Chromosomal Copy Number Research Articles

The Independent Adverse Prognostic Significance of 1q21 Gain/Amplification in Newly Diagnosed Multiple Myeloma Patients

Background Multiple myeloma (MM) is a hematological malignancy with proliferation of monoclonal plasma cells which has complex genetics and prognostic heterogeneity. It involves a variety of primary genetic abnormalities, including t (4; 14), t (11; 14), t (14,16) and hyperdiploid karyotypes, as well as secondary genetic abnormalities, including chromosome copy number abnormalities (1q21+, 1p-, 13q-, 17p-, etc) and secondary translocations like MYC. As the most common secondary genetic event, 1q21+ occurs in about 30-40% of NDMM patients while the proportion is especially higher in Chinese population, accounting for 59.7% (96/161) by a domestic study in 2022, it is closely related to the situation that MM patients may have long disease duration and late stage when initial diagnosed in China. Despite its frequency, considerable debate remains regarding to the prognostic impact of 1q21+ in NDMM, 1q21 status hasn't been included among the high-risk markers listed by the IMWG R-ISS stage, and neither in Chinese guidelines. If 1q21+ is included as one of the poor prognostic factors, it will greatly increase the proportion of high-risk patients in NDMM patients. So, our center has begun to take 1q21 seriously as a heterogeneity problem for clinical discussion. Methods 248 NDMM patients admitted in The First Affiliated Hospital of Soochow University from September 01, 2018 to August 31, 2021 of a VRD clinical study, with VRD in combination with ASCT or VRD treatment for 8 cycles, were retrospectively analyzed. 135 cases (54.4%) had 1q21+ by CD38-sorted fluorescence in situ hybridization (FISH), among which 153 patients were compared for the involved genes by CytoScan, focusing on the clinical characteristics, treatment response and prognostic significance by combining FISH and CytoScan technology complementary. Results Among the 248 NDMM patients, 54.4% patients with 1q21+ were more likely to have anemia, hypoalbuminemia, renal insufficiency, high lactate dehydrogenase and high proportion of R-ISS-III stage, and are more likely to be combined with 13q14 deletion, t(4;14), and 1p deletion，and they are all at middle-risk or high-risk groups defined by Prognostic Index score. Multivariate analysis showed that 1q21 gain/amplification was an independent adverse prognostic factor (PFS HR=2.358, 95%CI 1.286-4.324, P=0.006; OS HR=2.598, 95%CI 1.050-6.425, P=0.039). 1q21+ subgroup had an inferior outcome (PFS P=0.0133, OS P=0.0293) (Figure 1a). According to the mSMART3.0 stages, double-hit and triple-hit group had the worst prognosis, with a median PFS of 22 months and a median OS of 32 months. Both PFS and OS were clinically significant (P<0.0001), as shown in Figure 1b. Furthermore 1q21 amplification with more than 4 copy numbers had a shorter PFS than 1q21 gain with 3 copy numbers (24 months vs not reached, P=0.0403), but the OS difference hasn't been not clinically significant (Figure 1c). The positive proportion of 1q21 had no effects on prognosis，while the status of 1q21 in the main clone rather than in the subclone defined by CytoScan when the proportion of abnormal clone populations are differed more than 30%, was an adverse factor affecting the prognosis (PFS P=0.0172, OS P=0.1260). (Figure 1d). ASCT plays an important role in the prognostic survival of 1q21 gain and amplification patients (P<0.05). Outcomes of 1q have been widely discussed in CRD/CTD scheme of Myeloma XI, KRd/KCD scheme of FORTE, VRD/PAD of HOVON65 and so on, our study focusing on the patients with VRD +/- ASCT is also unique, demonstrating that the prognosis of 1q21+ in VRD scheme is also poor, but ASCT can overcome the adverse effects significantly. Conclusions Patients with 1q21+ have clinically significant end-stage organ damage and higher tumor burden, more likely to combine 13q14-, t(4;14), 1p32- and other cytogenetic abnormalities. 1q21+ is an independent high-risk cytogenetic factor for poor prognosis in NDMM patients, of which 4 or more copy numbers and main clone position significantly associated with prognosis results. By combining FISH, CytoScan and other technologies with related abnormalities such as 1q21+，17p-, 1p32-，t (4;14), t(14;16), etc., clinical characteristics, efficacy evaluation, and prognosis can be analyzed more accurately, aiming to identify patients with poor prognosis earlier and enhancing the possibility of moving forward to the treatment strategy based on risk stratification in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Position of MYC Rearrangements in the Genomic Landscape of Diffuse Large B-Cell Lymphoma

Introduction High grade B-cell lymphoma with MYC and BCL2 and/or BCL6 translocations (HGBL-double hit (DH)/-triple hit (TH) is the most strictly defined molecular class of diffuse large B-cell lymphoma (DLBCL) and is characterized by MYC plus BCL2 rearrangements with- or without BCL6 rearrangement. HGBL DH/TH have an inferior prognosis under standard DLBCL treatment compared to patients without MYC-rearrangements and currently few effective second-line options exist. Still, some HGBL DH/TH respond well to treatment, highlighting the need for further refined markers predictive of treatment resistance and relapse. To distinguish different DLBCL subtypes, two independent DNA based classifications were proposed by the Dana Farber Cancer institute (DFCI) and the National Cancer Institute (NCI, Lymphgen), that distinguish 5- and 7 DLBCL subtypes, respectively. These subtypes are characterized by distinct pathological mechanisms and outcomes. However, in both classifications the number of MYC rearranged cases was insufficient to accurately determine the position of MYC rearrangements in the genomic landscape of DLBCL. We collected a large DLBCL genomic dataset enriched for MYC rearranged cases to evaluate the hypothesis that HGBL DH/TH, or subsets of HGBL DH/TH define a separate molecular class. Methods High quality next generation sequencing (NGS) data for i) mutations, ii) genome-wide chromosomal copy numbers, and iii) MYC, BCL2 and BCL6 rearrangements was assembled for a set of N=706 DLBCL samples from 4 study cohorts (HOVON-84, HOVON-130, PETAL trials and the DFCI dataset) that included N=102 cases with MYC rearrangements including single-hit (SH), HGBL DH and TH. All samples were assigned to genomic subtypes according to published algorithms. Results Clustering all samples following the DFCI approach identified 5 clusters in concordance with published results (Figure 1). We find that MYC SH cases (N=36) are scattered throughout the 5 DFCI subtypes, while the majority of HGBL DH-BCL2/TH cases (N=41/50) fall together in subtype C3, a germinal center B-cell (GCB) rich subtype. Inspection of the genetic features of this HGBL and GCB rich subtype shows that it has many of the genetic characteristics corresponding to transformed follicular lymphomas. DH-BCL6 cases were few (N=16) and predominantly clustered in C1. When classifying samples according to Lymphgen, again we find that the MYC SH cases are scattered throughout the 7 subtypes, while HGBL-DH/TH with BCL2 rearrangement are classified as EZB (N=40/50), substantiating earlier interpretations. DH-BCL6 cases were mostly classified as BN2. Conclusion We recapitulate the 2 DNA-classification methods with a data set enriched for MYC rearranged cases (102/706 samples) and demonstrate that HGBL DH-BCL2/TH form a sub-cluster within DFCIs C3, and Lymphgen's EZB respectively, with genomic characteristics of transformed follicular lymphoma. DH-BCL6 cases were few, but come together in both classifications, DFCI C1 and Lymphgen BN2, consistent with the WHO-HAEM5, in which DH-BCL6 is classified as a separate subtype. MYC SH are not assigned to a specific subtype, also consistent with the WHO-HAEM5. These results provide the currently most robust molecular basis for stratification of DLBCL cases with MYC rearrangement. Figure 1. Cluster heatmap of 706 DLBCLs of which 102 have MYC rearrangements. Clustering was performed by non-negative matrix factorization (NMF) according to DFCI. Top: Classification of samples according to MYC status, Lymphgen assignment, and cell of origin (COO). Right: Genomic alterations (copy number alterations, CNAs; mutations and translocations) enriched in NMF clusters C1 to C5. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SURG-13. SYSTEMATIC REVIEW AND META-ANALYSIS OF IMPACT OF EXTENT OF RESECTION ON SURVIVAL ON GLIOBLASTOMA, IDH-WILDTYPE, WHO GRADE 4 (WHO 2021)

Abstract BACKGROUND Glioblastoma diagnostic criteria have been redefined with the 5th edition of WHO classification of tumors of the central nervous system. Glioblastomas are defined as IDH wildtype diffuse astrocytic glioma tumors with one of the following features: microvascular proliferation, or necrosis, or TERT promoter mutation, or EGFR gene amplification, or +7/-10 chromosome copy number changes. The aim of this study is to establish the impact of extent of resection in overall survival (OS) and progression free survival (PFS) in glioblastoma, IDH-wildtype (WT), WHO grade 4 (WHO 2021). METHODS Systematic literature search was performed using the following databases: PubMed, Web of Science, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews and ClinicalTrials.gov to identify studies comparing OS and PFS after gross total resection (GTR) vs subtotal resection (STR) or biopsy for glioblastoma IDH-WT. Prognostic hazard ratios (HR) for OS and PFS were analyzed using a random-effects model. RESULTS We identified 1439 publications. Nine studies met inclusion/exclusion criteria. 788 patients underwent GTR out of 1818. The meta-analysis showed a significant increase in OS and PFS duration when undergoing GTR for glioblastoma IDH-WT with a median OS of 20 months 95% CI (17-25) compared to 12 months 95% CI (9-15) for STR or biopsy and a median PFS of 11 months 95% CI (9-12) for GTR compared to 7 months 95% CI (5-7) for STR or biopsy respectively. GTR showed a 49% significant reduction of mortality risk HR=0.51 95%CI (0.42-0.59) and a 44% significant reduction of progression risk HR=0.56 95%CI (0.41-0.71) compared to STR or biopsy. CONCLUSIONS This systematic review indicates that GTR may be associated with improved OS and PFS compared to STR or biopsy for Glioblastoma, IDH-WT, WHO grade 4 (WHO 2021). However, this is limited by variable study design and significant clinical and methodological heterogeneity among studies.

Variation of Female Pronucleus Reveals Oocyte or Embryo Chromosomal Copy Number Variations.

The characteristics of the human pronuclei (PNs), which exist 16-22 h after fertilization, appear to serve as good indicators to evaluate the quality of human oocyte and embryo, and may reflect the status of female and male chromosome composition. Here, a quantitative PN measurement method that is generated by applying expert experience combined with deep learning from large annotated datasets is reported. After mathematic reconstruction of PNs, significant differences are obtained in chromosome-normal rate and chromosomal small errors such as copy number variants by comparing the size of the reconstructive female PN. After integrating the whole procedure of PN dynamics and adjusting for errors that occur during PN identification, the results are robust. Notably, all positive prediction results are obtained from the female propositus population. Thus, the size of female PNs may mirror the internal quality of the chromosomal integrity of the oocyte. Embryos that develop from zygotes with larger female PNs may have a reduced risk of copy number variations.

ODP469 Coexistence of two thyroid malignancies with Hurthle cell and Papillary thyroid carcinoma

Abstract Background The simultaneous presentation of mul tiple thyroid malignancies is a rare occurrence with each type of thyroid cancer having different cellular origins, histopathological appearance, and clinical course. Collision tumors representtwo histologically different tumor types occurring at the same anatomic region. When diagnosed within 6 months of the original they are also referred to as synchronous tumors. Papillary thyroid carcinoma (PTC) is the most common (80-85%) differentiated thyroid carcinomas (DTC), followed by follicular thyroid carcinoma (FTC) (10-15%), and Hurthle cell carcinoma (3–5%). We report a rare case of a patient with dual thyroid malignancy containing both PTC and Hurthle cell carcinoma in different thyroid lobes. Case presentation Patient is a 50 year old female with past medical history of hypertension and family history of thyroid disease in her sister and cousins, who presented with thyroid gland enlargement. Patient denied any hyper/hypothyroidism or compressive symptoms. Labs showed normal TSH and free T4. Thyroid ultrasound revealed a heterogeneous nodule with microcalcification in the right thyroid lobe and three well circumscribed left mid pole nodules. FNA thyroid biopsy of the right thyroid nodule demonstrated many Hurthle cells with uniform nuclei. The genomic classification identified multiple chromosomal copy number alterations consistent with a clonal neoplasm with intermediate-high risk for cancer, although no point mutations or gene fusions were found. Patient initially underwent right hemi-thyroidectomy and isthmectomy. Pathology confirmed two well circumscribed angioinvasive Hurthle cell carcinoma measuring 3.4×2.1×2. 0 cm and 1.6×1.5×1.4 cm with multiple foci of capsular invasion oncocytic features along with a 0.2 cm papillary thyroid microcarcinoma without extrathyroidal extension. After three weeks, patient proceeded with completion left thyroidectomy and showed a 1.2×1.1×1. 0 cm well circumscribed, partially encapsulated nodule, consistent with Papillary thyroid carcinoma (PTC). Lymph nodes were negative for metastasis. Patient received whole body I-131 scan and radioiodine ablation therapy. She was subsequently placed on Levothyroxine for thyroid hormone replacement and remained euthyroid without cancer recurrence. Conclusion Collision tumors account for 1% of thyroid malignancies with the most commonly reported combination being PTC and medullary thyroid carcinoma. Interestingly, this case presented with a rare finding of collision and synchronous tumors in the form of PTC and Hurthle cell carcinoma which was known to associate with increased chromosomal copy number instead of point genetic mutation. Previous literature have described collision effect, stem cell, hostage, and single progenitor theories in an attempt to explain the origin of these phenomena with possible molecular mechanisms. Clinicians should be aware that the dual thyroid malignancy with different histogenetic origin may co-exist in the same patient. The therapeutic strategy should be tailored based on each thyroid cancer variant with correlated biological behavior, clinical and genetic features. Presentation: No date and time listed

Spatial heterogeneity in DNA methylation and chromosomal alterations in diffuse gliomas and meningiomas

AbstractAdult-type diffuse gliomas and meningiomas are the most common primary intracranial tumors of the central nervous system. DNA methylation profiling is a novel diagnostic technique increasingly used also in the clinic. Although molecular heterogeneity is well described in these tumors, DNA methylation heterogeneity is less studied. We therefore investigated the intratumor genetic and epigenetic heterogeneity in diffuse gliomas and meningiomas, with focus on potential clinical implications. We further investigated tumor purity as a source for heterogeneity in the tumors. We analyzed genome-wide DNA methylation profiles generated from 126 spatially separated tumor biopsies from 39 diffuse gliomas and meningiomas. Moreover, we evaluated five methods for measurement of tumor purity and investigated intratumor heterogeneity by assessing DNA methylation-based classification, chromosomal copy number alterations and molecular markers. Our results demonstrated homogeneous methylation-based classification of IDH-mutant gliomas and further corroborates subtype heterogeneity in glioblastoma IDH-wildtype and high-grade meningioma patients after excluding samples with low tumor purity. We detected a large number of differentially methylated CpG sites within diffuse gliomas and meningiomas, particularly in tumors of higher grades. The presence of CDKN2A/B homozygous deletion differed in one out of two patients with IDH-mutant astrocytomas, CNS WHO grade 4. We conclude that diffuse gliomas and high-grade meningiomas are characterized by intratumor heterogeneity, which should be considered in clinical diagnostics and in the assessment of methylation-based and molecular markers.

59. Inferring copy number events in Glioblastoma using spatial gene expression data

<h3>Background</h3> Glioblastoma (GBM) is the most common malignant brain tumour in adults, with inevitable treatment resistance and poor outcomes. Genomic instability of GBM gives rise to large-scale chromosomal copy number variants (CNVs) that drive resistance. Genetically distinct clones in GBM are conventionally investigated using whole-genome sequencing (WGS), but recent bioinformatic approaches can infer CNVs using transcriptomics data only. <h3>Purpose</h3> To investigate whether patient-derived xenograft (PDX) mouse models of GBM exhibit clonal diversity as a function of space (across tumour regions) and time (early to late time points). <h3>Methods</h3> PDX data from 3 cell lines representing the tumour core, vascular regions, and infiltrating edge of one GBM patient's tumour were profiled at early and late time points in disease progression, using the 10X Genomics Visium platform for spatial transcriptomics. The computational tool inferCNV was used to identify CNVs. <h3>Results</h3> Successfully identified large-scale CNVs were concordant with events detected in matched WGS data. Additionally, we identified a minor subclone not detectable by WGS that expands in multiple PDX mice indicating it is pre-existing in the patient-derived cell lines. Further analysis revealed localization of this subclone to the tumour core region suggesting a microenvironment-specific adaptation. <h3>Conclusions</h3> We show that spatial gene expression data can be leveraged to infer CNVs in PDX models of GBM that correspond to known events from bulk WGS data. Subclones present below the limit of WGS detection can also be inferred. Charting the spatially resolved position of these clones in vivo supports the use of PDX models in GBM.

THE NATURE OF EMBRYONIC MOSAICISM ACROSS AGE AND CLINICS: AN ANALYSIS OF 21,345 PGT-A CYCLES

Preimplantation genetic testing for aneuploidy (PGT-A) was developed to prevent transferring embryos with chromosomal abnormalities. However, current genetic testing is sensitive enough to detect mosaic abnormalities, or non-uniform chromosomal copy number differences within a subset of trophectoderm cells. An absolute threshold for clinically significant mosaicism remains poorly defined and clinics differ in their management of mosaic embryo results. Here we share our efforts to uncover clinically relevant features of mosaicism via in depth analysis of a multinational database.

53. Clinical whole exome/whole transcriptome (WES/WTS) analysis detects copy number and structural rearrangements important

<h3>Background</h3> Chromosomal copy number alterations (CNAs) and structural rearrangements (SR) are a key part of risk stratification and management of patients with MM. We developed a clinical paired tumor/normal WES/WTS assay that detects CNAs at one-copy difference from focal to whole genome, and gene fusion at 20% and 10% limit of detection, respectively. <h3>Patients and Methods</h3> We performed WES/WTS analysis utilizing DNA/RNA extracted from CD138+ enriched cells and buccal swab from 92 MM patients. Plasma cell content was not available for most cases. We compared the detection of CNAs, and SR with other techniques currently in use including FISH, arrays and molecular analysis. <h3>Results</h3> Molecular alterations (MA) were present in 82/92(89.1%) cases, whereas 65/92(69.5%) cases had CNAs. In cases with CNAs (indicating sufficient tumor content), deletions of (1p32), (1p12), or both were detected in 7(10.8%), 12(18.5%), and 5(7.7%) cases, respectively. Gain of (1q22), and hyperdiploidy were detected in 19(29.2%) and 32(49.2%), respectively. Deletions of 13q, 14q, 16q, and 17p(TP53) were detected in 32(49.2%), 14(21.6%), 16(24.6%), and 13(20%) cases, respectively. In cases with MA, 10/82(10.8%), and 3(3.6%) cases had TP53 mono and bi-allelic inactivation, respectively. Translocations (4;14)(p16;q32)(IGH::NSD2/FGFR3) and t(14;16)(q32;q23)(IGH::MAF) were detected in 11(11.9%), and 3(3.7%) cases with MA, respectively. Overexpression of CCND1 (research use only) was observed in 8/82(9.7%) cases. <h3>Discussion</h3> Our WES/WTS detected abnormalities in this series similarly to what has been reported by techniques currently needed in combination such as FISH, arrays or molecular analysis. By incorporating RNA expression analysis, WES/WTS may reliably detect all clinically-relevant alterations in MM.

Clonal somatic copy number altered driver events inform drug sensitivity in high-grade serous ovarian cancer

Chromosomal instability is a major challenge to patient stratification and targeted drug development for high-grade serous ovarian carcinoma (HGSOC). Here we show that somatic copy number alterations (SCNAs) in frequently amplified HGSOC cancer genes significantly correlate with gene expression and methylation status. We identify five prevalent clonal driver SCNAs (chromosomal amplifications encompassing MYC, PIK3CA, CCNE1, KRAS and TERT) from multi-regional HGSOC data and reason that their strong selection should prioritise them as key biomarkers for targeted therapies. We use primary HGSOC spheroid models to test interactions between in vitro targeted therapy and SCNAs. MYC chromosomal copy number is associated with in-vitro and clinical response to paclitaxel and in-vitro response to mTORC1/2 inhibition. Activation of the mTOR survival pathway in the context of MYC-amplified HGSOC is statistically associated with increased prevalence of SCNAs in genes from the PI3K pathway. Co-occurrence of amplifications in MYC and genes from the PI3K pathway is independently observed in squamous lung cancer and triple negative breast cancer. In this work, we show that identifying co-occurrence of clonal driver SCNA genes could be used to tailor therapeutics for precision medicine.

Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.

Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.

Patient experience with non-invasive prenatal testing (NIPT) as a primary screen for aneuploidy in the Netherlands

BackgroundNon-invasive prenatal testing (NIPT) as a screening method for trisomy 21 and other chromosomal abnormalities has been adopted widely across the globe. However, while many clinical validation studies have been performed, less is known regarding the patient experience with NIPT. This study explored how individuals experience NIPT in a pre- and post-test setting, where NIPT is broadly available as a primary screening method with the option of reporting beyond common trisomies.MethodsParticipants were recruited using social media with a strategy designed to select individuals who had the option to have NIPT as part of the TRIDENT-2 study (In the Netherlands, NIPT is only available within the TRIDENT studies executed by the NIPT consortium. This research was done independently from the NIPT consortium.) in the Netherlands. The study used online questionnaires and semi-structured interviews. Both were developed around a patient experience framework consisting of seven themes: information, patient as active participant, responsiveness of services, lived experience, continuity of care and relationships, communication, and support.ResultsOverall, 4539 questionnaire responses were analyzed and 60% of the respondents had experienced NIPT. Of those, 1.7% received a high-risk result for trisomy or another chromosomal copy number variant (referred to as an “additional finding”). Overall, participants felt they had received sufficient information and had control over their decision regarding whether or not to choose NIPT. The vast majority of respondents who had NIPT were positive about their experience and would use it again. Those with results showing an increased probability for trisomy or additional findings were more likely to report negative feelings such as tension and anxiety, and less likely to feel that they had been sufficiently prepared for the implications of their results.ConclusionsThe patient experience with first-tier NIPT in the Netherlands was largely positive. Areas for improvement included counseling on the implications of screening and the different possible outcomes of NIPT, including additional findings that may be uncovered by expanding NIPT beyond the common trisomies. The experiences reported in this study may be useful for other countries intending to implement NIPT.

Combining bulk RNA-sequencing and single-cell RNA-sequencing data to reveal the immune microenvironment and metabolic pattern of osteosarcoma

Background: Osteosarcoma (OS) is a kind of solid tumor with high heterogeneity at tumor microenvironment (TME), genome and transcriptome level. In view of the regulatory effect of metabolism on TME, this study was based on four metabolic models to explore the intertumoral heterogeneity of OS at the RNA sequencing (RNA-seq) level and the intratumoral heterogeneity of OS at the bulk RNA-seq and single cell RNA-seq (scRNA-seq) level. Methods: The GSVA package was used for single-sample gene set enrichment analysis (ssGSEA) analysis to obtain a glycolysis, pentose phosphate pathway (PPP), fatty acid oxidation (FAO) and glutaminolysis gene sets score. ConsensusClusterPlus was employed to cluster OS samples downloaded from the Target database. The scRNA-seq and bulk RNA-seq data of immune cells from GSE162454 dataset were analyzed to identify the subsets and types of immune cells in OS. Malignant cells and non-malignant cells were distinguished by large-scale chromosomal copy number variation. The correlations of metabolic molecular subtypes and immune cell types with four metabolic patterns, hypoxia and angiogenesis were determined by Pearson correlation analysis. Results: Two metabolism-related molecular subtypes of OS, cluster 1 and cluster 2, were identified. Cluster 2 was associated with poor prognosis of OS, active glycolysis, FAO, glutaminolysis, and bad TME. The identified 28608 immune cells were divided into 15 separate clusters covering 6 types of immune cells. The enrichment scores of 5 kinds of immune cells in cluster-1 and cluster-2 were significantly different. And five kinds of immune cells were significantly correlated with four metabolic modes, hypoxia and angiogenesis. Of the 28,608 immune cells, 7617 were malignant cells. The four metabolic patterns of malignant cells were significantly positively correlated with hypoxia and negatively correlated with angiogenesis. Conclusion: We used RNA-seq to reveal two molecular subtypes of OS with prognosis, metabolic pattern and TME, and determined the composition and metabolic heterogeneity of immune cells in OS tumor by bulk RNA-seq and single-cell RNA-seq.

Exploration the global single-cell ecological landscape of adenomyosis-related cell clusters by single-cell RNA sequencing

Background: Adenomyosis (AM) is a common benign uterine disease that threatens the normal life of patients. Cells associated with microenvironmental immune ecology are crucial in AM, although they are not as well understood at the cellular level. Methods: Single-cell sequencing (scRNA-seq) data were used to construct an AM global single-cell map, to further identify relevant cell clusters and infer chromosomal copy number variation (CNV) in AM samples. The biological functions of cell clusters were explored and cellular evolutionary processes were inferred by enrichment analysis and pseudotime analysis. In addition, a gene regulatory network (GRN) analysis was constructed to explore the regulatory role of transcription factors in AM progression. Results: We obtained the expression profiles of 42260 cells and identified 10 cell clusters. By comparing the differences in cell components between AM patients and controls, we found that significant abundance of endometrial cells (EC), epithelial cells (Ep), endothelial cells (En), and smooth muscle cells (SMC) in AM patients. Cell clusters with high CNV levels possessing tumour-like features existed in the ectopic endometrium samples. Moreover, the Ep clusters were significantly involved in leukocyte transendothelial cell migration and apoptosis, suggesting an association with cell apoptosis and migration. En clusters were mainly involved in pathways in cancer and apoptosis, indicating that En has certain malignant features. Conclusion: This study identified cell clusters with immune-related features, investigated the changes in the immune ecology of the microenvironment of these cells during AM, and provided a new strategy for the treatment of AM.

136P Paths of chromosomal instability and copy number alteration in circulating tumor cells of progressing early-stage breast cancer patients

Sequencing of circulating tumor cells (CTC) allows capturing genetic diversity by copy number alterations (CNA) and measuring chromosomal instability (CIN) at single-cell level during cancer treatment and progression.

FACTORS ASSOCIATED WITH PREGNANCY LOSS AFTER SINGLE EUPLOID EMBRYO TRANSFER

Clinical pregnancy loss (CPL) is often attributed to chromosomal copy number errors in the human embryo. When undergoing in vitro fertilization (IVF), preimplantation genetic testing for aneuploidy (PGT-A) can be utilized to select euploid embryos for transfer, thus significantly decreasing the risk of miscarriage. Surprisingly, pregnancy loss after single euploid embryo transfer (SEET) can still occur. This study sought to identify patient and cycle-specific factors associated with CPL after a SEET.

Current Perspectives of Prenatal Cell-free DNA Screening in Clinical Management of First-Trimester Septated Cystic Hygroma

First-trimester septated cystic hygroma occurs in approximately 1 in 268 pregnancies and has long been associated with a markedly increased risk of fetal aneuploidy and, among euploid fetuses, an increased risk of structural anomalies primarily affecting the cardiac and skeletal systems. Invasive prenatal diagnosis - chorionic villus sampling and/or amniocentesis - encompasses the time-honored clinical tools for the next step in management following prenatal sonographic diagnosis of first-trimester septated cystic hygroma. Currently, prenatal cell-free DNA (cfDNA) screening for fetal aneuploidy with select microdeletions is gradually replacing the considerably less sensitive, and labor-intensive combined first-trimester screening. These new technologies have opened potential new venues in the clinical management of this ominous late first-trimester sonographic diagnosis. Advances in cfDNA technologies are now permitting detection of chromosomal copy number variants (CNV) larger than 7Mb across genome and select serious single-gene disorders (mainly impacting skeletal and neurological development), affecting quality of life and may benefit from medical and/or surgical management. This commentary will address the available non-invasive prenatal screening technologies, which clearly enhance immediate genetic analysis modalities applicable in the presence of the complex sonographic finding of first-trimester septated cystic hygroma.

Analysis of chromosomal copy number variations among 163 fetuses with echogenic bowel by using CNV-seq technology

To assess the value of low-depth whole-genome copy number variation sequencing (CNV-seq) for the analysis of chromosomal copy number variations among fetuses with echogenic bowel (EB). A total of 163 fetuses were included in this study. Amniotic fluid (162 cases) or chorionic villi (1 case) were collected and subjected to CNV-seq for the analysis of CNVs. Thirteen (8.0%) pathogenic CNVs were detected, including 9 (5.5%) aneuploidies and 4 (2.4%) CNVs. The detection rate of the isolated EB group and combined EB group were 1.7% (1/58) and 11.4% (12/105), respectively. There was a significant difference between the two groups (P < 0.05). A Xp22.1 duplication was detected in both groups, and the fetuses were predicted as female DMD carriers and born healthy. Nine cases of aneuploidies and 2 (likely) pathogenic CNVs were identified in the combined EB group, all of them have warranted induced labor. The prevalence of chromosomal aneuploidies and pathogenic CNVs in fetuses with combined EB was much higher than isolated EB, and most of them may warrant termination of pregnancy. Compared with isolated EB, more attention should be paid to combined EB, for which prenatal diagnosis and genetic counseling should be carried out in time.

JS04.4.A Beyond β-catenin: Genetic alterations ofTP53 andOTX2 and older age indicate increased risk of relapse in WNT medulloblastomas

Abstract Background This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of age, TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers. Material and Methods In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Association with prognosis was evaluated in 133 patients with follow-up data from the HIT2000 medulloblastoma trial, HIT registries, and the NOA-07 trial. Results CTNNB1 mutations were present in 92.2% of the samples. APC mutations were found in 6.8% (13 samples). One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, and more frequent in children compared to adolescents/adults (≥16 years). Adolescents/adults showed worse overall survival (OS; p=0.009) compared to children, but not worse progression-free survival (PFS; p=0.106). With an age cut-off at 18 years, no survival difference was found. Also adolescents alone (16-20 years) had worse OS (p=0.003) compared to children, whereas in patients ≥21 (n=12 adults with PFS/OS data) no tumor progression/relapse occurred. Only one adult died due to therapy-related complications. WNT-MB patients with tumors harboring TP53 mutations (24/133, 18.1%) showed significant worse PFS (p=0.001), which was also found in children and adolescents individually (p=0.004, resp. p=0.017). Gains of the OTX2 locus on chromosome 14q found in 40.2% (35/87) of samples were independent of TP53 mutations and also associated with poor PFS and OS (p=0.034, resp. p=0.016). Individual analyses of OTX2 gains within age groups showed only worse OS in children (p=0.012). Multivariate Cox regression analysis for PFS identified both genetic alterations, but not age, as independent prognostic markers. For OS, multivariate analysis found OTX2 gains and older age as independent prognostic markers. Conclusion Our data suggest that adolescent patients with WNT-MB and those patients carrying TP53 mutations or OTX2 gains - independent of age - are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

Genome-wide analysis reveals allelic variation and chromosome copy number variation in paromomycin-resistant Leishmania donovani.

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite's genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene's upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant.