Detection Of Copy Number Alterations Research Articles

CNAsim: improved simulation of single-cell copy number profiles and DNA-seq data from tumors.

CNAsim is a software package for improved simulation of single-cell copy number alteration (CNA) data from tumors. CNAsim can be used to efficiently generate single-cell copy number profiles for thousands of simulated tumor cells under a more realistic error model and a broader range of possible CNA mechanisms compared with existing simulators. The error model implemented in CNAsim accounts for the specific biases of single-cell sequencing that leads to read count fluctuation and poor resolution of CNA detection. For improved realism over existing simulators, CNAsim can (i) generate WGD, whole-chromosomal CNAs, and chromosome-arm CNAs, (ii) simulate subclonal population structure defined by the accumulation of chromosomal CNAs, and (iii) dilute the sampled cell population with both normal diploid cells and pseudo-diploid cells. The software can also generate DNA-seq data for sampled cells. CNAsim is written in Python and is freely available open-source from https://github.com/samsonweiner/CNAsim.

Shallow whole-genome sequencing of bone marrow aspirates in myelodysplastic neoplasms: A retrospective comparison with cytogenetics.

Copy number alterations (CNA) are powerful prognostic markers in myelodysplastic neoplasms (MDS) and are routinely analyzed by conventional cytogenetic analysis (CCA) on bone marrow (BM). Although CCA is still the gold standard, it requires extensive hands-on time and highly trained staff for the analysis, making it a laborious technique. To reduce turn-around-time per case, shallow whole genome sequencing (sWGS) technologies offer new perspectives for the diagnostic work-up of this disorder. We compared sWGS with CCA for the detection of CNAs in 33 retrospective BM samples of patients with MDS. Using sWGS, CNAs were detected in all cases and additionally allowed the analysis of three cases for which CCA failed. The prognostic stratification (IPSS-R score) of 27 out of 30 patients was the same with both techniques. In the remaining cases, discrepancies were caused by the presence of balanced translocations escaping sWGS detection in two cases, a subclonal aberration reported with CCA that could not be confirmed by FISH or sWGS, and the presence of an isodicentric chromosome idic(17)(p11) missed by CCA. Since sWGS can almost entirely be automated, our findings indicate that sWGS is valuable in a routine setting validating it as a cost-efficient tool.

Abstract 6220: Development of a mutation profiling NGS assay to facilitate clinical decisions in hematologic malignancies

Abstract Introduction: Accurate diagnosis, prognosis and treatment of hematologic disorders from both myeloid and lymphoid origin requires assessing somatic mutation status in an increasingly large number of genes. Here we present the results of the assay development and validation of a targeted next generation sequencing panel which provides full exon sequencing of 141 genes associated with myeloid and lymphoid malignancies, as well as the ability to detect clinically relevant copy number alterations (CNAs) and certain sub-gene level CNAs. Methods: Based on review of clinical guidelines, 141 genes including 72 myeloid and 84 lymphoid associated genes were selected. The assay interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The assay also identifies CNAs in 16 genes, FLT3 internal tandem duplicates (ITDs), and select non-coding pathogenic variants. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood or bone marrow followed by sequencing on Illumina sequencers. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/Indels or digital multiplexed ligation-dependent probe amplification for CNAs. In addition, analytical sensitivity was assessed using cell line dilution series and specificity by reference samples from Genome in a Bottle (GIAB). Small indels were defined as <25bp while long indels are ≥ 25bp. Results: Interim estimates of positive percentage agreement (PPA) and specificity have been determined for SNV/Indels, FLT3-ITDs, and CNVs, with further testing ongoing. PPA of SNVs was 100% (218/218 SNVs; n = 134 samples). PPA was also 100% for both small indels (21/21; n = 14 samples) and long indels (6/6; n = 6 samples). A dilution series of 8 positive clinical samples at 4 different dilutions showed SNV and small indel detection of 100% at allele frequency (AF) ≥ 3%. Long indel detection was 87.5% at AF ≥ 5%. FLT3-ITD was evaluated with 2 cell line samples at 5 different dilutions with 100% detection at AF ≥ 5%. PPA of CNAs were 95.7% (157/164) in 46 clinical samples. CNA detection was also evaluated with 8 cell line samples at 4 different dilutions showing 92.3% detection at a copy number ratio threshold ± 0.15. A preliminary estimate of specificity was determined using 4 replicates of the GIAB sample, NA12878, and was >99.99% for SNVs and small indels (n=6.9 × 105). Finally, a model for gender classification was created using a training cohort of 70 clinical samples. The performance was evaluated on a test set of 144 clinical samples with 100% PPA (144/144). Conclusions: Taken together, these data demonstrate that the developed NGS assay is a sensitive, specific, and accurate assay with high clinical utility to inform treatment decision making in hematologic malignancies. Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Stan Letovsky, Li Cai, Eric A. Severson, Shakti Ramkissoon, Anjen Chenn, Marcia Eisenberg, Eyad Almasri, Taylor Jensen. Development of a mutation profiling NGS assay to facilitate clinical decisions in hematologic malignancies [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 6220.

Combined low-pass whole genome and targeted sequencing in liquid biopsies for pediatric solid tumors

We designed a liquid biopsy (LB) platform employing low-pass whole genome sequencing (LP-WGS) and targeted sequencing of cell-free (cf) DNA from plasma to detect genome-wide copy number alterations (CNAs) and gene fusions in pediatric solid tumors. A total of 143 plasma samples were analyzed from 19 controls and 73 patients, including 44 bone or soft-tissue sarcomas and 12 renal, 10 germ cell, five hepatic, and two thyroid tumors. cfDNA was isolated from plasma collected at diagnosis, during and after therapy, and/or at relapse. Twenty-six of 37 (70%) patients enrolled at diagnosis without prior therapy (radiation, surgery, or chemotherapy) had circulating tumor DNA (ctDNA), based on the detection of CNAs from LP-WGS, including 18 of 27 (67%) patients with localized disease and eight of 10 (80%) patients with metastatic disease. None of the controls had detectable somatic CNAs. There was a high concordance of CNAs identified by LP-WGS to CNAs detected by chromosomal microarray analysis in the matching tumors. Mutations identified in tumor samples with our next-generation sequencing (NGS) panel, OncoKids®, were also detected by LP-WGS of ctDNA in 14 of 26 plasma samples. Finally, we developed a hybridization-based capture panel to target EWSR1 and FOXO1 fusions from patients with Ewing sarcoma or alveolar rhabdomyosarcoma (ARMS), respectively. Fusions were detected in the plasma from 10 of 12 patients with Ewing sarcoma and in two of two patients with ARMS. Combined, these data demonstrate the clinical applicability of our LB platform to evaluate pediatric patients with a variety of solid tumors.

Circulating Tumor DNA Is Prognostic in Intermediate-Risk Rhabdomyosarcoma: A Report From the Children's Oncology Group.

Novel biomarkers are needed to differentiate outcomes in intermediate-risk rhabdomyosarcoma (IR RMS). We sought to evaluate strategies for identifying circulating tumor DNA (ctDNA) in IR RMS and to determine whether ctDNA detection before therapy is associated with outcome. Pretreatment serum and tumor samples were available from 124 patients with newly diagnosed IR RMS from the Children's Oncology Group biorepository, including 75 patients with fusion-negative rhabdomyosarcoma (FN-RMS) and 49 with fusion-positive rhabdomyosarcoma (FP-RMS) disease. We used ultralow passage whole-genome sequencing to detect copy number alterations and a new custom sequencing assay, Rhabdo-Seq, to detect rearrangements and single-nucleotide variants. We found that ultralow passage whole-genome sequencing was a method applicable to ctDNA detection in all patients with FN-RMS and that ctDNA was detectable in 13 of 75 serum samples (17%). However, the use of Rhabdo-Seq in FN-RMS samples also identified single-nucleotide variants, such as MYOD1L122R, previously associated with prognosis. Identification of pathognomonic translocations between PAX3 or PAX7 and FOXO1 by Rhabdo-Seq was the best method for measuring ctDNA in FP-RMS and detected ctDNA in 27 of 49 cases (55%). Patients with FN-RMS with detectable ctDNA at diagnosis had significantly worse outcomes than patients without detectable ctDNA (event-free survival, 33.3% v 68.9%; P = .0028; overall survival, 33.3% v 83.2%; P < .0001) as did patients with FP-RMS (event-free survival, 37% v 70%; P = .045; overall survival, 39.2% v 75%; P = .023). In multivariable analysis, ctDNA was independently associated with worse prognosis in FN-RMS but not in the smaller FP-RMS cohort. Our study demonstrates that baseline ctDNA detection is feasible and is prognostic in IR RMS.

BIOS-05. SANGER SEQUENCING AND COPY-NUMBER ALTERATION ANALYSIS WITH MLPA CAN CLINICALLY CLASSIFY IDH-WILD-TYPE LOWER-GRADE ASTROCYTOMA

Abstract The IDH-wild-type lower-grade astrocytomas are a heterogeneous entity. According to 2021 WHO classification, IDH-wild-type astrocytomas with any of the following factors show poor prognosis: combination of chromosome 7 gain and 10 loss (+7/-10), and/or EGFR amplification, and/or TERT promoter (TERTp) mutation. Multiplex ligation-dependent probe amplification (MLPA) can detect copy number alterations with a reasonable cost. The purpose of this study was to find precise and cost-effective method for stratifying the prognosis of IDH-wild-type astrocytomas. Sanger sequencing, MLPA, and quantitative methylation-specific PCR were performed for 42 IDH-wild-type lower-grade astrocytomas surgically treated at Kyoto University Hospital, and overall survival was analyzed for 40 patients. Of the 42 IDH-wild-type astrocytomas, 21 were classified as grade 4 in 2021 WHO classification and all of them had either TERTp mutation or EGFR amplification. Kaplan-Meier analysis confirmed the prognostic significance of 2021 WHO Classification, and WHO grade was also prognostic. Cox regression hazard model identified PTEN loss and PDGFRA amplification as independent significant prognostic indicators (Risk ratio of 9.75, p&amp;lt; 0.001 and 13.9, p=0.002). The classification recommended by 2021 WHO Classification could be completed using Sanger sequencing and MLPA. Survival analysis revealed PTEN and PDGFRA were significant prognostic factors for IDH-wild-type lower-grade astrocytomas.

36. Clinical utility of copy number alteration analysis in the evaluation of Melanocytic Lesions for diagnosis and prognosis

Melanocytic lesions range from benign to highly aggressive malignancies. Although the diagnosis is often straightforward histologically, ambiguous cases provide a dilemma with critical clinical implications. Copy number alteration (CNA) analysis, from formalin-fixed paraffin-embedded tissue, has emerged over the last two decades as a valuable tool for diagnosis of histologically challenging melanocytic lesions. Numerous studies, with various microarray platforms, demonstrate that the number of CNAs in malignant melanomas is higher than in benign melanocytic nevi with proposed cutoffs for adequate sensitivity and specificity. Targeted fluorescence in situ hybridization panels, utilizing probes to detect the CNAs most commonly seen in melanoma, are sufficient for diagnostic purposes. Furthermore, studies determined that CNAs have prognostic significance. Both the number of CNAs and detection of specific CNAs are helpful for risk stratification. To provide evidence-based recommendations for clinical utility of CNAs in melanocytic lesions, the Cancer Genomics Consortium Working Group for Melanocytic Lesions assessed 410 peer-reviewed articles selected from an extensive Ovid search of publications from 1998 through 2021. The group determined the power of CNAs for 1) aiding diagnosis, 2) stratifying risk, and 3) guiding clinical management. We present the evidence base for the clinical utility of CNA assessment in the workup of melanocytic lesions including ocular, mucosal, and cutaneous melanomas with characteristic findings in various subtypes. This working group has synthesized the findings to establish consensus recommendations for the interpretation of CNAs in melanocytic lesions and incorporation into standard clinical laboratory testing.

Read Count Patterns and Detection of Cancerous Copy Number Alterations in Plasma Cell-Free DNA Whole Exome Sequencing Data for Advanced Non-Small Cell Lung Cancer.

Plasma cell-free DNA (cfDNA) sequencing data have been widely studied for early diagnosis and treatment response or recurrence monitoring of cancers because of the non-invasive benefits. In cancer studies, whole exome sequencing (WES) is mostly used for discovering single nucleotide variants (SNVs), but it also has the potential to detect copy number alterations (CNAs) that are mostly discovered by whole genome sequencing or microarray. In clinical settings where the quantity of the acquired blood from the patients is limited and where various sequencing experiments are not possible, providing various types of mutation information such as CNAs and SNVs using only WES will be helpful in the treatment decision. Here, we questioned whether the plasma cfDNA WES data for patients with advanced non-small cell lung cancer (NSCLC) could be exploited for CNA detection. When the read count (RC) signals of the WES data were investigated, a similar fluctuation pattern was observed among the signals of different samples, and it can be a major challenge hindering CNA detection. When these RC patterns among cfDNA were suppressed by the method we proposed, the cancerous CNAs were more distinguishable in some samples with higher cfDNA quantity. Although the potential to detect CNAs using the plasma cfDNA WES data for NSCLC patients was studied here, further studies with other cancer types, with more samples, and with more sophisticated techniques for bias correction are required to confirm our observation. In conclusion, the detection performance for cancerous CNAs can be improved by controlling RC bias, but it depends on the quantity of cfDNA in plasma.

Optical genome mapping identifies clinically relevant genomic rearrangements in prostate cancer biopsy sample

BackgroundProstate cancer (PCa) is characterized by complex genomic rearrangements such as the ETS oncogene family fusions, yet the clinical relevance is not well established. While paneled genetic tests of DNA repair genes are recommended in advanced PCa, conventional genomic or cytogenetic tools are not ideal for genome-wide screening of structural variations (SVs) such as balanced translocation due to cost and/or resolution issues.MethodsIn this study, we tested the feasibility of whole-genome optical genomic mapping (OGM), a newly developed platform for genome-wide SV analysis to detect complex genomic rearrangements in consecutive unselected PCa samples from MRI/US-fusion targeted biopsy.ResultsWe tested ten samples, and nine (90%) passed quality check. Average mapping rate and coverage depth were 58.1 ± 23.7% and 157.3 ± 97.7×, respectively (mean ± SD). OGM detected copy number alterations such as chr6q13 loss and chr8q12-24 gain. Two adjacent tumor samples were distinguished by inter/intra-chromosomal translocations, revealing that they’re from the same ancestor. Furthermore, OGM detected large deletion of chr13q13.1 accompanied by inter-chromosomal translocation t(13;20)(q13.1;p13) occurring within BRCA2 gene, suggesting complete loss of function.ConclusionIn conclusion, clinically relevant genomic SVs were successfully detected in PCa samples by OGM. We suggest that OGM can complement panel sequencing of DNA repair genes BRCA1/2 or ATM in high-risk PCa.

Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing

The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy.Graphical

Genomic copy number alterations as biomarkers for triple negative pregnancy-associated breast cancer

BackgroundPABC, commonly defined as breast cancer diagnosed during or ≤ 1 year after pregnancy, accounts for 7% of all breast cancers in women ≤ 45 years. Compared to age-matched non-PABC patients, PABC is characterized by a particularly aggressive histopathologic profile with poorly differentiated and estrogen- and progesterone receptor negative tumors and associated high mortality rates. This study assessed the genomic background of triple-negative PABC tumors by detection of copy number alterations (CNAs).MethodsMLPA was used to compare CNAs in breast cancer-associated chromosomal loci between triple-negative PABC- and subtype-matched non-PABC patients. Both CNA patterns were evaluated by cluster analysis; associations between individual gene CNAs, pathological characteristics and survival were explored.ResultsTriple-negative PABC tumors exhibited unique CNAs compared to non-PABC tumors, including enrichment for TOP2A copy number loss, an independent predictor of worse overall survival (HR 8.96, p = 0.020). Cluster analysis based on CNA profiles identified a triple-negative PABC-subgroup with a particularly poor prognosis, characterized by chromosome 8p copy number loss. Individual gene CNAs analysis revealed that FGFR1 copy number loss on chromosome 8p11.23 was an independent predictor of poor outcome in multivariate analysis (HR 3.59, p = 0.053) and predicted the development of distant metastases (p = 0.048).ConclusionThis study provides novel insights into the biology of triple-negative PABC tumors suggesting that CNAs, particularly 8p loss and TOP2A loss, are involved in the development of breast cancer during pregnancy. FGFR1 loss and TOP2A loss seem to be promising new biomarkers that independently identify subgroups of PABC patients with poor prognosis. These genomic biomarkers may provide clues for personalized therapy.

P456: A LONG READ SEQUENCING AND CRISPR-CAS9 BASED APPROACH FOR RAPID COPY-NUMBER ALTERATION AND STRUCTURAL VARIATION DETECTION IN HEMATOLOGIC MALIGNANCIES

Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions. Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit). Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints. Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics.

Abstract 2949: Enabling single cell analysis of copy number variation in breast cancer

Abstract Breast cancer is arguably one of the solid tumors most comprehensively analyzed by massively parallel sequencing. Although the repertoire of clonal somatic genetic alterations in primary and metastatic breast cancers has been characterized, a complete characterization of the subclonal genetic alterations present in these cancers, as well as their clonal composition and clonal dynamics remain to be fully defined. Addressing these knowledge gaps is germane to the successful personalization of treatments for breast cancer patients. Single cell sequencing is uncovering many insights into the clonal evolution and heterogeneity of breast cancers and how this can be leveraged for clinical and therapeutic benefit. Bulk next generation sequencing has primarily focused on mutation profiling; however, there are several lines of evidence to demonstrate the biological and clinical importance of accurately detecting copy number alterations. Previous single cell sequencing studies focusing on copy number alterations were based on whole-genome amplification methods or could not concurrently define the repertoire of somatic mutations and copy number alterations in single cells. Here we report on a novel single cell DNA sequencing approach leveraging the Tapestri device and Insights bioinformatic pipeline to detect both copy number alterations and somatic mutations in individual cells derived from breast cancer specimens. The identification of targeted somatic genetic alterations at single cell resolution provides novel insights about the development, progression and therapeutic resistance of breast cancers, and enables the development of novel diagnostic tools and predictive biomarkers. Citation Format: Saurabh Parikh, Arnaud Da Cruz Paula, Shirin Issa Bhaloo, Alex Li, Britta Weigelt, Khushali Patel, Ania Wronski, Anup Parikh, Khushali Patel, Jorge Reis-Filho. Enabling single cell analysis of copy number variation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2949.

Identification of IKZF1plus microdeletion profile in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) using single nucleotide polymorphism (SNP) array

Genetic abnormalities detected in the leukemic genome became essential prognostic factors stratifying the intensity of treatment in children with acute lymphoblastic leukemia (ALL). However, only selected copy number alterations are independently associated with the clinical course of disease. Deletions within the &lt;i&gt;IKZF1&lt;/i&gt; gene significantly confer to poor dzieprognosis of ALL, particularly when cooccurred with deletions of &lt;i&gt;CDKN2A/ B&lt;/i&gt;, &lt;i&gt;PAX5&lt;/i&gt;, and genes from PAR1 region and the concomitant lack of ERG gene deletion is present. In the project, high-density SNP array were tested on 195 childhood BCP-ALL samples to optimize the detection of copy number alterations within selected gene loci. Developed experimental protocol and data analysis enabled identification of clonal and mosaic forms of &lt;i&gt;IKZF1&lt;/i&gt; deletions.

Shallow whole-genome sequencing: a useful, easy to apply molecular technique for CNA detection on FFPE tumor tissue-a glioma-driven study.

Copy number alterations (CNAs) have increasingly become part of the diagnostic algorithm of glial tumors. Alterations such as homozygous deletion of CDKN2A/B, 7+/ 10- chromosome copy number changes or EGFR amplification are predictive of a poor prognosis. The codeletion of chromosome arms 1p and 19q, typically associated with oligodendroglioma, implies a more favorable prognosis. Detection of this codeletion by the current diagnostic standard, being fluorescence in situ hybridization (FISH), is sometimes however subject to technical and interpretation problems. In this study, we evaluated CNA detection by shallow whole-genome sequencing (sWGS) as an inexpensive, complementary molecular technique. A cohort of 36 glioma tissue samples, enriched with "difficult" and "ambiguous" cases, was analyzed by sWGS. sWGS results were compared with FISH assays of chromosomes 1p and 19q. In addition, CNAs relevant to glioblastoma diagnosis were explored. In 4/36 samples, EGFR (7p11.2) amplifications and homozygous loss of CDKN2A/B were identified by sWGS. Six out of 8 IDH-wild-type glioblastomas demonstrated a prognostic chromosome 7/chromosome 10 signature. In 11/36 samples, local interstitial and terminal 1p/19q alterations were detected by sWGS, implying that FISH's targeted nature might promote false arm-level extrapolations. In this cohort, differences in overall survival between patients with and without codeletion were better pronounced by the sequencing-based distinction (likelihood ratio of 7.48) in comparison to FISH groupings (likelihood ratio of 0.97 at diagnosis and 1.79 ± 0.62 at reobservation), suggesting sWGS is more accurate than FISH. We recognized adverse effects of tissue block age on FISH signals. In addition, we show how sWGS reveals relevant aberrations beyond the 1p/19q state, such as EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10, and homozygous loss of CDKN2A/B. The findings presented by this study might stimulate implementation of sWGS as a complementary, easy to apply technique for copy number detection.

Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine.

The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine. We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions. ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%. Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.

FACETS: Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing.

Clinical sequencing studies routinely involve molecular profiling of patients for mutations and copy number alterations. However, detection of "actionable" aberrations to guide treatment decisions require accurate, tumor purity-, ploidy-, and clonal heterogeneity-adjusted integer copy number calls. In this chapter, we describe the FACETS algorithm, an Allele-Specific Copy Number (ASCN) analysis tool with a broad application to whole-genome, whole-exome, as well as targeted panel sequencing platforms to annotate the genome for the detection of copy number alterations including homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH) events, allele-specific gains/amplifications, and cellular fraction profiles.We will describe some methodological details on joint segmentation of total and allele-specific copy number, on the estimation of integer copy number calls adjusting for tumor purity, ploidy, and intratumor heterogeneity, along with comprehensive output and integrated visualization. We also provide a tutorial on the installation, application, and tips to run and interpret FACETS.

Copy Number Alterations in Hepatoblastoma: Literature Review and a Brazilian Cohort Analysis Highlight New Biological Pathways

Hepatoblastoma (HB) is a rare embryonal tumor, although it is the most common pediatric liver cancer. The aim of this study was to provide an accurate cytogenomic profile of this type of cancer, for which information in cancer databases is lacking. We performed an extensive literature review of cytogenetic studies on HBs disclosing that the most frequent copy number alterations (CNAs) are gains of 1q, 2/2q, 8/8q, and 20; and losses at 1p and 4q. Furthermore, the CNA profile of a Brazilian cohort of 26 HBs was obtained by array-CGH; the most recurrent CNAs were the same as shown in the literature review. Importantly, HBs from female patients, high-risk stratification tumors, tumors who developed in older patients (> 3 years at diagnosis) or from patients with metastasis and/or deceased carried a higher diversity of chromosomal alterations, specifically chromosomal losses at 1p, 4, 11q and 18q. In addition, we distinguished three major CNA profiles: no detectable CNA, few CNAs and tumors with complex genomes. Tumors with simpler genomes exhibited a significant association with the epithelial fetal subtype of HBs; in contrast, the complex genome group included three cases with epithelial embryonal histology, as well as the only HB with HCC features. A significant association of complex HB genomes was observed with older patients who developed high-risk tumors, metastasis, and deceased. Moreover, two patients with HBs exhibiting complex genomes were born with congenital anomalies. Together, these findings suggest that a high load of CNAs, mainly chromosomal losses, particularly losses at 1p and 18, increases the tendency to HB aggressiveness. Additionally, we identified six hot-spot chromosome regions most frequently affected in the entire group: 1q31.3q42.3, 2q23.3q37.3, and 20p13p11.1 gains, besides a 5,3 Mb amplification at 2q24.2q24.3, and losses at 1p36.33p35.1, 4p14 and 4q21.22q25. An in-silico analysis using the genes mapped to these six regions revealed several enriched biological pathways such as ERK Signaling, MicroRNAs in Cancer, and the PI3K-Akt Signaling, in addition to the WNT Signaling pathway; further investigation is required to evaluate if disturbances of these pathways can contribute to HB tumorigenesis. The analyzed gene set was found to be associated with neoplasms, abnormalities of metabolism/homeostasis and liver morphology, as well as abnormal embryonic development and cytokine secretion. In conclusion, we have provided a comprehensive characterization of the spectrum of chromosomal alterations reported in HBs and identified specific genomic regions recurrently altered in a Brazilian HB group, pointing to new biological pathways, and relevant clinical associations.

PATH-48. RAPID-CNS2: RAPID COMPREHENSIVE ADAPTIVE NANOPORE-SEQUENCING OF CNS TUMORS, A PROOF OF CONCEPT STUDY

Abstract BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSIONS RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array- based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to &amp;lt; 12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

Accurate Prediction of Chromosome-Level CNVs from Targeted NGS

Background: Aneuploidy and large-scale Copy Number Variations (CNVs) are prominent features of cancer cells. While Fluorescence in situ hybridization (FISH) and conventional cytogenetics (CC) are the gold standard for detecting aneuploidy and CNVs, NGS-based assays are currently used for high-resolution detection of copy number alterations assessing the whole genome. However, although an increasing number of NGS-based tools have been developed for detecting aneuploidy or CNVs from whole genome or exome sequencing data, only a limited number of options are available for targeted gene panels. Despite mechanisms provided to establish normal profiles for a specific panel, the accuracy of these tools at the chromosome level suffer when only a small number of regions are targeted on each chromosome. Here we leveraged on a custom amplicon based NGS assay designed to detect somatic alterations (SNVs and indels) in 297 hematological cancer relevant genes, previously validated in our clinical laboratory. We introduce a simple approach to accurately predict chromosome-level CNVs such as monosomy and trisomy for a targeted gene panel, commonly used in a clinical setting.Methods: Mutation profiles, including SNVs, INDELs, and structural changes, were interrogated with an in-house bioinformatics pipeline that utilized PureCN and CNVkit algorithms to detect structural changes. The first step consists of finding optimal panel-specific decision thresholds for gains and losses at the gene level. This step was performed using an independent set of 1,314 clinical samples sequenced with the NeoType® Heme assay developed by NeoGenomics Laboratories, Inc. for which at least one FISH test was performed in addition to the sequencing. Three genes (ATM, TP53, and NF1) were used to find optimal decision thresholds based on the FISH result for these markers. These thresholds are used afterward to predict a gain or a loss for any other gene in the panel. The second step consists of predicting the chromosome-level gain or loss based on the individual predictions at the gene level by simply observing the frequency of targeted genes on the corresponding chromosome predicted as either gained or lost by the first step approach. The 19, 7, and 18 targeted genes in the NGS panel (Table 1) were respectively used to predict monosomy 7, trisomy 8, and trisomy 12 in a second set of over 7,000 clinical samples with known ploidy for chromosomes with clinically relevant ploidy abnormalities in hematological malignancies.Results: Evaluation of the first stage gene-level CNV prediction on 1,314 clinical samples shows a concordance rate of 97.95% between NGS and FISH results on ATM, TP53, and NF1. When we evaluated the second stage chromosome-level CNV prediction in clinical samples sequenced using the same targeted panel and assessed by FISH for chromosome-level variation on chromosomes 7, 8 and 12 (Table 1), a heatmap of the predicted Log 2 ratios for each sample and targeted gene from the first step shows a clear distinctive signal between aneuploidy and diploid samples (Figure 1). At the chromosome level, the concordance rate between the final prediction and the FISH results is consistently observed above 93% (Table 2). Roughly 50% of the 12, 78, and 40 discordant calls for monosomy 7, trisomy 8, and trisomy 12, respectively captured by FISH but not by NGS can be explained by low tumor content (less than 20%) in the tested samples. The concordance rate between NGS and FISH is consistently observed above 96% when leaving these samples aside. Note that results in Table 2 are obtained using all samples to decide the optimal decision threshold for the chromosome-level prediction, but are found identical when using a leave-one-out evaluation procedure, and nearly identical when using a repeated cross-validation procedure.Conclusion: This study demonstrates that chromosome-level CNVs can be accurately predicted in hematologic malignancies even when the number of targeted genes on a given chromosome is low. Despite the simplicity of the approach, the two stages bioinformatics pipeline based on an ensemble method allowed us to gain between 8% and 46% accuracy compared to relying only on the prediction of a single tool like PureCN. Samples with low tumor content remain, however, a difficult case to tackle with bulk NGS as it is difficult to distinguish a CNV from the natural variability of the sequencing coverage. [Display omitted] DisclosuresNam: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.