Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (n = 20) and non-colorectal cancers (n = 8); and healthy volunteers (n = 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (P = .001) and normal donors (P = .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; P = .027).

Baseline plasma fibrinogen level did not show prognostic significance for DLBCL patients, although it was associated with patients' clinical features and laboratory parameters. Being simple, cheap and widely available laboratory test, its use should be encouraged routinely in clinical practice to precisely clarify its predictive merit.

Background: Invasive aspergillosis (IA) is a leading cause of death among immunocompromised patients, particularly those with hematological malignancies. The use of galactomannan (GM) antigen as a biological marker for screening of IA in high-risk patients is attractive and non-invasive tool that detect evidence of IA prior to the appearance of clinical manifestations. Objectives: The aim of this study was to compare the diagnostic value of the conventional blood culture technique to the serological detection of GM antigen using ELISA for screening of IA in neutropenic patients with hematological malignancies. Methods: Forty patients with haematological malignancies from those admitted to the Clinical Oncology Department of Menoufia University Hospitals (MUH) were enrolled and classified to have either proven (5/40; 12.5%), probable (10/40; 25%) or possible (25/40; 62.5%) invasive aspergillosis based on the clinical criteria provided by the European Organization for research and treatment of Cancer (EORTC) and Mycoses Study Group (MSG). Blood samples were collected from all participants and subjected to conventional blood culture for isolation and identification of Aspergillus spp. ELISA technique was applied for serological detection of GM antigen in the patients’ serum samples. Results: The sensitivity, specificity, PPV(positive predictive value)  and NPV (negative predictive value of GM antigen ELISA testing were 100%, 74%, 36% and 100% respectively for both proven and probable cases. On applying the principle of test in series (the patient is positive if positive in both culture and the GM test), the results were improved to 100% sensitivity, 100% specificity, 100% PPV, 100% NPV and 100% accuracy. Galactomannan antigen testing proved excellent sensitivity (80%) compared to other clinical features and radiological criteria for diagnosis of probable aspergillosis and proved to be a good negative test. Conclusion: With conjunction of clinical and radiological signs, Aspergillus GM test can assist physicians in the of diagnosis of IA in patients with hematological malignancies to allow initiation of effective antifungal therapy which is ultimately important in high-risk populations.

BACKGROUND: Tumor-specific methylation changes in DNA CpG sites commonly occur in cancer and are believed to drive oncogenesis through gene silencing. Detection of methylation changes in circulating cell-free DNA (cfDNA) can offer a novel approach for cancer diagnostics. METHODS: Plasma samples from healthy controls and from patients with advanced colorectal and non-colorectal cancers were included in the study. Bisulfite conversion of cfDNA extracted from plasma was performed using EZ DNA Methylation Lightning Kit (Zymo Research) and was followed by library preparation using Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) and target enrichment using xGen Hybridization Capture for NGS Kit (IDT). Targeted methylation sequencing was done using NextSeq500 mid-output flow cell (300 cycles) (Illumina). Detection rates of methylation ratios in colorectal cancer samples were compared to non-colorectal cancers and healthy controls. RESULTS: First, we reviewed methylation changes in nearly 9,000 CpG sites in colorectal cancer (through TCGA database) and healthy controls. Subsequently, 32 CpG sites with greater than 50% methylation ratio in colorectal cancer and less than 1% methylation ratio in healthy controls were selected to develop targeted methylation sequencing based cfDNA assay. The assay was performed in 32 plasma samples from 20 individuals with advanced colorectal cancer who had tumor KRAS mutation, 8 individuals with advanced non-colorectal cancer who had tumor KRAS mutation (ovarian, n=2; endometrial, n=2; pancreatic, n=2; and lung cancer, n=2), and 4 healthy controls. Colorectal cancer specific methylation changes in cfDNA were detected in 85% (17/20) of colorectal cancer patients with a specificity of 92%. In colorectal cancer patients with confirmed KRAS mutation in cfDNA, methylation changes were detected in 92% (11/12) in comparison to 75% (6/8) in colorectal cancer patients without KRAS mutation in cfDNA. Median methylation ratio for target CpG sites was higher in colorectal cancer patients compared to patients with non-colorectal cancers and healthy controls (p<0.001). In 17 colorectal cancer patients with plasma samples collected before initiation of systemic cancer therapy, detection of methylation changes in cfDNA was associated with a shorter median progression-free survival compared to no detection (PFS; 8 weeks versus 54 weeks; p=0.027). CONCLUSIONS: Targeted methylation sequencing of cfDNA demonstrated high sensitivity and specificity for detection of colorectal cancer-specific cfDNA. Colorectal cancer patients with methylated cfDNA had shorter PFS while on cancer therapy. Citation Format: Mohamed A. Gouda, Dzifa Y. Duose, Morten Lapin, Stephanie Zalles, Helen J. Huang, Yuanxin Xi, Xiaofeng Zheng, Amira I. Aldesoky, Alshimaa M. Alhanafy, Mohamed A. Shehata, Jing Wang, Scott Kopetz, Funda Meric-Bernstam, Ignacio I. Wistuba, Rajyalakshmi Luthra, Filip Janku. Mutation-agnostic detection of colorectal cancer-specific cell-free DNA using targeted methylation sequencing [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 5152.

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