistance [Burger et al, Nat Comm 2016]. CLL cells are known to cycle between the peripheral blood and protective microenvironmental niches, which is a process modulated by BTKi. We applied CXCR4/CD5 expression as a read out to these migration patterns [Chen et al, Leukemia 2016], and observed that MUT cells were comparatively enriched in CXCR4lowCD5hi CLL, which is associated with recent emigration from the stromal niche and increased BCR signaling (Fig. 1E). Conversely, WT cells were the majority of CXCR4hiCD5low CLL, which is associated with a resting peripheral cellular state and BCR downregulation, suggesting WT quiescence with limited stromal support. To explore the phenotypic changes associated with BTK mutations, we first measured the activity of reported BTKi response expression signatures. WT cells showed higher mean response scores compared to MUT (Fig. 1F), demonstrating that the WT cells preserve BTKi responsiveness. To unbiasedly interrogate transcriptional differences, we applied a set of 336 predefined gene modules representing B-cell cellular functions and processes. Enriched modules in MUT cells implicated restoration of B-cell receptor (BCR) signaling and recovery of CLL cell identity (NF-kB, IRF4, CD40, Fig. 1G), a finding also supported by de novo differential gene expression analysis (Fig. 1H), and gene set enrichment of differentially expressed genes (Fig. 1I). In contrast, WT cells showed increases in modules related to quiescence, hypoxia, cellular stress (HIF-1α, XBP1) and terminal B-cell development (Blimp-1). Intriguingly, changes in gene targets associated with restoration of NOTCH1 and IL4 activity were also observed. As these pathways are impaired in patients responding to BTKi and implicated in resistance [Del Papa et al, CCR 2019; Chen et al, ASH 2019], their re-emergence may reflect a cytoprotective strategy. In summary, we utilized multi-omics single-cell sequencing to identify the distinct transcriptional programs of admixed MUT and WT cells in subclonal BTKC481 progression. MUT cells showed robust escape from BTKi inhibition via increased immune receptor signaling and restoration of CLL cell identity program, while WT cells demonstrated a signature of hypoxia and stress response, with NOTCH1 and IL4 activation implicated as mechanisms of clonal persistence. Disclosures Trisal: Celgene: Current Employment, Current equity holder in publicly-traded company. Gandhi:Celgene: Current Employment, Current equity holder in publicly-traded company. Wiestner:Pharmacyclics LLC, an AbbVie Company, Acerta, Merck, Nurix, Verastem, and Genmab: Research Funding; NIH: Patents & Royalties: NIH. Allan:Acerta, Genentech, Abbvie, Sunesis, Ascentage, Pharmacyclics, Janssen, AstraZeneca, BeiGene: Consultancy; Celgene, Genentech, Janssen, TG Therapeutics: Research Funding; Abbvie, Janssen, AstraZeneca, Pharmacyclics: Honoraria. Furman:Genentech: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy, Research Funding; Acerta: Consultancy; Verastem: Consultancy; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy, Research Funding; Incyte: Consultancy; Janssen: Consultancy, Speakers Bureau; Oncotarget: Consultancy; Loxo Oncology: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.

Prognostic Impact of Pre-Transplant Chromosomal Aberrations Detected By SNP-Array in Patients Undergoing Unrelated Donor Hematopoietic Cell Transplant for Acute Myeloid Leukemia

<h3>Background</h3> High-resolution genome-wide SNP-arrays detect large chromosomal aberrations including copy-neutral loss of heterozygosity (CNLOH), which is not captured in conventional cytogenetics. <h3>Methods</h3> We used SNP-array genotyping data generated by the DISCOVeRY-BMT study to detect chromosomal aberrations in pre-HCT blood samples from 1,974 acute myeloid leukemia (AML) patients. Clinical data and blood samples were available through the Center for International Blood and Marrow Transplant Research. We used Cox proportional hazard model for statistical analyses. <h3>Results</h3> AML patients in this study received unrelated donor HCT between 2000-2011 at a median age of 48.2 (range=0.6-78.0) years. About 53% of the patients were males, 7.4% had therapy-related disease, and 73% were in complete remission. Chromosomal aberrations were detected in 14.4% of the patients (Figure 1). Most common aberrations in patients with advanced disease at HCT (n=536) were copy losses in chr7 (5.8%), chr5 (4.5%), CNLOH in the following: chr13 (4.7%), chr11 (3.9%), or chr17 (3.5%), and copy gain in chr8 (3.5%). Common aberrations in patients in complete remission (n=1438) were copy-loss in chr7 (1.0%), chr5 (0.7%), and CNLOH in chr9 (0.9%) or in chr17 (0.8%) (Figure 2). Among patients with normal cytogenetics at diagnosis (n=572), 27.5% received HCT in advanced disease (of them 8.3% had chr13-CNLOH). In multivariable models including commonly detected aberrations and adjusted for important factors (footnote Figure 3), chr13-CNLOH was associated with an approximately 3-fold increased risk of leukemia relapse and post-HCT mortality in patients with advanced disease (relapse: HR=2.67, 95% CI=1.67-4.26; mortality: HR=2.79, 95% CI=1.81-4.28, p<0.0001). In contrast, chr17-CNLOH was associated with a statistically significant risk of post-HCT mortality in patients with remission (HR=2.85, 95% CI=1.51-5.37, p=0.001); the association with relapse was not statistically significant (HR=1.84, p=0.21). Having aberrations in ≥3 chromosomes was associated with a statistically significant excess risk of mortality in patients with advanced disease (HR=1.59, 95% CI=1.07-2.36, p=0.02) (Figure 3). In patients with normal cytogenetics at diagnosis, chr13-CNLOH pre-HCT refined risk stratification of patients with advanced disease; compared to patients in remission, the HR for OS in patients with advanced disease in the presence or absence of chr13-CNLOH= 5.7 vs. 1.7, respectively, p<0.0001 for both; HR=3.6, p=0.0004 when comparing presence or absence of chr13-CNLOH in patients with advanced disease. <h3>Conclusion</h3> Pre-HCT CNLOH in chr13 or chr17 are associated with inferior post-HCT survival in AML; possibly as a consequence of high risk of post-HCT leukemia relapse. CNLOH in chr13 or chr17 may provide new genomic prognostic markers that guide patient risk stratification and possibly therapeutic options.

Whole-genome analysis uncovers recurrent IKZF1 inactivation and aberrant cell adhesion in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematological malignancy with a poorly understood pathobiology and no effective therapeutic options. Despite a few recurrent genetic defects (eg, single nucleotide changes, indels, large chromosomal aberrations) have been identified in BPDCN, none are disease‐specific, and more importantly, none explain its genesis or clinical behavior. In this study, we performed the first high resolution whole‐genome analysis of BPDCN with a special focus on structural genomic alterations by using whole‐genome sequencing and RNA sequencing. Our study, the first to characterize the landscape of genomic rearrangements and copy number alterations of BPDCN at nucleotide‐level resolution, revealed that IKZF1, a gene encoding a transcription factor required for the differentiation of plasmacytoid dendritic cell precursors, is focally inactivated through recurrent structural alterations in this neoplasm. In concordance with the genomic data, transcriptome analysis revealed that conserved IKZF1 target genes display a loss‐of‐IKZF1 expression pattern. Furthermore, up‐regulation of cellular processes responsible for cell‐cell and cell‐ECM interactions, which is a hallmark of IKZF1 deficiency, was prominent in BPDCN. Our findings suggest that IKZF1 inactivation plays a central role in the pathobiology of the disease, and consequently, therapeutic approaches directed at reestablishing the function of this gene might be beneficial for patients.

Array Comparative Genomic Hybridization Analysis Reveals Significantly Enriched Pathways in Canine Oral Melanoma.

Human Mucosal Melanoma (hMM) is an aggressive neoplasm of neuroectodermal origin with distinctive features from the more common cutaneous form of malignant melanoma (cMM). At the molecular level, hMMs are characterized by large chromosomal aberrations rather than single-nucleotide mutations, typically observed in cMM. Given the scarcity of available cases, there have been many attempts to establish a reliable animal model. In pet dogs, Canine Oral Melanoma (COM) is the most common malignant tumor of the oral cavity, sharing clinical and histological aspects with hMM. To improve the knowledge about COM's genomic DNA alterations, in the present work, formalin-fixed, paraffin-embedded (FFPE) samples of COM from different European archives were collected to set up an array Comparative Genomic Hybridization (aCGH) analysis to estimate recurrent Copy Number Aberrations (CNAs). DNA was extracted in parallel from tumor and healthy fractions and 19 specimens were successfully submitted to labeling and competitive hybridization. Data were statistically analyzed through GISTIC2.0 and a pathway-enrichment analysis was performed with ClueGO. Recurrent gained regions were detected, affecting chromosomes CFA 10, 13 and 30, while lost regions involved chromosomes CFA 10, 11, 22, and 30. In particular, CFA 13 showed a whole-chromosome gain in 37% of the samples, while CFA 22 showed a whole-chromosome loss in 25%. A distinctive sigmoidal trend was observed in CFA 10 and 30 in 25 and 30% of the samples, respectively. Comparative analysis revealed that COM and hMM share common chromosomal changes in 32 regions. MAPK- and PI3K-related genes were the most frequently involved, while pathway analysis revealed statistically significant perturbation of cancer-related biological processes such as immune response, drug metabolism, melanocytes homeostasis, and neo-angiogenesis. The latter is a new evidence of a significant involvement of neovascularization-related pathways in COMs and can provide the rationale for future application in anti-cancer targeted therapies.

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

Chromosomal Instability and Karyotype Correction in Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) are a promising source of cells for regenerative medicine, study of the pathogenesis of various diseases, screening of pharmacological drugs, and other clinical and basic research. However, the maintenance of the genetic stability of the cells during reprogramming, long-term culture, and directed differentiation is necessary for the use of iPSCs. Large chromosomal aberrations affect the quality of iPSCs most adversely, so the review focuses on the analysis of chromosomal abnormalities, including the recurrent aneuploidy; the sources of its origin, the effect of reprogramming, and long-term culture on the accumulation of chromosome aberrations are discussed. Cases of spontaneous correction of the iPSCs karyotype and the possibility of induced correction of the large chromosomal abnormalities by removing or silencing the extra homologue are considered.

Widely metastatic IDH1-mutant glioblastoma with oligodendroglial features and atypical molecular findings: a case report and review of current challenges in molecular diagnostics

BackgroundGliomas with 1p/19q-codeletion as well as mutation of isocitrate dehydrogenase (IDH) 1 are typically characterized as oligodendrogliomas with comparatively good response to treatment with radiation and chemotherapy.Case presentationWe present the case of a 28-year-old man with an IDH1 and TP53 mutant high grade glioma with abnormalities in chromosomes 1 and 19 suggestive of anaplastic oligodendroglioma that rapidly progressed to widespread metastatic disease. Biopsy of a liver lesion confirmed metastasis of the patient’s known brain primary and chemotherapy with temozolomide was initiated. The patient’s rapidly growing tumor burden with fulminant liver failure and tumor lysis led to multisystem failure of which the patient died. Further molecular testing illustrated features more consistent with glioblastoma: multiple large chromosomal aberrations including loss of whole chromosome 1 and 2q; gain/amplification of MYCN, MET, and CDK4; loss of CDKN2A/B; and an ATRX mutation.ConclusionThis case illustrates the importance of higher level molecular diagnostic testing for patients with particularly aggressive disease progression that is not concordant with standard prognoses. Additional data on cases with atypical alterations of 1p and 19q are needed to better understand the distinct biology of these cancers so that appropriate therapies can be developed.

Next Generation Sequencing of Sporadic Vestibular Schwannoma: Necessity of Biallelic NF2 Inactivation and Implications of Accessory Non-NF2 Variants.

1) Describe the genetic alterations discovered in a series of sporadic vestibular schwannomas (VS). 2) Identify if more clinically aggressive variants possess different genetic alterations compared to more indolent-behaving VS. Fresh frozen tumor and matched peripheral blood leukocytes from 23 individuals with sporadic VS were analyzed using whole-exome sequencing, tumor whole transcriptome expression profiling (mRNA-Seq), and tumor mate-pair analysis. Source cases included tumors with fast preoperative growth, giant tumors in young patients, tumors with macrocystic change, recurrent tumors following radiation or microsurgery, and indolent small tumors with minimal or no growth before surgery. Somatic and germ-line alterations of the NF2 gene and beyond the NF2 locus were identified and analyzed using complementing analyses. Biallelic somatic events involving the NF2 gene were discovered in every analyzed tumor specimen with no concurrent NF2 variants identified in matching peripheral blood specimens. Thirteen tumors showed loss of one chromosome 22 (ch22), 4 tumors showed copy-neutral 22q loss of heterozygosity, and 31 unique small variants in the NF2 gene were discovered. Of the latter, 10 were essential splice site, 11 frame shift, 7 stop gain, 2 missense, and 1 in-frame mutation. No other common or recurring NF2 mutations were identified. However, several other notable large chromosomal aberrations were discovered including 2 tumors with loss of a chromosome 21, 3 with loss of an X or Y chromosome, 1 with copy-neutral loss of heterozygosity in chromosome 15, and 1 with loss of 18p and 16q. All of these other major chromosomal abnormalities only occurred in tumors demonstrating a more aggressive phenotype. To date, few studies have used whole-exome sequencing, mate-pair analysis, and RNA-seq to profile genome-wide alterations in sporadic VS. Using high-throughput deep sequencing, "two-hit" alterations in the NF2 gene were identified in every tumor and were not present in peripheral blood supporting that all events were somatic. Type of NF2 gene alteration and accessory mutations outside the NF2 locus may predict phenotypic expression and clinical course.

Abstract 5383: Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor

Abstract Uterine leiomyosarcoma (ULMS) is a highly aggressive smooth muscle tumor with poorly understood pathogenesis. Although ULMS is believed to develop as a de novo tumor, it has been suggested that in some cases uterine leiomyoma (UL) could serve as a precursor lesion for the pathogenesis. ULs are extremely common tumors affecting up to 70% of women. Whereas 90% of ULs are classified as conventional ULs, 10% of the tumors represent subtype histopathologies, some of which have features associated with malignancy. These subtypes include tumors with bizarre nuclei and increased cellularity. In this study, we investigated the genetic and molecular background of one ULMS showing simultaneously mixed histopathologies of UL with bizarre nuclei and cellular UL. The study material consisted of four formalin-fixed paraffin-embedded samples having distinct areas of ULMS, UL with bizarre nuclei, and cellular UL. Fallopian tube sample was used as a normal control. Libraries for exome sequencing were prepared with KAPA Library Preparation Kit and subjected to exome capture with NimbleGen SeqCap EZ System (Roche). Paired-end sequencing was performed with HiSeq2500 (Illumina). SNP genotyping data was produced with Infinium Omni2.5-8 Kit (Illumina). Immunohistochemistry was performed to proteins commonly displaying aberrant expression in UL and/or ULMS. Telomere-specific fluorescence in situ hybridization was conducted to detect alternatively lengthened telomeres (ALT), a feature often associated with ULMS pathogenesis. Exome sequencing data and protein expression analyses revealed that all tumor compartments were negative for UL driver alterations, including MED12 mutations, HMGA2 overexpression and biallelic FH inactivation. Data from exome sequencing indicated that ULMS shared the largest number of variants with UL with bizarre nuclei, including the pathogenic splice site mutation c.994-1G&amp;gt;A in tumor protein 53 (TP53). SNP genotyping data showed identical and large chromosomal aberrations in 6q, 9p, 10q and 19q in ULMS and UL with bizarre nuclei. No such aberrations were shared between ULMS and cellular UL. All tumor compartments shared changes in chromosomes 1q, 2p, 12q, 13q, 17q and 17p. ULMS showed prominent ALT, whereas UL with bizarre nuclei had a weaker ALT phenotype. Cellular UL had normal telomere lengths. In conclusion, these results suggest that the genome, including telomeres, of UL with bizarre nuclei resembles that of ULMS. Large genomic events have probably occurred in UL with bizarre nuclei during tumor evolution, leading to unstable chromosomes, which in turn favored the development of ULMS. Also, all tumor compartments displayed shared genetic variation, suggesting a common evolutionary origin. Understanding the pathogenesis of ULMS and the malignant potential of ULs is clinically highly relevant, as it may improve the diagnosis, prevent malignant transformation and enable the design of new treatments. Citation Format: Netta Mäkinen, Terhi Ahvenainen, Pernilla von Nandelstadh, Ralf Bützow, Pia Vahteristo. Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5383.

Genome instability: a conserved mechanism of ageing?

DNA is the carrier of genetic information and the primary template from which all cellular information is ultimately derived. Changes in the DNA information content through mutation generate diversity for evolution through natural selection but are also a source of deleterious effects. It has since long been hypothesized that mutation accumulation in somatic cells of multicellular organisms could causally contribute to age-related cellular degeneration and death. Assays to detect different types of mutations, from base substitutions to large chromosomal aberrations, have been developed and show unequivocally that mutations accumulate in different tissues and cell types of ageing humans and animals. More recently, next-generation sequencing-based methods have been developed to accurately determine the complete landscape of base substitution mutations in single cells. The first results show that the somatic mutation rate is much higher than the germline mutation rate and that base substitution loads in somatic cells are high enough to potentially affect cellular function.

The Multiple Myeloma Genome Project: Development of a Molecular Segmentation Strategy for the Clinical Classification of Multiple Myeloma

IntroductionSegmenting multiple myeloma (MM) into subgroups with distinct pathogenesis and clinical behavior is important in order to move forward with advancements in therapy and implement a targeted therapy approach. Current technologies have elucidated five major translocation groups, which have a varying effect on prognosis: t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20) along with recurrent copy number changes including deletion of CDKN2C (1p32.3) and TP53 (17p13.1) as well as gain or amplification of 1q21. However, minor translocation and mutational groups are poorly described because sample numbers are limited in small datasets. The availability of multiple sets of high quality mutation data associated with clinical outcomes has provided a unique opportunity in MM whereby clustering mutational data with chromosomal aberrations in the context of gene expression we can develop a molecular classification system to segment the disease into therapeutically meaningful subgroups. The Multiple Myeloma Genome Project (MGP) is a global collaborative initiative that aims to develop a molecular segmentation strategy for MM to develop clinically relevant tests that could improve diagnosis, prognosis, and treatment of patients with MM.Materials and methodsWe have established a set of 2161 patients for which whole exome sequencing (WES; n=1436), Whole Genome Sequencing (WGS; n=708), targeted panel sequencing (n=993) and expression data from RNA-Seq and Gene Expression arrays (n=1497) were available. These data were derived from the Myeloma XI trial (UK), Intergroupe Francophone du Myeloma/Dana-Faber Cancer Institute (MA), The Myeloma Institute (AR) and the Multiple Myeloma Research Foundation (IA1 - IA8).We assembled all data on a secure site and analyzed it using a streamlined and consistent pipeline using state of the art tools. First, BAM were converted to FASTQ using Picard tools v2.1.1 to extract read sequences and base quality scores. Next, all reads were realigned to the human genome assembly hg19 using BWA-mem. Duplicate marking and sorting was performed using Picard tools v2.1.1. For QAQC we use FASTQC and Picard tools. We identified somatic single nucleotide variants and indels with Mutect2 using default parameters. Translocations and large chromosomal aberrations were identified using MANTA and breakdancer and inferred copy number abnormalities and homozygous deletions using Sequenza v2.1.2 and ControlFreeC.ResultsWe have begun to integrate these diverse large genomic datasets with various correlates. Samples were stratified by RNA-seq expression values and WES/WGS to identify the main cytogenetic groups with high concordance. In addition to the main translocation groups, translocations into MAFA, t(8;14), were detected in 1.2% of samples by both RNA-seq and WES/WGS. RNA-seq also detected fusion transcripts, including the known Ig-WHSC1 transcript in t(4;14). However, a proportion of identified in-frame fusion genes involved kinase domains consistent with activation of the Ras/MAPK pathway, which may be clinical targets for therapy. The main recurrent mutations included KRAS and NRAS, and negative regulators of the NF-κB pathway. In addition we identified recurrent copy number abnormalities and examined the interaction of these with mutations. This highlighted the interaction of the recurrent changes at 1p, 13q, and 17p with mutation of genes located within these regions, specifically indicating bi-allelic inactivation of CDKN2C, RB1 and TP53. Using WGS and RNA-Seq data we identified recurrent translocations and fusion genes that can be used to instruct therapy. Based on these data and the presence of homogeneous inactivation of key tumor expressed genes we will present clinically relevant clusters of MM that can form the basis of future risk and molecular targeted trials. Interaction of mutation with expression patterns has identified distinct expression signatures associated with mutational groups.ConclusionsWe have established the largest repository of molecular profiling data in MM along with associated clinical outcome data. Integrated analyses of these are enabling generation of clinically meaningful disease segments associated with differing risk. The MGP intends to build a global network by expanding collaboration with leading MM centers around the world and incorporating additional datasets through current and new collaborations. DisclosuresMavrommatis:Discitis DX: Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Ashby:University of Arkansas for Medical Sciences: Employment. Ortiz:Celgene: Employment. Towfic:Celgene: Employment, Equity Ownership; Immuneering Corp: Equity Ownership. Amatangelo:Celgene: Employment, Equity Ownership. Yu:Celgene: Employment, Equity Ownership. Avet-Loiseau:celgene: Consultancy; janssen: Consultancy; sanofi: Consultancy; amgen: Consultancy. Jackson:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Thakurta:Celgene: Employment, Equity Ownership. Munshi:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Chromosome therapy: Potential strategies for the correction of severe chromosome aberrations.

Large chromosomal aberrations occur commonly during development, resulting in complex and multisystem diseases. In spite of this high frequency, there are currently no means for correcting these disorders due to their complexity and involvement of multiple genes. Recently, several new approaches have been devised that target whole chromosomes in vitro, which are collectively referred to as "Chromosome Therapies." These include silencing and selection for loss of the extra chromosome in trisomies, promotion of euploidy in an aneuploid culture, and forced loss and replacement of a chromosome. Here, we provide a review of Chromosome Therapy, and discuss potential directions for these methods clinically, as well as research applications and cellular models that can be made using these technologies. © 2016 Wiley Periodicals, Inc.

A 220-Gene Targeted Next-Generation Sequencing Panel for the Detection of Variants in Diffuse Large B-Cell Lymphoma, Follicular Lymphoma, and Mantle Cell Lymphoma: Application to a Cohort of 85 Formalin-Fixed Paraffin-Embedded Diffuse Large B-Cell Lymphoma Biopsies

Background: Recent advancements in comprehensive sequencing technologies has enabled researchers to uncover numerous somatic variants within many disease types, but their respective contributions to disease pathogenesis and potential roles as outcome biomarkers are less well-defined. Even fewer studies have integrated somatic mutation events with genomic gain and loss events of respective genes, mostly due to the need to utilize a second platform to robustly assess genomic copy number. To further understand the roles of these genomic aberrations in the disease biology of mature B-cell neoplasms, we developed a next-generation sequencing (NGS) panel of 220 genes for profiling of Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), and secondarily for Chronic Lymphocytic Leukemia (CLL).Methods: The 220 genes represented in the panel included genes based on reported frequency of mutations, targets of therapeutic agents, involvement in disease-enriched pathways, prognostic value, and associated with targeted therapy resistance. Also included were genes that mapped to sites of genomic gain and loss commonly observed in these neoplasms. Of all the genes, 163 were enriched for DLBCL, 144 for FL, 44 for MCL, and 107 for CLL. Probes were designed to cover the coding exons of the 220 genes using a hybrid-capture approach (Nimbledesign, Roche, Inc.), with a total capture size of 0.92 Mb comprising 4086 target regions. Optimization of DNA fragmentation and library preparation was performed for DNA extracted from formalin-fixed paraffin-embedded (FFPE) biopsy material, being the most common intended tissue type and also for which tumor burden could be assessed. Sequencing was performed using a Miseq sequencer (Illumina, Inc.) with the goal to enable detection of somatic variants down to an allele variant frequency (AVF) of 5%. CLC Genomics Workbench (Qiagen, Inc.) was used for alignment and variant calling. Filtering was performed to exclude synonymous, intronic, and untranslated region variants, variants within homopolymer regions (>6), and variants detected below 5% AVF.Results: The average depth of coverage achieved across an initial sample set of over 50 FFPE DLBCL specimens was approximately 350X, with only one sample having more than 10% of the target regions not achieving a coverage of 60X or greater (within 95% of the target region). As part of a proof-of-concept study, we sequenced a cohort of 85 DLBCL FFPE samples from a single institution from patients undergoing biopsy as part of their routine clinical care. Clinical outcome, cell-of-origin subtype (by the Hans method) and genomic gain/loss data by array-CGH were available. All studies were performed with IRB approval. Of the 85 cases, 80 had also been submitted to bi-directional Sanger's sequencing for Exons 5-8 of TP53 to establish accuracy of variant detection. Of cases completed to date, additional TP53 mutations have been detected by NGS due to an AVF below that detectable by Sangers or only detected in one direction. In addition, of the 14 cases completely sequenced and filtered to date, EZH2 variants in four samples have been detected in the GCB subtype while four CD79B variants were in samples of the non-GCB subtype, consistent with previously reported associations. Furthermore, six of the eight samples which harbored an EZH2 or CD79B variants had mutations within the catalytic domain at Y646 (EZH2) or the ITAM residue Y196 (CD79B), both of which have clinical implications. Initial assessment of the ability of the panel to infer copy number showed concordance with aCGH in detecting large chromosomal aberrations. The sensitivity and specificity of this approach is currently being optimized.Conclusion: Overall then, a hybrid-capture NGS panel has been developed, targeting genes enriched for involvement in the most frequent subtypes of mature B-cell neoplasms and optimized for application to FFPE biopsy specimens. While emerging pathogenomic patterns observed in the cases sequenced to date have confirmed previously observed patterns, completion of the NGS analysis of the 85 DLBCL cohort will permit more complex integrated analysis of somatic variants and genomic gain/loss with pathologic and clinical outcome data. DisclosuresFriedman:Cancer Genetics Inc.,: Employment, Equity Ownership. Guttapalli:Cancer Genetics, Inc.: Employment, Equity Ownership. Ma:Cancer Genetics, Inc.: Employment, Equity Ownership. Thodima:Cancer Genetics, Inc.: Employment, Equity Ownership. Kamalakaran:Cancer Genetics, Inc.: Employment, Equity Ownership. Houldsworth:Cancer Genetics, Inc.: Employment, Equity Ownership.

The Genomic and Epigenomic Landscapes of Blast Crisis Transformation in Chronic Myeloid Leukemia

The transition from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is characterized by reprogramming of the CML transcriptome (Radich et al. PNAS 2006), and shortened survival. Current models propose genomic instability as causal in BC transformation with enhanced DNA damage and impaired DNA repair inducing genetic mutations (ranging from large chromosomal aberrations to point mutations), altered gene function, and eventually BC transformation (Perrotti et al. JCI 2010). Consistent with this model are the phenomena of BC clonal evolution, and the increased frequency of ABL kinase domain mutations found in BC. Because different mutational processes are associated with distinct cancer-specific mutation signatures (Alexandrov et al. Nature 2013), this model also predicts the existence of a CML-specific mutation signature. In addition, recent work has highlighted the importance of epigenetic alterations in hematologic malignancies (Shih et al., Nat. Rev. Cancer, 2012). However, we lack a complete understanding of the type or frequency of genetic alterations in BC, and the relative contribution of genetic vs. epigenetic events in reprogramming the BC transcriptome.To address these knowledge gaps, we analyzed the CML progression genome, epigenome, and transcriptome in 12 CP/BC sample pairs. Whole-genome sequencing revealed the CML genome to be relatively stable with respect to structural variations, indels, and somatic single nucleotide variants. The average number of nonsynonymous coding mutations per BC genome was 5, placing the BC coding genome in the same mutation frequency range as AML and ALL genomes (Alexandrov et al. Nature 2013). In addition, we identified a novel mutation signature in all CML samples suggesting a CML-specific mutational process. 1175 genes were 'hit' by genomic, mostly copy number, alterations in >1 sample, and included TCR genes and Ikaros (IKZF1) among lymphoid BC pairs. Only 21 recurrently altered genes were affected by somatic SNVs or indels, with resistance-associated ABL1 mutations being commonest.We next used DNA methylation arrays to assess the BC epigenome, and found 20,651 CpG sites (out of 455,187) to be hyper-methylated, and 3225 to be hypo-methylated in BC compared to CP. Combined methylome and transcriptome analysis demonstrated an inverse relationship between methylation and expression changes at a subset of CpG sites enriched at promoters. Genes with increased methylation/decreased expression or decreased methylation/increased expression included those involved in cell cycle control/heme biosynthesis, and molecular mechanisms of cancer/G-protein coupled receptor signaling/MAPK signaling respectively. Unsupervised methylation-based clustering segregated samples into CP, lymphoid BC and myeloid BC groups, recapitulating expression-based clustering, and further supporting a functional role for DNA methylation in BC transcriptional reprogramming.We next performed an integrative analysis by combining the genome, methylome, and transcriptome datasets, and included data from 34 additional CML samples. Top ranking candidate genes included epigenetic modifiers, and hematopoetic differentiation- and stem cell-related genes. Functional analysis of candidate genes and epigenetic processes using genetic and epigenetic drug-based approaches are ongoing.In summary, we conclude that: 1. The genomic and epigenomic landscapes in BC are characterized by a modest number of recurring events in the former, but consistent and striking differences in the latter, 2. The BC methylome is functionally associated with the robust gene expression changes found in BC, and 3. Epigenetic modifier drugs may be of use in reversing the gene expression changes characteristic of BC. DisclosuresChuah:Children International: Honoraria; Novartis: Honoraria; Bristol Meyers Squibb: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau.