Tumor-derived DNA Research Articles

Analysis of cell-free DNA (cfDNA) fragmentomes and prediction scores across distinct molecular subtypes.

e15029 Background: Liquid biopsies have the advantage of being convenient in collecting tumor-derived DNA for clinical use. Genome-wide analysis of cell-free DNA (cfDNA) fragmentomes has emerged as a promising approach to detect lung cancer based on the examination of sequencing features other than actionable mutations. Here we report initial analyses of the DELFI (DNA evaluation of fragments for early interception) technology in patients enrolled in the Dutch Lung Cancer Early Molecular Assessment (LEMA) trial (NCT02894853), a prospective, observational, and multicenter study, to evaluate fragmentation patterns in genotyped blood samples with actionable alterations irrespective of disease stage and histology subtype. Methods: Plasma samples from 576 patients with an established diagnosis of lung cancer but awaiting the start of definitive treatment were processed and examined using low-coverage whole-genome sequencing. A lasso model that incorporated genome-wide fragmentation profiles was trained on an internal Delfi Diagnostics cohort and used to generate patient-specific DELFI scores for participants in LEMA. Results: Enrollees comprised 531 individuals with non-small cell lung cancer, 38 with small-cell lung cancer, and 7 with unknown lung cancer type. Most patients were diagnosed with late-stage disease (28% stage I, 10% stage II, 26% stage III, and 36% stage IV). Current smokers (N = 238; 41%) and former smokers (N = 295, 51%) outnumbered never smokers (N = 43; 8%). Analysis showed a stepwise increase of the DELFI scores from T1 to T4 and N0 to N1-3 disease, associating aberrant fragmentation profiles with the overall tumor burden. Small-cell carcinomas and squamous cell carcinomas presented with higher DELFI scores than adenocarcinomas ( p< 1e-7, generalized linear model). Patients without a smoking history had similar scores to those who had a previous history of smoking. When adjusted for stage, mutation status in ALK, BRAF, EGFR, ERBB2, KRAS, MET, RET, and ROS were not associated with DELFI scores ( p= 0.5, likelihood ratio test). Similarly, PD-L1 staining did not significantly affect DELFI scores when a 1% cutoff was applied to cases with PD-L1 staining data ( p= 0.07, generalized linear model). Conclusions: Our results reveal that DELFI scores increase stepwise with stage and are higher in small-cell and squamous cell carcinomas. The LEMA study reinforces the potential usefulness of genome-wide cfDNA fragmentation profiling as a liquid biopsy approach for identifying patients with lung cancer regardless of smoking status, somatic driver mutation, and PD-L1 expression scores.

P-96 Circulating tumor-derived DNA (ctDNA) clearance in patients with locally advanced rectal cancer treated with multimodal treatment

P-96 Circulating tumor-derived DNA (ctDNA) clearance in patients with locally advanced rectal cancer treated with multimodal treatment

Topology of cell-free DNA from tumour cells influences its immunogenicity in macrophages and dendritic cells in vitro

Abstract Activation of DNA-sensing pathways can induce an anti-tumour immune response, but the mechanisms by which tumour-derived cell-free DNA (cfDNA) activates pro-inflammatory signaling is not well-understood. In particular, the topological features of cfDNA that contribute to DNA sensor activation in phagocytes are unknown. As such, we investigated how tumour-derived cfDNA topology influences DNA sensor activation in macrophages and dendritic cells. We treated a murine colon adenocarcinoma cell line (MC38) with chemotherapy (topotecan or staurosporine) for varying durations to induce cfDNA release. We then harvested cfDNA, separated the protected (membrane-bound) and accessible (non-membranous) fractions, and quantified nuclear and mitochondrial cfDNA abundance. The cfDNA fractions were then added to murine RAW 264.7 macrophages or bone marrow-derived dendritic cells (BMDCs), and a panel of inflammatory markers was measured by RT-qPCR. We found that accessible cfDNA from MC38 cells treated with chemotherapy for 48h increased Il6expression in RAW 264.7, but other inflammatory markers were not elevated. Interestingly, both nuclear and mitochondrial cfDNA abundance directly correlated with Il6expression. Surprisingly, cfDNA did not induce pro-inflammatory signalling in BMDCs. These results demonstrate that tumour-derived cfDNA immunogenicity depends on its mechanistic origins, abundance, and the immune cell type to which it is exposed. We are currently examining the impact of other cfDNA biophysical properties on macrophage activation, as well as elucidating the DNA sensing pathways responsible for inducing Il6expression. Our findings will help uncover the role of cfDNA in mediating anti-tumour immunity. E.Z. Malkin is supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-175873) through CIHR. S.V. Bratman is supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre and the Dr. Mariano Elia Chair in Head and Neck Cancer Research at University Health Network and the University of Toronto.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors.

Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

Abstract CT270: Immunogenicity of PGV_001 neoantigen vaccine in a Phase-I clinical trial, across various types of cancers in adjuvant setting

Abstract Introduction: Immunotherapies such as checkpoint blockade, have demonstrated remarkable clinical efficacy yet a large percentage of patients do not respond, potentially due to a paucity of pre-existing immune priming against neoantigens. We developed a personalized genome vaccine (PGV_001) platform to generate neoantigen vaccines targeting each patient’s unique mutanome. Primary objectives of the study were to determine 1) the safety and tolerability; 2) the feasibility of PGV_001 production and administration; and 3) the immunogenicity of PGV_001. Secondary objectives included immunophenotyping vaccine driven cellular and soluble immune milieu in peripheral blood. We previously reported on the clinical efficacy, and here we report, analysis of vaccine-driven immune responses in all treated patients. Methods: The study (Trial Registration NCT02721043) enrolled patients with resected malignancies, including Head and neck squamous cell carcinomas, breast cancer and bladder cancer, or, in the case of multiple myeloma treated with autologous stem cell transplant; all patients determined to have a high risk of disease recurrence (>30% over 5 years). Tumor-derived and germline RNA and DNA was sequenced to predict neoantigens utilizing our custom computation pipeline, OpenVax. Approximately 10 peptides were synthesized per patient, and a mixture of these peptides was administered as 10 subcutaneous and intradermal vaccines over 27 weeks in combination with poly-ICLC and helper Tetanus peptide as adjuvants. Immune responses were analyzed utilizing assays including IFN-gamma ELISPOT, antigen specific T cell expansion followed by flow cytometry, etc. Results: In total 148 neoantigen peptides were manufactured for 15 patients. Overall, 136 PGV_001 doses were administered to 13 patients. Vaccine-specific T cell immunity was observed against multiple vaccine neoepitopes in all 13 subjects. Of the peptides administered, 45% of vaccine antigens (57/126) induced de novo immunity, starting as early as Week8 and often sustaining past last vaccination. Notably, while the vaccine driven T cell immunity was CD4 T cell dominant, most evaluated subjects also displayed vaccine induced polyfunctional CD8-T cell responses. Additional studies are ongoing to define qualities of reactive T cells, evaluate vaccine-induced humoral responses and probe the circulating inflammatory immune milieu. These will be presented. Conclusions: We have established a platform for generating personalized neoantigen vaccines. 100% of the vaccinated patients developed an immune response specific to the vaccine neoantigens predicted by OpenVax. Subjects who received treatment experienced mild Grade 1 or 2 adverse reactions as per the CTEP v 4.0 NCI CTCAE. This vaccine trial reached the primary endpoint of safety, tolerability, feasibility and immunogenicity. Based on the PGV_001 platform two clinical trials, one in patients with glioblastoma multiforme (NCT03223103) in combination with TT fields and second in patients with urothelial carcinoma (NCT03359239) in combination with Atezolizumab have been performed. Data from these trials is under evaluation. Citation Format: Mansi Saxena, Thomas Marron, Julia Kodysh, Alex Rubinsteyn, John Finnigan, Ana Blasquez, Marcia Meseck, Tim O'Donnell, Daniela Delbeau, Mathew Galsky, Deborah Doroshow, Brett Miles, Krzysztof Misiukiewicz, Hanna Irie, Amy Tiersten, Samir Parekh, Marshall Posner, Andrea Wolf, John Mandeli, Rachel Brody, Sacha Gnjatic, Eric Schadt, Philip Friedlander, Nina Bhardwaj. Immunogenicity of PGV_001 neoantigen vaccine in a Phase-I clinical trial, across various types of cancers in adjuvant setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT270.

Abstract 2287: Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA

Abstract Purpose: Extracellular vesicles (EVs) have been shown to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform circulating cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated whether EVs could be a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. Methods: evDNA from both DNase-treated and untreated circulating EVs, enriched from patients with advanced pancreatic cancer (n=10), was analyzed by fluorometry and digital droplet PCR to determine the level of evDNA inside and outside (surface-bound) the EVs. To also determine if choice of methodology affected the results, EVs were isolated using four different small EV isolation methods (differential ultracentrifugation, size-exclusion chromatography, affinity purification and precipitation) and differential centrifugation for isolation of large EVs. Western blotting, dynamic light scattering and transmission electron microscopy confirmed the enrichment of EVs. Results: Our results indicated that EVs enriched from patient blood samples contained significantly lower amounts of DNA compared to cfDNA (p < 0.001); >50-fold lower for untreated sEVs and >1000-fold lower for DNase-treated EVs. Thus, most of the detected vesicle-associated DNA seemed to be located outside of the vesicles. Further, tumor-derived DNA in EVs was barely detectable and the tumor-derived fraction in all evDNA samples was similar to that of cfDNA. Conclusion: Our results demonstrated that evDNA do not add any extra information compared with cfDNA as a source of tumor-derived DNA, at least not in patients with advanced pancreatic cancer. Citation Format: Morten Lapin, Kjersti Tjensvoll, Karoline Nedrebø, Eline Taksdal, Hans Janssen, Bjørnar Gilje, Oddmund Nordgard. Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA [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 2287.

Abstract 3553: Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes

Abstract Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high-yield liquid biopsy enabling in vivo detection of tumor-derived cell-free DNA (cfDNA) thus overcoming the contraindication of biopsy. Somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on RB1 gene, have been able to be identified in the same clinical samples but with two separate sequencing runs. In this study, we first developed a single targeted sequencing method to identify both SCNAs and RB1 SNVs. With this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye. Materials and Methods: 11 paired AH and Rb tumor samples were included in the study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated with QIAgen commercial kits. DNAs were constructed into whole genome libraries followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MycN genes, the all-exon regions of BCOR and CREBBP genes, and a whole genome CNV backbone. Libraries were submitted to Illumina HiSeq 4000 platform for fastq data generation with 2 × 150bp mode. The bioinformatics pipeline was optimized to generate SCNA profiles from targeted sequencing reads, along with SNV calling. Results: For SCNA profiles, 11/11 AH samples (100%) and 8/11 tumor samples (72.72%) have positive RB-SCNA signatures. Strong concordances were observed between AH and tumor SCNA profiles (median = 90.1%) with targeted sequencing reads. In total, 9 disease-driving RB1 SNVs were identified in 6 patients (54.5%). 7/9 (77.8%) of the variants were shared between AH and tumor samples, while the AH and tumor each contained one unique SNV. In all SNVs, the AH displayed a higher allele frequency. Notably, 4/11 samples have focal RB1 gene deletion detected with SCNA profiling, which may explain the difficulties of RB1 SNV detection in some samples. Further, one case was driven by a MYCN gene amplification with no RB1 alterations. Conclusions: This study presented highlights the utility of a single method to call both SVNs and SCNAs in a single clinical sample, with enriched tumor information detected in AH compared with tumor. This study further strengthens the utility of AH as a liquid biopsy platform for Rb eyes. Citation Format: Liya Xu, Mike J. Schmidt, Rishvanth K. Prakabar, Peter Kuhn, James Hicks, Jesse Berry. Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes. [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 3553.

Abstract 3346: Evaluation of a serum-based test integrating tumor tissue and gut microbiome derived metabolites for diagnosis of advanced colorectal adenoma

Abstract Background: Detection and endoscopic excision of colorectal precancerous lesions (advanced adenoma, AA) has been recognized as an effective way to prevent the occurrence of colorectal cancer (CRC) and reduce CRC induced mortality. Current DNA-based (mutations or methylations) tests exhibited compromised efficiency for AA diagnosis due to the late appearance of tumor-derived DNA biomarkers into stool or blood samples during tumorigenesis. Here, by integrating metabolic biomarkers associated with tumor tissue and colorectal neoplasia related gut microbiota, we established the serum metabolites-based test and examined its ability to detect advanced adenoma patients from normal, hyperplastic polyps and low-risk adenoma individuals. Methods: Consensus participants undergoing coloscopy were enrolled in this prospective study, and the result of colonoscope examination and histopathology from biopsied lesions were used as gold standard (ChiCTR2200058078). Serum metabolites were quantified by pseudo-targeted metabolomics based on LC-MS/MS method. By integrating abundances of these serum metabolites associated with tumor tissue and colorectal neoplasia related gut microbiota, a diagnostic model was established, and its performance for detecting advanced adenoma was validated using leave-one-out cross-validation. Results: In total, 803 participants, including 178 normal individuals, 167 patients with hyperplastic polyps, 203 patients with low-risk colorectal adenoma and 255 advanced colorectal adenoma patients, were enrolled in this study and subjected to the serum metabolites-based test. The diagnostic model achieved an AUC of 0.80 (95% CI: 0.77-0.83) for advanced adenoma, with a sensitivity of 44.7% at 88.9% specificity. Additionally, we also separately analyzed its performances for advanced adenoma with different histological subtypes, achieving a sensitivity of 37.0% for tubulous adenoma and a sensitivity of 53.3% for adenoma with villous structure, respectively. As for advanced adenoma with high-grade dysplasia, the model could achieve a sensitivity of 61.2%. In comparation, sensitivities of all current tests for advanced colorectal adenoma, either fecal or blood based, were less than 45% at a similar specificity. Conclusions: Based on integrating metabolic biomarkers associated with both tumor tissue and colorectal neoplasia related gut microbiota, we developed a novel blood-based test that can detect advanced colorectal adenoma with higher accuracy than all current fecal or blood-based tests. Citation Format: Jingyuan Xiang, Longsong Li, Xingting Guo, Xiaowei Li, Kai Lin, Liuyang Yang, Xudong Dai, Enqiang Linghu, Ningli Chai. Evaluation of a serum-based test integrating tumor tissue and gut microbiome derived metabolites for diagnosis of advanced colorectal adenoma [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 3346.

One toolkit to bring them all, and in silico analyze them.

One toolkit to bring them all, and in silico analyze them.

Specific activation of cGAS-STING pathway by nanotherapeutics-mediated ferroptosis evoked endogenous signaling for boosting systemic tumor immunotherapy

Activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway could effectively initiate antitumor immunity, but specific activation of STING pathway is still an enormous challenge. Herein, a ferroptosis-induced mitochondrial DNA (mtDNA)-guided tumor immunotherapy nanoplatform (designated as HBMn-FA) was elaborately developed for activating and boosting STING-based immunotherapy. On the one hand, the high-levels of reactive oxygen species (ROS) in tumor cells induced by HBMn-FA-mediated ferroptosis elicited mitochondrial stress to cause the release of endogenous signaling mtDNA, which specifically initiate cGAS-STING pathway with the cooperation of Mn2+. On the other hand, the tumor-derived cytosolic double-stranded DNA (dsDNA) from debris of death cells caused by HBMn-FA further activated the cGAS-STING pathway in antigen-presenting cells (e.g., DCs). This bridging of ferroptosis and cGAS-STING pathway could expeditiously prime systemic antitumor immunity and enhance the therapeutic efficacy of checkpoint blockade to suppress tumor growth in both localized and metastatic tumor models. The designed nanotherapeutic platform paves the way for novel tumor immunotherapy strategies that are based on specific activation of STING pathway.

Genome-wide analysis of aberrant position and sequence of plasma DNA fragment ends in patients with cancer.

Genome-wide fragmentation patterns in cell-free DNA (cfDNA) in plasma are strongly influenced by cellular origin due to variation in chromatin accessibility across cell types. Such differences between healthy and cancer cells provide the opportunity for development of novel cancer diagnostics. Here, we investigated whether analysis of cfDNA fragment end positions and their surrounding DNA sequences reveals the presence of tumor-derived DNA in blood. We performed genome-wide analysis of cfDNA from 521 samples and analyzed sequencing data from an additional 2147 samples, including healthy individuals and patients with 11 different cancer types. We developed a metric based on genome-wide differences in fragment positioning, weighted by fragment length and GC content [information-weighted fraction of aberrant fragments (iwFAF)]. We observed that iwFAF strongly correlated with tumor fraction, was higher for DNA fragments carrying somatic mutations, and was higher within genomic regions affected by copy number amplifications. We also calculated sample-level means of nucleotide frequencies observed at genomic positions spanning fragment ends. Using a combination of iwFAF and nine nucleotide frequencies from three positions surrounding fragment ends, we developed a machine learning model to differentiate healthy individuals from patients with cancer. We observed an area under the receiver operative characteristic curve (AUC) of 0.91 for detection of cancer at any stage and an AUC of 0.87 for detection of stage I cancer. Our findings remained robust with as few as 1 million fragments analyzed per sample, demonstrating that analysis of fragment ends can become a cost-effective and accessible approach for cancer detection and monitoring.

Aberrant circulating tumor DNA methylation and exosomal microRNA biomarkers for early detection of colorectal cancer.

Colorectal cancer (CRC) became the third most commonly diagnosed malignancy and the second leading cause of cancer death in 2020. However, the rates of early screening and early diagnosis for CRC remain unsatisfactory. Thus, it is essential to explore the initiating factors of CRC and strategies for its early diagnosis. Research progress in liquid biopsy has led to the finding that circulating tumor-derived DNA (ctDNA) and exosomes play vital roles in early detection of CRC. THE APPLICATIONS OF LIQUID BIOPSY FOR EARLY DETECTION OF COLORECTAL CANCER: Moreover, the increased understanding of epigenetics has highlighted the role of ctDNA methylation in CRC carcinogenesis, and the detection of aberrant ctDNA methylation markers is a feasible strategy for diagnosis of early-stage CRC. Among exosomal markers, microRNAs (miRNAs) are abundant and are the most researched. Upregulated or downregulated expression of exosome-derived miRNAs can indicate the occurrence of early-stage CRC. The current research progress on aberrant ctDNA methylation and tumor exosomal miRNA biomarkers in early detection of CRC is summarized in this review, and the advantages and shortcomings of the methods are discussed.

A perifériás vérben keringő szabad DNS molekuláris vizsgálata epeúti malignitásokban

Tumor-derived free-circulating DNA in peripheral blood allows the study of the molecular genetic profile in cholangiocarcinomas and even the effective monitoring of the response to chemotherapy. The use of a liquid biopsy is a favourable solution, as repeated invasive histological sampling is much more practicable and avoidable. The efficiency of liquid biopsy-based sequencing increases with tumor progression and thus with the release of larger amounts of free DNA. In the present study, clinically relevant point mutations were detected from both histological and liquid biopsy specimens of bile duct tumors. During next-generation sequencing, histological and DNA samples obtained during liquid biopsy from 33 patients were analyzed using a 67-gene solid tumor panel. In our prospective study, we present a minimally invasive approach to identify molecular genetic changes in cholangiocarcinoma and gallbladder tumors. The diagnostic application of free DNA reflects the spatial heterogeneity of tumors, making it a new approach to precision oncology treatments. Orv Hetil. 2022; 163(50): 1982-1991.

The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients.

Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of specific cancer-associated mutations and holds great promise for the development of minimally invasive diagnostic tests. Furthermore, serial monitoring of ctDNA over the course of systemic and targeted therapies not only allows unparalleled efficacy assessments but also enables the identification of patients who are at risk of progression or recurrence. Droplet digital PCR (ddPCR) is a powerful technique for the detection and monitoring of ctDNA. Due to its relatively high accuracy, sensitivity, reproducibility, and capacity for absolute quantification, it is increasingly used as a tool for managing cancer patients through liquid biopsies. In this review paper, we gauge the clinical utility of ddPCR as a technique for mutational profiling in breast cancer patients and focus on HER2, PIK3CA, ESR1, and TP53, which represent the most frequently mutated genes in breast cancers.

MPC-20 CLINICAL AND MOLECULAR FEATURES OF GLIOBLASTOMAS ARISING IN THE SETTING OF LI FRAUMENI SYNDROME

Abstract Introduction Li Fraumeni Syndrome (LFS) is a rare autosomal dominant tumor predisposition syndrome caused by germline alterations of the TP53. Brain tumors are included in LFS core cancers. Choroid plexus carcinomas and medulloblastomas tend to present in pediatric LFS patients, while gliomas tend to present in adult LFS patients. There are few previous reports about gliomas arising in the setting of LFS. Here, we report clinical and molecular features of three cases of glioblastoma arising in the setting of LFS. Results Mean age at diagnosis was 40 years (range: 32-45). Two patients were male. Two patients had medical history of multiple tumors and family history of brain tumors. Magnetic resonance imaging of each patients revealed a left cerebellar peduncle lesion, a right parietal lobe lesion and two lesions in the pons and right parietal lobe, respectively. Pathological diagnoses of all cases were glioblastomas, IDH-wildtype. All patients underwent chemoradiotherapy after surgery. Mean progression free survival was 10.4 months (range: 5.1-17.0). All patients were admitted to palliative care mean 17.3 months (range: 14.4-21.4) after diagnosis. All patients had heterozygous germline mutations of TP53. Genetic analysis of tumor tissues revealed TP53 mutation and PDGFRA amplification in all patients. In all cases, mutation spots of TP53 in tumor-derived DNA were consistent with those of germ line mutations. In all cases, copy number alteration of TP53 gene in tumor-derived DNA were not detected. EGFR amplification, the combination of gain of chromosome 7 and loss of chromosome 10 (7+/10-) and mutations of IDH, H3F3A, BRAF genes and TERT gene promoter were not detected in all tumor-derived DNA. Conclusion We experienced three cases of glioblastoma, IDH-wildtype arising in the setting of LFS. Clinical courses of all cases were poor. Notably, all tumor tissues harbored TP53 mutation and PDGFRA amplification, while no tumor tissues harbored TERTp mutation, EGFR amplification and 7+/10-.

The Profiling of Circulating Tumor DNA Liquid Biopsy in Children and Adolescent Lymphoblastic Lymphoma

Backgrounds: Next-generation sequencing (NGS) based on liquid biopsy has been an emerging technology to identify tumor-specific genetic aberrations in adult lymphoma. However, there were few studies on the genomic profiling of plasma circulating tumor-derived DNA (ctDNA) in pediatric and adolescent lymphoblastic lymphoma (LBL). Methods: Paraffin-embedded tissue (FFPE), plasma and bone marrow samples from the newly diagnosed patients were collected and sequenced by 475 genes panel before, during and post of treatment. Clinical stage system, risk stratification and treatment of lymphoblastic lymphoma according to modified BFM-95 protocol. Results: A total of 68 patients (50 T-LBL, 18 B-LBL) from 13 centers in China were included in this study, including 49 male and 19 female, 3 patients in the low-risk group, 30 in the intermediate-risk group, and 19 in the high-risk group, and 16 patients were not stratified. The baseline detection rate of tissue, plasma and bone marrow of the T-ALL/LBL patients were 96% (24/25), 97.14% (34/35), and 75% (12/16), respectively. The mean mutation abundance in baseline tissue (34.12%) was significantly higher than in plasma (15.16%) and bone marrow (13.93%). Regarding sample consistency compared with tissue, the sensitivity of baseline plasma and bone marrow were 73.83% (79/107, N=18) and 70.77% (46/65, N=10), respectively. Compared with plasma, the sensitivity of mutation detection in bone marrow samples was 67.29% (72/107%, N=14). There were no significant correlation between ctDNA mutation abundance, concentration and risk stratification, stage, and SUV of PET/CT. The most common molecular abnormality detected in tissue and plasma was NOTCH1 mutation (52.2% in tissue, 66.7% in plasma), and the mutation types were mainly missense mutation, deletion, and frameshift mutation, and also found rare NOTCH1 gene fusions (IKZF2: exon5~NOTCH1: exon28, NOTCH1: exon 30~IKZF2: exon6). Other high-frequency gene variants were CDKN2A (tissue 30.4%, plasma 18.2%), CDKN2B (tissue 30.4%, plasma 21.2%), JAK1 (tissue 34.8%, plasma 15.2%), BCL11B (tissue 4.3%, plasma 18.2%) FBXW7 (tissue 13%, plasma 21.2%), PTEN (tissue 13%, plasma 12.1%) and so on (Figure 1). A total of 24 patients underwent dynamic monitoring of two or more liquid biopsies (Figure 2), and all patients were positive in baseline plasma ctDNA. At the last follow-up time, 11 of 24 patients were at least 2 times positive for ctDNA examination, of whom 5 patients with tumor recurrence, progression or poor prognosis (3 cases poor response to induction IA chemotherapy with tumor shrunk less than 70%, 2 cases with tumor progression and 1 case with tumor relapsed) and 1 patient died of infection during consolidation treatment and 4 cases lost to follow-up. Twelve of 13 patients whose plasma ctDNA cleared at the second examination time achieved PR with tumors shrunk more than 70% after induction IA treatment and none of the 13 patients relapsed or disease progression. In B-LBL, the baseline detection rate of tissue, plasma and bone marrow were 75% (9/12), 100% (12/12), and 66.67% (4/6), respectively. The top five gene variants were CDKN2A (26.7%), FLT3 (26.7%), NRAS (26.7%), KRAS (20%), NTRK3 (20%). Conclusion: Plasma ctDNA testing by NGS was practicable in pediatric and adolescent lymphoblastic lymphoma and could well reflect the clinical efficacy and detect tumor recurrence. Nevertheless, it is necessary to expand the sample size and extend the follow-up time to explore the value of liquid biopsy in pediatric and adolescent lymphoblastic lymphoma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Determinants of Resistance to Engineered T-Cell Therapies Targeting CD19 in Large B-Cell Lymphomas

Background: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have activity in patients with relapsed/refractory large B-cell lymphoma (rrLBCL), but over half of patients ultimately relapse. We originally applied cell-free DNA (cfDNA) analysis to a cohort (n=65) of patients receiving axicabtagene ciloleucel (axi-cel) to identify determinants of resistance and characterize molecular thresholds predictive of treatment failure. Here, we extend these analyses to an additional independent cohort (n=73) of patients to validate these thresholds and refine genomic alterations associated with resistance. Methods: We developed a novel hybrid capture strategy targeting 187 protein coding genes, all T-cell receptor (TCR) loci, and axi-cel retroviral vector. This approach allows for simultaneous evaluation of circulating tumor-derived DNA (ctDNA), CAR19-derived cfDNA (cfCAR) and T-cell receptor rearrangements (cfTCR) from blood plasma. We applied this platform to profile serial samples from discovery (n=65) and validation (n=73) cohorts of rrLBCL patients receiving CAR19 therapy (total n=128). Pretreatment and relapse tumor tissue was also profiled when available. A previously determined optimized pretreatment (Day 0) ctDNA threshold to stratify event free survival (EFS) was revalidated in the discovery cohort using bootstrap resampling (2.5 log10 haploid genome equivalents per milliliter). Results: The median follow-up was 36.4 months in the discovery cohort, and 12.5 months in the validation cohort. 55% (36/65) and 58% (42/73) of patients in the discovery and validation cohorts had progressed following CAR19, respectively. In both cohorts, patients with high pretreatment ctDNA demonstrated inferior EFS (Discovery: p<0.001, Validation, p=0.002). Similarly, a ~300-fold (2.5 log10) decrease in ctDNA levels at Day 28 (Major Molecular Response - MMR) post CAR19 infusion was identified as the optimal post-treatment change to stratify EFS, and patients in both cohorts who achieved MMR had significantly superior EFS (Discovery: p<0.001, Validation, p=0.028). When considering ctDNA, cfTCR and cfCAR as continuous variables in the discovery cohort, higher pre- and post-treatment ctDNA levels were prognostic of EFS at all assayed time-points, as were pre-treatment LDH, week 1 cfCAR19 and week 4 cfTCR levels (Fig. A). Forward stepwise multivariable regressions identified week 4 ctDNA level (HR=2.2, p<0.001) and week 1 cfCAR19 level (HR=0.6, p=0.017) as the optimal independent covariates for predicting outcomes (Fig. A). A Cox proportional hazards model incorporating these two variables was trained on the discovery cohort (CAR Outcome Score) and was validated to be prognostic for outcomes in the validation cohort (HR=2.7, p<0.001). Genomic alterations in several genes were significantly associated with inferior outcomes in both cohorts, including PAX5, IRF8, TMEM30A, PPM1D and TP53 (Fig. B). Additional, non-stereotyped, emergent alterations were noted in relapsing patients in other genes associated with immunological escape, including mutations in CD19 and amplifications in the PD-L1 locus. CIBERSORTx was used to characterize intratumoral cell type composition, which was found to be influenced by tumor genotypes. Further, CAR19 persistence in tumors at relapse was associated with specific changes in the tumor microenvironment, including regulatory T-cell expansion and B-cell depletion. Conclusions: Pre-treatment and dynamic ctDNA levels predict treatment failure of LBCL after CAR19 therapy, and a novel CAR outcome score incorporating week 4 ctDNA and week 1 cfCAR19 levels is strongly predictive of outcome. Genomic determinants of resistance to CAR19 therapy are diverse, including recurrent mutations in genes controlling B-cell identity (IRF8, PAX5), and those influencing the composition of the tumor microenvironment and heterotypic cell-cell interactions (TMEM30A). Additional immunological escape mechanisms arise under selective pressure from CAR19 cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract PR013: High sensitive mutation detection in liquid biopsy with duodenal fluid captures genetic mutations associated with pancreatic carcinogenesis

Abstract Background: Blood-based liquid biopsy for comprehensive genomic profiling is available for cancer patients; however, given the limited amount of tumor-derived DNA in plasma, it remained challenging to detect pancreatic ductal adenocarcinoma (PDA) at earlier stages. Here, we evaluated the mutation detection in DNA from the duodenal fluid as an alternative biospecimen obtained by a less-invasive approach relative to endoscopic biopsy. Methods: In 300 patients of PDA, IPMN, or other pancreatic diseases in seven institutions, we collected duodenal fluid containing pancreatic juice from the duodenal lumen through the endoscopic channel. Plasma was also collected, and DNA was extracted from both samples, and molecular barcode sequencing, which enabled the highly sensitive detection of low- frequent mutation, targeting five pancreatic cancer-associated genes, namely, KRAS, GNAS, TP53, CDKN2A, and SMAD4, were performed. Detected mutations were evaluated for concordance with surgically resected tissue or needle biopsy. Results: DNA extracted from the duodenal fluids (DFDNA) had a much higher yield (avg. 25.4 ng/ml) than plasma cell-free DNA (avg. 1.3 ng/ml). While plasma cfDNA had a typical fragmentation pattern with a peak at 160 bp, DFDNA has a variety of fragmentation patterns. Hotspot mutations detected in the duodenal fluids showed as much as 51.4% concordance with a 0.2 % the limit of detection (LOD) for its variant allele frequency in PDA-associated mutations in the DFDNA (n=109) with the mutations in PDA lesion, whereas only 25% concordance with 0.4% LOD in the plasma cfDNA (n=104). In contrast to plasma cfDNA, where liquid-tissue matches were limited to advanced stages of cancer, DFDNA assay enabled the higher concordance through a wide range of PDA stages, even Stage 0 (83.3%) or I (45.5%) UICC 8th, or IPMN cases, and allowed to detect of additional mutations that were originated from multicentric/concomitant lesions in the pancreas. Conclusions: Duodenal fluid-derived DNA can be a potent biospecimen to detect mutations with higher yield and sensitivity than plasma cfDNA. Although validation of the duodenal fluid collection and methods up to more sensitive analysis is required to meet the standards of practical testing, it has the potential to detect fast-stage pancreatic disease and the detection of lesions that were not visible on pathological images and is expected to lead to the implementation of highly sensitive liquid biopsy. Citation Format: Yusuke Ono, Kenji Takahashi, Hidenori Karasaki, Yusuke Mizukami. High sensitive mutation detection in liquid biopsy with duodenal fluid captures genetic mutations associated with pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR013.

Clinical utility of plasma cell-free DNA in gliomas.

Noninvasive molecular profiling of tumors using plasma-based next-generation sequencing (NGS) is increasingly used to aid in diagnosis, treatment selection, and disease monitoring in oncology. In patients with glioma, however, the plasma cell-free DNA (cfDNA) tumor fraction, defined as the fractional proportion of circulating tumor-derived DNA (ctDNA) relative to total cfDNA, is especially low, in large part due to the blood-brain barrier. As a result, commercial plasma-based NGS assays, designed to screen for a small number of actionable genomic alterations, are not sensitive enough to guide the management of patients with glioma. As this has been long recognized in neuro-oncology, significant research efforts have been undertaken to improve the sensitivity of plasma ctDNA detection in patients with glioma and to understand the biology and clinical relevance of non-tumor-derived cfDNA, which makes up most of the total cfDNA pool. Here, we review key recent advances in the field of plasma cfDNA analysis in patients with glioma, including (1) the prognostic impact of pre-treatment and on-treatment total plasma cfDNA concentrations, (2) use of tumor-guided sequencing approaches to improve the sensitivity of ctDNA detection in the plasma, and (3) the emergence of plasma cfDNA methylomics for detection and discrimination of glioma from other primary intracranial tumors.

Towards clinical implementation of circulating tumor DNA in metastatic prostate cancer: Opportunities for integration and pitfalls to interpretation.

Plasma circulating tumor DNA (ctDNA) represents short fragments of tumor-derived DNA released into the bloodstream primarily from cancer cells undergoing apoptosis. In metastatic castration-resistant prostate cancer (mCRPC), characterizing genomic alterations in ctDNA identifies mutations, copy number alterations, and structural rearrangements with predictive and prognostic biomarker utility. These associations with clinical outcomes have resulted in ctDNA increasingly incorporated into routine clinical care. In this review, we summarize current and emerging applications for ctDNA analysis in metastatic prostate cancer, including outcome prediction, treatment selection, and characterization of treatment resistance. We also discuss potential pitfalls with interpreting ctDNA findings, namely false negatives arising from low tumorcontent and optimal assay design, including correction for clonal hematopoiesis of indeterminate potential and germline variants. Understanding the influence of these limitations on interpretation of ctDNA results is necessary to overcome barriers to clinical implementation. Nevertheless, as assay availability and technology continue to improve, recognizing both opportunities and shortcomings of ctDNA analysis will retain relevance with informing the implementation of precision-oncology initiatives for metastatic prostate cancer.