Chromosomal Copy Number Variations Research Articles

Abstract IA005: Tumor cell-based liquid biopsy to characterize prostate cancer

Abstract Circulating Tumor Cells (CTCs) hold the promise of comprehensive yet noninvasive molecular characterization of cancer cells during the course of tumor evolution and progression. To dissect the epigenetic profile of prostate CTCs, we undertook paired DNA methylation and RNA sequencing of single CTCs isolated from blood specimens of patients with prostate cancer. Across the genome, our study identified 40 core Partial Methylation Domains (PMDs) that arise early in tumorigenesis and are shared by all individual CTCs across multiple patients. Key among these is a single genomic locus harboring multiple interferon-inducible genes along with all the CD1 gene family members, implicated in innate immunity. Based on mouse models, we propose that early biallelic silencing of multiple genes residing within a single PMD may contribute to the escape from immune surveillance in prostate and potentially other cancers. Studies of CTC heterogeneity within individual patients are limited by the very small number of cells that can be isolated from standard 10ml blood volumes. To enable such analyses, we applied a new high throughput microfluidic platform, capable of processing an entire leukapheresis product, interrogating 2-3 L of blood volume equivalent. Applying this technology to patients with metastatic prostate cancer, we successfully enriched thousands of CTCs from individual cases, enabling the characterization of intra-patient heterogeneity at the level of cell morphology and marker expression, chromosome copy number variation and exome-wide genotyping, and transcriptionally-defined tumor cell subpopulations. Such high-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies. Citation Format: Daniel A Haber, Shyamala Maheswaran, Hongshan Guo, Joanna Vuille, Shih-Bo Huang, Ben S Wittner, Lecia V Sequist, Richard J Lee, Patricia AR Brunker, David T Miyamoto, Avanish Mishra, Mehmet Toner. Tumor cell-based liquid biopsy to characterize prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA005.

PATH-21. SINGEL CELL TRANSCRIPTOMICS OF SPINAL EPENDYMOMA SUBTYPES IDENTIFIES INTRATUMORAL HETEROGENEITY

Abstract Spinal ependymomas comprise glial neoplasms that are classified by histological and molecular features. Two of the respective types, myxopapillary ependymomas (MPE) and spinal ependymomas (SP-EPN) are largely understudied. For each of these, our group identified clinically relevant subtypes with poorer (MPE-A; SP-EPN-A) and more favorable progression-free survival (MPE-B; SP-EPN-B) employing global methylation and transcriptomic profiling. As cellular origin and pathogenesis of these tumors are largely unknown, therapeutic options for patients with unfavorable subtypes are unavailable. We performed single cell transcriptomic sequencing of 12 formalin-fixed and paraffin-embedded spinal ependymomas including all MPE and SP-EPN subtypes and obtained a median of 10,091 cells per tumor after quality control. Uniform Manifold Approximation and Projection (UMAP) analysis revealed intertumoral heterogeneity as most samples formed separate clusters with tumors of the same subtype located in proximity. The expression of literature-defined neoplastic consensus cell states was examined for each cell of the data set and revealed intratumoral heterogeneity. Tumors expressed predominantly two programs, being a ciliated (ependymal-like) cell state (61% of all neoplastic cells) and the astrocytic state (31 % of all neoplastic cells). Interestingly, third and fourth most abundant states (mesenchymal and hypoxia) were almost exclusively expressed in MPE-A and SP-EPN-A. Furthermore, distinct clones of chromosomal copy number variations were identified across all tumors. These clones can be distinguished in UMAP representation of MPE single cell transcriptomes and to a lesser extend in SP-EPN. Next, we sought to characterize the resemblance of integrated tumor subtypes to normal spinal cord cell populations and observed a high similarity to human spinal cord ependymal cells. All spinal ependymoma subtypes display intratumoral heterogeneity with respect to the transcriptomic cell state resemblance of the neoplastic cells. Together with the transcriptomic similarity to ependymal and astrocytic cells, our data suggest the tumor origin within the astro-ependymal lineage.

Prenatal diagnosis and postnatal follow-up of 15 fetuses with 16p13.11 microduplication syndrome.

The clinical phenotypes of 16p13.11 microduplication syndrome have been extensively reported in previous studies, mostly about adults and children, with limited information available on fetal cases. This study aims to explore the genotype-phenotype correlation of fetuses with 16p13.11 microduplication syndrome and analyze the characteristics of prenatal diagnosis indications and provide clinical information for prenatal and postnatal genetic counseling. We conducted a retrospective analysis of 3,451 pregnant women who underwent invasive prenatal diagnosis for SNP array between January 2018 and December 2022 at the Jinan Maternal and Child Health Hospital. Descriptive statistical analysis was performed on the prenatal diagnosis indications, pedigree analysis, pregnancy outcomes and postnatal follow-up of 15 fetuses with 16p13.11 microduplication syndrome. SNP array revealed that 15 fetuses had duplications in the 16p13.11 region with varying prenatal diagnosis indications. Among the cases, 6/15 exhibited ultrasound abnormalities, 5/15 had abnormal chromosomal copy number variations as indicated by non-invasive prenatal testing (NIPT), one case involved advanced maternal age, and 3/15 had other abnormalities. 16p13.11 microduplication syndrome was closely related to ultrasound abnormalities, especially structural abnormalities and soft marker anomalies (abnormal ultrasonic soft indicators), while the indication of NIPT could improve the detection rate of copy number variations (CNVs) in this region. Only 7/15 fetuses underwent pedigree verification, with one case of de novo 16p13.11 microduplication, and the others inherited from one parent. Pregnancy was terminated in 2/15 cases and the outcome of one case is unknown due to loss to follow-up. Among the remaining cases, only one case exhibited a ventricular septal defect, while another presented with omphalocele. No other obvious abnormalities were reported postnatally. The prenatal phenotypes of fetuses with 16p13.11 microduplication were highly associated with ultrasound abnormalities but lacked specificity. Comprehensive genetic tracing, outcome analysis, and follow-up are essential for providing accurate prenatal and postnatal genetic counseling.

Bovine aortic arch: a potential indicator that may not serve in prenatal diagnosis - a study based on fetal anatomy, genetics, and postnatal clinical outcomes

ObjectiveTo investigate the structural abnormalities, genetic results, and postnatal clinical outcomes of fetuses with bovine aortic arch (Bovine Aortic Arch, BAA) to provide a basis for prenatal counseling and management.MethodsA retrospective analysis was conducted on 216 fetuses diagnosed with bovine aortic arch through prenatal ultrasound screening at the First Affiliated Hospital of Anhui Medical University and the No.901 Hospital of the Joint Service of the People’s Liberation Army from January 2019 to February 2023. Their family history of genetic diseases, prenatal screening results, and postnatal follow-up data were collected. The fetuses were divided into an isolated BAA group (n = 192) and a non-isolated BAA group (n = 24). Chromosomal karyotyping and copy number variation (CNV) testing were conducted, and statistical analysis was performed using SPSS 22.0 software.ResultsOf the 216 fetuses with BAA, 192 were isolated BAA (88.89%), and 24 were non-isolated BAA (11.11%). Among the isolated BAA fetuses, only 1 case (0.52%) had chromosomal karyotype and pathogenic CNV abnormalities. Among the non-isolated BAA fetuses, 4 cases (16.67%) had chromosomal or CNV abnormalities, but the overall risk was low. The postnatal outcomes of isolated BAA fetuses were good (99.48%), while 79.17% of non-isolated BAA fetuses had good postnatal outcomes.ConclusionMost BAA fetuses are isolated, with a very low incidence of chromosomal abnormalities and pathogenic CNVs, and have good postnatal outcomes. The clinical value of isolated BAA is limited, and invasive prenatal diagnosis is not recommended for low-risk populations. Prenatal screening should focus on the risk of concurrent severe structural anomalies and chromosomal abnormalities.

B-213 Including Turner Syndrome Screening in Newborn Sequencing by Using NGS Read Counts to Infer Sex Chromosomes Copy Number: Leveraging Existing Data to Enhance Early Diagnosis

Abstract Background Turner Syndrome (TS) can lead to heart defects, short stature, and premature ovarian failure. Early diagnosis can improve outcomes, but routine screening is not standard, often delaying diagnosis until symptoms prompt karyotype analysis. Newborn NGS sequencing offers potential for early detection of genetic disorders and targets numerous gene regions on autosomes and sex chromosomes. It could be utilized to deduce sexual chromosomal copy number variations, including not only chrX monosomy but also its partial deletions and structural alterations, and chrY, which increases the risk of gonadoblastoma in TS patients. Thus, this study aims to validate a TS screening approach using NGS read count data from newborn sequencing to develop a strategy for early detection. Methods DNA samples with established chromosomal microarray ChrX and ChrY copy-number assessments were used. The cohort included 13 males (46,XY), 10 females (46,XX), 5 TS cases (45,X), 5 mosaic TS cases (2 with chrY, 3 without), 1 TS case with Isochromosome Xq, 8 Klinefelter Syndrome cases (47,XXY), and one case (48,XXYY). For NGS, a focused newborn sequencing panel targeting 388 genes derived from the larger xGEN Inherited Disease Panel (Integrated DNA Technologies) was employed. The larger panel covers 2,968 chrX regions, 53,455 autosomal regions, and 135 chrY regions. Sample processing utilized the xGEN DNA Library Prep and Hybridization Capture (Integrated DNA Technologies) and sequencing on NextSeq-500 (Illumina). The alignment was conducted with Dragen Enrichment v.3.9.5 and read count analysis with Samtools v.1.19.2. ChrX and ChrY copy numbers were assessed by comparing chrX and chrY read count ratios to autosomal read counts. Hierarchical clustering grouped samples based on ratio similarities, aiding result attribution. To identify structural alterations in each arm of chrX, every tested chrX region was analyzed for read count ratio calculation and subjected to circular binary segmentation using the DNAcopy R package. The observed sex chromosome counts were compared to expected results to validate the approach. Results Observed chrX/autosome and chrY/autosome ratio ranges matched the expected sex chromosome counts: 0.99-1.03 and 0.82-1.02 for 46,XY; 1.92-1.97 and 0.00-0.01 for 46,XX; 1.00-1.02 and 0.00-0.00 for 45,X; 1.15-1.46 and 0.00-0.00 for mosaic TS without chrY; 0.98-1.06 and 0.27-0.32 for mosaic TS with chrY; 2.45 and 0.0 for TS with Isochromosome Xq; 1.82-2.01 and 0.87-0.95 for 47,XXY; and 2.02 and 1.96 for 48,XXYY. Hierarchical cluster analysis grouped these values into three branches: one for 48,XXYY, one for 47,XXY, 46,XX, and Isochromosome Xq, and one for all other TS samples and 46,XY. TS and 47,XXY samples were further divided into subbranches from 46,XX and 46,XY. Circular binary segmentation of chrX read counts provided a visual profile of the copy number for each arm of chrX, explaining structural alterations in out-of-pattern chrX dosages, such as 2.45, 1.15, and 1.46. Conclusions There was complete agreement between the NGS read count-based assessment of sex chromosome copy number and the expected results for 46,XY, 46,XX, 45,X, TS with mosaic and structural variants, 47,XXY, and 48,XXYY samples. This suggests that TS can be effectively screened using newborn sequencing NGS data.

Single-cell RNA sequencing explored potential therapeutic targets by revealing the tumor microenvironment of neuroblastoma and its expression in cell death

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in childhood and is closely related to the early development and differentiation of neuroendocrine (NE) cells. The disease is mainly represented by high-risk NB, which has the characteristics of high mortality and difficult treatment. The survival rate of high-risk NB patients is not ideal. In this article, we not only conducted a comprehensive study of NB through single-cell RNA sequencing (scRNA-seq) but also further analyzed cuproptosis, a new cell death pathway, in order to find clinical treatment targets from a new perspective.Materials and MethodsThe Seurat software was employed to process the scRNA-seq data. This was followed by the utilization of GO enrichment analysis and GSEA to unveil pertinent enriched pathways. The inferCNV software package was harnessed to investigate chromosomal copy number variations. pseudotime analyses involved the use of Monocle 2, CytoTRACE, and Slingshot software. CellChat was employed to analyze the intercellular communication network for NB. Furthermore, PySCENIC was deployed to review the profile of transcription factors.ResultUsing scRNA-seq, we studied cells from patients with NB. NE cells exhibited superior specificity in contrast to other cell types. Among NE cells, C1 PCLAF + NE cells showed a close correlation with the genesis and advancement of NB. The key marker genes, cognate receptor pairing, developmental trajectories, metabolic pathways, transcription factors, and enrichment pathways in C1 PCLAF + NE cells, as well as the expression of cuproptosis in C1 PCLAF + NE cells, provided new ideas for exploring new therapeutic targets for NB.ConclusionThe results revealed the specificity of malignant NE cells in NB, especially the key subset of C1 PCLAF + NE cells, which enhanced our understanding of the key role of the tumor microenvironment in the complexity of cancer progression. Of course, cell death played an important role in the progression of NB, which also promoted our research on new targets. The scrutiny of these findings proved advantageous in uncovering innovative therapeutic targets, thereby bolstering clinical interventions.

Prenatal diagnosis of fetuses with 15q11.2 BP1-BP2 microdeletion in the Chinese population: a seven-year single-center retrospective study

BackgroundThe 15q11.2 BP1-BP2 microdeletion syndrome is associated with developmental delays, language impairments, neurobehavioral disorders, and psychiatric complications. The aim of the present study was to provide prenatal and postnatal clinical data for 16 additional fetuses diagnosed with the 15q11.2 BP1-BP2 microdeletion syndrome in the Chinese population.MethodsA total of 5,789 pregnancy women that underwent amniocentesis were enrolled in the present study. Both karyotype analysis and chromosomal microarray analysis (CMA) were conducted on these subjects to detect chromosomal abnormalities and copy number variants (CNVs). Whole exome sequencing (WES) was performed to investigate sequence variants in subjects with clinical abnormalities after birth.ResultsSixteen fetuses with 15q11.2 BP1-BP2 microdeletion were identified in the present study, with a detection rate of 0.28% (16/5,789). The 15q11.2 BP1-BP2 microdeletion fragments ranged from 311.8 kb to 849.7 kb, encompassing the NIPA1, NIPA2, CYFIP1, and TUBGCP5 genes. The follow-up results regarding pregnancy outcomes showed that five cases opted for pregnancy termination, while the remaining cases continued with their pregnancies. Subsequent postnatal follow-up indicated that only one case with the 15q11.2 BP1-BP2 microdeletion displayed neurodevelopmental disorders, demonstrating an incomplete penetrance rate of 9.09% (1/11).ConclusionThe majority of fetuses with the 15q11.2 microdeletion exhibit typical features during early childhood, indicating a low penetrance and mild impact. Nonetheless, pregnancies involving fetuses with the 15q11.2 microdeletion require thorough prenatal counseling. Additionally, enhanced supervision and extended postnatal monitoring are warranted for those who choose to proceed with their pregnancies.

Mosaic variegated aneuploidy in development, ageing and cancer.

Mosaic variegated aneuploidy (MVA) is a rare condition in which abnormal chromosome counts (that is, aneuploidies), affecting different chromosomes in each cell (making it variegated) are found only in a certain number of cells (making it mosaic). MVA is characterized by various developmental defects and, despite its rarity, presents a unique clinical scenario to understand the consequences of chromosomal instability and copy number variation in humans. Research from patients with MVA, genetically engineered mouse models and functional cellular studies have found the genetic causes to be mutations in components of the spindle-assembly checkpoint as well as in related proteins involved in centrosome dynamics during mitosis. MVA is accompanied by tumour susceptibility (depending on the genetic basis) as well as cellular and systemic stress, including chronic immune response and the associated clinical implications.

Genetic analysis of a child with mos 46,X,psu idic(X)(q21.3)[40]/45,X[3]

To explore the correlation between structural chromosomal abnormality and clinical characteristics of a child featuring gonadal dysplasia. A 13-year-old child who was admitted to Lianyungang Maternal and Child Health Care Hospital on February 7, 2023 for primary amenorrhoea and occasional abdominal pain was selected as the study subject. Clinical data of the child was collected, and peripheral blood samples of the child and her parents were collected. G-banding chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out. "Pseudodual centromere isochromosome X" and "psu idic(X)" were used as keywords to search the CNKI, Wanfang and PubMed databases, and the search period was set as from January 1, 2002 to June 1, 2023. Relevant literature on the structural abnormality of X chromosome was searched and analyzed retrospectively. The child has a height of 153 cm and weighed 45 kg. She has no obvious facial dysmorphism. Laboratory tests showed that she had higher FSH and luteinizing hormone, and lower E2. Ultrasonography showed that she had small ovaries and rudimentary uterus. She was found to have a karyotype of 46,X,psu idic(X)(q21.3)[40]/mos 45,X[3], whilst both of her parents had a normal karyotype. CNV-seq showed that she had a 63.27 Mb deletion in Xq21.32q28 and a 91.59 Mb duplication in Xp22.33q21.32 (mosaicism rate = 74%). A total of 11 relevant literature were retrieved. Clinical phenotypes of patients with similar structural chromosomal abnormalities were diverse, which was closely related to the mosaicism rate of the 45,X karyotype and the location of the breaking point. 46,X,psu idic(X)(q21.3)/45,X probably underlay the dysplasia of uterus and ovary and sex hormone abnormalities in this child, while her height was spared. Deletion of Xq21.32q28 is a key factor leading to Turner syndrome-like phenotype such as rudimentary uterus and ovarian dysplasia.

Medulloblastomas Initiated by Homologous Recombination Defects in Mice

Germline mutations of homologous-recombination (HR) genes are among the top contributors to medulloblastomas. A significant portion of human medulloblastomas exhibited genomic signatures of HR defects. We asked whether ablation of Brca2, Palb2, and their related Brca1 and Bccip genes, in the mouse brain can differentially initiate medulloblastomas. We established conditional knockout mouse models of these HR-genes, and a conditional knockdown of Bccip (shBccip-KD). Deletion of any of these genes led to microcephaly and neurological defects, with Brca1- and Bccip- producing the worst. Trp53 co-deletion significantly rescued the microcephaly with Brca1, Palb2, and Brca2 deficiency, but it exhibited limited impact on Bccip- mice. For the first time, inactivation of either Brca1 or Palb2 with Trp53 was found to induce medulloblastomas. Bccip/Trp53 deletions failed, despite that shBccip-CKD was highly penetrative, to induce medulloblastomas. The tumors displayed diverse immunohistochemical features and chromosome copy number variation. While there were widespread upregulations of cell proliferative pathways, most of the tumors expressed biomarkers of the Sonic Hedgehog subgroup. The MBs developed from Brca1-, Palb2-, and Brca2- mice were highly sensitive to a PARP inhibitor, but not the ones from shBccip-CKD mice. Our models recapitulate the spontaneous medulloblastoma development with high penetrance and narrow time-window, providing ideal platforms to test therapeutic agents with the ability to differentiate HR defective and proficient tumors.

Correlation between maternal serum biomarkers and the risk of fetal chromosome copy number variants: a single-center retrospective study.

To explore the association between the concentration of maternal serum biomarkers and the risk of fetal carrying chromosome copy number variants (CNVs). Pregnant women identified as high risk in the second-trimester serological triple screening and underwent traditional amniotic fluid karyotype analysis, along with comparative genomic hybridization array (aCGH)/copy number variation sequencing (CNV-seq), were included in the study. We divided the concentration of serum biomarkers, free beta-human chorionic gonadotropin (fβ-hCG), alpha fetoprotein (AFP) and unconjugated estriol (uE3), into three levels: abnormally low, normal and abnormally high. The prevalence of abnormally low, normal and abnormally high serum fβ-hCG, AFP and uE3 levels in pregnant women with aberrant aCGH/CNV-seq results and normal controls was calculated. Among the 2877 cases with high risk in the second-trimester serological triple screening, there were 98 chromosome abnormalities revealed by karyotype analysis, while 209 abnormalities were detected by aCGH/CNVseq (P＜0.001) . The carrying rate of aberrant CNVs increased significantly when the maternal serum uE3 level was less than 0.4 multiple of median (MoM) of corresponding gestational weeks compared to normal controls, while the carrying rate of aberrant CNVs decreased significantly when the maternal serum fβ-hCG level was greater than 2.5 MoM compared to normal controls. No significant difference was found in the AFP group. Low serum uE3 level (<0.4 MoM) was associated with an increased risk of aberrant CNVs.

Metastatic Leiomyoma With Malignant Transformation Harboring RAB2A-PLAG1 Fusion-A Case Report and Review With Molecular Analysis.

Metastasizing leiomyoma is a rare condition characterized by the development of benign-appearing smooth muscle neoplasms at extrauterine sites in patients with a history of uterine leiomyoma. These lesions occur most commonly in the lung, with the abdominopelvic and mediastinal lymph nodes being other reported sites. Malignant transformation of metastasizing leiomyoma is extremely rare, with only a few cases described in the literature. We describe a case of metastasizing leiomyoma with malignant transformation in a middle-aged Asian lady, who developed pulmonary metastatic foci 12 years after surgical excision of the original uterine leiomyomata. Molecular analysis showed a common RAB2A-PLAG1 fusion gene and identical single nucleotide variants in both tumor foci, with significantly more pronounced segmental chromosomal copy number variations in one focus showing high-grade features. A comprehensive review of the literature lends support to the hypothesis that the original leiomyomata and the metastatic foci are clonally related, with high-grade features being associated with more complex genomic signatures.

Copy Number Profiling Implicates Thin High-Grade Squamous Intraepithelial Lesions as a True Precursor of Cervical Human Papillomavirus-Induced Squamous Cell Cancer

Full-thickness high-grade squamous intraepithelial lesions (HSIL) are precursors of invasive cervical squamous cell carcinoma (SCC). The World Health Organization and Lower Anogenital Squamous Terminology Standardization Project for human papilloma virus (HPV)–associated lesions divide full-thickness HSIL of the cervix into thin HSIL with thickness of 1 to 9 cell layers and the typical full-thickness HSIL of >10 cell layers. Although HPV oncogene transcripts and p16ink4a overexpression, as markers of transforming HPV infection, are detectable in thin HSIL, the biological significance of thin HSIL in cervical carcinogenesis remains poorly understood. To further characterize thin HSIL, we performed a comparative study of chromosomal copy number variations (CNV), an analysis of dysregulated genes present in the segments with CNV, and a generalized genetic complexity calculation for 31 thin HSIL, 31 thick HSIL, 24 microinvasive SCC (pT1a SCC), and 22 highly invasive SCC samples. Thin HSIL share various CNV and specific dysregulated gene pathways with thick HSIL and invasive SCC. Thin HSIL exhibited an average CNV of 11.6% compared with 14.1% for thick HSIL, 15.5% for pT1a SCC, and 26.6% for highly invasive SCC. The CNV included gains at 1q and 3q (40% and 43%, respectively), partial loss of 3p, and loss of chromosomes 11 (18%), 16 (50%), 20 (35%), and 22 (40%). Pathways affected solely in thin HSIL were those enhancing immune evasion and primarily involved the (interleukin) IL6, IL21, and IL23 genes. ILs are transiently upregulated in response to infection and play a crucial role in mounting antitumor T-cell activity. Deregulation reflects an attempt by the HPV to evade the initial immune response of the host. The primary pathways shared by thick HSIL and invasive SCC were interactions between lymphoid and nonlymphoid cells, NOTCH2 signaling, tight junction interactions (primarily of the claudin family), and FGR2 alternative splicing. Our results show that thin HSIL carry similar genetic changes as thick HSIL and SCC, indicating that thin HSIL are true precursor lesions that can progress to thick HSIL and SCC.

O-035 Euploidy’s hidden potential: emerging insights from abnormal fertilized embryos

Abstract Study question Can pregnancies be achieved from abnormally fertilized embryos? Summary answer After conducting parental origin PGTA testing to confirm ploidy status, achieving pregnancy and live births becomes feasible. What is known already A major limitation in IVF is the availability of viable embryos per cycle. Particularly relevant for patients with poor prognosis in response to controlled ovarian stimulation. In these couples, the limited number of oocytes results in a significant reduction of the chance to achieve a live birth. In IVF, zygotes displaying successful extrusion of the 2PB and 2PN are considered “normally fertilized” and cultured further to be used for treatment. In contrast, zygotes showing a single or more than 2PN are deemed abnormally fertilized. However, some can ultimately develop into normal embryos, which may lead to live birth. Study design, size, duration This is a single centre retrospective observational study of 125 blastocysts classified as abnormal through standard fertilization check post-IVF (50 embryos) and ICSI (75 embryos) Embryos were subjected to genetic testing utilizing a validated targeted next-generation sequencing-based PGT-A protocol. The protocol's proven efficacy lies in distinguishing between haploid, diploid and triploid samples, a distinction made possible by the parallel analysis of genotyping data and chromosome copy number variations. Participants/materials, setting, methods The participants in this study were couples who underwent IVF cycles using PGT-A between 2021-2024. Embryos were divided into two groups based on their fertilization method Group A involved blastocysts derived from In vitro fertilization (IVF) = 50. Classified 1PN, micro 3PN, 3PN, and 4PN. Group B blastocysts derived from Intracytoplasmic sperm injection Intracytoplasmic sperm injection (ICSI) =75 Classified 1PN, micro 3PN, 3PN, and 4PN. Main results and the role of chance Group A: Involved embryos resulting from IVF; 54% were diploid, with 15% being euploid from 3PN. From 1PN, 92% were diploid, and 54% were euploid. Micro PN had a 100% diploid rate, of which 33% were euploid. No diploid embryos were reported from 4PN fertilization. Group B: Involved embryos resulting from ICSI; 36% were diploid, and 14% were euploid from 3PN. From 1PN, 53% were diploid, and 40% were euploid. Micro PN had a 100% diploid rate, with 23% being euploid. 4PN had a 50% diploid rate and no euploid embryos. On the other hand, 0.4% (17/4822) of 2PN-classified embryos were reported as haploid, and 1.4% (68/4822) of the 2PN-classified embryos were reported as polyploid. Eighty-nine percent of haploidy and polyploidy in ICSI originated maternally, while 71% of polyploidy in IVF originated paternally and only one embryo was haploid resulted from IVF was maternally originated. Remarkably, three live births were reported: one from 1PN, one from Micro PN, and the third from 3PN. Limitations, reasons for caution The data are observational and were retrospectively analyzed, so unknown confounders could not be assessed. Wider implications of the findings To our knowledge, this is the most extensive cohort study analyzing embryos resulting from abnormal fertilization. It has implications for clinical practice and gives an extra chance for patients, particularly those with a low number of eggs, in response to controlled ovarian stimulation. Trial registration number n/a

Comparison of the diagnostic significance of cerebrospinal fluid metagenomic next-generation sequencing copy number variation analysis and cytology in leptomeningeal malignancy

BackgroundDiagnosis and monitoring of leptomeningeal malignancy remain challenging, and are usually based on neurological, radiological, cerebrospinal fluid (CSF) and pathological findings. This study aimed to investigate the diagnostic performance of CSF metagenomic next-generation sequencing (mNGS) and chromosome copy number variations (CNVs) analysis in the detection of leptomeningeal malignancy.MethodsOf the 51 patients included in the study, 34 patients were diagnosed with leptomeningeal malignancies, and 17 patients were diagnosed with central nervous system (CNS) inflammatory diseases. The Sayk’s spontaneous cell sedimentation technique was employed for CSF cytology. And a well-designed approach utilizing the CSF mNGS-CNVs technique was explored for early diagnosis of leptomeningeal malignancy.ResultsIn the tumor group, 28 patients were positive for CSF cytology, and 24 patients were positive for CSF mNGS-CNVs. Sensitivity and specificity of CSF cytology were 82.35% (95% CI: 66.83-92.61%) and 94.12% (95% CI: 69.24-99.69%). In comparison, sensitivity and specificity of CSF mNGS-CNV were 70.59% (95% CI: 52.33-84.29%) and 100% (95% CI: 77.08-100%). There was no significant difference in diagnostic consistency between CSF cytology and mNGS-CNVs (p = 0.18, kappa = 0.650).ConclusionsCSF mNGS-CNVs tend to have higher specificity compared with traditional cytology and can be used as a complementary diagnostic method for patients with leptomeningeal malignancies.

MDB-105. CONSTRUCTION OF AN INTERACTIVE MEDULLOBLASTOMA VISUALIZATION PORTAL FROM MOLECULAR AND CLINICAL DATA DERIVED FROM THREE MULTI-INSTITUTIONAL PROTOCOLS (ACNS0331, SJMB03, ACNS0332) TO COMPARE, CONTRAST, AND INVESTIGATE COHORTS AS A MEANS TO REFINE THERAPY

Abstract BACKGROUND Medulloblastoma (MB) patients display a heterogeneous mix of molecular and clinical characteristics that influence tumor behavior and survival. We hypothesized that if these multiple characteristics could be incorporated into an organized, searchable, and interactive dataset, this would advance our understanding and improve treatment of this disease. Consequently, we organized multifaceted datasets into an interactive portal equipped with cutting-edge visualization tools. METHODS Clinical and molecular data from the ACNS0331, SJMB03, and ACNS0332 protocols were collated and harmonized. A data dictionary was built to capture demographics, treatment, histology, methylation classification, mutations, chromosomal copy number variations and outcomes. To display the data, a software framework was applied. Five visualization methods were implemented (summary plots, sample view, scatter plots, sample matrix, and survival plots) in an interconnected manner such that these “cross-talk” with each other. RESULTS Data from 898 patients with MB were incorporated: 131 WNT, 151 SHH, 220 G3, 396 G4. The portal features enabled the creation of custom cohorts that could be instantly queried by any of the dictionary terms. For example, cohorts could be customized to include only cases with a mutation of the user’s choice and analyzed by molecular group, gender, age at diagnosis, or treatment given. Survival of patients with SHH-MB harboring TP53 mutations could be compared to patients with WNT-MB harboring TP53 mutations. Moreover, the influence of MB subtype (e.g. G34_I-VIII), focal amplifications (MYC, MYCN) and whole chromosome aberration phenotype on survival could be queried across treatment regimens accounting for metastatic status and/or additional variables of the user’s choice. CONCLUSION The integration of molecular and clinical data from multiple trials into an interactive visualization data portal presents an unprecedented resource for research and clinical practice. This portal facilitates answering highly specific questions about mutational frequency and patient outcome by molecular and clinical risk factors.

Whole-exome-based single nucleotide variants and copy number analysis for prenatal diagnosis of compound heterozygosity of SMPD4.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder. These variants have been identified in a group of children with neurodevelopmental disorders with microcephaly, arthrogryposis, and structural brain anomalies. SMPD4 encodes a sphingomyelinase that hydrolyzes sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes. For the efficient prenatal diagnosis of rare and undiagnosed diseases, the parallel detection of copy number variants (CNVs) and single nucleotide variants using whole-exome analysis is required. A physical examination of the parents was performed. Karyotype and whole-exome analysis were performed for the fetus and the parents. A fetus with microcephaly and arthrogryposis; biallelic null variants (c.387-1G>A; Chr2[GRCh38]: g.130142742_130202459del) were detected by whole-exome sequencing (WES). We have reported for the first time the biallelic loss-of-function mutations in SMPD4 in patients born to unrelated parents in China. WES could replace chromosomal microarray analysis and copy number variation sequencing as a more cost-effective genetic test for detecting CNVs and diagnosing highly heterogeneous conditions.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal ‘M1-like’ phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Prenatal diagnosis and pregnancy outcomes in fetuses with ventriculomegaly.

Genetic etiology plays a critical role in fetal ventriculomegaly (VM). However, the studies on chromosomal copy number variants (CNVs) in fetal VM are limited. This study aimed to investigate the chromosomal CNVs in fetuses with mild to moderate VM, and explore its genotype-phenotype correlation. A total of 242 fetuses with mild to moderate VM detected by prenatal ultrasound were enrolled in our study from October 2018 to October 2022. All cases underwent chromosomal microarray analysis (CMA) and G-banding simultaneously. All VM cases were classified different subgroups according to the maternal age, severity, VM distribution and presence/absence of other ultrasound abnormalities. The pregnancy outcomes and health conditions after birth were followed up. We also performed a pooled analysis regarding likely pathogenic and pathogenic CNVs (LP/P CNVs) for VM. The detection rate of chromosomal abnormalities by karyotyping was 9.1% (22/242), whereas it was 16.5% (40/242) when CMA was conducted (P < 0.05). The total detection rate of chromosomal abnormalities by karyotyping and CMA was 21.1% (51/242). A 12.0% incremental yield of CMA over karyotyping was observed. The detection rate of total genetic variants in fetuses with bilateral VM was significantly higher than in fetuses with unilateral VM (30.0% vs. 16.7%, P = 0.017). No significant differences were discovered between isolated VM and non-isolated VM, or between mild and moderate VM, or between advanced maternal age (AMA) and non-AMA (all P > 0.05). 28 fetuses with VM were terminated and 214 fetuses were delivered: one presented developmental delay and one presented congenital heart disease. The VM cases with both positive CMA and karyotypic results had a higher rate of termination of pregnancy than those with either a positive CMA or karyotypic result, or both negative testing results (P < 0.001). The combination of CMA and karyotyping should be adopted to improve the positive detection rate of chromosomal abnormalities for VM. The total genetic abnormalities detected using both techniques would affect the final pregnancy outcomes. LP/P CNVs at 16p11.2, 17p13, and 22q11.21 were identified as the top three chromosomal hotspots associated with VM, which would enable genetic counselors to provide more precise genetic counseling for VM pregnancies.