Methylation-specific qPCR for the EBV C promoter to quantify EBV methylation

The underlying mechanism of the protective and suppressive role of NKT cells in human tumor immunosurveillance remains to be fully elucidated. We show that the frequencies of CD8(+) NKT cells in patients with EBV-associated Hodgkin's lymphoma or nasopharyngeal carcinoma are significantly lower than those in healthy EBV carriers. These CD8(+) NKT cells in tumor patients are also functionally impaired. In human-thymus-severe combined immunodeficient (hu-thym-SCID) chimeras, EBV challenge efficiently promotes the generation of IFN-gamma-biased CD8(+) NKT cells. These cells are strongly cytotoxic, drive syngeneic T cells into a Th1 bias, and enhance T-cell cytotoxicity to EBV-associated tumor cells. Interleukin-4-biased CD4(+) NKT cells are predominately generated in unchallenged chimeras. These cells are noncytotoxic, drive syngeneic T cells into a Th2 bias, and do not affect T-cell cytotoxicity. In humanized xenogeneic tumor-transplanted hu-thym-SCID chimeras, adoptive transfer with EBV-induced CD8(+) NKT cells significantly suppresses tumorigenesis by EBV-associated malignancies. EBV-induced CD8(+) NKT cells are necessary and sufficient to enhance the T-cell immunity to EBV-associated malignancies in the hu-thym-SCID chimeras. CD4(+) NKT cells are synergetic with CD8(+) NKT cells, leading to a more pronounced T-cell antitumor response in the chimeras cotransferred with CD4(+) and CD8(+) NKT cells. Thus, immune reconstitution with EBV-induced CD8(+) NKT cells could be a useful strategy in management of EBV-associated malignancies.

<div>Abstract<p>The underlying mechanism of the protective and suppressive role of NKT cells in human tumor immunosurveillance remains to be fully elucidated. We show that the frequencies of CD8<sup>+</sup> NKT cells in patients with EBV-associated Hodgkin's lymphoma or nasopharyngeal carcinoma are significantly lower than those in healthy EBV carriers. These CD8<sup>+</sup> NKT cells in tumor patients are also functionally impaired. In human-thymus-severe combined immunodeficient (hu-thym-SCID) chimeras, EBV challenge efficiently promotes the generation of IFN-γ–biased CD8<sup>+</sup> NKT cells. These cells are strongly cytotoxic, drive syngeneic T cells into a Th1 bias, and enhance T-cell cytotoxicity to EBV-associated tumor cells. Interleukin-4–biased CD4<sup>+</sup> NKT cells are predominately generated in unchallenged chimeras. These cells are noncytotoxic, drive syngeneic T cells into a Th2 bias, and do not affect T-cell cytotoxicity. In humanized xenogeneic tumor-transplanted hu-thym-SCID chimeras, adoptive transfer with EBV-induced CD8<sup>+</sup> NKT cells significantly suppresses tumorigenesis by EBV-associated malignancies. EBV-induced CD8<sup>+</sup> NKT cells are necessary and sufficient to enhance the T-cell immunity to EBV-associated malignancies in the hu-thym-SCID chimeras. CD4<sup>+</sup> NKT cells are synergetic with CD8<sup>+</sup> NKT cells, leading to a more pronounced T-cell antitumor response in the chimeras cotransferred with CD4<sup>+</sup> and CD8<sup>+</sup> NKT cells. Thus, immune reconstitution with EBV-induced CD8<sup>+</sup> NKT cells could be a useful strategy in management of EBV-associated malignancies. [Cancer Res 2009;69(20):7935–44]</p></div>

<div>Abstract<p>The underlying mechanism of the protective and suppressive role of NKT cells in human tumor immunosurveillance remains to be fully elucidated. We show that the frequencies of CD8<sup>+</sup> NKT cells in patients with EBV-associated Hodgkin's lymphoma or nasopharyngeal carcinoma are significantly lower than those in healthy EBV carriers. These CD8<sup>+</sup> NKT cells in tumor patients are also functionally impaired. In human-thymus-severe combined immunodeficient (hu-thym-SCID) chimeras, EBV challenge efficiently promotes the generation of IFN-γ–biased CD8<sup>+</sup> NKT cells. These cells are strongly cytotoxic, drive syngeneic T cells into a Th1 bias, and enhance T-cell cytotoxicity to EBV-associated tumor cells. Interleukin-4–biased CD4<sup>+</sup> NKT cells are predominately generated in unchallenged chimeras. These cells are noncytotoxic, drive syngeneic T cells into a Th2 bias, and do not affect T-cell cytotoxicity. In humanized xenogeneic tumor-transplanted hu-thym-SCID chimeras, adoptive transfer with EBV-induced CD8<sup>+</sup> NKT cells significantly suppresses tumorigenesis by EBV-associated malignancies. EBV-induced CD8<sup>+</sup> NKT cells are necessary and sufficient to enhance the T-cell immunity to EBV-associated malignancies in the hu-thym-SCID chimeras. CD4<sup>+</sup> NKT cells are synergetic with CD8<sup>+</sup> NKT cells, leading to a more pronounced T-cell antitumor response in the chimeras cotransferred with CD4<sup>+</sup> and CD8<sup>+</sup> NKT cells. Thus, immune reconstitution with EBV-induced CD8<sup>+</sup> NKT cells could be a useful strategy in management of EBV-associated malignancies. [Cancer Res 2009;69(20):7935–44]</p></div>

To assess plasma Epstein-Barr virus (EBV) DNA as a biomarker of tumour burden at diagnosis and during therapy in children with Hodgkin lymphoma. Case-control study, with prospective follow-up of the Hodgkin lymphoma cohort (2007-2012). Pediatric Hematology Oncology unit of a tertiary care hospital in Delhi. Thirty children with Hodgkin lymphoma and 70 sex and age-matched controls (benign lymphadenopathy 19, non-lym-phoid malignancy 29, Burkitt lymphoma 5, healthy children 17). Positive EBV-staining on immunohistochemistry was defined as EBV-associated Hodgkin lymphoma. Plasma EBV real-time quantitative polymerase chain reaction (PCR) was tested at presentation, after first and last chemotherapy cycles, and on follow-up. Plasma EBV quantitative PCR was compared between cases and controls. Its kinetics was assessed during and after chemotherapy. EBV quantitative PCR was positive in 19 (63%) Hodgkin lymphoma cases (range 500 to 430,000 copies/mL), with 87.5% accuracy (kappa=0.69) as compared with EBV immunohistochemistry. Sensitivity and specificity of the quantitative PCR were 87.5% and 81.8%, respectively. Only boys showed positive EBV immunohistochemistry and,or quantitative PCR positivity. All controls were quantitative PCR negative. All quantitative PCR positive cases with follow up blood sample showed EBV clearance after the first cycle. A quantitative PCR negative case in long-term remission became positive at relapse. EBV status did not influence survival. Plasma EBV-DNA, detectable in EBV-associated Hodgkin lymphoma, becomes undetectable early after initiating therapy. It can be used as a biomarker of treatment response in EBV-associated Hodgkin lymphoma.

The Epstein-Barr Virus (EBV) Encoded Oncoprotein LMP1 Mediates Down Regulation of Shelterin Proteins, Formation of Telomere Aggregates and Multinuclearity.

Epstein–Barr virus (EBV) is associated with around one-third of Hodgkin's lymphoma (HL) cases and this association is believed to be causal. In these EBV-associated cases, there is a clonal EBV infection within tumors, and EBV genomes and gene products are detectable in Hodgkin and Reed-Sternberg (HRS) cells. The proportion of EBV-associated HL in any population varies with age, sex, ethnicity and histologic subtype. Two population-based epidemiologic studies have examined risk factor profiles in HL with cases stratified according to EBV status. For EBV-associated HL cases, there is a small peak in incidence in young adults (15 – 24 years) and a second larger peak in older adults. By contrast, HL that is not associated with EBV (EBV-negative HL) accounts for the major part of the young adult incidence peak after which the incidence of this disease entity then declines. Prior infectious mononucleosis (IM) is associated with an increased risk of developing HL, and there is a specific, probably causal, association between previous IM and young adult EBV-associated HL. We therefore believe that the small peak in the incidence of EBV-associated HL in young adults is real and related to late infection by EBV. EBV-associated HL in childhood and young adults, therefore, appears to follow primary infection by the virus. At the time of diagnosis, EBV-associated HL patients have an increased frequency of circulating EBV-infected cells compared to patients with EBV-negative HL and normal controls. The EBV is present in memory B cells and most probably reflects increased viral replication at another site, such as the oropharynx. EBV genomes are detectable in the serum and plasma of EBV-associated HL cases. The origin of EBV genomes in serum/plasma differs in different disease states; in HL viral genomes are present as naked DNA and are probably shed from tumors. EBV genome copy number in serum/plasma may provide an indication of tumor burden and may prove to be a useful marker for monitoring HL patients. The etiology of EBV-negative HL remains unknown and, while the involvement of an infectious agent may be suspected, none has yet been identified. Overall, epidemiologic studies support the idea that HL can be divided into two etiologic subgroups on the basis of EBV status and suggest that EBV-associated cases can be further divided into three groups related to age at diagnosis.

Introduction: Promoter methylation (PM) is a critical epigenetic mechanism that is involved in gene silencing and is associated with key pathways in cancer progression. Traditionally, PM detection has relied on single-gene testing of tissue biopsies, which are invasive, less clinically accessible, and impractical for tracking tumor evolution. Here, we evaluate PM within clinical multi-modal assessments using Guardant Infinity smart liquid biopsy platform, providing an innovative approach for monitoring epigenetic alterations. Methods: Data across >17k samples, including >5k cancer-free samples, and cancer-positive samples from >100 cancer types were used to define promoter regions associated with hypermethylation in cancer-related genes. PM regions were refined to ensure (a) the methylation signal is present exclusively in cancer and low in cancer-free samples, and (b) a negative correlation between gene expression and methylation in The Cancer Genome Atlas Program (TCGA) cancer samples. PM regions are quantified relative to constitutively hypermethylated control regions, measuring gene PM relative to total cfDNA. Analytical accuracy was demonstrated using an orthogonal Enzymatic Methyl Sequencing (EM-seq) assay on a cohort of clinical cfDNA and contrived samples. Limit of detection, precision, and limit of blank were established based on cancer cfDNA samples, cell line dilutions, and healthy donor samples. Gene PM prevalence was assessed in a test cohort of >7k samples across multiple cancer types to compare the relative prevalence to published tissue data sets. Results: Guardant Infinity PM limit of detection (>95% variant level) was established at PM mutant allele frequency (MAF) 1.6% across a panel of promoter genes. PM above the limit of detection is highly concordant with orthogonal EM-Seq, with 98.2% positive and 98% negative concordance. MLH1 PM was identified in 54% of patients with colorectal cancer (CRC) and 100% of patients with endometrial cancer (EC) who also had MSI-H detected. MLH1 PM in our cohort of CRC showed significant enrichment for BRAF V600E (36%, p<0.001). High prevalence of BRCA1 PM was observed in breast (BC) and ovarian cancer (OC), consistent with what is observed in literature. Enrichment of BRCA1 PM in BC was observed with loss of heterozygosity (LOH) (6%, p=0.02). The relative PM prevalence for individual genes across tumor types is largely consistent with what has been reported in tissue datasets. Conclusion: PM in liquid biopsy is consistent with tissue-based findings and correlates with underlying tumor characteristics, such as MSI-H and LOH events, thus demonstrating Guardant Infinity’s ability to identify potentially actionable PM patterns in a non-invasive manner. This analysis represents a fraction of the PM landscape observed, highlighting the broader potential of methylation as a novel biomarker for cancer detection and characterization. Citation Format: Janice A. Patterson, Martina Lefterova, Brooke Overstreet, Colby Jenkins, Katie Quinn, Darya Chudova. Analytical validation of cancer gene promoter methylation detection in cfDNA liquid biopsy assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3255.

75. Development of an LMP-Specific T Cell Bank for Third Party Use as a Curative Strategy Post-Transplant Lymphoproliferative Disease After Solid Organ Transplant

Inborn errors of immunity (IEIs) are caused by deleterious variants in immune-related genes. ASXL1 is an epigenetic modifier not previously linked to an IEI. Clonal hematopoiesis and hematologic neoplasms often feature somatic ASXL1 variants, and Bohring-Opitz syndrome, a neurodevelopmental disorder, is caused by heterozygous truncating ASXL1 variants. We present an IEI caused by biallelic germline missense variants in ASXL1. The patient had a history of hematologic abnormalities and viral-associated complications, including chronic macrocytosis, persistent vaccine-strain rubella granulomas, and EBV-associated Hodgkin lymphoma. Immunophenotyping revealed loss of B cells, hypogammaglobulinemia, and impairments in cytotoxic T and NK cell populations. T cells exhibited skewing toward an exhausted memory phenotype, global DNA methylation loss, and increased epigenetic aging. These aberrations were ameliorated by wild-type ASXL1 transduction, confirming the patient variants' pathogenicity. This study defines a novel human IEI caused by ASXL1 deficiency, a diagnosis that should be considered in individuals with chronic viral infections, viral-associated malignancies, and combined immune deficiency.

EBV-Specific Cytotoxic T Lymphocytes by LMP2a- and EBNA3a-Specific T Cell Receptor Gene Transfer for Adoptive Therapy in EBV-Associated Hodgkin's Disease and Post-Transplant Lymphoma.

Previously, we demonstrated that when two human hepatoma cell lines, Hep3B and HepG2, were exposed to gemfibrozil, a hypolipidemic drug, a 2-fold induction in apolipoprotein A-I (apoA-I) mRNA levels resulted. To determine if mRNA stabilization was responsible for the changes in apoA-I mRNA levels, the half-lives for apoA-I mRNA were measured in the presence of actinomycin D with and without gemfibrozil. These experiments revealed no differences in stability. However, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was increased 2-fold in gemfibrozil-treated cells. Transient transfection experiments also indicated that the induction of apoA-I mRNA level in response to gemfibrozil is mediated at the transcriptional level. We have identified two copies of the "drug-responsive element" (DRE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gemfibrozil. Using gel mobility shift assays with a synthetic DRE oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2 cells to gemfibrozil resulted in strong induction of a protein-DNA complex. The formation of this complex is highly sequence-specific as indicated by the DNA competition experiments. The drug-inducible nuclear proteins bind to the DRE of the human apoA-I gene with an apparent Kd of 4.1 nM. Methylation interference experiments have localized the contact sites of nuclear factors to the DRE region. Southwestern blot analyses have identified two groups of drug-inducible nuclear proteins with molecular masses of approximately 30 and 15 kDa. When a copy of synthetic DRE oligonucleotide was inserted upstream of the thymidine kinase promoter and luciferase reporter construct, a significant 2-fold induction in luciferase activity was observed in the presence of gemfibrozil following transient transfection of two human hepatoma cell lines, HepG2 and Hep3B. However, a plasmid containing one copy of mutated apoA-I-DRE oligomer did not confer responsiveness to gemfibrozil treatment. Furthermore, pGL2 (apoA-I -250 mutant DRE), which carried an internal mutation of the DRE in the human apoA-I proximal promoter region, showed no increase in luciferase activity in response to gemfibrozil. These results implicate protein-DNA interactions at the DRE region in the transcriptional induction of human apoA-I gene expression by gemfibrozil.

Introduction: Promoter methylation is a hallmark of cancer and holds promise as a biomarker for early cancer detection, prognostication, treatment guidance, and response monitoring. This study explores the utility of GRAIL’s ctDNA-based targeted methylation assay in detecting gene-specific promoter methylation signals and assessing their potential as cancer biomarkers. Methods: A comprehensive literature review was conducted to identify genes with promoter methylation proposed as a cancer biomarker, focusing on those covered by GRAIL's ctDNA targeted methylation assay. We then prioritized genes implicated in multiple cancer types across several studies. As a proof-of-concept, four genes (EYA4, SFRP1, BNIP3, ASCL1) were evaluated using the ctDNA targeted methylation data from the Circulating Cell-free Genome Atlas (CCGA; NCT02889978) study. Contrived samples with known methylation fractions were utilized to establish the limit of detection (LoD) and linearity. Our analysis included 6127 CCGA cancer samples, spanning 21 cancer types, and 2323 non-cancer samples. We estimated the fraction of methylated fragments in each promoter region for all samples and the prevalence of promoter methylation for each cancer type. A Cox proportional hazard model was employed to evaluate the association of promoter methylation for a given gene with 5-year overall survival, adjusting for cancer stage. Results: EYA4 promoter methylation was prevalent in colorectal (39%,108/279, 95% CI: 32%-44%) and liver/bile duct cancer (68%,47/69, 95% CI: 56%-79%). Significant association of EYA4 promoter methylation with survival was observed in colorectal cancer (HR=2.79, p<0.001) and in the pan-cancer setting (HR=1.71, p<0.001). SFRP1 promoter methylation was significantly associated with poorer survival in lung (HR=1.62, p=0.006), colorectal (HR=1.96,p=0.036) and stomach cancers (HR=2.79, p=0.048). BNIP3 promoter methylation, reportedly associated with chemoresistance, was prevalent in colorectal (37%, 104/279, 95% CI: 32%-43%) and pancreatic (34%, 70/205, 95% CI: 23%-41%) cancer, with significant association with worse survival (pancreatic: HR=2.72, p<0.001; colorectal: HR=1.98, p=0.009). ASCL1 promoter methylation correlated with clinical HPV status of HPV-associated cancers, with higher fraction of methylated fragments in HPV-positive head and neck and cervical cancers (p=0.015). Conclusion: This proof-of-concept study underscores the potential of GRAIL’s ctDNA-based targeted methylation assay in detecting clinically informative promoter methylation signals. It also demonstrates the value of the CCGA cohort in facilitating biomarker discovery in plasma samples across multiple cancer types. Further validation and investigation in therapeutic settings are necessary to fully leverage promoter methylation as a biomarker in precision oncology. Citation Format: Margaret Antonio, Eran Mick, Roham Razaghi, Chun Zhang. Promoter methylation as a cancer biomarker: insights from ctDNA-based targeted methylation data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1943.

Deoxyuridine triphosphatase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and pyrophosphate thus preventing the incorporation of uracil into replicating DNA. Previous studies of several virus models have suggested that viral dUTPases may be required for virus replication in resting cells whereas in proliferating cells cellular dUTPase may substitute for a mutant viral protein. Using monoclonal antibodies and immunohistochemistry, Epstein-Barr virus-associated non-neoplastic and neoplastic diseases were studied for the expression of viral and human dUTPases. Oral hairy leukoplakia, an AIDS-associated lesion of the tongue, is known to support EBV replication in the upper epithelial cell layers. In agreement with this, strong focal expression of EBV dUTPase was detected in the upper epithelial cell layers of oral hairy leukoplakia whereas expression of human dUTPase was confined to the basal proliferative cell compartment. Furthermore, in infectious mononucleosis tonsils, rare scattered small lymphoid cells expressed EBV dUTPase, consistent with the expression pattern of other EBV lytic cycle antigens. These findings are in agreement with the notion that EBV replicates in resting cells. Three EBV-associated tumours, Hodgkin lymphoma, Burkitt lymphoma and nasopharyngeal carcinoma, lacked detectable expression of EBV dUTPase, in agreement with the notion that EBV infection is largely latent in these tumours. By contrast, expression of human dUTPase was observed regularly in these tumours. These results suggest that EBV dUTPase may be a suitable target for anti-viral therapy and that inhibitors of human dUTPase should prove useful for the treatment of human tumours, including EBV-associated cancers.

Eukaryotic cells possess several distinct mismatch repair pathways. A mismatch can be introduced in retroviral double-stranded DNA by a pre-existing mutation within the primer binding site (PBS) of the viral RNA genome. In order to evaluate mismatch repair of retroviral double-stranded DNA, Moloney leukemia virus (MLV)-based vectors with a mutation in their PBS were used to infect mismatch repair-competent as well as mismatch repair-deficient cell lines. If the target cells were capable of repairing the mismatch before an infected cell divided, the mismatch within the PBS could be repaired to the wild-type or mutant PBS. If the target cells were unable to repair the mismatch, half the cells in the colony should contain the mutant PBS while the other half should contain the wild-type PBS. To evaluate these predictions, individual colonies were isolated and analyzed by PCR. Almost all mismatch-deficient cell colonies analyzed (cell lines HCT 116 and PMS2-/-) contained both the wild-type and mutated PBS, therefore, mismatches within retroviral double-strand DNA could not be repaired by the mismatch-deficient cells. In contrast, mismatches in approximately 25% of the mismatch repair-competent cell clones analyzed (cell lines HeLa and PMS2+/+) were repaired, while 75% were not. Therefore, the cellular mismatch repair system is able to repair mismatches within viral double-stranded DNA, but at a low frequency.

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer