Copy Number Profiles Research Articles

A pan-cancer landscape of pathogenic somatic copy number variations

ObjectiveCopy number variations (CNVs) play crucial roles in physiological and pathological processes, including cancer. However, the functional implications of somatic CNVs in tumor progression and evolution remain unclear. This study focuses on identifying CNV alterations with high pathogenic potential that drive and sustain tumorigenesis, distinguishing them from passenger alterations that accumulate during tumor growth. Our goal is to explore the variability of CNVs across different tumor types and infer their impact on tumor cell functions. MethodsStarting from 7352 copy number profiles across 33 different cancer types, we infer the pathogenicity of each CNV and perform both intra- and inter-tumor analyses to predict the functional impact of different genomic patterns. We evaluate the actionability of genes belonging to altered regions and we correlate the presence of pathogenic regions with genome instability patterns and patients’ survival. ResultsOur analysis uncovered large heterogeneity among different tumors suggesting in many cases distinct genetic drivers of tumorigenesis. Recurrent genomic alterations frequently coincide with dysfunctional homologous recombination pathways and negative regulation of the immune system. In certain tumors, the number of pathogenic CNVs emerged as a prognostic biomarker, highlighting their significance in cancer progression. ConclusionThis study contributes to elucidate the functional impact of pathogenic CNVs in tumor progression and sheds light on their potential as prognostic markers in specific cancer types.

Transcriptomic subtyping of malignant peripheral nerve sheath tumours highlights immune signatures, genomic profiles, patient survival and therapeutic targets

Malignant peripheral nerve sheath tumour (MPNST) is an aggressive orphan disease commonly affecting adolescents or young adults. Current knowledge of molecular tumour biology has been insufficient for development of rational treatment strategies. We aimed to discover molecular subtypes of potential clinical relevance. Fresh frozen samples of MPNSTs (n=94) and benign neurofibromas (n=28) from 115 patients in a European multicentre study were analysed by DNA copy number and/or transcriptomic profiling. Unsupervised transcriptomic subtyping was performed and the subtypes characterized for genomic aberrations, clinicopathological associations and patient survival. MPNSTs were classified into two transcriptomic subtypes defined primarily by immune signatures and proliferative processes. "Immune active" MPNSTs (44%) had sustained immune signals relative to neurofibromas, were more frequently low-grade (P=0.01) and had favourable prognostic associations in a multivariable model of disease-specific survival with clinicopathological factors (hazard ratio 0.25, P=0.003). "Immune deficient" MPNSTs were more aggressive and characterized by proliferative signatures, high genomic complexity, aberrant TP53 and PRC2 loss, as well as high relative expression of several potential actionable targets (EGFR, ERBB2, EZH2, KIF11, PLK1, RRM2). Integrated gene-wise analyses suggested a DNA copy number-basis for proliferative transcriptomic signatures in particular, and the tumour copy number burden further stratified the transcriptomic subtypes according to patient prognosis (P<0.01). Approximately half of MPNSTs belong to an "immune deficient" transcriptomic subtype associated with an aggressive disease course, PRC2 loss and expression of several potential therapeutic targets, providing a rationale for molecularly-guided intervention trials. Research grants from non-profit organizations, as stated in the Acknowledgements.

Circulating tumor DNA-based copy-number profiles enable monitoring treatment effects during therapy in high-grade serous carcinoma

Circulating tumor DNA (ctDNA) analysis has emerged as a promising tool for detecting and profiling longitudinal genomics changes in cancer. While copy-number alterations (CNAs) play a major role in cancers, treatment effect monitoring using copy-number profiles has received limited attention as compared to mutations. A major reason for this is the insensitivity of CNA analysis for the real-life tumor-fraction ctDNA samples. We performed copy-number analysis on 152 plasma samples obtained from 29 patients with high-grade serous ovarian cancer (HGSC) using a sequencing panel targeting over 500 genes. Twenty-one patients had temporally matched tissue and plasma sample pairs, which enabled assessing concordance with tissues sequenced with the same panel or whole-genome sequencing and to evaluate sensitivity. Our approach could detect concordant CNA profiles in most plasma samples with as low as 5% tumor content and highly amplified regions in samples with ∼1% of tumor content. Longitudinal profiles showed changes in the CNA profiles in seven out of 11 patients with high tumor-content plasma samples at relapse. These changes included focal acquired or lost copy-numbers, even though most of the genome remained stable. Two patients displayed major copy-number profile changes during therapy. Our analysis revealed ctDNA-detectable subclonal selection resulting from both surgical operations and chemotherapy. Overall, longitudinal ctDNA data showed acquired and diminished CNAs at relapse when compared to pre-treatment samples. These results highlight the importance of genomic profiling during treatment as well as underline the usability of ctDNA.

OR01-03 DNA Copy Number Profiles In Recurrent And Stable Non-functioning Pituitary Adenomas

Abstract Disclosure: G. Johannsson: None. M. Persson: None. S. Schlaffer: None. A. Fehr: None. C. Örndal: Employee; Self; Unilabs AB. W. Seager: None. M. Buchfelder: None. G. Stenman: None. D.S. Olsson: Consulting Fee; Self; Ipsen, Novo Nordisk, Sandoz. Grant Recipient; Self; Pfizer Global R&amp;D. Employee; Self; AstraZeneca. Background: Residual tumors are common after primary surgery in patients with non-functioning pituitary adenoma (NFPA), 30-50% of these will experience tumor progression. Tumor control is of vital importance for patient outcome since tumor progression is strongly associated with excess morbidity and mortality. The aim of the study was to explore new potential biomarkers of tumor progression by studying the DNA copy number profiles of NFPAs with either a stable residual tumor behavior or marked tumor progression. Methods: In this case-controlled study, 72 patients operated for NFPA were selected. The progression group (n = 39) included patients [age at diagnosis (mean ± SD) 41.3±14.0 years] with marked tumor progression (30 patients required ≥3 surgical procedures). The stable group (n = 33; 61.1±10.2 years) had either a stable residual tumor without evidence of tumor progression during follow-up (n = 25) or no recurrence during follow-up (n = 8). The mean follow-up time in the stable group was 6.2 years (range 3.6-15 years). ArrayCGH analysis was performed on DNAs isolated from fresh frozen tumor tissue using the Human Genome CGH Microarray 244K and 180K oligonucleotide arrays. Copy number alterations (CNAs) were validated using FISH on formalin-fixed paraffin-embedded sections. Results: CNAs were identified in 29 tumors (40%). A total of 183 CNAs were detected in the 72 tumors, including 75 segmental alterations and 108 gains and losses of whole chromosomes and chromosome arms. The average number of CNAs per tumor was 2.5 (SD 5.9). CNAs were more common in the progression group compared to the stable group (Mean no. of CNAs 3.7 vs 1.2; P = 0.060). Twenty-four tumors (72%) in the stable group had normal profiles whereas only 19 (49%) in the progression group lacked CNAs. Eighteen CNAs were recurrent, defined as minimal common regions of deletion or gain in ≥4 tumors. The most frequently lost region was 11q21-q23.3 (n = 10). Deletions involving 10q23.21-q23.3 were seen in both the progression group (n = 5) and the stable group (n = 2) and included an approximately 2.8 Mb minimal common region harboring the tumor suppressor gene PTEN. The most frequent gains involved whole chromosomes 5 (n = 8), 7 (n = 10) and 12 (n = 7). Recurrent CNAs involving 10q (PTEN probe), 11q (MAML2 probe), and chromosomes 5 and 7 were confirmed using FISH. Conclusion: In this explorative study, we found a higher number of CNAs in NFPAs with marked tumor progression compared to stable adenomas without the need for reintervention during the long-term follow-up. Interestingly, segmental losses of chromosome 10q23.21-q23.3, including the tumor suppressor gene PTEN, were overrepresented in patients with marked tumor progression. Presentation: Thursday, June 15, 2023

PGT-A mosaicism based on NGS intermediate copy numbers: is it time to stop reporting them?

Mosaicism represents a genuine real phenomenon, but its high prevalence and undisclosed clinical significance, stress the burden on genetic counseling and the management of PGT-A results. Even though the assumption of mosaicism from NGS intermediate chromosome copy number profiles may represent a reasonable interpretation, other potential technical reasons, including amplification bias, contamination, biopsy technique, or the analysis algorithms,may constitute alternative explanations. Thresholds confining mosaicism ranges are established according to models employing mixtures of normal and abnormal cells with steady conditions of quantity and quality which are unable to reflect the full extent of variability present in a trophectoderm (TE) biopsy specimen. When the concordance of TE with the ICM is considered, mosaic TE biopsies poorly correlate with the chromosomal status of the remaining embryo, displaying mostly ICM aneuploidy in cases of TE high-range mosaics diagnosis and euploidy when mosaicism grade in TE is less than 50% (low-mid range mosaicism), which implies an evident overestimation of mosaicism results. Indeed, a binary classification of NGS profiles that excludes mosaic ranges, including only euploid and aneuploid diagnosis, provides higher specificity and accuracy in identifying abnormal embryos and discarding them. As intermediate copy number profiles do not represent strong evidence of mosaicism but only an inaccurate and misleading assumption, and considering thatno increased risk has been reported in the offspring, until diagnosis specificity is improved and its clinical implications are determined, laboratories should considerlimiting predictions to euploid and aneuploidand stop reporting mosaicism.

Cracking the pattern of tumor evolution based on single-cell copy number alterations.

Copy number alterations (CNAs) are a key characteristic of tumor development and progression. The accumulation of various CNAs during tumor development plays a critical role in driving tumor evolution. Heterogeneous clones driven by distinct CNAs have different selective advantages, leading to differential patterns of tumor evolution that are essential for developing effective cancer therapies. Recent advances in single-cell sequencing technology have enabled genome-wide copy number profiling of tumor cell populations at single-cell resolution. This has made it possible to explore the evolutionary patterns of CNAs and accurately discover the mechanisms of intra-tumor heterogeneity. Here, we propose a two-step statistical approach that distinguishes neutral, linear, branching and punctuated evolutionary patterns for a tumor cell population based on single-cell copy number profiles. We assessed our approach using a variety of simulated and real single-cell genomic and transcriptomic datasets, demonstrating its high accuracy and robustness in predicting tumor evolutionary patterns. We applied our approach to single-cell DNA sequencing data from 20 breast cancer patients and observed that punctuated evolution is the dominant evolutionary pattern in breast cancer. Similar conclusions were drawn when applying the approach to single-cell RNA sequencing data obtained from 132 various cancer patients. Moreover, we found that differential immune cell infiltration is associated with specific evolutionary patterns. The source code of our study is available at https://github.com/FangWang-SYSU/PTEM.

P18.04.B SINGLE CELL TRANSCRIPTOMIC ANALYSIS IDENTIFIES DISTINCT INTRATUMORAL SUBCLONES IN MENINGIOMA

Abstract BACKGROUND Meningiomas arise from the leptomeninges of the brain and spinal cord. Despite being the most common intracranial tumor in adults, risk stratification remains challenging. Currently, tumors are graded based on histology as well as molecular markers. In recent years, several epigenomic classification systems with a correlation to patient outcome have been proposed for meningiomas. However, these classification systems do not discriminate between effects of the tumor microenvironment and the tumor itself or consider distinct intratumoral subpopulations with different phenotypes. To address this, we set out to investigate intratumoral differences and correlated them with gene expression differences. MATERIAL AND METHODS We applied single nuclei RNA sequencing (10X Genomics 3’) to 30 meningioma samples to obtain transcriptomic profiles on single cell level. Copy number variations were estimated for single cells based on the transcriptomic profiles to identify clonal differences within a given tumor. RESULTS Based on aberrations in the copy number profile, we were able to identify distinct subclones within the same tumor, which could in many cases be connected to a change in the transcriptomic profile of the respective subclone. Different patterns of subclonal diversity were observed between tumor grades. Further insight into the subpopulations was used to infer a novel model for prognostication which was validated by methylation data. CONCLUSION Altogether, these results demonstrate that diverse subclones co-exist within meningioma, especially within higher-grade tumors. Effects of meningioma subclones on tumor growth and their relevance for clinical risk prediction remain to be elucidated.

OS13.5.A MOLECULAR CHARACTERISTICS AND PROGNOSTIC BIOMARKERS OF CENTRAL NEUROCYTOMA

Abstract BACKGROUND Central neurocytomas (CN) are intraventricular brain tumors predominantly arising in young adults. Long-term prognosis is favorable in the majority of cases, whereas a considerable proportion of patients experience disease progression. Progression risk is currently assessed morphologically based on the presence of histological signs of atypia and/or an elevated ki67 proliferation index (&amp;gt;3%). The molecular profile and etiology of CN remains elusive and molecular biomarkers for risk stratification have not been identified yet. MATERIAL AND METHODS We analyzed a retrospective multi-institutional cohort of 108 CNs by genome-wide DNA methylation profiling. DNA whole exome sequencing was performed in 12 cases. Follow-up information was available for 45 patients. RESULTS The histological diagnosis of neurocytoma was epigenetically confirmed in 96 cases. 12/108 cases (11%) were assigned to another methylation class based on the v12.5 Heidelberg Brain Tumor Classifier. Median age at diagnosis was 29 years, distribution among sexes was equal. Based on morphology and immunohistochemistry, tumors were diagnosed as classical CN (n = 71), CN with increased proliferation (n = 10) and atypical CN (n = 15). In 49/72 cases (68%, NA=24), an elevated Ki67 above 3% was reported. DNA whole exome sequencing revealed no recurrent genetic alterations. Most copy number profiles were balanced (n = 87), while some cases (n = 9) showed individual chromosomal alterations (e.g. chr. +5, n = 3). Tumor recurrence was observed in 16/45 (36%, NA = 51) of cases. Median progression-free survival (PFS) after the initial diagnosis was 72 months, none of the patients died (observation time: range 3 - 141 months). In 16 cases, surgery was followed by adjuvant radiotherapy, predominantly in patients with subtotal resection (STR: n = 11, 69%; GTR: n = 5). There was no significantly different outcome between classical and atypical CNs (n = 45; median PFS 73 months vs. 26 months, p-value = 0.067) and cases with low vs. elevated Ki67 (n = 45; median PFS NA vs. 72 months, p-value = 0.44). CONCLUSION Preliminary data of our study indicate that histological diagnosis and Ki67 proliferation rate were not associated with PFS in epigenetically defined neurocytomas. Further research using clinical and molecular covariates is necessary to identify novel biomarkers to predict recurrence risk and benefit from adjuvant radiotherapy.

OS12.5.A HIGH-GRADE GLIOMAS OCCURRING IN TEENAGERS AND YOUNG ADULT PATIENTS COMPRISE NOVEL MOLECULAR SUBGROUPS

Abstract BACKGROUND High-grade gliomas (HGG) arise in any central nervous system location, in adults and children, with a poor prognosis. HGGs in teenagers and young adults (TYA) are understudied; this project aimed to characterise these tumours and identify potential therapeutic targets. MATERIAL AND METHODS HGG samples (n=195, FFPE/FF, 13-30 years) were collected from national/international collaborators, excluding well-characterised entities (histone/IDH-mutant). DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package), n=195) classified cases against a reference cohort. Calibrated scores guided workflows to characterise mutational landscapes including RNA-based ArcherDx fusion panel (n=112), whole exome sequencing (n=84), and histological reviews in parallel. Well-characterised entities (n=50, including IDH-mutant tumours (n=18), PXA (n=12)) were excluded. RESULTS Of the cases scoring &amp;gt;0.6, 64% classified as paediatric-type subgroups (pedHGG_RTK1A/B/C (30%), pedHGG_MYCN (10%), pedHGG_A/B (7%), pedHGG_RTK2A (6%)). 29% classified as subgroups more frequently seen in adults (GBM_MES (12%), GBM_RTK1/2 (3%)). 54% classified with a score &amp;lt;0.6. 38% cases were assigned to novel, recently identified, poorly-characterised subgroups with distinct methylation profiles and molecular features (HGG_B, HGG_E, ANTCON, GBM_CBM). Frequency comparisons to publicly available methylation data showed GBM_MES_ATYP tumours are TYA age-specific, in contrast to adult age-specific GBM_MES_TYP. Copy number profiling identified frequent changes across the cohort, including chromosomal gains (chr1q (54%), chr2 (22%), chr7 (41%)) and losses (chr10 (40%), chr13 (64%)). Focal amplifications included PDGFRA (12%), CDK4 (7%), MYCN/ID2 (3%), MYC (2%) and EGFR (0.7%) and the most common focal deletion was CDKN2A/p16 (12%). The most frequent glioma-associated somatic variants included TP53, PDGFRA, EGFR, and NF1. BCOR alterations were enriched compared to adult and paediatric reference cohorts. 10% cases showed a hypermutator phenotype, enriched in HGG_E. CDK4 amplifications were recurrent in GBM_RTK1 and GBM_CBM. Histology showed variable cytological and architectural features within novel subgroups. CONCLUSION TYA HGG comprise novel subgroups with distinct methylation profiles and molecular characteristics, representing opportunities to refine future treatment.

Gene expression and copy number profiling of follicular lymphoma biopsies from patients treated with first-line rituximab without chemotherapy

The Nordic Lymphoma Study Group has performed two randomized clinical trials with chemotherapy-free first-line treatment (rituximab +/- interferon) in follicular lymphoma (FL), with 73% of patients alive and 38% without any need of chemotherapy after 10.6 years median follow-up. In order to identify predictive markers, that may also serve as therapeutic targets, gene expression- and copy number profiles were obtained from 97 FL patients using whole genome microarrays. Copy number alterations (CNAs) were identified, e.g. by GISTIC. Cox Lasso Regression and Lasso logistic regression were used to determine molecular features predictive of time to next therapy (TTNT). A few molecular changes were associated with TTNT (e.g. increased expression of INPP5B, gains in 12q23/q24), but were not significant after adjusting for multiple testing. Our findings suggest that there are no strong determinants of patient outcome with respect to GE data and CNAs in FL patients treated with a chemotherapy-free regimen (i.e. rituximab +/– interferon).

Loss of Chromosome 3q Is a Prognostic Marker in Fusion-Negative Rhabdomyosarcoma.

Soft tissue sarcomas (STS) are rare mesenchymal neoplasms that frequently show complex chromosomal aberrations such as amplifications or deletions of DNA sequences or even whole chromosomes. We recently found that gain of chromosome (chr) 8 is associated with worse overall survival (OS) in STS as a group. We therefore aimed to investigate the overall copy number profile of rhabdomyosarcoma (RMS) to evaluate for prognostic signatures. Fluorescence in situ hybridization (FISH) testing was performed on a cohort of STS to assess for chr8 gain. Copy number variation (CNV) data from the National Cancer Institute were analyzed to assess for prognostically significant CNV aberrations in FOXO1 fusion-negative (FN)- versus fusion-positive (FP)-RMS. FISH testing was performed on a cohort of FN-RMS to assess for chr3q loss and correlate with outcomes. Chr8 gain is a highly prevalent CNV in embryonal RMS and shows slightly improved prognosis. Meanwhile, loss of chr3q was associated with worse outcome in FN-RMS compared with FP-RMS. The pathogenesis of STS including FN-RMS remains poorly understood, emphasizing the need for new therapeutic advances and adequate risk stratification. Our data demonstrate that loss of chr3q is associated with poor OS in FN-RMS, supporting it as an important tool for risk stratification.

Pan-Cancer Analysis of Copy-Number Features Identifies Recurrent Signatures and a Homologous Recombination Deficiency Biomarker to Predict Poly (ADP-Ribose) Polymerase Inhibitor Response.

Copy-number (CN) features reveal the molecular state of cancers and may have predictive and prognostic value in the treatment of cancer. We sought to apply published CN analysis methods to a large pan-cancer data set and characterize ubiquitous CN signatures across tumor types, including potential utility for treatment selection. We analyzed the landscape of CN features in 260,333 pan-cancer samples. We examined the association of 10 signatures with genomic alterations and clinical characteristics and trained a machine learning classifier using CN and insertion and deletion features to detect homologous recombination deficiency signature (HRDsig) positivity. Clinical outcomes were assessed using a real-world clinicogenomic database (CGDB) of comprehensive genomic profiling linked to deidentified, electronic health record-derived clinical data. CN signatures were prevalent across cancer types and associated with diverse processes including focal tandem duplications, seismic amplifications, genome-wide loss of heterozygosity (gLOH), and HRD. Our novel HRDsig outperformed gLOH in predicting BRCAness and effectively distinguished biallelic BRCA and homologous recombination-repair wild-type (HRRwt) samples pan-tumor, demonstrating high sensitivity to detect biallelic BRCA in ovarian (93%) and other HRD-associated cancers (80%-87%). Pan-tumor prevalence of HRDsig was 6.4%. HRRwt cases represented a significant fraction of the HRDsig-positive cohort, likely reflecting a population with nongenomic mechanisms of HRD. In ovarian and prostate CGDBs, HRDsig identified more patients than gLOH and had predictive value for poly (ADP-ribose) polymerase inhibitor (PARPi) benefit. Tumor CN profiles are informative, revealing diverse processes active in cancer. We describe the landscape of 10 CN signatures in a large pan-cancer cohort, including two associated with HRD. We trained a machine learning-based HRDsig that robustly identified BRCAness and associated with biallelic BRCA pan-tumor, and was predictive of PARPi benefit in real-world ovarian and prostate data sets.

KDM2B-Rearranged Soft Tissue Sarcomas Expand the Concept of BCOR-Associated Sarcoma

Sarcomas with BCOR genetic alterations (BCOR-associated sarcomas) represent a recently recognized family of soft tissue and bone tumors characterized by BCOR fusion, BCOR internal tandem duplication, or YWHAE::NUTM2B fusion. Histologically, the tumors demonstrate oval to spindle cell proliferation in a variably vascular stroma and overexpression of BCOR and SATB2. Herein, we describe 3 soft tissue sarcomas with KDM2B fusions that phenotypically and epigenetically match BCOR-associated sarcomas. The cases included 1 infant, 1 adolescent, and 1 older patient. All tumors showed histologic findings indistinguishable from those of BCOR-associated sarcomas and were originally diagnosed as such based on the phenotype. However, none of the tumors had BCOR or YWHAE genetic alterations. Instead, targeted RNA sequencing identified in-frame KDM2B::NUTM2B, KDM2B::CREBBP, and KDM2B::DUX4 fusions. KDM2B fusions were validated using reverse-transcription PCR, Sanger sequencing, and in situ hybridization assays. Genome-wide DNA methylation analysis matched all 3 tumors with BCOR-associated sarcomas using the Deutsches Krebsforschungszentrum (DKFZ) classifier and t-distributed stochastic neighbor embedding analysis. One localized tumor showed a flat genome-wide copy number profile, and the patient remained disease-free after treatment. The other tumors showed multiple copy number alterations, including MDM2/CDK4 amplification and/or CDKN2A/B loss, and both tumors metastasized, leading to the patient’s death in one of the cases. When tested using KDM2B immunohistochemistry, all 3 KDM2B-rearranged sarcomas showed diffuse strong staining, and all 13 sarcomas with BCOR genetic alterations also demonstrated diffuse, strong, or weak staining. By contrast, among 72 mimicking tumors, only a subset of synovial sarcomas showed focal or diffuse weak KDM2B expression. In conclusion, our study suggests that KDM2B-rearranged soft tissue sarcomas belong to the BCOR-associated sarcoma family and expand its molecular spectrum. This may be related to the known molecular relationship between KDM2B and BCOR in the polycomb repressive complex 1.1. Immunohistochemical analysis of KDM2B is a potentially valuable diagnostic tool for BCOR-associated sarcomas, including those with KDM2B rearrangement.

Whole exome sequencing and MicroRNA profiling of lung adenocarcinoma identified risk prediction features for tumors at stage I and its substages

ObjectivesAbout 20% of stage I lung adenocarcinoma (LUAD) patients suffer a relapse after surgical resection. While finer substages have been defined and refined in the AJCC staging system, clinical investigations on the tumor molecular landscape are lacking. Materials and methodsWe performed whole exome sequencing, DNA copy number and microRNA profiling on paired tumor-normal samples from a cohort of 113 treatment-naïve stage I Taiwanese LUAD patients. We searched for molecular features associated with relapse-free survival (RFS) of stage I or its substages and validated the findings with an independent Caucasian LUAD cohort. ResultsWe found sixteen nonsynonymous mutations harbored at EGFR, KRAS, TP53, CTNNB1 and six other genes associated with poor RFS in a dose-dependent manner via variant allele fraction (VAF). An index, maxVAF, was constructed to quantify the overall mutation load from genes other than EGFR. High maxVAF scores discriminated a small group of high-risk LUAD at stage I (median RFS: 4.5 versus 69.5 months; HR = 10.5, 95% CI = 4.22–26.12, P < 0.001). At the substage level, higher risk was found for patients with high maxVAF or high miR-31; IA (median RFS: 32.1 versus 122.8 months, P = 0.005) and IB (median RFS: 7.1 versus 26.2, P = 0.049). MicroRNAs, miR-182, miR-183 and miR-196a were found correlated with EGFR mutation and poor RFS in stage IB patients. ConclusionDistinctive features of somatic gene mutation and microRNA expression of stage I LUAD are characterized to complement the survival prognosis by substaging. The findings open up more options for precision management of stage I LUAD patients.

Copy number architectures define treatment-mediated selection of lethal prostate cancer clones

Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance (p-values 3.07 × 10−8 and 6.4 × 10−4). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories.

Genomic analysis of spinal meningiomas: correlation with histopathological grade.

Spinal meningiomas are one of the most common primary intradural tumors of the adult spine. Spinal meningiomas typically have a benign course with low rates of recurrence. Recent advances in genomic profiling have provided valuable information on meningioma biology and natural history, but these studies have focused primarily on cranial meningiomas. Chromosomal copy number analysis of meningiomas has been shown to be a valuable molecular profiling technique for distinguishing benign from aggressive tumors. The Integrated Grade for Meningioma is a novel grading scheme that uses mitotic index and copy-number profile to identify aggressive tumors at high risk for recurrence. The integrated grade has been shown to be a better predictor of tumor behavior than WHO grade alone. The objective of this study was to evaluate the chromosomal copy-number profile of spinal meningiomas, and to correlate these findings with the assigned WHO grades. The authors evaluated 94 spinal meningiomas treated surgically at their institution between 2002 and 2022. The histopathological results including WHO grade, mitotic index, presence of atypical features, and MIB-1 index were recorded. Chromosomal copy number as determined by institutional whole-genome DNA copy-number profiling was available for 57 tumors. The WHO grades of the cohort consisted of 81 (86%) WHO grade 1 tumors and 13 (14%) WHO grade 2 tumors. In tumors for which copy-number profiling was available, 44 (77%) of 57 demonstrated loss of 22q/NF2. Notably absent were frequent high-risk copy number alterations including loss of 1p, 3p, 4p/q, 6p/q, 10p/q, 14q, 18p/q, 19p/q, and focal loss of CDKN2A on 9p. Of the 9 WHO grade 2 tumors for which copy-number profiling was available, 6 tumors were reclassified to a lower risk profile (integrated grade 1). This analysis suggests that spinal meningiomas exhibit overwhelmingly indolent biology, as supported by their benign integrated grade. These findings have implications in the surgical management of these patients in relation to the need for complete Simpson grade I resection, as well as the potential avoidance of adjuvant therapy following surgery in the setting of otherwise frequently benign pathology.

Assessing consistency of calculating homologous recombination deficiency (HRD) score using different segmentation method.

89 Background: A number of similar tests were developed since FDA approval of using niraparib in HRD positive (HRD score &gt;=42) patients. However, various factors can substantially influence HRD score estimation. We hereby evaluated the concordancy of bioinformatics pipelines, especially different copy number variation (CNV) algorithms, in inferring HRD scores. Methods: A total of 195 ovarian cancer patients enrolled from April 2021 to Dec 2022 were used for retrospective analysis in this study. Both tumor tissue and blood samples were sequenced with Topgen HRD panel, which covered ~35,000 single nucleotide polymorphisms (SNPs). Sequence data were trimmed and aligned to hg19 reference genome. Two commonly used CNV pipeline (i.e. Sequenza, and PureCN which have tumor-normal mode and tumor-only mode) were adopted to generate allele specific copy number (ASCN) profile. HRD scores were then inferred from ASCN profile using R package scarHRD. Pearson correlation was used for evaluating correlation between HRD scores generated with different algorithms. Paired T-test and Wilcoxon signed-rank test was used in comparing differences in HRD scores and segment counts from different algorithms. Percentage agreement was evaluated based on cutoff of HRD score &gt;=42. Results: HRD scores produced from Sequenza and PureCN (tumor-normal mode) showed significant correlation (R = 0.83, P value &lt; 0.001). Sequenza tended to over-segment than PureCN (median segment counts: 139 vs 82, P value &lt; 0.001) and have higher HRD scores (mean HRD scores: 55.0 vs 23.5, P value &lt; 0.001). Using Sequenza, HRD-positivity was observed in 70.0% of the total population, whereas PureCN classified only 39.0% as HRD-positive. HRD scores inferred from two modes of PureCN showed improved correlation (R = 0.89, P value &lt; 0.001). Tumor-only mode generated higher mean HRD score than tumor-normal mode (42.7 vs 36.0, p-value &lt; 0.001). Prevalence of HRD-positivity was 47.7% using tumor-only mode. Using tumor-normal mode as the reference test, the percentages of agreement were shown in the Table. Conclusions: Comparison of different CNV algorithms demonstrated significant variances in HRD scores estimation. Despite good correlation between different pipelines was observed on HRD scores, the estimated prevalence of HRD-positive patients varied significantly from 39% to 70%. Our results urge the necessity of harmonisation on bioinformatics pipeline during HRD score calculation. [Table: see text]

Circulating tumour DNA (ctDNA) burden quantified through low-pass whole genome sequencing as an early pharmacodynamic biomarker of therapeutic response in patients enrolled on phase 1 trials.

23 Background: Early phase trials are conducted to determine the recommended dose and safety of an investigational therapy. Targeted therapies and immunotherapy may not always have dose-dependent toxicities. Therefore, dose finding is increasingly reliant on optimal biological dosing. Pharmacodynamic biomarkers are increasingly important to demonstrate proof of concept. Using ctDNA allows for dynamic and early tumour assessments compared to imaging. We sought to evaluate ctDNA as an early pharmacodynamic biomarker of therapeutic response. Methods: Patients (pts) enrolled on phase 1 trials at the National Cancer Centre Singapore were prospectively consented to collect serial blood samples at defined timepoints (pre-treatment, mid-cycle 1, end of cycle 1, and with subsequent imaging evaluations). ctDNA burden was quantified using low pass whole genome sequencing (lpWGS) with both established (ichorCNA) and novel (cell-free DNA degradation) bioinformatic analyses of ctDNA burden, and targeted panel sequencing. Early kinetics of ctDNA burden prior to imaging evaluation was evaluated as response (reduction in ctDNA burden) versus progression (increase in ctDNA burden). Results: Between May 2021 to Oct 2022, 68 plasma samples (median 3, range 2-5) were collected from 22 pts. Median age was 56 years (range 37-77) and 73% were male. Pts were enrolled on trials of targeted therapy (55%), immunotherapy (40%) or combinations of both (5%). Common tumour types were lung (36%), colorectal (23%) and pancreatic (9%) cancer. Best response on phase 1 trial as per imaging and RECIST v1.1 was partial response (PR) in 9%, stable disease (SD) in 45%, progressive disease (PD) in 27% and not evaluable (NE; taken off trial early due to toxicity) in 18%. Quantification of ctDNA burden using cell-free DNA fragment length pattern demonstrated enhanced limit of detection in detecting baseline ctDNA compared to cell-free DNA copy number profile based ichorCNA method (100% vs 59%). For pts on targeted therapies, early kinetics of ctDNA burden indicated ctDNA response in 9/13 pts, with subsequent imaging showing PR (n=2), SD (n=2), PD (n=2) or NE (n=3). In the remaining 4/13 pts on targeted therapies with early ctDNA progression, subsequent imaging showed SD (n=3) or NE (n=1). For pts on immunotherapy, early ctDNA progression was seen in 8/10 pts, with subsequent imaging showing SD (n=5) and PD (n=3). Patient enrolment and sample analysis is ongoing to further validate these initial findings and updated data will be presented. Conclusions: Early ctDNA evaluation via lpWGS may represent an early pharmacodynamic biomarker of therapeutic response. ctDNA has the potential to provide early proof of concept, particularly for targeted therapies, that may influence subsequent decisions for dose determination.

Implementation of DNA Methylation Array Profiling in Pediatric Central Nervous System Tumors: The AIM BRAIN Project: An Australian and New Zealand Children's Haematology/Oncology Group Study

DNA methylation array profiling for classifying pediatric central nervous system (CNS) tumors is a valuable adjunct to histopathology. However, unbiased prospective and interlaboratory validation studies have been lacking. The AIM BRAIN diagnostic trial involving 11 pediatric cancer centers in Australia and New Zealand was designed to test the feasibility of routine clinical testing and ran in parallel with the Molecular Neuropathology 2.0 (MNP2.0) study at Deutsches Krebsforschungszentrum (German Cancer Research Center). CNS tumors from 269 pediatric patients were prospectively tested on Illumina EPIC arrays, including 104 cases co-enrolled on MNP2.0. Using MNP classifier versions 11b4 and 12.5, we report classifications with a probability score ≥0.90 in 176 of 265 (66.4%) and 213 of 269 (79.2%) cases, respectively. Significant diagnostic information was obtained in 130 of 176 (74%) for 11b4, and 12 of 174 (7%) classifications were discordant with histopathology. Cases prospectively co-enrolled on MNP2.0 gave concordant classifications (99%) and score thresholds (93%), demonstrating excellent test reproducibility and sensitivity. Overall, DNA methylation profiling is a robust single workflow technique with an acceptable diagnostic yield that is considerably enhanced by the extensive subgroup and copy number profile information generated by the platform. The platform has excellent test reproducibility and sensitivity and contributes significantly to CNS tumor diagnosis.

Melt-Encoded-Tags for Expanded Optical Readout in Digital PCR (METEOR-dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling.

Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.