Fragmentomics of plasma mitochondrial and nuclear DNA inform prognosis in COVID-19 patients with critical symptoms.

BACKGROUND: The mortality rate of COVID-19 patients with critical symptoms is reported to be 40.5%. Early identification of patients with poor progression in the critical cohort is essential to timely clinical intervention and reduction of mortality. Although older age, chronic diseases, have been recognized as risk factors for COVID-19 mortality, we still lack an accurate prediction method for every patient. This study aimed to delve into the cell-free DNA fragmentomics of critically ill patients, and develop new promising biomarkers for identifying the patients with high mortality risk. METHODS: We utilized whole genome sequencing on the plasma cell-free DNA (cfDNA) from 33 COVID-19 patients with critical symptoms, whose outcomes were classified as survival (n = 16) and death (n = 17). Mitochondrial DNA (mtDNA) abundance and fragmentomic properties of cfDNA, including size profiles, ends motif and promoter coverages were interrogated and compared between survival and death groups. RESULTS: Significantly decreased abundance (~ 76% reduction) and dramatically shorter fragment size of cell-free mtDNA were observed in deceased patients. Likewise, the deceased patients exhibited distinct end-motif patterns of cfDNA with an enhanced preference for "CC" started motifs, which are related to the activity of nuclease DNASE1L3. Several dysregulated genes involved in the COVID-19 progression-related pathways were further inferred from promoter coverages. These informative cfDNA features enabled a high PPV of 83.3% in predicting deceased patients in the critical cohort. CONCLUSION: The dysregulated biological processes observed in COVID-19 patients with fatal outcomes may contribute to abnormal release and modifications of plasma cfDNA. Our findings provided the feasibility of plasma cfDNA as a promising biomarker in the prognosis prediction in critically ill COVID-19 patients in clinical practice.

The Next Frontier for Colorectal Cancer Screening: Blood-Based Tests.

Colorectal cancer is associated with substantial morbidity and mortality worldwide. Detection of early colorectal cancer is possible through approved screening tests but overall adherence is quite low. Implementation of effective noninvasive options could increase screening uptake and prevent a significant number of deaths. Noninvasive early cancer detection can potentially be achieved using a liquid biopsy. In this issue of Cancer Research, Cao and colleagues report on a novel multidimensional fragmentomics assay, named FRAGTECT, for colorectal cancer detection in circulating cell-free DNA with promising results. See related article by Cao et al., p. 3286.

Application of graft-derived cell-free DNA for solid organ transplantation.

Monitoring the status of grafts and the occurrence of postoperative complications, such as rejection, is crucial for ensuring the success and long-term survival of organ transplants. Traditional histopathological examination, though effective, is an invasive procedure and poses risks of complications, making frequent use impractical. In recent years, graft-derived cell-free DNA (gd-cfDNA) has emerged as a promising non-invasive biomarker. It not only provides early warnings of rejection and other types of graft injury but also offers important information about the effectiveness of immunosuppressive therapy and prognosis. gd-cfDNA shows potential in the monitoring of organ transplants. The early, real-time information on graft injury provided by gd-cfDNA facilitates timely individualized treatment and improves patient outcomes. However, the progress of research on gd-cfDNA varies across different organs. Therefore, this article will comprehensively review the application and findings of gd-cfDNA in monitoring various solid organs, discussing the advantages, limitations, and some future research directions to aid in its clinical application.

Evaluation of a plasma cell-free DNA methylation test for colorectal cancer diagnosis: a multicenter clinical study.

BACKGROUND: A blood-based diagnostic test is a promising strategy for colorectal cancer (CRC). The MethyDT test (IColohunter), which detects methylation levels of NTMT1 and MAP3K14-AS1, exhibited potential in discriminating CRC, but its clinical performance needs to be validated in large-scale populations. METHODS: A multicenter, double-blinded, cross-sectional study that enrolled 1194 participants was performed. Plasma samples were collected to detect methylation levels of NTMT1 and MAP3K14-AS1 using quantitative methylation-specific PCR with the MethyDT test, and the accuracy was further evaluated by Sanger sequencing. RESULTS: The sensitivities of the MethyDT test for detecting CRC, early stages of CRC (I and II), advanced adenoma (AA), and high-grade intraepithelial neoplasia (HGIN) were 91.2% (95% confidence interval [CI], 88.4-94.0), 87.4% (95% CI, 82.5-92.2), 43.5% (95% CI, 35.7-51.4), and 72.7% (95% CI, 57.5-87.9), respectively. The specificities for participants with non-AA, interfering diseases (ID), and no evidence of disease (NED) were 85.0% (95% CI, 78.8-91.3), 93.7% (95% CI, 91.4-95.9) and 97.3% (95% CI, 90.5-99.7), respectively, and its overall specificity for all-controls was 92.4% (95% CI, 90.3-94.4). The consistency of the MethyDT test with pathology for CRC was high with a kappa value of 0.830 (95% CI, 0.795-0.865). Additionally, the MethyDT test was comparable to Sanger sequencing for detecting methylation with kappa values > 0.97. CONCLUSIONS: The MethyDT test demonstrates excellent sensitivity and specificity for CRC and high consistency with Sanger sequencing for methylation, suggesting it may serve as a potential noninvasive diagnostic tool for the detection of CRC. TRIAL REGISTRATION: This clinical trial has been registered in ClinicalTrials.gov (NCT05508503).

Histone modifications of circulating nucleosomes are associated with changes in cell-free DNA fragmentation patterns.

The analysis of tissues of origin of cell-free DNA (cfDNA) is of research and diagnostic interest. Many studies focused on bisulfite treatment or immunoprecipitation protocols to assess the tissues of origin of cfDNA. DNA loss often occurs during such processes. Fragmentomics of cfDNA molecules has uncovered a wealth of information related to tissues of origin of cfDNA. There is still much room for the development of tools for assessing contributions from various tissues into plasma using fragmentomic features. Hence, we developed an approach to analyze the relative contributions of DNA from different tissues into plasma, by identifying characteristic fragmentation patterns associated with selected histone modifications. We named this technique as FRAGmentomics-based Histone modification Analysis (FRAGHA). Deduced placenta-specific histone H3 lysine 27 acetylation (H3K27ac)-associated signal correlated well with the fetal DNA fraction in maternal plasma (Pearson's r = 0.96). The deduced liver-specific H3K27ac-associated signal correlated with the donor-derived DNA fraction in liver transplantation recipients (Pearson's r = 0.92) and was significantly increased in patients with hepatocellular carcinoma (HCC) (P < 0.01, Wilcoxon rank-sum test). Significant elevations of erythroblasts-specific and colon-specific H3K27ac-associated signals were observed in patients with ß-thalassemia major and colorectal cancer, respectively. Furthermore, using the fragmentation patterns from tissue-specific H3K27ac regions, a machine learning algorithm was developed to enhance HCC detection, with an area under the curve (AUC) of up to 0.97. Finally, genomic regions with H3K27ac or histone H3 lysine 4 trimethylation (H3K4me3) were found to exhibit different fragmentomic patterns of cfDNA. This study has shed light on the relationship between cfDNA fragmentomics and histone modifications, thus expanding the armamentarium of liquid biopsy.

A promising application of kidney-specific cell-free DNA methylation markers in real-time monitoring sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (SI-AKI) is a common clinical syndrome that is associated with high mortality and morbidity. Effective timely detection may improve the outcome of SI-AKI. Kidney-derived cell-free DNA (cfDNA) may provide new insight into understanding and identifying SI-AKI. Plasma cfDNA from 82 healthy individuals, 7 patients with sepsis non-acute kidney injury (SN-AKI), and 9 patients with SI-AKI was subjected to genomic methylation sequencing. We deconstructed the relative contribution of cfDNA from different cell types based on cell-specific methylation markers and focused on exploring the association between kidney-derived cfDNA and SI-AKI.Based on the deconvolution of the cfDNA methylome: SI-AKI patients displayed the elevated cfDNA concentrations with an increased contribution of kidney epithelial cells (kidney-Ep) DNA; kidney-Ep derived cfDNA achieved high accuracy in distinguishing SI-AKI from SN-AKI (AUC = 0.92, 95% CI 0.7801-1); the higher kidney-ep cfDNA concentrations tended to correlate with more advanced stages of SI-AKI; strikingly, SN-AKI patients with potential kidney damage unmet by SI-AKI criteria showed higher levels of kidney-Ep derived cfDNA than healthy individuals. The autonomous screening of kidney-Ep (n = 24) and kidney endothelial (kidney-Endo, n = 12) specific methylation markers indicated the unique identity of kidney-Ep/kidney-Endo compared with other cell types, and its targeted assessment reproduced the main findings of the deconvolution of the cfDNA methylome. Our study first demonstrates that kidney-Ep- and kidney-Endo-specific methylation markers can serve as a novel marker for SI-AKI emergence, supporting further exploration of the utility of kidney-specific cfDNA methylation markers in the study of SI-AKI.

Liquid biopsy for CNS lymphoma: CSF exosomes and CSF exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b are promising biomarkers for diagnosis.

BACKGROUND: Central nervous system lymphoma (CNSL) is a devastating disease with a poor prognosis. Early diagnosis, monitoring of the treatment response, and outcome prediction carry the utmost importance in the management of patients with CNSL. Surgical biopsy is the gold standard for tissue diagnosis, however, this procedure has potential complications. Therefore, there is a need for a method that provides information about diagnosis and patient monitoring to avoid surgical risks. The study aimed to investigate potential diagnostic biomarkers for patients with CNSL. METHODS AND RESULTS: Patients with secondary CNSL were included in this study. Serum and cerebrospinal fluid (CSF) samples were collected before treatment and after completion of the treatment. Cell-free DNA (cfDNA), exosomes, free and exosomal microRNA (miR)-15a, miR-21, miR-155, miR-210, and miR-19b in both serum and CSF were examined, and they were compared with the controls. Also, their levels before and after treatment were compared. Nine patients with the diagnosis of secondary CNSL were reviewed. cfDNA, miR-15a, and miR-155 in serum, and exosome in CSF were found to be significantly higher in CNSL patients compared to the controls. Exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b in CSF were found to be significantly higher in CNSL patients compared to controls, whereas their levels in serum were not significantly high. CONCLUSIONS: Our findings suggested that exosomes and exosomal miR-15a, miR-21, miR-155, miR-210 and miR-19b in CSF would be promising biomarkers for the diagnosis of patients with CNSL. Further studies are needed to confirm our findings.

5-Hydroxymethylcytosine in circulating cell-free DNA as a potential diagnostic biomarker for SLE.

BACKGROUND: SLE is a complex autoimmune disease with heterogeneous manifestations and unpredictable outcomes. Early diagnosis is challenging due to non-specific symptoms, and current treatments only manage symptoms. Epigenetic alternations, including 5-Hydroxymethylome (5hmC) modifications, are important contributors to SLE pathogenesis. However, the 5hmC modification status in circulating cell-free DNA (cfDNA) of patients with SLE remains largely unexplored. We investigated the distribution of 5hmC in cfDNA of patients with SLE and healthy controls (HCs), and explored its potential as an SLE diagnosis marker. METHODS: We used 5hmC-Seal to generate genome-wide 5hmC profiles of plasma cfDNA and bioinformatics analysis to screen differentially hydroxymethylated regions (DhMRs). In vitro mechanistic exploration was conducted to investigate the regulatory effect of CCCTC-binding factor (CTCF) in 5hmC candidate biomarkers. RESULTS: We found distinct differences in genomic regions and 5hmC modification motif patterns between patients with SLE and HCs, varying with disease progression. Increased 5hmC modification enrichment was detected in SLE. Additionally, we screened 151 genes with hyper-5hmC, which are significantly involved in SLE-related processes, and 5hmC-modified BCL2, CD83, ETS1 and GZMB as SLE biomarkers. Our findings suggest that CTCF regulates 5hmC modification of these genes by recruiting TET (ten-eleven translocation) protein, and CTCF knockdown affected the protein expression of these genes in vitro. CONCLUSIONS: Our findings demonstrate the increased 5hmC distribution in plasma cfDNA in different disease activity in patients with SLE compared with HCs and relating DhMRs involved in SLE-associated pathways. Furthermore, we identified a panel of SLE relevant biomarkers, and these viewpoints could provide insight into the pathogenesis of SLE.

DNA demethylation triggers cell free DNA release in colorectal cancer cells.

BACKGROUND: Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. METHODS: We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. RESULTS: Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. CONCLUSIONS: Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

5-Hydroxymethylated Biomarkers in Cell-Free DNA Predict Occult Colorectal Cancer up to 36 Months Before Diagnosis in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.

PURPOSE: Using the prostate, lung, colorectal, and ovarian (PLCO) Cancer Screening Trial samples, we identified cell-free DNA (cfDNA) candidate biomarkers bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that detected occult colorectal cancer (CRC) up to 36 months before clinical diagnosis. MATERIALS AND METHODS: We performed the 5hmC-seal assay and sequencing on ≤8 ng cfDNA extracted from PLCO study participant plasma samples, including n = 201 cases (diagnosed with CRC within 36 months of blood collection) and n = 401 controls (no cancer diagnosis on follow-up). We conducted association studies and machine learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. RESULTS: We successfully obtained 5hmC profiles from these decades-old samples. A weighted Cox model of 32 5hmC-modified gene bodies showed a predictive detection value for CRC as early as 36 months before overt tumor diagnosis (training set AUC, 77.1% [95% CI, 72.2 to 81.9] and validation set AUC, 72.8% [95% CI, 65.8 to 79.7]). Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and race/ethnicity, and significantly outperformed risk factors such as age and obesity (assessed as BMI). Finally, when splitting cases at median weighted prediction scores, Kaplan-Meier analyses showed significant risk stratification for CRC occurrence in both the training set (hazard ratio, [HR], 3.3 [95% CI, 2.6 to 5.8]) and validation set (HR, 3.1 [95% CI, 1.8 to 5.8]). CONCLUSION: Candidate 5hmC biomarkers and a scoring algorithm have the potential to predict CRC occurrence despite the absence of clinical symptoms and effective predictors. Developing a minimally invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient outcomes.

Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

Usefulness of newer testing modalities for the accurate diagnosis of culture-negative endocarditis.

A woman in her 80s with a history of congestive heart failure, atrial arrhythmia treated with atrioventricular nodal ablation and permanent pacemaker (PPM) placement, mitral valve disease status post-repair and colon cancer status post-treatment was admitted for further evaluation of severe dyspnea on exertion. Imaging revealed vegetation on both the prosthetic mitral valve and the PPM lead. Blood cultures were collected without growth, so a cell-free DNA Karius test was performed, which can detect over 1000 pathogens and has a sensitivity between 87% and 93%. Testing returned positive results for Streptococcus bovis subspecies pasteurianus Given its association with colorectal cancer, abdominal imaging and an endoscopic biopsy were performed, showing recurrent colonic malignancy. The patient underwent a right colon resection prior to cardiac intervention. This report describes the clinical application of the novel cell-free DNA Karius test, which led to the diagnosis of recurrent colon cancer associated with S. pasteurianus endocarditis.

Extracting regulatory active chromatin footprint from cell-free DNA.

Cell-free DNA (cfDNA) has emerged as a pivotal player in precision medicine, revolutionizing the diagnostic and therapeutic landscape. While its clinical applications have significantly increased in recent years, current cfDNA assays have limited ability to identify the active transcriptional programs that govern complex disease phenotypes and capture the heterogeneity of the disease. To address these limitations, we have developed a non-invasive platform to enrich and examine the active chromatin fragments (cfDNAac) in peripheral blood. The deconvolution of the cfDNAac signal from traditional nucleosomal chromatin fragments (cfDNAnuc) yields a catalog of features linking these circulating chromatin signals in blood to specific regulatory elements across the genome, including enhancers, promoters, and highly transcribed genes, mirroring the epigenetic data from the ENCODE project. Notably, these cfDNAac counts correlate strongly with RNA polymerase II activity and exhibit distinct expression patterns for known circadian genes. Additionally, cfDNAac signals across gene bodies and promoters show strong correlations with whole blood gene expression levels defined by GTEx. This study illustrates the utility of cfDNAac analysis for investigating epigenomics and gene expression, underscoring its potential for a wide range of clinical applications in precision medicine.

Mutational spectrum of breast cancer by shallow whole-genome sequencing of cfDNA and tumor gene panel analysis.

Breast cancer (BC) has different molecular subgroups related to different risks and treatments. Tumor biopsies for BC detection are invasive and may not reflect tumor heterogeneity. Liquid biopsies have become relevant because they might overcome these limitations. We rationalize that liquid cfDNA biopsies through shallow whole genome sequencing (sWGS) could improve the detection of tumor alterations, complementing the genomic profiling. We evaluated the feasibility to detect somatic copy number alterations (SCNAs) in BC using shallow whole genome sequencing (sWGS) in cfDNA from archived samples from National Cancer Institute of Colombia patients. We sequenced tumor tissues from 38 BC patients with different molecular subtypes using a gene panel of 176 genes significantly mutated in cancer, and by liquid biopsies using sWGS on 20 paired samples to detect SCNAs and compare with the tumor samples. We identified an extensive intertumoral heterogeneity between the molecular subtypes of BC, with a mean tumor load of 602 mutations in the gene panel of tumor tissues. There was a 12.3% of concordance in deletions in the cfDNA-tumor pairs considering only the genes covered by the panel encompassing seven genes: BRCA1, CDK12, NF1, MAP2K4, NCOR1, TP53, and KEAP1 in three patients. This study shows the feasibility to complement the genomic analysis of tumor tissue biopsies to detect SCNA in BC using sWGS in cfDNA, providing a wider identification of potential therapeutic targets.

Genomic profiling of cell-free DNA from dogs with benign and malignant tumors.

OBJECTIVE: Cancer is currently the most common cause of death in adult dogs. Like humans, dogs have a one-third chance of developing cancer in their lifetime. We used shallow whole-genome sequencing (sWGS) to analyze blood cell-free DNA (cfDNA) from four tumor-bearing dogs (one with benign and three with malignant tumors) and 38 healthy dogs. RESULTS: Similar to the results observed in the healthy dogs, no copy number aberration (CNA) was detected in the dog with benign lipomas, and the distribution of cfDNA fragment size (FS) closely resembled that of the healthy dogs. However, among the three dogs diagnosed with malignant tumors, two dogs exhibited varying degrees and quantities of CNAs. Compared to the distribution of FS in the healthy dogs, the cancer dogs exhibited a noticeable shift towards shorter lengths. These findings indicated that CNA and FS profiles derived from sWGS data can be used for non-invasive cancer detection in dogs.

Noninvasive prenatal screening and diagnosis of two fetuses with Williams syndrome in a cohort of 19,607 pregnancies.

BACKGROUND: This study aimed to evaluate the efficiency of noninvasive prenatal screening (NIPS) technology in screening for microdeletions in the 7q11.23 region. METHODS: 19,607 pregnant women underwent NIPS in our hospital. Maternal peripheral cell-free foetal DNA (cffDNA) was routinely screened for aneuploidy by cffDNA enrichment and simultaneously analyzed for pathogenic copy number variants (CNVs). The Williams syndrome (WS) 7q11.23 region was targeted in this study. Chromosomal microarray analysis (CMA) was used to verify the screen-positive samples. RESULTS: The mean concentration of cffDNA before and after enrichment increased from 9.44% to 19.32%, with a statistically significant difference. Two out of 19,607 samples tested for CNVs were found to have a heterozygous deletion at the 7q11.23 region, indicating a high risk for WS. CMA results confirmed the 1.5 megabase (Mb) deletions at the 7q11.23 region in amniotic fluid samples. One of the two WS foetuses had a small left ventricle by ultrasound screening, and the other did not have a significant cardiovascular abnormality phenotype. CONCLUSIONS: NIPS screening for Williams syndrome can be achieved by enriching cell-free foetal DNA and improving bioinformatic analysis algorithms.

Liquid biopsy: paving a new avenue for cancer research.

The current constraints associated with cancer diagnosis and molecular profiling, which rely on invasive tissue biopsies or clinical imaging, have spurred the emergence of the liquid biopsy field. Liquid biopsy involves the extraction of circulating tumor cells (CTCs), circulating free or circulating tumor DNA (cfDNA or ctDNA), circulating cell-free RNA (cfRNA), extracellular vesicles (EVs), and tumor-educated platelets (TEPs) from bodily fluid samples. Subsequently, these components undergo molecular characterization to identify biomarkers that are critical for early cancer detection, prognosis, therapeutic assessment, and post-treatment monitoring. These innovative biosources exhibit characteristics analogous to those of the primary tumor from which they originate or interact. This review comprehensively explores the diverse technologies and methodologies employed for processing these biosources, along with their principal clinical applications.

Large language model produces high accurate diagnosis of cancer from end-motif profiles of cell-free DNA.

Instruction-tuned large language models (LLMs) demonstrate exceptional ability to align with human intentions. We present an LLM-based model-instruction-tuned LLM for assessment of cancer (iLLMAC)-that can detect cancer using cell-free deoxyribonucleic acid (cfDNA) end-motif profiles. Developed on plasma cfDNA sequencing data from 1135 cancer patients and 1106 controls across three datasets, iLLMAC achieved area under the receiver operating curve (AUROC) of 0.866 [95% confidence interval (CI), 0.773-0.959] for cancer diagnosis and 0.924 (95% CI, 0.841-1.0) for hepatocellular carcinoma (HCC) detection using 16 end-motifs. Performance increased with more motifs, reaching 0.886 (95% CI, 0.794-0.977) and 0.956 (95% CI, 0.89-1.0) for cancer diagnosis and HCC detection, respectively, with 64 end-motifs. On an external-testing set, iLLMAC achieved AUROC of 0.912 (95% CI, 0.849-0.976) for cancer diagnosis and 0.938 (95% CI, 0.885-0.992) for HCC detection with 64 end-motifs, significantly outperforming benchmarked methods. Furthermore, iLLMAC achieved high classification performance on datasets with bisulfite and 5-hydroxymethylcytosine sequencing. Our study highlights the effectiveness of LLM-based instruction-tuning for cfDNA-based cancer detection.