Search

Uveal melanomas are rare tumors arising from melanocytes that reside in the eye. Despite surgical or radiation treatment, approximately 50% of patients with uveal melanoma will progress to metastatic disease, most often to the liver. Cell-free DNA (cfDNA) sequencing is a promising technology due to the minimally invasive sample collection and ability to infer multiple aspects of tumor response. We analyzed 46 serial cfDNA samples from 11 patients with uveal melanoma over a 1-year period following enucleation or brachytherapy (n = ∼4/patient) using targeted panel, shallow whole genome, and cell-free methylated DNA immunoprecipitation sequencing. We found detection of relapse was highly variable using independent analyses (P = 0.06–0.46), whereas a logistic regression model integrating all cfDNA profiles significantly improved relapse detection (P = 0.02), with greatest power derived from fragmentomic profiles. This work provides support for the use of integrated analyses to improve the sensitivity of circulating tumor DNA detection using multi-modal cfDNA sequencing.Significance:Here, we demonstrate integrated, longitudinal cfDNA sequencing using multi-omic approaches is more effective than unimodal analysis. This approach supports the use of frequent blood testing using comprehensive genomic, fragmentomic, and epigenomic techniques.

Supplemental Figure 3

Uveal melanomas are rare tumors arising from melanocytes that reside in the eye. Despite surgical or radiation treatment, approximately 50% of patients with uveal melanoma will progress to metastatic disease, most often to the liver. Cell-free DNA (cfDNA) sequencing is a promising technology due to the minimally invasive sample collection and ability to infer multiple aspects of tumor response. We analyzed 46 serial cfDNA samples from 11 patients with uveal melanoma over a 1-year period following enucleation or brachytherapy (n = ∼4/patient) using targeted panel, shallow whole genome, and cell-free methylated DNA immunoprecipitation sequencing. We found detection of relapse was highly variable using independent analyses (P = 0.06–0.46), whereas a logistic regression model integrating all cfDNA profiles significantly improved relapse detection (P = 0.02), with greatest power derived from fragmentomic profiles. This work provides support for the use of integrated analyses to improve the sensitivity of circulating tumor DNA detection using multi-modal cfDNA sequencing.Significance:Here, we demonstrate integrated, longitudinal cfDNA sequencing using multi-omic approaches is more effective than unimodal analysis. This approach supports the use of frequent blood testing using comprehensive genomic, fragmentomic, and epigenomic techniques.

differentially methylated probes

Peer reviewed: True, Funder: Scholarship Award from The Ministry of Education, Saudi Arabia, Funder: FRQS and Quebec Breast Cancer Foundation, Funder: Fonds de recherche du Québec, McGill University, FANCI was recently identified as a new candidate ovarian cancer (OC)-predisposing gene from the genetic analysis of carriers of FANCI c.1813C>T; p.L605F in OC families. Here, we aimed to investigate the molecular genetic characteristics of FANCI, as they have not been described in the context of cancer. We first investigated the germline genetic landscape of two sisters with OC from the discovery FANCI c.1813C>T; p.L605F family (F1528) to re-affirm the plausibility of this candidate. As we did not find other conclusive candidates, we then performed a candidate gene approach to identify other candidate variants in genes involved in the FANCI protein interactome in OC families negative for pathogenic variants in BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, and FANCI, which identified four candidate variants. We then investigated FANCI in high-grade serous ovarian carcinoma (HGSC) from FANCI c.1813C>T carriers and found evidence of loss of the wild-type allele in tumour DNA from some of these cases. The somatic genetic landscape of OC tumours from FANCI c.1813C>T carriers was investigated for mutations in selected genes, copy number alterations, and mutational signatures, which determined that the profiles of tumours from carriers were characteristic of features exhibited by HGSC cases. As other OC-predisposing genes such as BRCA1 and BRCA2 are known to increase the risk of other cancers including breast cancer, we investigated the carrier frequency of germline FANCI c.1813C>T in various cancer types and found overall more carriers among cancer cases compared to cancer-free controls (p = 0.007). In these different tumour types, we also identified a spectrum of somatic variants in FANCI that were not restricted to any specific region within the gene. Collectively, these findings expand on the characteristics described for OC cases carrying FANCI c.1813C>T; p.L605F and suggest the possible involvement of FANCI in other cancer types at the germline and/or somatic level.

SummaryDespite advances in cancer therapeutics, early detection is often the best prognostic indicator for survival (1). People with Li-Fraumeni syndrome harbor a germline pathogenic variant in the tumor suppressor geneTP53(2) and face a near 100% lifetime risk of developing a wide spectrum of, often multiple, cancers (3).TP53mutation carriers routinely undergo intensive surveillance protocols which, although associated with significantly improved survival, are burdensome to both the patient and the health care system (4). Liquid biopsy, the analysis of cell-free DNA fragments in bodily fluids, has become an attractive tool for a range of clinical applications, including early cancer detection, because of its ability to provide real-time holistic insight into the cellular milieu (5). Here, we assess the efficacy of a multi-modal liquid biopsy assay that integrates a targeted gene panel, shallow whole genome, and cell-free methylated DNA immunoprecipitation sequencing for the early detection of cancer in a cohort of Li-Fraumeni syndrome patients: 196 blood samples from 89 patients, of which 26 were pediatric and 63 were adults. Our integrated analysis was able to detect a cancer-associated signal in 79.4% of samples from patients with active cancer, a 37.5% – 58.8% improvement over each individual analysis. Through analysis of patient plasma at cancer negative timepoints, we were able to detect cancer-associated signals up to 16 months prior to occurrence of cancer as detected by conventional clinical modalities in 17.6% ofTP53mutation carriers. This study provides a framework for the integration of liquid biopsy into current surveillance methods for patients with Li-Fraumeni syndrome.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer., The genomic characterisation of nasopharyngeal carcinoma (NPC) remains crucial. Here, the authors perform whole-genome sequencing for 70 NPCs with EBV gene expression, report the somatic alterations and EBV-mediated effects converging on NF-κB activation and immune escape and identify targetable homozygous MTAP deletions.

Purpose: An expected outcome following germline genome sequencing in oncology is the discovery of ‘secondary findings’ (SFs). SFs comprise pathogenic(P)/likely P (LP) germline variants in cancer genes not typically associated with the presenting cancer, in addition to germline variants of uncertain significance (VUS) to the patient’s cancer. Due to the rarity of childhood cancers and a dearth of studies analyzing SFs, many pediatric SFs are categorized as VUS without clinical interpretation. Interpreting SFs poses significant challenges: VUSs and other SFs are frequently not included in clinical molecular reports, and even when reported (often through research), their clinical utility and long-term impact on patient health are unclear. However, we know VUSs can have clinical importance because some VUSs, when investigated thoroughly, have been reclassified as pathogenic predictors of significant health conditions in children. We hypothesize that an in-depth characterization of the landscape of germline SFs/VUSs across a diverse pediatric cancer cohort will reveal new roles of these genes and mutations in pediatric cancers. Methods: To explore germline SFs in pediatric cancer patients, we analyzed germline whole-genome sequencing (WGS) data for patients with rare, relapsed, refractory, and metastatic childhood cancers enrolled in the SickKids Cancer Sequencing Program (KiCS). We developed a custom analysis pipeline to identify germline single-nucleotide variants and indels deemed SFs, auto-classify their pathogenicity (ex. P, LP, or VUS) using CharGer, filter for PanCanAtlas-indicated cancer predisposition genes, and sort the remaining variants by cancer and non-cancer associations. Results: The KiCS cohort (n = 511) encompassed over 133 different tumor types; the median age of participants was 14 years (SD = 10.27) and 55% of patients were male. Ongoing work in our lab will catalogue the frequency and distribution of SFs in KiCS and analyze germline variants by subgroup (gene, tumor subtype, stage, demographics, gene function). We will also compare SF prevalence in KiCS to the general population using the gnomAD dataset. Results from preliminary analyses of this cohort will be presented. Significance: SFs/VUSs are under-utilized in cancer management. This work advances the holistic understanding of germline genomics in pediatric oncology and the roles of SFs in disease. Future studies will evaluate SFs by patient ancestry and validate cancer associations through the evaluation of allelic imbalance/loss of heterozygosity in matched tumor genomes. Citation Format: Safa Majeed, Stephenie Prokopec, Brianne Laverty, Vallijah Subasri, Michael Taylor, Yvonne Bombard, Trevor Pugh, Adam Shlien, Anita Villani, David Malkin. Investigating secondary findings in a pediatric cancer cohort: preliminary findings. [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 6545.

Background: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by a germline likely/pathogenic variant in BRCA1 or BRCA2, leading to increased risk of developing breast, ovarian, prostate, and pancreatic cancers. While HBOC patients often undergo regular surveillance including breast imaging, other cancers have no effective screening or biomarkers. One emerging technology is the analysis of circulating tumor DNA (ctDNA), fragments of DNA that are shed from the tumor into the bloodstream. In sporadic cancer, several studies have demonstrated the advantages and sensitivity of cell-free whole genome sequencing (cfWGS) compared to targeted panel sequencing. However, the use of cfWGS for cancer detection in HBOC has not been explored. Methods: Matched tumor, germline, and plasma were collected from 5 HBOC patients (3 breast, 2 ovarian) as part of the Canada-wide CHARM consortium (https://charmconsortium.ca). Tumor (40x), germline (20x), and plasma (20x) underwent whole genome sequencing (WGS) and subsequent variant calling pipeline using an ensemble of 6 variant callers. Insertions, deletions, and structural variants were not included in downstream analyses. Plasma tumor fraction prediction and copy number alterations were performed using ichorCNA. Detection of tumor associated mutations in plasma was compared to MRDetect software. Results: Variant calling identified an average of 11,170 tumor (7,649-16,706) and 1,215 (544-3,205) plasma SNVs per patient. Somatic BRCA1/2 mutations were detected in 0/5; whereas deletion of the intact BRCA1/2 allele was identified in 5/5 patients. An average of 24.6% (8.3%-75.9%) of plasma mutations where shared with SNVs identified in matched tumor. Targeted panel deep-sequencing (20,000X) was only able to detect mutations in TP53 in 3/5 cases (0.8% - 8.6%). Plasma derived copy number alterations correlated well with tumor derived copy number alterations in 3/5 cases and ichorCNA predicted tumor fractions ranged from 0.038 to 0.196. Conclusions: Traditionally, targeted panel sequencing has been used to identify tumor associated mutations in plasma. While this technique is sensitive, major disadvantages include the narrow breadth of the captured regions and the inability to detect deletion events such as those that occur in BRCA1/2. Here we show that plasma WGS is effective for detecting tumor associated mutations in HBOC and is more sensitive than targeted panel sequencing. We are currently expanding our cohort to an additional 20 matched tumor pairs, including patients with pre-diagnosis plasma timepoints. Citation Format: Derek Wong, Adriana Aguilar-Mahecha, Josiane Lafleur, Clarissa Chan, Kirsten Farncombe, Maia Norman, Leslie Oldfield, Prabhjit Basra, Stephanie Pederson, Johanna Wellum, Stephenie Prokopec, Mark Basik, Raymond H. Kim, Trevor Pugh. Cell-free whole genome sequencing for the detection of cancer in patients with germline BRCA1/2 mutations [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 538.

Array QA/QC (Female Cohort). To verify data quality, the distributional homogeneity of arrays (A) pre- and (B) post- RMA processing was assessed. In addition, (C) RNA degradation was evaluated across probes for each array and (D) the inter-array correlation was examined to identify potential outliers; all arrays appeared highly similar and none were excluded from downstream analyses. (PDF 397Â kb)

Universally TCDD-Responsive Genes in Multiple Biological Contexts. Datasets from multiple TCDD toxicity studies in rodents were integrated together to visualize transcriptomic alterations of (top) “AHR-core” genes and TCDD-responsive genes in different biological contexts. Two groups of genes were identified as being associated with (middle) sensitive (log2|fold-change| > 1 and p adj 1 and p adj

Overlap of Enriched Pathways. Pathway enrichment analysis was performed using GOMiner software. Analyses were performed for each sex/dose combination. Enriched gene ontologies were compared between male and female mice following treatment with (A) 125, (B) 250, (C) 500 and (D) 1000Â Îźg/kg TCDD. (PDF 126Â kb)

TCDD-Responsive Genes in Male or Female Mice Examined in Multiple Biological Contexts. Datasets from multiple TCDD toxicity studies in rodents were integrated together to visualize transcriptomic alterations of TCDD-responsive genes (log2|fold-change| > 1 and p adj

Array QA/QC (Male Cohort). To verify data quality, the distributional homogeneity of arrays (A) pre- and (B) post-RMA processing was assessed. In addition, (C) RNA degradation was evaluated across probes for each array and (D) the inter-array correlation was examined to identify potential outliers; all arrays appeared highly similar and none were excluded from downstream analyses. (PDF 418Â kb)

Transcriptomic Overlap. Venn diagrams display the number of significantly altered genes (log2|fold-change| > 1 and p adj

Additional file 6: Table S3. Univariate Analysis for Disease-free Survival.

Supplementary data. Supplementary information to the results. (DOCX 1249Â kb)

Additional file 5: Table S2. Univariate Analysis for Overall Survival.

Table S1. Publications using BPG. (DOC 67 kb)

Supplementary data. Supplementary information to the results. (DOCX 1249Â kb)

Table S1. Publications using BPG. (DOC 67 kb)

Additional file 4: Table S1. Summary of TP53 mutations.

Additional file 4: Table S1. Summary of TP53 mutations.

Additional file 5: Table S2. Univariate Analysis for Overall Survival.

Additional file 6: Table S3. Univariate Analysis for Disease-free Survival.