Chromosomal Copy Number Research Articles

P-713 Preimplantation DNA methylation screening (PIMS) illustrates the outcome of assisted reproductive technology

Abstract Study question It is unknown whether using DNA methylation can improve the outcome of ART and can exclude the imprinted gene diseases. Summary answer Our study provides a criterion on selecting blastocyst according to DNA methylome, which can improve live birth rate and decrease birth defect rate. What is known already How to select the embryos is one key factor that can determine the successful rate of ART. As aneuploidy exists in 20-30% of blastocysts, preimplantation genetic screening (PGS) has been widely used, which increases the live birth rate. Unfortunately, the live birth rate remains around 50% even with the help of PGS. DNA methylation is known to play important function during embryogenesis. Previous study has shown that a large proportion of human embryos have abnormal DNA methylome. However, whether DNA methylation pattern can affect the clinical outcome of ART has not been investigated in clinics. Study design, size, duration To determine whether PIMS method can increase live birth rate during ART, the whole genome DNA methylation of 3-5 biopsied cells from trophectoderm were measured. The methylation level and chromosome copy number variation (CNV) were analyzed by using the methylome data. elective single embryo transfer (eSET) will be performed for embryos with euploid chromosome. The live birth rate, pregnant rate and abortion rate will be examined. 182 families including 800 blastocysts were enrolled in PIMT. Participants/materials, setting, methods Eligible couples include advanced maternal age, recurrent pregnancy loss with unknown reason, recurrent implantation failure with unknown reason, severe oligoteratozoospermia. Eligible couples must have two or more good-quality blastocysts. We call a blastocyst as good quality if the blastocyst is 4BC or better according to the Gardner morphologic criteria on day 5-6 of embryo culture. Main results and the role of chance No one has ever performed clinical trial to investigate the impact of DNA methylation of blastocyst on the outcome of artificial assisted reproductive (ART). Here, we performed PIMS using the biopsied trophectoderm of 800 blastocysts. Chromosome copy number variation and DNA methylation pattern were analyzed by using methylome data. 162 of euploid embryos were transferred. Our data show that significant variation of DNA methylation level of blastocysts can be observed in both younger women (£37 years old) and older women (>37 years). Blastocysts with global methylation level around 0.26 have the highest birth and pregnancy rate, but the lowest abortion rate. The higher the difference methylation value from 0.26 is, the lower the birth rate becomes. Furthermore, by using artificial intelligence method according to different methylated regions between the embryos succeeding in live-birth and those failing in live-birth, we can robustly predict the clinical outcome of blastocysts. PIMS method can find the abnormality of DNA methylation states in the imprinted control regions which can cause the imprinted diseases. Our study provides a criterion on selecting blastocyst according to DNA methylome during ART, which can potentially improve the live birth rate and decrease the birth defect rate. Limitations, reasons for caution Because of the low coverage of single cell DNA methylome, PIMS method cannot cover all imprinted control regions. Wider implications of the findings PIMS method can potentially find more human diseases which is caused by DNA methylation mutation. Trial registration number NCT03642574

P-708 Establishment of linkage phase, using Oxford Nanopore Technologies, for Preimplantation Genetic Testing of Coffin-Lowry syndrome with a de novo RPS6KA3 mutation

Abstract Study question There was a challenge to establishing haplotype for PGT in the family with de novo mutation because of the lack of information from affected members. Summary answer The ONT platform combined with MARSALA method can be used to perform PGT for DNM patients without the need for other samples as a reference. What is known already The proband was a 31-year-old female who was born with mental retardation and language delay. On physical examination, she was found to have distinctive Coffin-Lowry Syndrome features, such as distance between the eyes and low nasal bridge. However, the proband’s parents did not show any abnormalities. The couple underwent genetic counseling and whole-exome sequencing (WES). The results showed that a frameshift mutation (c.1496delG, p.Gly499Valfs*25) was identified in the RPS6KA3 gene, with a true mutation in the proband but no mutations in the parents. Study design, size, duration A couple requested genetic counseling at the reproductive center of Zhongshan Boai Hospital and underwent whole-exome sequencing (WES) in 2019. The proband then underwent PGT with Oxford Nanopore Technologies (ONT) and the MARSALA platform to block the transmission of the pathogenic mutation to her offspring in 2022. Participants/materials, setting, methods The proband underwent PGT to block the transmission of the pathogenic mutation to her offspring.We used long-read sequencing on the ONT platform to directly detect the mutation and nearby SNPs for construction of the haplotype in PGT. The MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus. Main results and the role of chance Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of the RPS6KA3 gene in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby. Limitations, reasons for caution This strategy has only been applied to one case and more cases with de novo mutaition are needed to prove the feasibility of this strategy for PGT-M. Wider implications of the findings This study is the first report of a de novo RPS6KA3 c.1496delG mutation in patient with CLS, which can expands the RPS6KA3 mutation spectrum and also provides a new strategy based on ONT and MARSALA to perform PGT-M with DNMs without the need for a pedigree sample as a reference. Trial registration number Not applicable

P-727 New methods reveal the true incidence of DNA contamination in PGT-A samples for the first time and avoid errors that could result in serious misdiagnoses

Abstract Study question What is the occurrence of contamination in embryo biopsy samples and could this lead to incorrect interpretation of preimplantation genetic testing for aneuploidy (PGT-A) results? Summary answer On average, contamination affects 0.4% of biopsy samples, but can be significantly more common in some clinics. Misdiagnosis can occur when contamination is not detected. What is known already Until recently, most commercially available platforms for PGT-A have utilised whole genome amplification followed by sequencing of a random selection of DNA fragments scattered across the genome using next generation sequencing (NGS). However, the simple quantitative measurements of DNA fragments derived from each chromosome, provided by such methods, cannot reveal when a biopsy sample is contaminated with non-embryonic DNA. Negative controls are seldom used during PGT-A and are inadequate as they do not evaluate contamination in the actual tube containing the biopsy specimen. The extent to which failure to detect DNA contamination is a problem for PGT-A is unknown. Study design, size, duration This was a retrospective study involving analysis of 49,287 trophectoderm biopsy samples that underwent PGT-A over a three-year period. Embryos found to have a contaminated biopsy specimen typically underwent a second biopsy. In such cases, results from the two samples were compared to ascertain whether the contaminated sample would have been misdiagnosed if the analysis had been restricted to examining only the relative chromosome copy number, as is the case for most NGS-based PGT-A methods. Participants/materials, setting, methods All trophectoderm biopsies underwent targeted DNA amplification and next generation sequencing using a highly validated PGT-A method that evaluates the relative chromosomal copy number, similar to traditional PGT-A methods, but combines this with analysis of variations in DNA sequence (single nucleotide polymorphisms - SNPs). The genotype of each SNP, and the relative quantity of DNA fragments containing each of the different alleles, allows detection of otherwise invisible states, such as triploidy, haploidy, and contamination. Main results and the role of chance From the 49,287 TE biopsies analysed, contamination with non-embryonic DNA was detected in 218 (0.44%). There was variation in the rates of contamination between the 25 clinics that provided samples, ranging between 0% and 1.5%. Additionally, one clinic had a contamination rate of 7.7%, but the number of biopsies derived from that site was considered too small for reliable evaluation (n = 26). 156 of the embryos with a contaminated biopsy specimen underwent secondary biopsy (71.5%), allowing the relative chromosome copy number result from the contaminated specimen to be compared to that obtained from an uncontaminated sample. The results were split into three categories: 1) no change in interpretation between the first (contaminated) and second biopsy specimens; 2) false positive – the contaminated sample was euploid but would have been wrongly interpreted as triploid and would have been erroneously discarded, potentially impacting the patients chances of achieving a pregnancy; 3) false negative – the contaminated sample was fully aneuploid but would have been incorrectly classified mosaic or euploid and could have been eligible for transfer, potentially leading to implantation failure or abnormal pregnancy. 19% of contaminated samples gave a false negative result, while 24% gave a false positive, appearing to be triploid. Limitations, reasons for caution It is not possible to determine the origin of contaminants with certainty without having DNA from the contamination source for comparison. Additionally, we were unable to conclude whether contamination is more likely to occur in IVF or ICSI cycles as only 3% of samples were fertilized using IVF. Wider implications of the findings Contamination detection during PGT-A is important to prevent misdiagnosis of embryos. Misclassification due to undetected contamination can lead to discard of potentially viable embryos. It can also lead to the transfer aneuploid embryos, wrongly classified as mosaic, which could lead to increased rates of implantation failure, miscarriage and aneuploid pregnancy. Trial registration number Not applicable

Genetic analysis of a child with SATB2‑associated syndrome and literature study.

The present study aimed to investigate clinical phenotype and genotype characteristics of a male child with SATB2-associated syndrome (SAS) and analyzed the relationship between these characteristics and the possible underlying genetic mechanism. His clinical phenotype was analyzed. Using a high-throughput sequencing platform, his DNA samples were subjected to medical exome sequencing, screened for suspected variant loci and analyzed for chromosomal copy number variations. The suspected pathogenic loci were verified by Sanger sequencing. He presented with phenotypic anomalies of delayed growth, delayed speech and mental development, facial dysmorphism showing the typical manifestation of SAS and motor retardation symptoms. Gene sequencing result analyses revealed a de novo heterozygous repeat insertion shift mutation in the SATB2 gene (NM_015265.3) c.771dupT (p.Met258Tyrfs*46), resulting in a frameshift mutation from methionine to tyrosine at the amino acid site 258 and a truncated protein with 46 amino acids missing. The parents showed no mutation at this locus. This mutation was identified as the nosogenesis of this syndrome in children. To the best of the authors' knowledge, this is the first report on this mutation. The clinical manifestations and gene variation characteristics of 39 previously reported SAS cases were analyzed together with this case. The findings of the present study suggested severely impaired language development, facial dysmorphism and varying degrees of delayed intellectual development as the characteristic clinical manifestations of SAS.

Immunohistochemical analysis of the tumor microenvironment in acral lentiginous melanoma (ALM).

e21588 Background: Acral lentiginous melanoma (ALM) is a rare subtype of melanoma occurring on the palms, soles and nail beds. Molecularly, ALM is characterized by a higher frequency of chromosomal structural and copy number variants, but lower tumor mutational burden (TMB). Meanwhile, immune checkpoint inhibitors (ICI) are less efficacious in this subtype. In order to better understand these differences in outcomes to ICI, we conducted an immunohistochemical analysis of ALM to explore putative immune biomarkers. Methods: Clinically annotated samples of ALM were identified from patients treated at the USC Keck School of Medicine between 2010-2018. Immunohistochemistry stains included CD8, PD-L1, CD56, CD68, FOXP3, CTLA-4, HLA-ABC and HLA-G, and were characterized based on the intensity of staining. The percentage of tumor cells and infiltrating immune cells (IC) within the tumor microenvironment for the aforementioned antigens were recorded. Data are reported as percent of total cells ( > 1%, > 5%, > 25%, > 50%) expressing marker in the entire sample. Results: A total of 28 ALM specimens from 21 different patients were analyzed, including 19 primary and 9 metastatic tumors. The median age of all patients was 67 years (range 32-92); the majority were men (66%) and Hispanic (66%). Primary ALM tumors were located more often on the plantar foot (62%), compared to the plantar hand (29%). The median tumor thickness was 2.8 mm (range 0.25 – 6.5 mm). Among all patients, 57% had localized disease, while 19% had regional metastases and 24% had distant metastases. CD8+ TIL cells were observed in 95% (18/19) of samples, with 68.4% (13/19) showing > 50% CD8+ staining on IC. PD-L1 expression > 1% on tumor cells was noted in 37% (10/27) of samples, and 19% (5/27) on IC; PD-L1 > 5% was noted in only 18.5% (5/27) on tumor cells and on 11% (3/27) on IC. Both CD56 and CD68 were noted to have at least 1% expression on IC among nearly all (94%) of samples analyzed (7/8 and 9/9, respectively). FoxP3 expression > 1% was noted on IC in 92.3% (24/26) of samples, and > 5% in 50% (13/26). CTLA-4 expression > 1% was observed among 81.5 % (22/27) of samples, while 63% (17/27) had expression > 5%; CTLA-4 expression > 50% was noted among 100% on melanophages (7/7 samples). HLA-ABC expression was detected in 100% of samples analyzed (10/10). The percentage of samples with HLA-G expression > 1% was 60.7% (17/28), while 28.6% (8/28) demonstrated > 5% expression. Further correlations with clinical outcomes will be reported. Conclusions: Our study suggests a unique immune tumor microenvironment in ALM, characterized by low PD-L1 expression, despite infiltration by CD8+, as well as CD56 and CD68 immune cells. Furthermore, the presence of FOXP3+ Tregs and HLA-G expression, both which mediate immune response evasion, may explain the poor outcomes of ICI therapy among these patients and warrant further investigation.

The association of centromere amplification and response to trastuzumab in HER2+ metastatic breast cancer.

1016 Background: Chromosomal instability and copy number aberration (CNA) burden can be associated with patients' survival in some cancers. Centromeres, chromosomal regions crucial for genomic stability, can be found altered by CNA in tumours but remain largely unexplored due to sequencing technologies. Using whole-genome sequencing (WGS) data from cases of long term, never relapsed HER2+ metastatic breast cancer (MBC) patients, also referred as exceptional responders (ExRs), we aim to better characterise the CNA profiles and underlying long-term survival of this “exceptional” cohort and investigate the centromeric and pericentromeric association with prognosis and treatment response. Methods: Two hundred and forty-three HER2+ MBC patients were enrolled in the HER2 Patients Project Database from St Vincent University Hospital, Dublin, Ireland. Eighty-five HER2+ MBC patients were identified as exceptional survivors (ExS) with an OS > 60 months (range 60-248 months), of which 28 never-relapsed and responded exceptionally (ExR) to trastuzumab. Patients with an OS < 60 months (N=158, range 0.2-59 months) were identified as non responders (NR). WGS was performed on 13 ExR tumours (primary or metastases) and matching control at a mean depth of 60X and 30X, respectively. Control-FREEC and CNVkit were used to characterise the CNA profiles and estimate the CNA burden. Samples from 10 NR patients were used for comparison with the ExR. Results: WGS analysis revealed that ExR samples are more impacted by gain rather than loss of copy overall, with a median fraction altered by gain of 0.24. No significant difference was observed between the ExR and NR in terms of CNA burden. However, a large fragment on chromosome 9 was amplified in 92% of ExR (12/13) which corresponded to the centromere (chr9q11) and a large heterochromatic block (chr9q12). A higher copy number status of this centromeric region was detected in the ExR, with a gain of at least 1 additional copy compared to the NR tumoral samples (P<0.001). The dichotomisation into high versus low copy number of the centromeric region of chromosome 9 was also observed in centromeres of chromosome 17 (P=0.009) and chromosome 19 (P<0.001). No case of polysomy was detected in our cohort and genes located near the centromeres, such as ERBB2 (amplified in both ExR and NR), were independent of the centromeres copy-number status. Conclusions: The identification of the genomic aberrations of these metastatic patients, treated with trastuzumab who never relapsed, increases our understanding of the mechanisms involved in MBC progression. Our results suggest that the centromere co-amplification of chr9q11-q12, chr17p11.1-q11.1 and chr19p11-q11 stratifies patients according to their OS. CNA status of the centromeric regions of chromosomes 9, 17 and 19 may therefore represent a novel prognostic predictor to trastuzumab response and new outcomes for patients.

Abstract 102: Transient chromosomal instability as a driver of ovarian cancer genome evolution

Abstract Chromosomal instability (CIN) is a major driver of tumor progression and treatment resistance in many cancers. CIN is characterized by ongoing chromosome missegregation, generating copy number heterogeneity that provides a substrate for natural selection. Although CIN has been well studied in model systems, the evolutionary dynamics and genomic impact of CIN is underexplored in primary tumors and metastases. Newly developed single cell genomics assays allow for the measurement of the whole genomes of single cells obtained from patient tissues, thus providing a direct view into the evolution of tumor genomes undergoing CIN. To study the single-cell and spatial variation of chromosomally unstable tumors, we performed multi-region analysis of 128 tumor samples in a cohort of 45 patients with high-grade serous ovarian cancer (HGSOC), an archetype of high CIN cancers. We performed single-cell whole genome sequencing (scWGS) and profiled a total of 70,644 single cells from 63 pre-treatment samples obtained across 43 patients. For each patient, we computed total and allele specific copy number per cell, and inferred the evolutionary history of each tumor, including clonal and subclonal whole genome doubling (WGD) events and cell-specific whole chromosome or arm level copy number changes, indicative of missegregations. To determine whether missegregated chromosomes fail to become part of the primary nucleus (PN) upon mitotic exit and become enclosed in a micronucleus (MN), we used high-resolution cGAS immunofluorescence (IF) microscopy to profile site-matched FFPE tissues and quantify spatially-resolved rates of MN formation and rupture across 128 site-matched samples. A majority of tumors were characterized by early clonal WGD (61%, 22/36 cases). The remaining patients exhibited subclonal WGD subpopulations averaging 10% of the patient’s cells in total. To understand the interplay between recent WGD and CIN across the cohort, we subset all patients cells into non-WGD, subclonal WGD, and clonal WGD and computed missegregation rates for these subsets. Non-WGD cells showed the lowest levels of missegregation. Subclonal WGD exhibited the highest rates of missegregation indicative of CIN following a recent WGD event. Clonal WGD exhibited intermediate levels of missegregation indicative of stabilization following a historical WGD event. CIN following WGD often resulted in a reduction in ploidy relative to the ancestral WGD cell. Using IF, we quantified the frequent breakdown of the MN membrane, and estimated spatially-resolved CIN rates by tracking localization of cGAS to the MN, which revealed elevated rates of MN formation and rupture. Through multimodal single cell measurements of HGSOC tumors, we were able to characterize genomic complexity from individual cell division errors in HGSOC, and shed light into how the transient processes governing ovarian cancer cell evolution impact the genomes of cell populations. Citation Format: Ignacio Vazquez-Garcia, Florian Uhlitz, Marc J. Williams, Jun Li, Hongyu Shi, Samuel Freeman, Matthew Myers, MSK SPECTRUM Consortium, Britta Weigelt, Dmitriy Zamarin, Andrew McPherson, Samuel F. Bakhoum, Sohrab P. Shah. Transient chromosomal instability as a driver of ovarian cancer genome evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 102.

Clinical and genetic characteristic in patients with disorders of sex development caused by Y chromosome copy number variant

Objective: To investigate the clinical phenotype and genetic characteristics of disorders of sex development (DSD) caused by Y chromosome copy number variant (CNV). Methods: A retrospective analysis was performed on 3 patients diagnosed with DSD caused by Y chromosome CNV admitted to the First Affiliated Hospital of Zhengzhou University from January, 2018 to September, 2022. Clinical data were collected. Clinical study and genetic test were performed by karyotyping, whole exome sequencing (WES), low coverage whole genome copy number variant sequencing (CNV-seq), fluorescence in situ hybridization (FISH) and gonadal biopsy. Results: The 3 children, aged 12, 9, 9 years, the social gender were all female, presented with short stature, gonadal dysplasia and normal female external genital. No other phenotypic abnormality was found except for case 1 with scoliosis. The karyotype of all cases were identified as 46, XY. No pathogenic vraiants were found by WES. CNV-seq determined that case 1 was 47, XYY,+Y(2.12) and case 2 was 46, XY,+Y(1.6). FISH concluded that the long arm of Y chromosome was broken and recombined near Yq11.2, and then produced a pseudodicentric chromosome idic(Y). The karyotype was reinterpreted as mos 47, X, idic(Y)(q11.23)×2(10)/46, X, idic(Y)(q11.23)(50) in case 1. The karyotype was redefined as 45, XO(6)/46, X, idic(Y)(q11.22)(23)/46, X, del(Y)(q11.22)(1) in case 2. 46, XY, -Y(mos) was found by CNV-seq in case 3, and the karyotype of 45, XO/46, XY was speculated. Conclusions: The clinical manifestations of children with DSD caused by Y chromosome CNV are short stature and gonadal dysgenesis. If there is an increase of Y chromosome CNV detected by CNV-seq, FISH is recommended to classify the structural variation of Y chromosome.

Prenatal detection of chromosomal abnormalities and copy number variants in fetuses with corpus callosum agenesis.

The corpus callosum is the main pathway that connects interhemispheric communication. Agenesis of corpus callosum (ACC) have not consistently detected replicate genetic risk factors, potentially due to Etiological heterogeneity of this trait. This study aimed to retrospectively analyze the molecular basis for the ACC and the potential genotyping-phenotyping association and provide the basis for genetic counselling. Karyotyping and chromosomal microarray analysis were performed for copy number variants. Three cases had 1p36 deletions, two cases had 2q31.2 and 2p16.3 microdeletions, one case had microdeletion of Xq26.3q27.1, five cases involved derived chromosomes due to unbalanced translocations. These cases had variable deletions and duplications with partial overlapping. Phenotypically, besides agenesis of corpus callosum and other brain morphological abnormalities as well as heart abnormalities. ACC may occur alone or be related to other abnormal clinical phenotypes, and its genetic mechanism is very complicated. These results revealed ACC is associated with a variety of chromosomal abnormalities. The findings of the present study expand the genotypes associated with ACC, and further delineation of the genotype-phenotype correlations for ACC. With current applications of chromosome microarray analysis, congenital submicroscopic copy-number variations in fetuses can be detected more effectively.

Replication Timing Aberration of KIF14 and MDM4 / PI3KC 2 β Alleles and Aneuploidy as Markers of Chromosomal Instability and Poor Treatment Response in Ewing Family Tumor Patients.

Replication timing of allelic gene pairs is strictly regulated according to expression, genome stability, and epigenetic changes, and tumorigenesis may be associated with changes in the allelic replication in various tumors. Our aim was to determine whether such alterations had a prognostic value in Ewing's family tumor (EFT) patients. The KIF14 and MDM4 / PI3KC 2β and the centromeric satellite sequence of chromosomes 8 and 12 were used for replication timing assessments. Aneuploidy was assessed by enumerating the copy numbers of chromosomes 8 and 12. Replication timing and aneuploidy were detected cytogenetically using multicolors fluorescence in situ hybridization assay applied in 135 EFT. Patients with trisomy 8 presented an association with an asynchronous replication pattern (SD) of MDM4 / PI3KC 2β genes ( p = 0.013). Trisomy 12 was associated with a synchronous pattern (DD) of KIF14 probe signals ( p = 0.04). The DD synchronous replication pattern of KIF14 showed a correlation with age ( p < 0.0001), and the SS synchronous replication pattern of the same locus showed a correlation with lung metastatic ( p = 0.012). The subgroup of patients presenting with multiplet signals of MDM4 / PI3KC 2β showed an association with treatment response ( p = 0.045) and age ( p = 0.033). Replication pattern of KIF14 may, significantly, be associated with chromosomal instability as MDM4 / PI3KC 2β may be a considerably new marker of poor treatment response in EFT patients.

The role of replication-induced chromosomal copy numbers in spatio-temporal gene regulation and evolutionary chromosome plasticity.

For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but on replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in Escherichia coli. Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen Dickeya dadantii and the biotechnologically relevant organism Vibrio natriegens. The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. A further extension to the bacterial kingdom indicates that chromosome evolution is governed by growth rate related transient copy numbers.

Diagnostic Value of Chromosomal Microarray Analysis for Fetal Congenital Heart Defects with Different Cardiac Phenotypes and Extracardiac Abnormalities.

(1) Background: The objective of this study was to investigate the diagnostic value of chromosomal microarray analysis (CMA) for congenital heart defects (CHDs) with different cardiac phenotypes and extracardiac abnormalities (ECAs) and to explore the pathogenic genetic factors of CHDs. (2) Methods: We collected fetuses diagnosed with CHDs by echocardiography at our hospital from January 2012 to December 2021. We analyzed the CMA results of 427 fetuses with CHDs. We then categorized the CHD into different groups according to two dimensions: different cardiac phenotypes and whether it was combined with ECAs. The correlation between the numerical chromosomal abnormalities (NCAs) and copy number variations (CNVs) with CHDs was analyzed. Statistical analyses, including Chi-square tests and t-tests, were performed on the data using IBM SPSS and GraphPad Prism. (3) Results: In general, CHDs with ECAs increased the detection rate for CA, especially the conotruncal defects. CHD combined with the thoracic and abdominal walls and skeletal, thymic and multiple ECAs, were more likely to exhibit CA. Among the CHD phenotypes, VSD and AVSD were associated with NCA, while DORV may be associated with NCA. The cardiac phenotypes associated with pCNVs were IAA (type A and B), RAA, TAPVC, CoA and TOF. In addition, IAA, B, RAA, PS, CoA and TOF were also associated with 22q11.2DS. The length distribution of the CNV was not significantly different between each CHD phenotype. We detected twelve CNV syndromes, of which six syndromes may be related to CHDs. The pregnancy outcome in this study suggests that termination of pregnancy with fetal VSD and vascular abnormality is more dependent on genetic diagnosis, whereas the outcome in other phenotypes of CHDs may be associated with other additional factors. (4) Conclusions: CMA examination for CHDs is still necessary. We should identify the existence of fetal ECAs and specific cardiac phenotypes, which are helpful for genetic counseling and prenatal diagnosis.

Primary papillary epithelial tumor of the sella and posterior pituitary tumor show similar (epi)genetic features and constitute a single neuro-oncological entity.

"Primary papillary epithelial tumor of the sella (PPETS)" is a recently described rare tumor entity of the central nervous system (CNS) with stereotypic location in the sella. Comprehensive molecular investigations and epigenetic profiles of PPETS have not been performed to date. We report a comprehensive clinical, histopathologic, and molecular assessment of 5 PPETS cases in comparison with a cohort composed of 7 choroid plexus papilloma (CPP), 7 central neurocytoma (CN), 15 posterior pituitary tumor (PPT) including 4 pituicytoma, 6 granular cell tumors of the sellar region (GCT), and 5 spindle cell oncocytoma. All PPETS had good outcomes. Immunohistochemically, PPETS tumors showed positive staining with TTF1, EMA, AE1/AE3, MAP2, and Vimentin, but were negatively stained with Syn, GFAP, CgA, and S100, and sporadically stained with Ki-67. In unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analyses of DNA-methylation data, PPETS and PPT tumors formed a distinct cluster irrespective of their histologic types. However, PPETS tumors did not cluster together with CPP and CN samples. Similar findings were obtained when our samples were projected into the reference cohort of the brain tumor classifier. Substantial fractions of the PPETS and PPT tumors shared broadly similar chromosomal copy number alterations. No mutations were detected using targeted next-generation sequencing. Though more cases are needed to further elucidate the molecular pathogenesis of these tumors, our findings indicate that PPETS and PPT tumors may constitute a single neurooncological entity.

Double-checking chromosome segregation.

Enduring chromosome segregation errors represent potential threats to genomic stability due to eventual chromosome copy number alterations (aneuploidy) and formation of micronuclei-key intermediates of a rapid mutational process known as chromothripsis that is found in cancer and congenital disorders. The spindle assembly checkpoint (SAC) has been viewed as the sole surveillance mechanism that prevents chromosome segregation errors during mitosis and meiosis. However, different types of chromosome segregation errors stemming from incorrect kinetochore-microtubule attachments satisfy the SAC and are more frequent than previously anticipated. Remarkably, recent works have unveiled that most of these errors are corrected during anaphase and only rarely result in aneuploidy or formation of micronuclei. Here, we discuss recent progress in our understanding of the origin and fate of chromosome segregation errors that satisfy the SAC and shed light on the surveillance, correction, and clearance mechanisms that prevent their transmission, to preserve genomic stability.

Abstract 3133: Loss of the Y chromosome as a candidate driver in solid tumors

Abstract It has long been recognized that certain cancer types afflict female and male patients disproportionately. The reasons for this disparity include differences in male and female physiology, effect of sex hormones, risk behavior and environmental exposures, and different copy number of the sex chromosomes X and Y. Loss of Y (LOY) is common in peripheral blood cells in aging men, and this phenomenon is associated with several diseases. However, the frequency and role of LOY in tumors is little understood. Here, we present a comprehensive catalog of LOY in about 5,000 primary tumors from male patients in the TCGA. We show that LOY rates vary by tumor type, and provide evidence for LOY being either a passenger or driver event depending on context. LOY in uveal melanoma specifically is associated with age, survival and is an independent predictor of poor outcome. LOY creates common dependencies on DDX3X and EIF1AX in male cell lines, suggesting that LOY generates unique vulnerabilities that could be therapeutically exploited. Citation Format: Meifang Qi, Esther Rheinbay, Andrew A. Lane, Jiali Pang, Irene Mitsiades. Loss of the Y chromosome as a candidate driver in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3133.

Abstract 92: Deploying spatial transcriptomics to inform intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples

Abstract Uveal Melanoma (UM) is a rare tumor characterized by mutually-exclusive activating mutations in GNAQ or GNA11, followed by a mutation event in BAP1, SF3B1 or EIF1AX. Notably, a large subset of patients present with a unique chromosome 3 copy-loss event, which is highly associated with metastatic progression in late-stage disease. To date, the biology underlying the causes and consequences of this unique copy-loss event (Monosomy 3 or M3), and its role in promoting metastases remains largely unexplored. This can be attributed to both the lack of preclinical models and in-depth characterization of paired primary and metastatic tumors in patients. To address this, we derived a preclinical isogenic model of chromosome 3 copy-loss through CRISPR-based centromere targeting in a well characterized Disomy 3 UM cell line. Isogenic Disomy (D3) and Monosomy (M3) clones derived from these efforts have enabled us to develop patient derived xenograft (PDX) models to compare D3 and M3 behavior in paired, in vivo, primary and metastatic settings. This modeling has enabled us to employ spatial transcriptomics in a PDX setting to characterize gene expression signatures across unique D3 and M3 UM clones. Investigation of metastatic UM tumor heterogeneity in our models enables us to characterize unique features of phenotypically transformed clones (e.g. depigmentation and growth advantage). We have also adapted existing methodology to infer chromosomal copy number events from spatial transcriptomics data to our PDX system, where we are able to overcome the lack of same-species microenvironment controls. Our methodology allows for deep characterization of sub-clonal heterogeneity in both D3 and M3 settings and informs on the unique biology underlying invasiveness and outgrowth of M3 tumors. Finally, we complement our preclinical analyses with an investigation of spatial heterogeneity in a patient cohort of paired primary and metastatic tumors. More broadly, comparing these D3 vs. M3 signatures provides an opportunity to expand on our knowledge of metastatic disease drivers and derive prognostic signatures associated with poor survival. Citation Format: Sanjana Srinivasan, Johnathon Rose, Joseph R. Daniele, Michael Peoples, Meng He, Rosalba Minelli, Chiu-Yi Liu, Jason Gay, Melinda Soeung, Khalida Wani, Luigi Perelli, Dmitriy Loza, Ningping Feng, Christopher P. Vellano, Joseph Marszalek, Giulio F. Draetta, P. Andrew Futreal, Scott E. Woodman, Alexander J. Lazar, Timothy Heffernan, Alessandro Carugo, Giannicola Genovese, Virginia Giuliani. Deploying spatial transcriptomics to inform intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 92.

Holoprosencephaly: Review of Embryology, Clinical Phenotypes, Etiology and Management.

Holoprosencephaly (HPE) is the most common malformation of the prosencephalon in humans. It is characterized by a continuum of structural brain anomalies resulting from the failure of midline cleavage of the prosencephalon. The three classic subtypes of HPE are alobar, semilobar and lobar, although a few additional categories have been added to this original classification. The severity of the clinical phenotype is broad and usually mirrors the radiologic and associated facial features. The etiology of HPE includes both environmental and genetic factors. Disruption of sonic hedgehog (SHH) signaling is the main pathophysiologic mechanism underlying HPE. Aneuploidies, chromosomal copy number variants and monogenic disorders are identified in a large proportion of HPE patients. Despite the high postnatal mortality and the invariable presence of developmental delay, recent advances in diagnostic methods and improvements in patient management over the years have helped to increase survival rates. In this review, we provide an overview of the current knowledge related to HPE, and discuss the classification, clinical features, genetic and environmental etiologies and management.

Genetic Status Affects Disease-Specific Mortality But Not the Incidence of Local Recurrence in Patients with Uveal Melanoma

Increased disease-specific mortality has been observed among patients with local recurrence (LR) from uveal melanoma (UM), but the underlying mechanism is unknown. The purpose of this study was to determine if copy number alterations of chromosomes 3 and/or 8q, at the time of diagnosis, increase the incidence of LR and if disease-specific mortality among patients with LR depends on the chromosome status of the primary tumor. Retrospective cohort study. The study included 239 consecutive patients with primary UM (choroidal or ciliary body) treated with Ruthenium-106 (Ru-106) brachytherapy from January 2009 to December 2019 at a single national referral center. Cox regression modeling and Kaplan-Meier analyses were used to assess the effect of the status of chromosomes 3 and 8q on the incidence of LR and disease-specific mortality after the event of LR. Multistate models were used to illustrate the probabilities over time of patients being alive and disease-free, alive with LR, dead from UM metastases, or dead from other causes split on the status of chromosomes 3 and 8q. Incidence of LR and disease-specific mortality. Local recurrence was observed in 42 patients (16%). Overall incidence of LR was not affected by aberrations of chromosomes 3 and/or 8q (P= 0.87). Although LR occurred earlier in patients with aberrations of chromosomes 3 and/or 8q compared with patients with a normal copy number of chromosomes 3 and 8q, the median time from primary diagnosis to LR was 1.6 years (interquartile range [IQR], 1.0-2.0) and 3.2 years (IQR, 2.1-5.0), respectively. Cox regression found LR to be an independent risk factor for disease-specific mortality (hazard ratio [HR], 2.7; 95% confidence interval [CI], 1.5-5.0) among all patients, but multistate models demonstrated a low risk of disease-specific death among patients with normal chromosomes 3 and 8q status, even after an LR. Copy number alterations of chromosome 3 and/or 8q in the primary UM did not increase the overall incidence of LR. However, the development of an LR enhanced the risk of disease-specific mortality among patients with copy number alterations of chromosomes 3 and/or 8q. Even after an LR, disease-specific mortality remained low among patients with normal copy numbers of chromosomes 3 and 8q. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

Genomic Profiling of the Craniofacial Ossifying Fibroma by Next-Generation Sequencing.

Ossifying fibroma (OF) of the craniofacial skeleton is a fibro-osseous lesion characterized by various patterns of bone formation in a cellular fibroblastic stroma. The molecular landscape of OF remains mostly unknown. There are a few known pathogenic abnormalities in OF, including HRPT2 mutations in conventional OF and SATB2 translocations in juvenile psammomatoid OF. On the other hand, conflicting reports exist regarding MDM2 gene amplification and chromosomal copy number alterations (CNA) in OF. Surgically removed biopsies and curettage specimens from OF patients were obtained. Clinical, radiographic, and pathologic features of tumors were reviewed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded blocks of tumor tissue. Capture-based DNA next-generation sequencing targeting the coding regions 529 cancer genes and select introns was performed. We identified 17 OF cases from 8 male and 8 female patients with mean age of 22years (range 1-58years). Nine case occurred in the gnathic bones and 8 in the extragnathic craniofacial bones. These cases included 3 juvenile psammomatoid OF, 6 conventional OF and 8 juvenile trabecular OF. Large-scale CNAs were present in 6 of 17 cases. Seven cases (41%) had focal amplifications including FOSB (n = 2, 11%), FOS (n = 4, 23%), COL1A1 (n = 4, 23%) and TBX3 (n = 5, 29%). Three cases (17%) had pathogenic CDC73 mutations. No cases showed focal MDM2 amplification. Here, we provided a comprehensive molecular characterization of OF that reveals a heterogeneous genetic profile with occasional large-scale CNAs (n = 6, 35%). FOS, FOSB, and TBX3 genes that regulate AP-1 transcriptional complex are frequently altered in OF (n = 7, 41%), chiefly in juvenile trabecular OF. These genes encode transcription factors that act as downstream effectors of the MAP kinase signaling pathway. MDM2 amplification is an exceedingly rare event in OF, if present at all, so identification of this event should continue to raise concern for low-grade gnathic osteosarcoma. In summary, our findings suggest that OF represents a heterogeneous group of tumors at the genetic level but dysregulation of the AP-1 pathway may play a role in pathogenesis of juvenile trabecular OF.