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Applications in Oncology

Apostle technologies have been applied in many world-class R&D studies, clinical laboratory settings, and public health response and surveillance.

This page lists some of the examples in Oncology.  (Note: The number in front of each publication may not be sequential as it indicates the serial number in the full publication list.)

For a complete list of applications citing Apostle technologies, including publications and customer testimonials, see References

Oncology-related Publications (2025)

96.  Cell Free DNA, Tumor Molecular Concordance and Clinical Correlates of Cancer Patients Treated in a Large Community Health Care Network. William A. LaFramboise, Patti Petrosko,  Phillip H. Gallo, et al. The Journal of Molecular Diagnostics (2025) Articles in Press. June 25, 2025. DOI: 10.1016/j.jmoldx.2025.05.007

(Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Blood collection, plasma processing and cell free DNA (cfDNA) purification was optimized to capture circulating tumor DNA (ctDNA) without blood cell background DNA among 874 cancer patients. cfDNA comprised predominantly mononucleosomal fragments (n=874; x¯ ± S.D. = 166 ± 5 bp) that generated comparably sized sequencing reads (x¯ ± S.D. = 162 ± 25 bp). Despite a vast range of cfDNA concentrations (0.50 to 1132.9 ng/ml) across 21 tumor types, matched tumor and blood specimens (n = 430 patients) revealed high concordance for coding (Med = 97%) and clinical oncogenic mutations (Med = 88% concordance). Therapeutically actionable mutations were identified in 233 patients by both assays while 126 patients had oncogenic mutations without an established pharmacotherapeutic agent. An additional 48 patients (11%) had actionable mutations detected only in cfDNA assays while 23 patients (5%) had mutations in tumor only. Concordance was high in both prevalent (lung, breast, colon) and rare tumors (appendiceal, sarcoma). Cell free DNA levels from diagnostic blood specimens were a strong indicator of patient survival duration independent of age, sex, tumor type and stage, demonstrative of a potentially important role as a prognostic biomarker. Mutations in established oncogenes and tumor suppressors were readily detectable across all tumor types in ctDNA indicating a diagnostic role for cfDNA from blood extending beyond the identification of companion therapeutics to patient screening and monitoring.


Materials and Methods section

Purification of Cell Free DNA from Plasma. Frozen plasma was thawed at room temperature (60 min) and the volume adjusted as needed (1X PBS, pH 7.4; cat. #10010-023, ThermoFisher) for purification of cell-free DNA (cfDNA) using the Apostle MiniMax High Efficiency cfDNA Isolation Kit (cat. #A17622-250, Beckman, Indianapolis, IN). 

95.  Integrated analysis of oral rinse-derived and plasma circulating tumour DNA for mutation profiling and outcome prediction with oral squamous cell carcinoma. Zhuo Chen, Lihuang Chen, Shuaize Li, et al. npj Precision Oncology. volume 9, Article number: 183 (2025)  (Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

This study evaluates the potential of oral rinse-derived and plasma circulating tumour DNA (ctDNA) in HPV-negative oral squamous cell carcinoma (OSCC), where early recurrence occurs in a significant proportion of patients, contributing to poor prognosis. Analysis of paired tissue, oral rinse, and plasma samples from 123 patients revealed ctDNA detection rates of 94.3% in oral rinse and 80.5% in plasma samples. Combined testing improved mutation detection sensitivity to 48.6%. A machine learning model integrating seven mutated genes (TP53, TERT, IKZF1, EP300, MYC, EGFR, PIK3CA) and clinical factors demonstrated robust prediction of recurrence (validation AUC: 0.854) and survival outcomes. Integration of pretreatment plasma ctDNA status further enhanced predictive performance. In longitudinal analysis, ctDNA detected recurrence approximately four months before clinical manifestation. These findings suggest that integrated ctDNA analysis offers improved mutation profiling and outcome prediction, potentially enabling earlier interventions in OSCC.


Methods section

Plasma samples were collected using Apostle tubes. All samples were transported at 2–8 °C and subsequently stored at -80 °C until analysis.

92.  Combined genomic profiling of cell-free DNA (cfDNA) and circulating tumor cell DNA (ctcDNA) in S1802, a prospective phase 3 trial for metastatic prostate cancer (mHSPC). Daniel Bsteh, Jonathan Buckley, Scott Mitchell, et al. Journal of Clinical Oncology. Volume 43, Number 16_suppl. Meeting Abstract: 2025 ASCO Annual Meeting. May 28, 2025 (Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 


Background: Parallel analysis of cfDNA and ctcDNA can yield expanded and complementary molecular insights. We developed HERCULES, a prostate cancer specific targeted amplicon sequencing panel capable of assessing single nucleotide variants (SNVs) and copy number variants (CNVs) in cfDNA and single CTCs obtained concurrently from a single tube of blood. Here we present results from the first 35 patients analyzed at multiple time points in S1802, an NCTN/SWOG randomized prospective phase 3 trial of standard systemic therapy +/- definitive treatment of the primary tumor in newly diagnosed mHSPC.

Methods: Blood was collected in Streck DNA preservative tubes at 4 prespecified time points: registration, randomization, post-definitive therapy, and progression. Plasma and single CTCs were isolated using the RareCyte platform, and DNA was extracted using the Apostle MiniMax cfDNA extraction kit (Beckman) or Single Cell Lysis Kit (Thermo Fisher). The HERCULES AmpliSeq-HD panel includes 35 prostate cancer -relevant genes. SNVs and CNVs were called using Ion Reporter Software (Thermo Fisher) and filtered for variants associated with clonal hematopoiesis of indeterminate significance (CHIP).

Results: A total of 85 samples were obtained from 35 patients at up to 4 different time points. CTCs were detected in 22(61%) patients, with median 3 CTCs/7.5ml (range 1-986). SNV limit of detection varied with input DNA, from 0.16% at >20 ng input to 0.91% with 1-5 ng input. Fewer than 5% of all SNVs were censored due to potential CHIP. Across all times points, SNVs and CNVs were detected in 32 and 20 patients, respectively. SNVs were detected both in cfDNA and in ctcDNA. Known prostate cancer driver variants were observed in AR, CTNNB1, FOXA1, SF3B1, TP53 and others. Recurrent CNVs were observed in AR, AR enhancer, FOXA1, CDK4, MYC and CHD1. In general, the 4th time point, representing transition to mCRPC, had twice the number of alterations as earlier mHSPC time points.

Conclusions: Analysis of the first 35 patients sequenced at multiple time points in S1802 demonstrates the feasibility of concurrent, same-sample genomic profiling of cfDNA and ctcDNA in a multi-center prospective phase 3 NCTN setting. The HERCULES sequencing workflow enables parallel processing of cfDNA and single-cell ctcDNA, with LOD that exceeds that of hybrid capture panels at these input levels. Presence of concordant and distinct alterations in cfDNA and CTCs is consistent with findings from prior small studies and underscores the potential value of analyzing both of these liquid biopsy components.

90. Integrating Plasma Cell-Free DNA Fragment End Motif and Size with Genomic Features Enables Lung Cancer Detection. Tae-Rim Lee,   Jin Mo Ahn, Junnam Lee , et al. Cancer Research (2025) 85 (9): 1696–1707. MAY 02 2025. (Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Early detection of lung cancer is important for improving patient survival rates. Liquid biopsy using whole-genome sequencing of cell-free DNA (cfDNA) offers a promising avenue for lung cancer screening, providing a potential alternative or complementary approach to current screening modalities. Here, we aimed to develop and validate an approach by integrating fragment and genomic features of cfDNA to enhance lung cancer detection accuracy across diverse populations. Deep learning–based classifiers were trained using comprehensive cfDNA fragmentomic features from participants in multi-institutional studies, including a Korean discovery dataset (218 patients with lung cancer and 2,559 controls), a Korean validation dataset (111 patients with lung cancer and 1,136 controls), and an independent Caucasian validation cohort (50 patients with lung cancer and 50 controls). In the discovery dataset, classifiers using fragment end motif by size, a feature that captures both fragment end motif and size profiles, outperformed standalone fragment end motif and fragment size classifiers, achieving an area under the curve (AUC) of 0.917. The ensemble classifier integrating fragment end motif by size and genomic coverage achieved an improved performance, with an AUC of 0.937. This performance extended to the Korean validation dataset and demonstrated ethnic generalizability in the Caucasian validation cohort. Overall, the development of a deep learning–based classifier integrating cfDNA fragmentomic and genomic features in this study highlights the potential for accurate lung cancer detection across diverse populations.


Significance: Evaluating fragment-based features and genomic coverage in cell-free DNA offers an accurate lung cancer screening method, promising improvements in early cancer detection and addressing challenges associated with current screening methods.


(Materials and Methods section)

cfDNA extraction, library preparation, and sequencing

cfDNA was isolated from 0.4 mL of plasma using the Apostle MiniMax Kit (Beckman Coulter) following the manufacturer’s guidelines.

88. Enzymatic profiling of cfDNA methylation for detection and monitoring of lung cancer. Abed Agbarya, Noa Gilat, Yael Michaeli, Jasline Deek, Assaf Grunwald, Suheil Artul, Rasha Khoury, Michael Peled,  Yuval Ebenstein. medRxiv, May 08, 2025. https://doi.org/10.1101/2025.05.08.25327181

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

We present a highly sensitive, low-cost approach for detecting lung cancer and monitoring response to therapy, based on sequencing-free detection of methylation biomarkers in cell-free DNA. An engineered methyltransferase is used to fluorescently label CpG sites. When applied to bisulfite-treated, PCR-amplified cell-free DNA, fluorescent reporters attach to all originally methylated sites, which can then be read on a standard hybridization microarray. In a proof-of-concept study involving 60 blinded participants, we distinguished cancer patients from healthy individuals with both sensitivity and specificity exceeding 90 %.


(Materials and Methods section)

Sample preparation, labeling and hybridization

Whole blood samples were collected in Streck tubes (STRECK). Plasma was separated and cfDNA was extracted with Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit according to manufacturer’s instructions. Following DNA extraction.

87. Analysis of the clinical application value of cfDNA methylation and fragmentation in early diagnosis of esophageal cancer. Xin Liu, Chen Liang, Lingwen Ding, et al. Genomics. Volume 117, Issue 3, May 2025, 111034 (Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background

This study explores the clinical value of cfDNA methylation and fragmentation for the early diagnosis of esophageal cancer using liquid biopsy.

Methods

Whole genome bisulfite sequencing and low-pass whole genome sequencing were utilized to detect cfDNA biomarkers, comparing 30 esophageal cancer patients with 10 healthy controls.

Results

Significant differences in cfDNA methylation and fragmentation were observed between cancerous and non-cancerous samples (p < 0.05). A volcano plot identified 822 differentially methylated markers (817 upregulated, 5 downregulated), with SOX17, SOX1, ZNF382, ZNF667-AS1, and TFPI2 highly associated with esophageal cancer. Fragmentation markers (EDM, FSD, FSR, TFBS, CNV) showed 95 % specificity and sensitivity, with EDM demonstrating the best performance.

Conclusion

Our study highlights the clinical potential of cfDNA methylation and fragmentation biomarkers for the early diagnosis of esophageal cancer.


(Materials and Methods section)

Sample collection, storage, and extraction

Peripheral blood samples (2 tubes, totaling 20 mL) were collected from each participant in Apostle tubes for the detection of methylation and fragmentation markers.

86. Longitudinal genomic profiling of chemotherapy-related CHIP variants in patients with ovarian cancer. Sara Corvigno, Jun Yao, Amma Asare, et al. Front Oncol. 2025 Apr 29;15:1538446. doi: 10.3389/fonc.2025.1538446 (Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Introduction

Clonal hematopoiesis (CH) is characterized by the presence of hematopoietic stem cells (HSCs) with the potential of clonally expanding and giving rise to hematological malignancies. Clonal hematopoiesis of indeterminate potential (CHIP) is the outgrowth of a single HSC clone with an acquired somatic mutation in the absence of hematological abnormalities. CHIP variants occur with a variant allele frequency (VAF) of at least 2% in peripheral blood. This definition does not account for less frequent mutations that give rise to hematopoietic clones. Previous studies indicate an association between CH and secondary hematologic malignancies in cancer patients who receive chemotherapy.

Methods

To discover novel candidate CHIP mutations, including those with extremely low VAFs, we performed an in-depth characterization of low-frequency CHIP variants in a highly selected group of patients with high-grade serous ovarian cancer (HGSC) before and after neoadjuvant chemotherapy (NACT). We performed comprehensive ultra-high-depth whole-exome sequencing of circulating free DNA (cfDNA) and matched white blood cell (WBC) DNA from pre- (n=9) and post-NACT (n=9) samples from HGSC patients who had excellent response (ER; n=4) or poor response (PR; n=5) to NACT.

Results

Variants present in both the WBC DNA and cfDNA from a patient were considered candidate CHIP variants. We identified 93,088 candidate CHIP variants in 13,780 genes. Compared with pre-NACT samples, post-NACT samples tended to have fewer CHIP mutations with VAFs of less than 5%, which may reflect the negative selective pressure of chemotherapy on rare hematopoietic clones. Finally, we identified CHIP variants in tumor samples matched to the liquid biopsies.

Discussion

Our innovative sequencing approach enabled the discovery of a large number of novel low-frequency candidate CHIP mutations, whose frequency and composition are affected by chemotherapy, in the cfDNA of patients with HGSC. The CHIP variants that were enriched after chemotherapy, if validated, might become essential predictive markers for therapy-related myeloid neoplasia.


(Materials and Methods section)

Isolation of cfDNA and WBC DNA and sequencing

cfDNA was extracted using the MiniMax High Efficiency Cell-Free DNA Isolation Kit (#A17622-50; Apostle) and then quantified using the Quant-iT PicoGreen dsDNA Assay Kit; ...

85. Diagnostic and prognostic potential of cell-free RNAs in cerebrospinal fluid and plasma for brain tumors. Jinyong Huang, Jinxia Zhou, Jun Wang, et al. npj Precision Oncology. 9, Article number: 123 (2025) (Download PDF)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Systematic assessment of the clinical applicability of cell-free RNAs (cfRNAs), which includes broader RNA categories beyond microRNAs, for patients with brain tumors remains largely unexplored due to the lack of sensitive profiling technologies. Our study endeavors to bridge this gap by utilizing an optimized cell-free transcriptome profiling technique that we have recently developed. We comprehensively profiled the cell-free transcriptome in plasma and cerebrospinal fluid (CSF) samples from a total of 85 patients with glioma, meningioma, or tumor-free central nervous system diseases. We identified 16 cfRNA signatures in CSF with robust performance in brain tumor detection (test set AUC = 0.94; validation set AUC = 1). The integration of CSF and plasma-derived cfRNAs outperformed individual analyses using either CSF or plasma candidates for the classification of glioma (test set AUC = 0.94; validation set AUC = 0.85) and meningioma (test set AUC = 0.92; validation set AUC = 0.83). Additionally, we identified 33 CSF and 3 plasma cfRNAs with prognostic significance for postoperative patient outcomes. Multivariate analysis showed that cfRNA-based risk scores (Hazard ratio=9.9) outperformed traditional risk factors in predicting recurrence-free survival. Importantly, our findings in liquid biopsies are consistent with results from primary tumor tissues. By delving into the diagnostic and prognostic implications of cfRNA signatures in CSF and plasma, our study paves the way for improved diagnostic precision and personalized therapeutic interventions for brain tumor patients.


(Methods section)

cfRNA extraction, library preparation, and sequencing

The extraction of cfRNA from 400 μl CSF samples was conducted using the RNAiso Plus total RNA extraction reagent (TaKaRa), following the manufacturer’s protocol. For the plasma samples, cfRNA extraction was carried out on 200 μl aliquoted plasma utilizing the Apostle MiniMax™ High Efficiency cfRNA Isolation Kit (Apostle) in accordance with the manufacturer’s protocol. 

93.  Multi-Omics Integration of Cfdna for Non-Invasive Early Detection of Esophageal Cancer in High-Risk Populations. Wenya Li, Youbin Cui, Jingcai Wu, et al. Available at SSRN: https://ssrn.com/abstract=5290998

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 


Esophageal cancer (ESCA) is a leading cause of cancer-related mortality in China, with over 95% of patients diagnosed at advanced stages, resulting in low survival rates. Early detection is critical for improving outcomes, yet current screening methods are invasive or lack sensitivity. We developed a non-invasive early-detection model by integrating circulating cell-free DNA (cfDNA) methylation and fragmentomics features. Using a three-layer stacked ensemble machine learning framework, we analyzed multicenter blood samples from 309 participants, including ESCA patients and high-risk individuals. The integrated model achieved an AUC of 0.994 (95% CI: 0.979–1.000) in validation, outperforming single-modality methods (methylation AUC: 0.969; fragmentomics AUC: 0.991). At 99% specificity, sensitivity reached 87.3%, with simulated screening showing a 24.8% increase in stage I detection. Stratified analyses confirmed robustness across demographics, risk factors, and tumor stages. Clinical benefit modeling projected a 56.3% stage I detection rate under ideal conditions, significantly improving survival outcomes compared to standard care. This study demonstrates the superiority of multi-omics integration for non-invasive ESCA screening in early diagnosis. The CoBFP-based methylation analysis and fragmentomics-enhanced model offer high accuracy (AUC 0.994) and clinical applicability for high-risk populations.

Methods section

2.2 Sample collection and cfDNA extraction

Two 10-mL tubes of peripheral blood from patients were collected using Apostle MiniMax cfDNA Blood Collection Tubes (Apostle) and were gently inverted 10 times to mix immediately.

82. Intestinal microbiota as biomarkers for different colorectal lesions based on colorectal cancer screening participants in community. Gairui Li, Dan Zhao, Binfa Ouyang, et al. Front. Microbiol. , 06 February 2025. Volume 16 - 2025 | https://doi.org/10.3389/fmicb.2025.1529858.               (Download PDF)

(Note: Apostle MinoGenomics technology is used in this study.) 

Abstract 

Introduction: The dysregulation of intestinal microbiota has been implicated in the pathogenesis of colorectal cancer (CRC). However, the utilization of intestinal microbiota for identify the lesions in different procedures in CRC screening populations remains limited.

Methods: A total of 529 high-risk individuals who underwent CRC screening were included, comprising 13 advanced adenomas (Aade), 5 CRC, 59 non-advanced adenomas (Nade), 129 colon polyps (Pol), 99 cases of colorectal inflammatory disease (Inf), and 224 normal controls (Nor). 16S rRNA gene sequencing was used to profile the intestinal microbiota communities. The Gut Microbiota Health Index (GMHI) and average variation degree (AVD) were employed to assess the health status of the different groups.

Results: Our findings revealed that the Nor group exhibited significantly higher GMHIs and the lowest AVD compared to the four Lesion groups. The model incorporating 13 bacterial genera demonstrated optimal efficacy in distinguishing CRC and Aade from Nor, with an area under the curve (AUC) of 0.81 and a 95% confidence interval (CI) of 0.72 to 0.89. Specifically, the 55 bacterial genera combination model exhibited superior performance in differentiating CRC from Nor (AUC 0.98; 95% CI, 0.96-1), the 25 bacterial genera combination showed superior performance in distinguishing Aade from Nor (AUC 0.95). Additionally, the 27 bacterial genera combination demonstrated superior efficacy in differentiating Nade from Nor (AUC 0.82). The 13 bacterial genera combination exhibited optimal performance in distinguishing Inf from Nor (AUC 0.71).

Discussion: Our study has identified specific microbial biomarkers that can differentiate between colorectal lesions and healthy individuals. The intestinal microbiota markers identified may serve as valuable tools in community-based CRC screening programs.


(Materials and Methods section)

DNA extraction, library construction and sequencing

Genomic DNA was extracted from stool samples utilizing the Apostle MiniGenomics Genomic DNA Extraction according to the manufacturer’s instructions. 


81. Highly specific multiplex DNA methylation detection for liquid biopsy of colorectal cancer. Dewen Zhu, Jinlei Li, Wenwen Zhang , Clinica Chimica Acta (2025) 565: 120026, doi: https://doi.org/10.1016/j.cca.2024.120026

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background

Circulating tumor DNA (ctDNA) has emerged as a useful biomarker for cancer detection and prognosis. In this study, we developed a strategy for developing a highly specific multiplex qPCR assay to detect methylated ctDNA in the blood of colorectal cancer (CRC) patients and investigated the potential use for the detection and prognosis of CRC.

Methods

Bisulfite conversion and amplicon sequencing were used to confirm potential CRC-specific DNA methylation markers. The selected DNA methylation candidates were validated by qMSP. The six best-performing markers were used to develop a new single-tube multiplex quantitative methylation-specific PCR assay (mqMSP). The mqMSP assay was applied to analyze plasma samples from 114 CRC patients, 47 patients with advanced adenoma, 45 patients with benign polyps, and 57 healthy controls. The clinical performance of the assay and associations with clinical outcomes were assessed.

Results

Six DNA methylation biomarkers were confirmed to be specifically hypermethylated in CRC tumor tissues. The newly developed mqMSP assay detected CRC with extremely high specificity (specificity of 98.2 %, with sensitivity of 67.5 %). The detection rate of ctDNA was significantly correlated with tumor size and clinical stage, with ctDNA methylation levels in the blood markedly increased with larger tumor size, poor differentiation, and advanced stage. Moreover, high preoperative methylated ctDNA level was associated with worse recurrence-free survival and overall survival.

Conclusion

We provided a strategy for identification of multiple highly-specific DNA methylation markers for designing multiplex DNA methylation assays for liquid biopsies of CRC. The newly developed assay has potential for CRC early detection, and prognosis.

(Materials and Methods section)

For plasma samples, 2-5 mL plasma was extracted using the Apostle MiniMax High-Efficiency cfDNA Isolation Kit (Apostle). 


Oncology-related Publications (2024)

80. A shared neoantigen vaccine combined with immune checkpoint blockade for advanced metastatic solid tumors: phase 1 trial interim results. Amy R. Rappaport, Chrisann Kyi, Monica Lane, et al. Nature Medicine   30, pages 1013–1022 (2024)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Therapeutic vaccines that elicit cytotoxic T cell responses targeting tumor-specific neoantigens hold promise for providing long-term clinical benefit to patients with cancer. Here we evaluated safety and tolerability of a therapeutic vaccine encoding 20 shared neoantigens derived from selected common oncogenic driver mutations as primary endpoints in an ongoing phase 1/2 study in patients with advanced/metastatic solid tumors. Secondary endpoints included immunogenicity, overall response rate, progression-free survival and overall survival. Eligible patients were selected if their tumors expressed one of the human leukocyte antigen-matched tumor mutations included in the vaccine, with the majority of patients (18/19) harboring a mutation in KRAS. The vaccine regimen, consisting of a chimp adenovirus (ChAd68) and self-amplifying mRNA (samRNA) in combination with the immune checkpoint inhibitors ipilimumab and nivolumab, was shown to be well tolerated, with observed treatment-related adverse events consistent with acute inflammation expected with viral vector-based vaccines and immune checkpoint blockade, the majority grade 1/2. Two patients experienced grade 3/4 serious treatment-related adverse events that were also dose-limiting toxicities. The overall response rate was 0%, and median progression-free survival and overall survival were 1.9 months and 7.9 months, respectively. T cell responses were biased toward human leukocyte antigen-matched TP53 neoantigens encoded in the vaccine relative to KRAS neoantigens expressed by the patients’ tumors, indicating a previously unknown hierarchy of neoantigen immunodominance that may impact the therapeutic efficacy of multiepitope shared neoantigen vaccines. These data led to the development of an optimized vaccine exclusively targeting KRAS-derived neoantigens that is being evaluated in a subset of patients in phase 2 of the clinical study. ClinicalTrials.gov registration: NCT03953235.

(Methods section)  

 cfDNA was extracted from the entire plasma volume of a single draw using the Apostle MiniMax cfDNA Isolation kit (ApostleBio) and quantified using the Qubit 1× dsDNA High Sensitivity Assay (Thermo Fisher Scientific).

79. Tumor- and circulating-free DNA methylation identifies clinically relevant small cell lung cancer subtypes. Simon Heeke, Carl M. Gay, Marcos R. Estecio, et al. Cancer Cell   Volume 42, Issue 2, Pages 225-237.e5 (2024)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy.

(Methods section)  

Critical commercial assays

Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit Apostle Bio A17622-250

Nucleic acid extraction

cfDNA was extracted using the Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Inc). 

78. Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification. Jun Wang, Jinyong Huang, Yunlong Hu, et al. Nature Communications   15, Article number: 156 (2024) 

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Cell-free RNAs (cfRNAs) offer an opportunity to detect diseases from a transcriptomic perspective, however, existing techniques have fallen short in generating a comprehensive cell-free transcriptome profile. We develop a sensitive library preparation method that is robust down to 100 µl input plasma to analyze cfRNAs independent of their 5’-end modifications. We show that it outperforms adapter ligation-based method in detecting a greater number of cfRNA species. We perform transcriptome-wide characterizations in 165 lung cancer, 30 breast cancer, 37 colorectal cancer, 55 gastric cancer, 15 liver cancer, and 133 cancer-free participants and demonstrate its ability to identify transcriptomic changes occurring in early-stage tumors. We also leverage machine learning analyses on the differentially expressed cfRNA signatures and reveal their robust performance in cancer detection and classification. Our work sets the stage for in-depth study of the cfRNA repertoire and highlights the value of cfRNAs as cancer biomarkers in clinical applications.

(Methods section)  cfRNA extraction

Frozen plasma samples were thawed on ice prior to cfRNA extraction. 200 μl of plasma samples were subjected to cfRNA extraction using the Apostle MiniMax High-Efficiency cfRNA Isolation Kit (Apostle), following the manufacturer’s protocol with minor modifications.

77. Extensive methylation analysis of circulating tumor DNA in plasma of patients with gastric cancer. Shinnosuke Nagano, Yukinori Kurokawa, Takaomi Hagi, et al. Scientific Reports 14, Article number: 30739 (2024). 

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

DNA methylation is known to be involved in tumor progression. This is the first study to perform an extensive methylation analysis of plasma circulating tumor DNA (ctDNA) using targeted bisulfite sequencing in gastric cancer (GC) patients to evaluate the usefulness of ctDNA methylation as a new biomarker. Sixteen patients who received chemotherapy for recurrent GC were included. After confirmation of the methylation status of 63 genes using the Cancer Genome Atlas (TCGA) dataset, the methylation status in paired tumor and non-tumor tissues and plasma were investigated using targeted bisulfite sequencing in these genes. Forty-four of the 63 genes were significantly hypermethylated in GC patients in the TCGA cohort. Of these 44 genes, hierarchical clustering showed that five (SPG20, FBN1, SDC2, TFPI2, SEPT9) were particularly hypermethylated in tumor compared to non-tumor tissues in our GC cohort. In plasma methylation analysis, patients with high methylation of these genes had significantly worse overall survival than those with low methylation (log-rank P = 0.009). In a patient who underwent blood sampling at multiple points, the methylation levels of these five genes varied closely with clinical tumor status. The plasma ctDNA methylation levels of these five genes could be useful as a noninvasive prognostic biomarker for GC.

(Methods section)

DNA preparation

DNA from plasma samples was isolated using the Apostle MiniMax™ High Efficiency cfDNA Isolation Kit (Beckman Coulter, CA, USA).


75. Comprehensive Longitudinal ctDNA Monitoring in Metastatic Cancer Patients Treated with an Individualized Neoantigen-directed Vaccine. Desiree Schenk, Matthew J. Davis, Rita Zhou, Alexis Mantilla, Madeline Galbraith, Oliver Spiro, Olivia Petrillo, Italo Faria do Valle, Andrew R. Ferguson, Karin Jooss, Ankur Dhanik. bioRxiv (2024), https://doi.org/10.1101/2024.12.04.626817

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Purpose Circulating-tumor DNA (ctDNA) is an emerging, minimally invasive diagnostic and prognostic biomarker for patients receiving a variety of cancer therapies. Comprehensive and robust longitudinal monitoring of ctDNA can provide an understanding of tumor burden, heterogeneity, and response or resistance to treatment.

Experimental Design ctDNA of 28 metastatic cancer patients receiving an individualized neoantigen-directed immunotherapy was monitored longitudinally, up to two years, using a unique hybrid next generation sequencing assay targeting tumor-informed and tumor-naïve variants. Patient-specific panels were designed targeting an average of 144 variants per patient. A tumor-naïve universal panel was also designed for inclusion with patient-specific panels to monitor recurrently mutated tumor hotspots (e.g., KRAS and TP53) and genes implicated in immunotherapy resistance (B2M, TAP1/2).

Results Analytical characterization of the assay established linearity with a mean variant allele frequency (VAF) ≥0.049%, and a variant-level limit of detection (LOD95) of 0.12%. Tumor-informed variants were detected in 26/28 patients, and de novo variants were observed in 25/28 patients. HLA LOH was also observed. Longitudinal ctDNA data provided key insights into patients’ responses to vaccine treatment.

Conclusions The hybrid design of the ctDNA monitoring assay provides the sensitivity and specificity required for evaluating patient samples undergoing individualized therapy. It provides an improved capability to understand patient response to experimental therapies and further supports the utility of ctDNA as a cancer biomarker.

(Materials and Methods section)

cfDNA extraction

cfDNA was extracted using the Apostle MiniMax High Efficiency cfDNA Extraction kit (Apostle Bio, San Jose, CA). 


74. Liquid Biopsy for Evaluating Mutations and Chromosomal Aberrations in Cerebrospinal Fluid from Patients with Primary or Metastatic Central Tumors. Ahmad Charifa, Sally Agersborg,  Arash Mohtashamian, Andrew Ip, Andre Goy, Maher Albitar, The Journal of Liquid Biopsy (2024), https://doi.org/10.1016/j.jlb.2024.100281

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background

Cytopathology analysis of cerebrospinal fluid (CSF) is limited in detecting tumors in patients with suspected primary or metastatic central nervous system (CNS) malignancy. We investigated the use of CSF liquid biopsy (LBx) to detect neoplastic processes in the CNS. Methods: Cell-free DNA (cfDNA) from the CSF of patients with suspected metastatic (N=106) or primary CNS (N=23) tumors was deep sequenced using a 302-gene panel.

Results

Four samples (3%) (3 metastatic and 1 primary) failed sequencing quality control criteria. Metastatic tumor was confirmed in 84 (82%) of the 103 patients suspected of metastatic tumor. Primary CNS tumor was confirmed in 11 of 22 (50%) patients suspected of CNS tumor. Chromosomal abnormalities were detected in 55 samples (54%). Germline mutations were detected in 23 (22%) patients with metastatic tumors and in 1 (5%) with a primary CNS tumor. Of the 29 patients with metastatic breast cancers, 2 (7%) had mutations in ESR1 and 9 (31%) had mutations in PIK3CA. Of the 21 patients with metastatic lung cancer, 9 (43%) had EGFR mutations and 5 (24%) had KRAS mutations. Upon comparing CSF LBx with peripheral blood LBx in 14 patients, 13 (93%) showed only CHIP and one patient showed CNS primary tumor mutation. Serial samples from 14 patients demonstrate that CSF LBx can be used for monitoring therapy efficacy.

Conclusions

LBx using CSF is clinically reliable and provides informative results in a substantial proportion of patients with metastatic CNS tumors and to a lesser degree in patients with primary CNS tumors.

(Materials and Methods section)

cfDNA and cfRNA Extraction

We used the Apostle MiniMax High-Efficiency total nucleic acid isolation Kit (Beckman Coulter, Brea, CA, USA) and followed the protocol recommended by the manufacturer as previously described in detail. After extraction, half of the cell-free total nucleic acid was treated with DNase to obtain cfRNA, and the other half was used for cfDNA analysis.


73. CancerSpot: A multi-cancer early detection test developed and validated on a retrospective cohort.  Swaraj Basu, Prakash Hiremath M, Nihesh Rathod, et al.  medRxiv (2024), doi: https://doi.org/10.1101/2024.12.03.24318395

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Next-generation sequencing (NGS) technologies have transformed biomarker discovery, enabling the detection of disease-associated markers at the earliest stages of illness. In this study, we introduce a blood-based, non-invasive test for multi-cancer detection using cell-free DNA (cfDNA) methylation sequencing. The test employs a novel methylation scoring system derived from sequencing data and integrates machine learning to analyze a retrospective cohort of newly diagnosed cancer cases and controls recruited from multiple centers across India. To enhance robustness, the study includes a substantial proportion of controls with habitual tobacco and alcohol use, ensuring the test’s resilience against confounding factors. The test’s accuracy was further validated through synthetic data augmentation, demonstrating reliability under conditions of random signal perturbation. At an approximate specificity of 97%, the assay achieves sensitivities of 79.3% for Stage I, 78.4% for Stage II, 78.4% for Stage III, and 86.8% for Stage IV cancers in an independent validation cohort. Additionally, the test demonstrates Top 2 Tissue of Origin (TOO) accuracies of 78.3% for Stage I, 79.3% for Stage II, 82.8% for Stage III, and 69.7% for Stage IV cancers. This blood-based test holds considerable promise for early cancer detection, offering a precise test for cancer screening.

(Methods and Materials section)

Methylation assay and sequencing

Cell-free DNA (cfDNA) was extracted from 4 mL of plasma using the Apostle MiniMax High Efficiency cfDNA Isolation Kit (Cat. No.: A17622-384) following the manufacturer’s protocol (Apostle Bio).


72. The pattern of homologous recombination associated genemutations in circulating tumor DNA and solid tumors from pre-treatment ovarian cancer patients reveals potentially usefulpatterns of mutation. John Nakayama, Stephanie Gaillard, Louis Gil Acevedo, et al. Gynecologic Oncology. Volume 190, Supplement 1, S177, November 2024 (Conference abstract)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Objectives

This study aimed to assess the ability of comprehensive genomic profiling to identify gene mutations associated with homologous recombination (HR) in the circulating tumor DNA and the solid tumor samples from pre-treatment ovarian cancer patients.

Methods

Samples from 29 ovarian cancer patients were selected from our internal biobank based on the availability of longitudinal samples, as this was the first step of a serial analysis. Comprehensive genomic profiling (CGP) was performed using a validated in-house pan-cancer panel (523 genes), including 25 HR genes, to compare precisely matched solid tumor and blood samples. Pre-treatment blood samples were collected in tubes containing a membrane stabilizer (Streck), and plasma was processed within 72 h to obtain germline-free samples. DNA was extracted from matching formalin-fixed paraffin-embedded tumor tissues and blood using magnetic beads (FormaPure XL, Apostle MiniMax, respectively), and CGP was performed on both samples using the same targeted panel (ct-DNA: NovaSeq6000; st-DNA: NextSeqDx550). The combination of HR mutations found in the solid tumor and blood sample was used in this analysis. Homologous recombination deficiency (HRD+) was defined as either germline or somatic BRCA mutation or positive HRD testing by a commercial vendor. HRD groups were compared using a t-test. A clinically actionable mutation was determined using the OncoKB database.

Results

There were 9 HRD-positive and 10 HRD-negative (all epithelial, stage III & IV) along with 10 HRD unknown patients (5 stage I & II including 1 serous borderline and 1 granulosa cell). Analysis revealed that regardless of stage or histology, every ovarian cancer patient has non-synonymous coding mutations in HR genes. There was a mean of 16.4 (SD: 4.7) coding variants per patient in the total population. A 4-gene (6 variant) signature captured all 29 patients with 18 sharing a BARD1 mutation (c.1518_1519delTGinsCT: Val507Leu) along with FANCA, ATR, and RAD51D single-nucleotide variants. When separated by HRD status, the mean number of mutations per patient was 18.0 (SD: 4.4) in the HRD-positive versus 16.7 (SD: 4.5) in the HRD- negative patients (P = 0.52). Clinically actionable mutations (PARP inhibitors) were identified in 7 of 9 HRD-positive and 4 of 10 HRD-negative patients.

Conclusions

These data show that non-synonymous mutations in HR genes are present in all ovarian cancer patients evaluated. The most common gene variants were found in BARD1, BRCA1, FANCA, ATM, and ATR. A 4-gene mutation signature of ATR, BARD1, FANCA, and RAD51 captured all 29 patients in the study across all stages. Four of 10 HRD-negative patients had clinically actionable mutations, which, if validated in future studies, could open new patients to PARP inhibitor therapy.

71. Comprehensive genomic profiling of circulating tumor DNA ishighly concordant with solid tumor DNA and identifies additionalactionable mutations among advanced ovarian and endometrialcancer patients. John Nakayama, Patricia Petrosko, Phillip Gallo, et al. Gynecologic Oncology. Volume 190, Supplement 1, S167, November 2024 (Conference abstract)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Objectives

This study aimed to determine the accuracy and concordance of circulating tumor DNA (ctDNA) blood samples in providing relevant diagnostic information for patients with advanced ovarian cancer

(OC) and endometrial cancer (EC).

Methods

Advanced OC and EC patients with time-matched, pre-treatment solid tumor and blood samples were identified from our internal biobank. Comprehensive genomic profiling (CGP) was performed using an in-house, validated pan-cancer panel (523 genes). Blood samples were collected using tubes containing a membrane stabilizer (Streck), with plasma processed within 72 hours. DNA was extracted from matching formalin-fixed paraffin-embedded (FFPE) tumor tissues and blood (ctDNA) for each patient using magnetic beads (FormaPure XL, Apostle MiniMax, respectively), and CGP was performed on both samples using the same targeted panel (ctDNA: NovaSeq6000; stDNA: NextSeqDx550). SNV and InDel variants were identified and classified from non-synonymous mutations for clinical actionability using the OncoKB database.

Results

Blood samples from 24 OC and 15 EC patients with stage II-IV disease prior were evaluated. Analysis revealed a mean of 167.2 (SD = 10.0) non-synonymous coding mutations in ovarian solid tumors (ST), with 97.4 % of these mutations (M = 163.0; SD = 10.7) detected in matched ctDNA specimens. Similarly, 181.7 (SD = 21.6) coding mutations were detected in endometrial cancers, with 97.6 % of these found in matched ctDNA specimens (M = 177.6; SD = 21.4). A mean of 19.7 and 14.5 additional mutations/patient in OC and EC samples, respectively, were detected only in the ctDNA as compared to ST specimens. When confined to oncogenic variants defined by OncoKB, 94.3 % (M = 7.0; SD = 2.2) of ovarian and 93.0 % (M = 6.8; SD = 1.8) of endometrial variants in the ST were detected by the ctDNA assay. The ctDNA assay identified an additional 5.0 and 3.1 mutations/patient in the OC and EC samples, respectively. Eleven OC patients were identified to have actionable mutations for targeted antineoplastic therapy using both assays, and an additional 4 patients were identified with actionable targets only in the ctDNA samples. Among EC patients, 7 were identified with actionable mutations in both blood and tumor samples, while only 1 patient had an actionable mutation based on the ctDNA assay alone. Only 1 patient among all 39 patients evaluated had an actionable mutation detected in the ST assay that was absent in the ctDNA assay.

Conclusions

A high concordance exists between paired ctDNA and ST assays from patients with advanced-stage OC and EC. We have demonstrated that ctDNA assays can detect additional actionable mutations not detected in the ST assays. These data provide evidence that ctDNA CGP assays detect additional clinically relevant variants beyond those found in ST assays alone and may be a useful addition to the current standard of ST testing.


70. Liquid Biopsy Assay to Detect Low Levels of ctDNA in Early-Stage Lung Cancer Samples. M. Smith, M. Salmans, M. Wang, et al.  Journal of Thoracic Oncology. Volume 19, Issue 10, Supplement, October 2024, Page S474 (Conference abstract)

(Note: Apostle MiniMax technology is used in this study.) 


Abstract 

Introduction

A fundamental challenge in technologies designed to analyze circulating tumor DNA (ctDNA) is the typically small fraction of cell-free DNA (cfDNA) in a blood sample that comes from tumor cells. This proportion of ctDNA is generally low in early-stage cancers, which are characterized by small, localized tumors, but increases as the cancer progresses and the tumors grow and metastasize. Therefore, the capability to reliably detect minimal quantities of ctDNA is crucial for technologies like liquid biopsies. These technologies are particularly focused on detecting cancers at an early stage, when the tumor size is small and the amount of ctDNA present in the bloodstream is minimal. Early detection is especially relevant in lung cancer, where American Cancer Society studies show that the 5-year survival rates plummet from 65% to 9% as the stage at diagnosis advances. Various approaches are being explored to increase the amounts of ctDNA available for analysis, including simply increasing the amount of blood required from the patient paired with ultra-deep targeted sequencing. A low-pass whole genome sequencing approach, in which global cancer-like patterns can be detected in minimal amounts of cfDNA, can provide a reliable indicator of tumor presence without requiring prohibitive amounts of input material. Here we demonstrate the ability of the Genece Health Lung Cancer Assay to leverage fragment and end-motif analysis of cfDNA to detect low levels of ctDNA from early-stage lung cancer samples, while only requiring minimal input material. With the cfDNA available from a single tube of blood from a patient, the Genece Health Lung Cancer Assay can detect all stages of cancer.

Methods

Plasma collected in Streck cfDNA BCT devices from 19 early-stage lung cancer samples (16 stage I samples & 3 stage II samples) were obtained from Discovery Life Sciences. Two plasma aliquots of each sample were extracted using the Apostle MiniMax High Efficiency cfDNA Isolation kit on the KingFisher instrument. Eluates were pooled, cfDNA was quantified using Agilent Tapestation, and then diluted in water to 2ng, 1ng, 0.5ng & 0.25ng, as possible, based on the measured cfDNA concentration.

Results

All early-stage lung cancer samples were detected neat, and subsequent dilution series of these early-stage samples established that an input of only 1ng cfDNA is required for utilization of the Genece Health Lung Assay.

Conclusions

Signal detection in these early-stage lung cancer samples, even after dilution, indicates that the Genece Health Lung Assay has a sufficiently low limit of detection to ensure highly sensitive performance in early-stage lung cancer samples with only a single tube of blood or less.

68. Evaluation and integration of cell-free DNA signatures for detection of lung cancer. Ruyue Xue, Xiaomin Li, Lu Yang, et al. Cancer Letters  Volume 604, 1 November 2024, 217216. https://doi.org/10.1016/j.canlet.2024.217216

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Cell-free DNA (cfDNA) analysis has shown potential in detecting early-stage lung cancer based on non-genetic features. To distinguish patients with lung cancer from healthy individuals, peripheral blood were collected from 926 lung cancer patients and 611 healthy individuals followed by cfDNA extraction. Low-pass whole genome sequencing and targeted methylation sequencing were conducted and various features of cfDNA were evaluated. With our customized algorithm using the most optimal features, the ensemble stacked model was constructed, called ESim-seq (Early Screening tech with Integrated Model). In the independent validation cohort, the ESim-seq model achieved an area under the curve (AUC) of 0.948 (95% CI: 0.915–0.981), with a sensitivity of 79.3% (95% CI: 71.5–87.0%) across all stages at a specificity of 96.0% (95% CI: 90.6–100.0%). Specifically, the sensitivity of the ESim-seq model was 76.5% (95% CI: 67.3–85.8%) in stage I patients, 100% (95% CI: 100.0–100.0%) in stage II patients, 100% (95% CI: 100.0–100.0%) in stage III patients and 87.5% (95% CI: 64.6%–100.0%) in stage IV patients in the independent validation cohort. Besides, we constructed LCSC model (Lung Cancer Subtype multiple Classification), which was able to accurately distinguish patients with small cell lung cancer from those with non-small cell lung cancer, achieving an AUC of 0.961 (95% CI: 0.949–0.957). The present study has established a framework for assessing cfDNA features and demonstrated the benefits of integrating multiple features for early detection of lung cancer.

(Methods section)  Sample collection and cfDNA extraction

Two 10-mL tubes of peripheral blood from patients were collected using Apostle MiniMax cfDNA Blood Collection Tubes (Apostle) and were gently inverted 10 times to mix immediately. The median time between blood collection and plasma isolation was <2 days (range: 1-5 days). Plasma was separated by two-step centrifugation process.30 Lung cancer and paired para-cancerous tissue samples were collected at the time of surgical resection from the First Affiliated Hospital of Zhengzhou University. Lung cancer tissues with less than 20% tumor cell content were excluded from the study. cfDNA was extracted from 8 to 10 mL of plasma using the Apostle MiniMax High Efficiency cfDNA Isolation Kit (Apostle) on an Apostle MagTouch 2000 Nucleic Acids Extraction Automation System (Apostle), and stored in LoBind tubes (Eppendorf AG). Genomic DNA (gDNA) was extracted from formalin-fixed paraffin-embedded (FFPE) tissue samples using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s protocol. The concentration and quality of cfDNA and gDNA were assessed using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) on a Qubit 4 Fluorometer (Thermo Fisher Scientific).

66. Impact of preanalytical factors on liquid biopsy in the canine cancer model. Kate Megquier, Christopher Husted, Justin Rhoades, et al. bioRxiv   July 30, 2024

(Note: Apostle MiniMax technology is used in this study.) 

Abstract While liquid biopsy has potential to transform cancer diagnostics through minimally-invasive detection and monitoring of tumors, the impact of preanalytical factors such as the timing and anatomical location of blood draw is not well understood. To address this gap, we leveraged pet dogs with spontaneous cancer as a model system, as their compressed disease timeline facilitates rapid diagnostic benchmarking. Key liquid biopsy metrics from dogs were consistent with existing reports from human patients. The tumor content of samples was higher from venipuncture sites closer to the tumor and from a central vein. Metrics also differed between lymphoma and non-hematopoietic cancers, urging cancer-type-specific interpretation. Liquid biopsy was highly sensitive to disease status, with changes identified soon after post chemotherapy administration, and trends of increased tumor fraction and other metrics observed prior to clinical relapse in dogs with lymphoma or osteosarcoma. These data support the utility of pet dogs with cancer as a relevant system for advancing liquid biopsy platforms.

(Methods section)  

Frozen plasma (up to 4mL) was extracted using the Apostle MiniMax cfDNA extraction kit (Apostle, LLC *) on a Biomek i7 Hybrid liquid handling system (Beckman Coulter) per manufacturer instructions. 

( * Note from Apostle team:  the company name should be Apostle, Inc.)

65. A Blood Hepatocellular Carcinoma Signature Recognizes Very Small Tumor Nodules with Metastatic Traits. Kun Chen, Junxiao Wang, Liping Jiang, et al. Journal of Clinical and Translational Hepatology  2024, doi: 10.14218/JCTH.2023.00559

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background and Aims

Hepatocellular carcinoma (HCC) cases with small nodules are commonly treated with radiofrequency ablation (RFA), but the recurrence rate remains high. This study aimed to establish a blood signature for identifying HCC with metastatic traits pre-RFA.

Methods

Data from HCC patients treated between 2010 and 2017 were retrospectively collected. A blood signature for metastatic HCC was established based on blood levels of alpha-fetoprotein and des-γ-carboxy-prothrombin, cell-free DNA (cfDNA) mutations, and methylation changes in target genes in frozen-stored plasma samples that were collected before RFA performance. The HCC blood signature was validated in patients prospectively enrolled in 2021.

Results

Of 251 HCC patients in the retrospective study, 33.9% experienced recurrence within 1 year post-RFA. The HCC blood signature identified from these patients included des-γ-carboxy-prothrombin ≥40 mAU/mL with cfDNA mutation score, where cfDNA mutations occurred in the genes of TP53, CTNNB1, and TERT promoter. This signature effectively predicted 1-year post-RFA recurrence of HCC with 92% specificity and 91% sensitivity in the retrospective dataset, and with 87% specificity and 76% sensitivity in the prospective dataset (n=32 patients). Among 14 cases in the prospective study with biopsy tissues available, positivity for the HCC blood signature was associated with a higher HCC tissue score and shorter distance between HCC cells and microvasculature.

Conclusions

This study established an HCC blood signature in pre-RFA blood that potentially reflects HCC with metastatic traits and may be valuable for predicting the disease’s early recurrence post-RFA.

(Methods section) 

Analysis of cfDNA mutations and methylation changes in stored plasma

Briefly, cfDNA was extracted from the plasma samples using the Apostle MiniMax cfDNA isolation kit. The cfDNA (5–40 ng) was digested using the methylation-sensitive restriction enzyme Hha I (R0139L, New England BioLabs) and subsequently ligated to customized MCP adapters with random DNA barcodes. 

64. Optimization of high-volume cell free DNA extraction and end-to-end automation for TSO 500 ctDNA library prep [abstract]. Nripesh Prasad, Rachel Rock, Rebecca Beatty, Melanie Robinson, Dineen Wildman, Michael Sykes, Boris Umylny, Thomas Halsey. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2298. 

(Note: Apostle MiniMax technology is used and compared in this study.) 

Abstract Liquid biopsy such as plasma, has emerged as one of the most valuable sample types for profiling circulating tumor DNA (ctDNA) for screening, response to treatment, and monitoring relapse in oncology patients. Due to the diffuse nature of ctDNA however, both extraction and library preparation must be optimized for the efficient downstream next-generation sequencing (NGS) application. We initially addressed the optimization of quality and quantity of cfDNA recovery from healthy and various oncology patients by evaluating QIAsymphony DSP Circulating DNA Kit and Apostle MiniMax cfDNA isolation kits. Overall, the cfDNA yields from Apostle MiniMax kit were approximately 12X higher. We then optimized high-volume automated cfDNA extraction from 1ml, 3ml and 6ml plasma samples using the Biomek i7 for Apostle MiniMax kit. Colorectal, Gastric and Ovarian cancer gave the highest yield of cfDNA whereas, Esophageal, Lung and Kidney cancers were the lowest cfDNA yielding samples. We then analyzed ctDNA from plasma from healthy donors, colorectal, kidney, Breast, and endometrial cancer patients along with Oncospan and Seraseq ctDNA mutation mix (5-0.5% AF) to evaluate the mutation detection efficacy, repeatability, and reproducibility by targeted NGS using TSO 500ctDNA assay ay 10ng, 20ng, and 30ng inputs. For library preparation batches of 48 samples were prepared using a fully automated processes for the library prep, Hybridization capture and post capture enrichment on the Beckman i5 robot. Libraries were sequenced on NovaSeq 6000 at 600M Paired-end depth. This method demonstrates an overall concordance of inter-run and intra-run accuracy of variant detection to be 90% demonstrating that this method run as described produces highly repeatable and reproducible variant detection to sub-1% allele frequencies. The inter-run, lot-to-lot, and operator-to-operator variability was ≥99%. Down-sample analysis demonstrated high levels of assay performance with sample inputs of 30 ng with ≥96% sample passing Illumina cut-off metrics at 150M PE read depth as compared to Illumina recommended 420M PE reads. Taken together, we provide a high-volume automated solution for cfDNA extraction from plasma samples, additionally these data demonstrate a robust, reproducible, and highly accurate targeted DNA sequencing method that can be used for low allele frequency variant detection in cfDNA.


63. Sequencing of cerebrospinal fluid cell-free DNA facilitated early differential diagnosis of intramedullary spinal cord tumors. Chai, et al. npj Precision Oncology volume 8, Article number: 43 (2024)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Pre-surgery differential diagnosis is valuable for personalized treatment planning in intramedullary spinal cord tumors. This study assessed the performance of sequencing cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) for differential diagnosis of these tumors. Prospectively enrolling 45 patients with intramedullary spinal cord lesions, including diffuse midline glioma (DMG), H3K27-altered (14/45), glioblastoma (1/45), H3-wildtype-astrocytoma (10/45), ependymoma (11/45), and other lesions (9/45), CSF samples were collected via lumbar puncture (41/45), intraoperative extraction (3/45), and Ommaya reservoir (1/45). Then, these samples underwent targeted sequencing along with paired tissue DNA. DMG, H3K27-altered patients exhibited a higher ctDNA positivity (85.7%, 12/14) compared to patients with H3-wildtype-astrocytoma (0/8, P = 0.0003), ependymoma (2/10, P = 0.003), and glioneuronal tumor (0/3, P = 0.009). The histological-grade-IV (P = 0.0027), Ki-67 index ≥10% (P = 0.014), and tumor reaching spinal cord surface (P = 0.012) are also associated with higher ctDNA positivity. Interestingly, for patients with TERT promoter mutant tumors, TERT mutation was detectable in the CSF cfDNA of one DMG case, but not other five cases with histological-grade-II tumors. Shared copy number variants were exclusively observed in DMG, H3K27-altered, and showed a strong correlation (Correlation = 0.95) between CSF and tissue. Finally, H3K27M mutations in CSF exhibited high diagnostic efficiency for DMG, H3K27-altered (Sensitivity = 85.7%, Specificity = 100.0%, AUC = 0.929). Notably, H3K27M was detectable in CSF from patients with recurrent tumors, making it easily applicable for postoperative monitoring. In conclusion, the molecular profile from ctDNA released into CSF of malignant tumors was more frequently detected compared to relatively benign ones. Sequencing of ctDNA in CSF exhibited high efficiency for the differential diagnosis of DMG, H3K27-altered.

(Methods section)  Circulating cell-free DNA (cfDNA) isolation from cerebrospinal fluid (CSF)

Then, circulating nucleic acid was extracted from CSF using the Apostle MiniMax High-Efficiency cfDNA Isolation Kit (Apostle, USA) following the manufacturer's instructions. 

62. A noninvasive multianalytical approach establishment for risk assessment and gastric cancer screening. Fan, et al. International Journal of Cancer, 2024; 154(6):1111-1123

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Effective screening and early detection are critical to improve the prognosis of gastric cancer (GC). Our study aims to explore noninvasive multianalytical biomarkers and construct integrative models for preliminary risk assessment and GC detection. Whole genomewide methylation marker discovery was conducted with CpG tandems target amplification (CTTA) in cfDNA from large asymptomatic screening participants in a high-risk area of GC. The methylation and mutation candidates were validated simultaneously using one plasma from patients at various gastric lesion stages by multiplex profiling with Mutation Capsule Plus (MCP). Helicobacter pylori specific antibodies were detected with a recomLine assay. Integrated models were constructed and validated by the combination of multianalytical biomarkers. A total of 146 and 120 novel methylation markers were found in CpG islands and promoter regions across the genome with CTTA. The methylation markers together with the candidate mutations were validated with MCP and used to establish a 133-methylation-marker panel for risk assessment of suspicious precancerous lesions and GC cases and a 49-methylation-marker panel as well as a 144-amplicon-mutation panel for GC detection. An integrated model comprising both methylation and specific antibody panels performed better for risk assessment than a traditional model (AUC, 0.83 and 0.63, P < .001). A second model for GC detection integrating methylation and mutation panels also outperformed the traditional model (AUC, 0.82 and 0.68, P = .005). Our study established methylation, mutation and H. pylori-specific antibody panels and constructed two integrated models for risk assessment and GC screening. Our findings provide new insights for a more precise GC screening strategy in the future.

(Methods section)  Blood sample collection and DNA extraction

The Apostle MiniMax cfDNA isolation kit (Apostle; San Jose, CA) was used for cfDNA extraction. 

61. Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA [abstract]. Benjamin B. Morris, Zhihui Zhang, Simon Heeke, et al. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A022.

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer. A key driver of this near universal lethality is SCLC’s recalcitrance to therapy. The standard of care for SCLC is a chemotherapy doublet of etoposide and cis/carboplatin in combination with immunotherapy (EP+IO). While most tumors respond to these therapies, >80% of SCLCs progress within one year of treatment. Additionally, the aggressive clinical course of SCLC has historically precluded collection of patient-matched treatment naïve and recurrent tumor tissue traditionally required to study evolution. In lieu of tissue, several groups have used circulating tumor DNA (ctDNA) to study SCLC evolution. However, these studies used targeted sequencing panels that analyze <0.04% of the SCLC genome. Using new methods to comprehensively trace how SCLCs rapidly evolve to therapy resistance is a significant unmet need.

Methods: Medical records were reviewed to identify treatment naïve SCLC patients treated at MD Anderson that had both pre- and post-EP+IO liquid biopsies collected under IRB approved protocols. ctDNA was isolated from plasma using a MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Bio). Germline DNA was extracted from buffy coat samples using a QIAamp DNA Blood Midi Kit (Qiagen). Deep whole genome sequencing (WGS) (>100X) of ctDNA and WGS of germline DNA (>60X) was performed using an Illumina NovaSeq X sequencer. Reads were aligned to the hg38 reference genome using BWA-mem. Somatic mutations were called using MuTect2, and clonal and subclonal copy number alterations were identified using Battenberg. DPClust was used to reconstruct cancer cell populations, leveraging mutation and copy number calls.

Results: We identified several deleterious genomic alterations impacting TP53 and RB1 tumor suppressor genes, known genetic drivers of SCLC development. We also detected recurrent, whole chromosome arm loss of heterozygosity events impacting many chromosomes in treatment naïve samples, some of which are known to be present in >80% of SCLCs. Additionally, we found that both treatment naïve and recurrent SCLCs were composed of multiple subclones, each defined by dozens to thousands of unique somatic mutations. COSMIC signature analysis identified high activity of tobacco carcinogens, APOBEC, and replicative-based processes across ctDNA samples. We also identified emergence of novel subclones carrying unique copy number alterations following EP+IO recurrence. Importantly, the affected regions encode key effectors that regulate anti-tumor immune responses and cell survival following DNA damage, both of which are intimately related to the mechanism of action of EP+IO.

Conclusions: Our data shows that deep WGS of ctDNA recapitulates known genetics of SCLC and allows for sensitive characterization of therapy resistant clones. Using this method, we have captured snapshots of both intrinsic and acquired resistance mechanisms to frontline EP+IO. Deep WGS of ctDNA is a promising approach to comprehensively dissect routes of SCLC evolution and therapy resistance.


Oncology-related Publications (2023)

58. Integrative modeling of tumor genomes and epigenomes for enhanced cancer diagnosis by cell-free DNA. Mingyun Bae, Gyuhee Kim, Tae-Rim Lee, et al. Nature Communications  14, Article number: 2017 (2023)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Multi-cancer early detection remains a key challenge in cell-free DNA (cfDNA)-based liquid biopsy. Here, we perform cfDNA whole-genome sequencing to generate two test datasets covering 2125 patient samples of 9 cancer types and 1241 normal control samples, and also a reference dataset for background variant filtering based on 20,529 low-depth healthy samples. An external cfDNA dataset consisting of 208 cancer and 214 normal control samples is used for additional evaluation. Accuracy for cancer detection and tissue-of-origin localization is achieved using our algorithm, which incorporates cancer type-specific profiles of mutation distribution and chromatin organization in tumor tissues as model references. Our integrative model detects early-stage cancers, including those of pancreatic origin, with high sensitivity that is comparable to that of late-stage detection. Model interpretation reveals the contribution of cancer type-specific genomic and epigenomic features. Our methodologies may lay the groundwork for accurate cfDNA-based cancer diagnosis, especially at early stages.

(Methods section)  cfDNA was extracted from 0.4 mL plasma ... and eluted in a final volume of 22 μL, using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (Apostle, US) according to the manufacturer’s instructions. 

56. Comprehensive Analysis of Cell-Free DNA Fragmentation Across Cancer Stages. Guo, et al. medRxiv, November 2023; https://doi.org/10.1101/2023.11.07.23298181

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Circulating cell-free DNA (cfDNA) in the bloodstream displays cancer-derived fragmentation patterns, offering a non-invasive diagnostic avenue for cancer patients. However, the association between cfDNA fragmentation patterns and cancer progression remains largely unexplored. In this study, we analyzed this relationship using 214 whole-genome cfDNA samples across seven solid cancer types and revealed that the relation between cfDNA fragmentation patterns and cancer stages vary across cancer types. Among them, cfDNA fragmentation patterns in colorectal cancer (CRC) showed a strong correlation with cancer stages. This finding is further validated with an independent targeted cfDNA dataset from 29 CRC samples. Inspired by these findings, we designed “frag2stage”, a machine learning model that exploits cfDNA fragmentation data to differentiate cancer stages of CRC. Evaluated on two independent cfDNA datasets, our model can distinguish cancer stages of CRC with the area under the curve (AUC) values ranging from 0.68 to 0.99. The results suggest that cfDNA fragmentation patterns might carry yet undiscovered genetic and epigenetic signals, highlighting their promising potential for broader diagnostic applications in oncology.

(Methods section)  CFDNA EXTRACTION AND NGS LIBRARY PREPARATION

Blood samples, about 10 ml from each individual, were collected using the Apostle MiniMax cf-DNA Blood Collection Tube (Apostle; San Jose, CA, USA) and processed within seven days from the collection. The cfDNA extraction from 2 to 5 ml of each plasma sample was conducted using the Apostle MiniMax High Efficiency cfDNA isolation kit (Apostle; San Jose, CA, USA), with adherence to the manufacturer’s protocol with slight modifications.

55. Concordance analysis of cerebrospinal fluid with the tumor tissue for integrated diagnosis in gliomas based on next-generation sequencing. Wang, et al. Pathology and Oncology Research, 26 September 2023 https://doi.org/10.3389/pore.2023.1611391

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Purpose: The driver mutations of gliomas have been identified in cerebrospinal fluid (CSF). Here we compared the concordance between CSF and tumor tissue for integrated diagnosis in gliomas using next-generation sequencing (NGS) to evaluate the feasibility of CSF detection in gliomas.

Patients and methods: 27 paired CSF/tumor tissues of glioma patients were sequenced by a customized gene panel based on NGS. All CSF samples were collected through lumbar puncture before surgery. Integrated diagnosis was made by analysis of histology and tumor DNA molecular pathology according to the 2021 WHO classification of the central nervous system tumors.

Results: A total of 24 patients had detectable circulating tumor DNA (ctDNA) and 22 had at least one somatic mutation or chromosome alteration in CSF. The ctDNA levels varied significantly across different ages, Ki-67 index, magnetic resonance imaging signal and glioma subtypes (p < 0.05). The concordance between integrated ctDNA diagnosis and the final diagnosis came up to 91.6% (Kappa, 0.800). We reclassified the clinical diagnosis of 3 patients based on the results of CSF ctDNA sequencing, and 4 patients were reassessed depending on tumor DNA. Interestingly, a rare IDH1 R132C was identified in CSF ctDNA, but not in the corresponding tumor sample.

Conclusion: This study demonstrates a high concordance between integrated ctDNA diagnosis and the final diagnosis of gliomas, highlighting the practicability of NGS based detection of mutations of CSF in assisting integrated diagnosis of gliomas, especially glioblastoma.

(Methods section)  DNA extraction and quantification

Cell-free DNA (cfDNA) was extracted using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (APOSTLE) according to the manufacturer’s instructions. 

54. Altered cfDNA fragmentation profile in hypomethylated regions as diagnostic markers in breast cancer.  Wang J, et al. Epigenetics & Chromatin. 16, Article number: 33 (2023) 

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background

Breast cancer, the most common malignancy in women worldwide, has been proven to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profiles. Nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although fragmentation pattern of cfDNA is determined by nuclease digestion of chromatin, structure of which may be affected by DNA methylation, whether cfDNA methylation and fragmentation are biologically related or not still remains unclear.

Methods

Improved cfMeDIP-seq were utilized to characterize both cfDNA methylation and fragmentation profiles in 49 plasma samples from both healthy individuals and patients with breast cancer. The feasibility of using cfDNA fragmentation profile in hypo- and hypermethylated regions as diagnostic markers for breast cancer was evaluated.

Results

Mean size of cfDNA fragments (100–220 bp) mapped to hypomethylated regions decreased more in patients with breast cancer (4.60 bp, 172.33 to 167.73 bp) than in healthy individuals (2.87 bp, 174.54 to 171.67 bp). Furthermore, proportion of short cfDNA fragments (100–150 bp) in hypomethylated regions when compared with it in hypermethylated regions was found to increase more in patients with breast cancer in two independent discovery cohort. The feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic regions for breast cancer diagnosis in validation cohort was evaluated. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity).

Conclusion

We identified enriched short cfDNA fragments after 5mC-immunoprecipitation (IP) in patients with breast cancer, and demonstrated the enriched short cfDNA fragments might originated from hypomethylated genomic regions. Furthermore, we proved the feasibility of using differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for breast cancer diagnosis.

(Methods section)  Sample collection and cfDNA extraction

cfDNA was extracted from plasma using MiniMax(TM) High Efficiency Cell-Free DNA Isolation Kit (Apostle, A17622-250) according to manufacturer’s instructions.

53. Combined detection of SDC2/ADHFE1/PPP2R5C methylation in stool DNA for colorectal cancer screening.  Li B, et al. Journal of Cancer Research and Clinical Oncology. June 03, 2023. https://doi.org/10.1007/s00432-023-04943-4

(Note: Apostle MiniGenomics technology is used in this study.) 

Abstract 

Background Colorectal cancer (CRC) is a disease of global concern, and its increasing incidence suggests the need for early and accurate diagnosis. The aim of this study was to investigate the value of combined detection of SDC2, ADHFE1 and PPP2R5C gene methylation in stool samples for early CRC screening.

Methods StoolsamplesfrompatientswithCRC(n=105),advancedadenoma(AA)(n=54),non-advancedadenoma(NA) (n = 57), hyperplastic or other polyps (HOP) (n = 47) or no evidence of disease (NED) (n = 100) were collected from Sep- tember 2021 to September 2022. The methylation levels of SDC2, ADHFE1 and PPP2R5C were quantified by quantitative methylation-specific polymerase chain reaction (qMSP), and faecal immunochemical testing (FIT) was performed. The diagnostic value was assessed using reporter operating characteristic (ROC) curve analysis.

Results The sensitivity of combined detection of SDC2/ADHFE1/PPP2R5C methylation in predicting CRC (0–IV) was 84.8%, the specificity was 98.0%, and the AUC was 0.930 (95% CI 0.889–0.970). Compared to FIT and serum tumour bio- markers, it showed better diagnostic performance for different stages of CRC.

Conclusion The results of this study verified that the methylation levels of SDC2, ADHFE1 and PPP2R5C in stool DNA were significantly increased in CRC patients. Combined detection of SDC2/ADHFE1/PPP2R5C methylation is a potential non-invasive diagnostic method for CRC and precancerous lesion screening.

Clinical trial registration Chinese Clinical Trials Registry, ChiCTR2100046662, registered on 26 May 2021, prospective registration.

(Methods section)  DNA isolation, bisulphite treatment and fluorescence‐based quantitative methylation‐specific polymerase chain reaction (qMSP)

Genomic DNA was isolated from stool samples using the Apostle MiniGenomics (TM) Stool Fast Kit (Apostle, A181206) and stored in the refrigerator at – 20 °C until further use. 

52. Combining cell-free RNA with cell-free DNA in liquid biopsy for hematologic and solid tumors. Maher Albitar, Hong Zhang, Ahmad Charifa, et al. Heliyon  9 (2023) e16261; May 16, 2023 

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Current use of liquid biopsy is based on cell-free DNA (cfDNA) and the evaluation of mutations or methylation pattern. However, expressed RNA can capture mutations, changes in expression levels due to methylation, and provide information on cell of origin, growth, and proliferation status. We developed an approach to isolate cell-free total nucleic acid (cfDNA) and used targeted next generation sequencing to sequence cell-free RNA (cfRNA) and cfDNA as new approach in liquid biopsy. We demonstrate that cfRNA is overall more sensitive than cfDNA in detecting mutations. We show that cfRNA is reliable in detecting fusion genes and cfDNA is reliable in detecting chromosomal gains and losses. cfRNA levels of various solid tumor biomarkers were significantly higher (P < 0.0001) in samples from solid tumors as compared with normal control. Similarly, cfRNA lymphoid markers and cfRNA myeloid markers were all higher in lymphoid and myeloid neoplasms, respectively as compared with control (P < 0.0001). Using machine learning we demonstrate cfRNA was highly predictive of diagnosis (AUC >0.98) of solid tumors, B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, and myeloid neoplasms. In evaluating the host immune system, cfRNA CD4:CD8B and CD3D:CD19 ratios in normal controls were as expected (median: 5.92 and 6.87, respectively) and were significantly lower in solid tumors (P < 0.0002). This data suggests that liquid biopsy combining analysis of cfRNA with cfDNA is practical and may provide helpful information in predicting genomic abnormalities, diagnosis of neoplasms and evaluating both the tumor biology and the host response.

(Methods section)  We used Apostle MiniMax High Efficiency cfRNA/cfDNA isolation kit and followed the recommended protocol. After extraction, half of the cfDNA was treated with DNase to obtain cfRNA and the other half was used for DNA studies. 

49. Cell-free chromatin immunoprecipitation can determine tumor gene expression in lung cancer patients. Christoffer Trier Maansson, Peter Meldgaard, Magnus Stougaard, Anders Lade Nielsen, Boe Sandahl Sorensen.  Molecular Oncology.  February 24, 2023. DOI: 10.1002/1878-0261.13394.

(Note: Apostle MiniMax technology is used in this study.)

Cell-free DNA (cfDNA) in blood plasma can be bound to nucleosomes that contain post-translational modifications representing the epigenetic profile of the cell of origin. This includes histone H3 lysine 36 trimethylation (H3K36me3), a marker of active transcription. We hypothesized that cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-modified nucleosomes present in blood plasma can delineate tumor gene expression levels. H3K36me3 cfChIP followed by targeted NGS (cfChIP-seq) was performed on blood plasma samples from non-small cell lung cancer patients (NSCLC, n = 8), small cell lung cancer patients (SCLC, n = 4) and healthy controls (n = 4). H3K36me3 cfChIP-seq demonstrated increased enrichment of mutated alleles compared to normal alleles in plasma from patients with known somatic cancer mutations. Additionally, genes identified to be differentially expressed in SCLC and NSCLC tumors had concordant H3K36me3 cfChIP enrichment profiles in NSCLC (sensitivity = 0.80) and SCLC blood plasma (sensitivity = 0.86). Findings here expand the utility of cfDNA in liquid biopsies to characterize treatment resistance, cancer subtyping, and disease progression.

(Material and Methods section) -Both the input as well as the cfChIP samples were purified using Apostle MiniMax High Efficiency cfDNA Isolation Kit (Beckman Coulter, Indianapolis, IN, USA) according to manufacturer’s instructions. 

48. Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples. Jin-Han Bae, Jay Jeong, Byung Chul Kim, et al.  Journal of Visualized Experiments.  January 13th, 2023. DOI: 10.3791/64325

(Note: Apostle MiniMax technology is used in this study.)

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.  We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

(Materials section) - Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit Apostle A17622-250 5 mL X 50 preps version

47. Comparison of cfDNA content in different cancer types with different extraction methods. Wenlong Zhang, Yanan Zhao, Jun Li, et al.  Journal of Clinical Oncology 41, no. 16_suppl (June 01, 2023) e15026-e15026.

(Note: Apostle MiniMax technology is used in this study.)

Background: ctDNA liquid biopsy technology has been widely used in the whole course of cancer management, from early screening and early diagnosis of tumors to tumor monitoring and medication guidance. However, the performances of ctDNA as a biomarker in these scenarios are different among different cancer types. One possible reason is that different types of cancer release different amounts of ctDNA or cfDNA, therefore, we explored the cfDNA levels in different cancer types in this study.

Methods: We collected 15913 peripheral blood samples from 14686 cancer patients with different clinicopathologies, 256 patients with pulmonary nodules, and 971 healthy people. We isolated their plasma and extracted the cfDNA using QIAamp Circulating Nucleic Acid Kit, Apostle MiniMax cfDNA Extraction Kit or HaploX magnetic bead method cfDNA extraction kit. We calculated the average concentration of cfDNA (ng/ml) for different groups.

Results: The number of samples of cervical cancer, liver cancer, glioma, colon cancer, prostate cancer, nasopharyngeal carcinoma, gallbladder carcinoma, melanoma, cholangiocarcinoma, colorectal cancer, breast cancer, gastric cancer, endometrial carcinoma, lung cancer, urothelial carcinoma, rectal cancer, ovarian cancer, healthy people, and pulmonary nodule patients are 93, 469, 113, 277, 139, 76, 89, 132, 171, 4010, 407, 547, 68, 7750, 46, 203, 96, 971 and 256, respectively. The cfDNA level of corresponding groups are 88.07, 79.51, 75.57, 63.58, 61.37, 59.09, 57.59, 57.16, 42.67, 34.73, 31.75, 31.37, 31.33, 29.84, 28.72, 28.69, 23.83, 17.82 and 12.54 ng/ml, respectively. The main groups are shown in the table below.

Conclusions: The cfDNA concentrations of healthy people or pulmonary nodule patients were significantly lower than that of tumor patients, and there were also significant differences between different types of cancer. What's interesting, the cfDNA concentrations of lung cancer patients, pulmonary nodule patients and healthy people were quite different. What’s more, there were differences between Colon cancer and Rectal cancer patients. So, the cfDNA concentration maybe a good dimention to be used in early cancer screening and tracing of tumor types.

Oncology-related Publications (2022)

46. Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results. Christine D. Palmer, Amy R. Rappaport, Matthew J. Davis, et al. Nature Medicine  volume 28, pages 1619–1629 (2022)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 1012 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.

(Methods section)  cfDNA was extracted from the entire plasma volume of a single draw using the Apostle MiniMax cfDNA Isolation kit (ApostleBio) 


45. Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection. Pei Wang, Qianqian Song, Jie Ren, et al. Science Translational Medicine 14, eabp8704 (2022) 23 November 2022

(Note: Apostle MiniMax technology is used in this study.) 

Cell-free DNA (cfDNA)–based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

(Methods Section) cfDNA was extracted from the plasma samples using the Apostle MiniMax cfDNA isolation kit (C43468, Apostle). 


42. Reliability of Cell-Free DNA and Targeted NGS in Predicting Chromosomal Abnormalities of Patients With Myeloid Neoplasms. Andrew Ip,  Alexandra Della Pia,  Gee Youn (Geeny) Kim,  Jason Lofters,  James Behrmann,   Dylon Patel,  Simone Kats,  Jeffrey Justin Estella,  Ivan De Dios,  Wanlong Ma,  Andrew L. Pecora,  Andre H. Goy,  Jamie Koprivnikar,  James K. McCloskey and Maher Albitar. Frontiers in Oncology June 14, 2022; https://doi.org/10.3389/fonc.2022.923809

(Note: Apostle MiniMax technology is used in this study.) 

Introduction: Cytogenetic analysis is important for stratifying patients with various neoplasms. We explored the use of targeted next generation sequencing (NGS) in detecting chromosomal structural abnormalities or copy number variations (CNVs) in patients with myeloid neoplasms.

Methods: Plasma cell-free DNA (cfDNA) from 2821 myeloid or lymphoid neoplasm patients were collected. cfDNA was sequenced using a 275 gene panel. CNVkit software was used for analyzing and visualizing CNVs. Cytogenetic data from corresponding bone marrow (BM) samples was available on 89 myeloid samples.

Results: Of the 2821 samples, 1539 (54.5%) showed evidence of mutations consistent with the presence of neoplastic clones in circulation. Of these 1539 samples, 906 (59%) showed abnormalities associated with myeloid neoplasms and 633 (41%) with lymphoid neoplasms. Chromosomal structural abnormalities in cfDNA were detected in 146 (16%) myeloid samples and 76 (12%) lymphoid samples. Upon comparison of the myeloid samples with 89 BM patients, NGS testing was able to reliably detect chromosomal gain or loss, except for fusion abnormalities. When cytogenetic abnormalities were classified according to prognostic classes, there was a complete (100%) concordance between cfDNA NGS data and cytogenetic data.

Conclusions: This data shows that liquid biopsy using targeted NGS is reliable in detecting chromosomal structural abnormalities in myeloid neoplasms. In specific circumstances, targeted NGS may be reliable and efficient to provide adequate information without the need for BM biopsy considering broad mutation profiling can be obtained through adequate sequencing within the same test. Overall, this study supports the use of liquid biopsy for early diagnosis and monitoring of patients with myeloid neoplasms.

(Methods Section) Sample Collection and cfDNA Isolation

We extracted cfDNA from 2821 peripheral blood samples using the Apostle MiniMax(TM) High Efficiency cfDNA Isolation Kit (San Jose, CA). These peripheral blood samples were collected in EDTA anticoagulant over approximately 18 months. DNA was extracted from separated plasma within 48 hours of collection. We tested these samples for the presence of circulating tumor DNA (ctDNA).


41. Identification of multi-omic biomarkers from Fecal DNA for improved Detection of Colorectal Cancer and precancerous lesions. Yujing Fang, Jiaxi Peng, Zhilong Li, et al. medRxiv. November 13, 2022.  https://doi.org/10.1101/2022.11.08.22282099

(Note: Apostle MiniGenomics technology is used in this study.) 

Background Timely diagnosis and intervention of colorectal cancer (CRC) at curable stages is essential for improving patient survival. Stool samples carry exfoliation of intestinal epithelium, therefore providing excellent opportunity for non-invasive diagnosis of CRC as well as precancerous lesions. In this study, we aimed to conduct multi-dimensional analysis of fecal DNA and investigate the utility of different types of biomarkers for CRC detection.

Method In this case-control study, we performed comprehensive analyses of the genomic, epigenomic, and metagenomic features of fecal DNA from CRC patients, individuals with advanced precancerous lesions (APLs) and controls. DNA methylation markers were identified by whole genome bisulfite sequencing of paired colorectal cancer and normal tissues. A multi-gene fecal DNA methylation test was then developed based on three marker genes (SDC2, ADHFE1 and PPP2R5C) using quantitative methylation-specific PCR (qMSP), and validated on fecal DNA samples. Genomic mutation profiles as well as microbiome signatures of fecal DNA were analyzed using high-throughput sequencing.

Results The methylation-based fecal DNA test demonstrated an overall sensitivity of 88% for CRC and 46.2% for APL respectively, and a specificity of 91.8% for controls. On the other hand, the mutation-based diagnostic model yielded limited sensitivity, and combined detection of methylation markers and mutation in fecal DNA did not improve the assay performance. Meanwhile, a diagnostic model based on the relative abundance of bacterial species showed inferior performance than the methylation-based model. Finally, integrated diagnostic model combining both methylation and microbial markers showed an enhanced performance (AUC= 0.95) compared to methylation markers alone.

Conclusions The multi-gene fecal DNA methylation test provided remarkable diagnostic performance for CRCs and APLs. Furthermore, multi-target assay integrating both methylation and microbial markers may further improve the diagnostic performance. Our findings may aid in the development of novel diagnostic tools for CRC.

(Methods section)

For stool samples, fecal DNA was isolated by Apostle MiniGenomics Stool Fast Kit (Apostle, A181206).



38. A method for early diagnosis of lung cancer from tumor originated DNA fragments using plasma cfDNA methylome and fragmentome profiles. Yeo Jin Kim, Hahyeon Jeona, Sungwon Jeon, et al.  Molecular and Cellular Probes Volume 66, December 2022

(Note: Apostle MiniMax technology is used in this study.) 

Abstract

Early detection is critical for minimizing mortality from cancer. Plasma cell-free DNA (cfDNA) contains the signatures of tumor DNA, allowing us to quantify the signature and diagnose early-stage tumors. Here, we report a novel tumor fragment quantification method, TOF (Tumor Originated Fragment) for the diagnosis of lung cancer by quantifying and analyzing both the plasma cfDNA methylation patterns and fragmentomic signatures. TOF utilizes the amount of ctDNA predicted from the methylation density information of each cfDNA read mapped on 6243 lung-tumor-specific CpG markers. The 6243 tumor-specific markers were derived from lung tumor tissues by comparing them with corresponding normal tissues and healthy blood from public methylation data. TOF also utilizes two cfDNA fragmentomic signatures: 1) the short fragment ratio, and 2) the 5′ end-motif profile. We used 298 plasma samples to analyze cfDNA signatures using enzymatic methyl-sequencing data from 201 lung cancer patients and 97 healthy controls. The TOF score showed 0.98 of the area under the curve in correctly classifying lung cancer from normal samples. The TOF score resolution was high enough to clearly differentiate even the early-stage non-small cell lung cancer patients from the healthy controls. The same was true for small cell lung cancer patients.

(Methods Section) Cell-free DNA was extracted from 3 to 5 ml of plasma using XXX method or Apostle MiniMaxTM High Efficiency Isolation Kit (Beckman Coulter Life Sciences, C40603), according to the manufacturers’ procedures. 



32. Response prediction and risk stratification of patients with rectal cancer after neoadjuvant therapy through an analysis of circulating tumour DNA. Liu W, Li Y, et al. EBioMedicine March 17, 2022; https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(22)00129-3/fulltext

(Note: Apostle MiniMax technology is used in this study.) 

Background - Multiple approaches based on cell-free DNA (cfDNA) have been applied to detect minimal residual disease (MRD) and to predict prognosis or recurrence. However, a comparison of the approaches used in different cohorts and studies is difficult. We aimed to compare multiple approaches for MRD analysis after neoadjuvant therapy (NAT) in patients with locally advanced rectal cancer (LARC).

Methods - Sixty patients with LARC from a multicentre, phase II/III randomized trial were included, with tissue and blood samples collected. For each cfDNA sample, we profiled MRD using 3 approaches: personalized assay targeting tumour-informed mutations, universal panel of genes frequently mutated in colorectal cancer (CRC), and low depth sequencing for copy number alterations (CNAs).

Findings - Positive MRD based on post-NAT personalized assay was significantly associated with an increased risk of recurrence (HR = 27.38; log-rank P < 0.0001). MRD analysis based on universal panel (HR = 5.18; log-rank P = 0.00086) and CNAs analysis (HR = 9.24; log-rank P = 0.00017) showed a compromised performance in predicting recurrence. Both the personalized assay and universal panel showed complementary pattern to CNAs analysis in detecting cases with recurrence and the combination of the two types of biomarkers may lead to better performance.

Interpretation - The combination of mutation profiling and CNA profiling can improve the detection of MRD, which may help optimize the treatment strategies for patients with LARC.


Oncology-related Publications (2021)


24. Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy. Shengnan Jin, Dewen Zhu, Fanggui Shao, Shiliang Chen, Ying Guo, Kuan Li, Yourong Wang, Rongxiu Ding, Lingjia Gao, Wen Ma, Tong Lu, Dandan Li, Zhengzheng Zhang, Suili Cai, Xue Liang, Huayu Song, Ling Ji, Jinlei Li, Zhihai Zheng, Feizhao Jiang, Xiaoli Wu, Ju Luan, Huxiang Zhang, Zhengquan Yang, Charles R. Cantor, Chang Xu, and Chunming Ding. PNAS February 2, 2021 118 (5) e2017421118; https://doi.org/10.1073/pnas.2017421118 

(Note: Apostle MiniMax technology is used in this study.) 

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery. 



23. A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers. Shengnan Jin, Qian Ye, Yanping Hong, Wenqing Dai, Chengliang Zhang, Weihao Liu, Ying Guo, Dewen Zhu, Zhengzheng Zhang, Shiliang Chen, Yourong Wang, Dandan Li, Wen Ma, Zhengquan Yang, Jinlei Li, Zhihai Zheng, Ju Luan, Xiaoli Wu, Feizhao Jiang, Chang Xu and Chunming Ding. Clinical Chemistry and Laboratory Medicine (CCLM). 2021; 59(1): 91–99

Objectives

Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.

Methods

We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.

Results

The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.

Conclusions

Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

(Methods section) For magnetic beads-based method, Apostle Stool gDNA Isolation Kit (APOSTLE) was used according to the manufacturer’s instructions. Either 0.2 g pellets or 0.2 mL supernatant from homogenized stool was mixed with 1 mL lysis buffer (APOSTLE) for DNA extractions.

Oncology-related Publications (2020)


17. Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples. Nicole Roseman, Shilpa Parakh, Hsiao-Yun Huang, Kevin Lai, Timothy Barnes, Lyn Lewis, Ushati Das Chakravarty, Anastasia Potts, Alisa Jackson, Amy Yoder, Jessica Sheu, Tzu-Chun Chen. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5863. 


Demonstrate performance of a complete automation and reagent workflow for analysis of cfDNA from bodily fluids. The efficient extraction of cfDNA from bodily fluids is a unique challenge due to the very low concentrations of nucleic acid. The extraction process along with library preparation is a laborious workflow, where human variability can lead to increased variability in the downstream analysis. Integrated DNA Technology (IDT) and Beckman Coulter (BC) have teamed up to provide a complete automation and reagent workflow for analysis of low frequency variants in cfDNA. The Apostle MiniMax™ High Efficiency Isolation Kit from BC provides complex, utilized magnetic nanoparticles to effectively capture cfDNA. IDT's library prep kit utilizes novel chemistry to maximize conversion, suppress adapter-dimer formation, reduce chimera rates, and facilitate double strand consensus analysis to call ultra-low frequency variants. Finally, IDT's xGen™ hybrid capture products maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. The Biomek i5 and i7 Hybrid workstations bring out the best performance from these reagents. The Biomek NGS workstations protocol is written with a modular design with safe stop points, making it customizable for each lab. The automated protocol uses Beckman's Demonstrated Method Interface tools which include: Biomek Method Launcher to run the method without going into Biomek software, Method Options Selector to choose the run parameters with a user friendly interface, Guided labware Setup to set the deck with labware based on the run parameters, DeckOptix Final Check software to help reduce deck setup errors. We demonstrate the performance of this complete workflow with a range of plasma inputs (4-8 mL). Using control samples with known variant frequencies, the workflow yields high library complexity, 100% positive predictive value, and reliable detection of <0.5% mutant allele frequency variants. With real cfDNA, the workflow demonstrates both high cfDNA and sequencing library yields along with high library complexity. The combination of these reagents on the Biomek workstations provides a robust and reproducible solution for the analysis of cfDNA.  


Oncology-related Publications (2019)


12. Correlation between mutations found in FFPE tumor tissue and paired cfDNA samples. (n=8) Niccum B., Saunders L., Hur A.,Patel A. The American Society of Human Genetics (ASHG). (abstract #1766). Houston, TX. Oct 15, 2019 

Liquid biopsies represent a promising area of facilitating cancer research as taking blood is less invasive than tumor biopsies. Cell-free DNA (cfDNA) consists of small (150 – 500 bp) DNA fragments that circulate in the blood. Levels of cfDNA tend to be low in healthy, non-pregnant patients and increased in patients with cancer, pregnancy, or extensive tissue damage. cfDNA is believed to be derived mostly from apoptotic cells and a source for biomarkers for a variety of diseases. 

As a non-invasive way to detect disease cfDNA is extracted from blood; however, there is some concern that cfDNA does not contain the same biomarkers as tumor tissue. Tumor tissue is typically removed and stored as formalin-fixed, paraffin-embedded tissue, a process that preserves the morphological structures well but chemically modifies and degrades the nucleic acids. 

Despite the difficulties, FFPE tissue (shown on the left) is often used to look for cancer-associated mutations; however it does not always correlate with the mutations seen in cfDNA. In this poster we present a comparison of matched FFPE and plasma samples to determine how many mutation are seen in both tissues. We also look at where the mutational mismatches appear in the chromosome. We found chromosomal regions have different mismatch rates, and we use this to draw conclusions about the best chromosomal locations for biomarkers. We also look at the different results that can come from using multiple different panels. 

Here we show that sequencing of cfDNA captures the majority of variants that are found when sequencing FFPE DNA. One result shown here is that more indels are identified using cfDNA than with FFPE DNA. This is especially shown here with breast tissue, while this is inconclusive with lung tissue. More samples should be tested before any conclusions can be derived. 

Another interesting finding is the distribution of variants across the genome. There is some indication that variants sequenced using cfDNA is correlated better to the variants found with either cfDNA or FFPE. This result could be used to better understand if there is bias that occurs when sequencing FFPE DNA and where this bias could be from such as cross-linking could be more apparent in parts of chromosomes. 



11. Comparison between mutation profiles of paired whole blood and cfDNA samples. Patel A., Saunders L., Hur A. The American Society of Human Genetics (ASHG). (abstract #1767). Houston, TX. Oct 15, 2019 

Liquid biopsies are increasingly becoming a tool of choice for researching cancer detection and monitoring. Cell-free DNA or cfDNA is simply small fragments of DNA circulating in bodily fluids. It is also known as circulating cell-free DNA (ccfDNA), circulating tumor DNA (ctDNA) and cell free-fetal DNA (cffDNA). Next-Gen Sequencing of cfDNA is coming into maturity as a non-invasive method to identify mutational profiles in many cancer types. 

A big question is how do you separate a germ line variant from a tumor variant. Understanding the difference in a patient sample can give a more thorough understanding of a variant that can be used to study a cancer type. An easy solution is to compare germ line variants from whole blood genomic DNA (gDNA). This would couple easily with plasma sample collection as plasma can be directly separated from a single sample point. 

Here we describe a simple method to isolate both gDNA and cfDNA from a donor blood sample and discuss the automation of both extractions. We show the efficacy of cfDNA as reliable biomarker analysis tool by comparing mutations in cfDNA vs whole blood. The study determines if the difference between tumor and germ line mutations can be established and the limitations. 

Due to larger volumes necessary to extract sufficient concentrations of cfDNA, automation can assist in the extraction. The Apostle MiniMax™ High Efficiency Cell-Free DNA (cfDNA) extraction kit was automated on the Biomek i-Series. It provides equal recovery of cfDNA as a manual extraction with much less hands on-time. The kit used in the study to extract whole blood, GenFind V3, has also been automated on the Biomek i-Series; allowing for reduced hands with the same quality results as a manual extraction. 

Conclusions

• Variants found only in cfDNA could be used as an initial screen for ctDNA analysis

• Apostle MiniMax™ and GenFind V3 can be used together to get a picture of germ line variants and cfDNA, potential ctDNA, variants

• These results show that a holistic view of a cancer subject can be gained by using one sample source, whole blood 



10. Correlation between mutations found in FFPE tumor tissue and paired cfDNA samples. Niccum B., Heath C., Saunders L., Hur A.,Patel A. Association for Molecular Pathology (AMP). (abstract #ST103). Baltimore, MD. November 7-9, 2019 

Liquid biopsies represent a promising area of facilitating cancer research as blood collection is less invasive than tumor biopsies. Cell free DNA (cfDNA) consists of small (150 – 500 bp) DNA fragments that circulate in the blood. cfDNA levels tend to be low in healthy, non-pregnant patients, and increase in patients with cancer, pregnancy, or extensive damage to tissue. cfDNA is believed to be derive mostly from apoptotic cells for which biomarkers for a variety of diseases have been found in cfDNA. 

FFPE tissue is often used to look for cancer-associated mutations despite invasiveness; however it does not always correlate with the mutations seen in cfDNA. In this poster we present a comparison of matched FFPE and plasma samples to determine how many mutation are seen in both tissues. We also look at where the mutational mismatches appear in the chromosome. Different chromosomal regions can have different mismatch rates, and we use this to draw conclusions about the best chromosomal locations for biomarkers. We automated from extraction through sequencing in collaboration with Swift biosciences. 

As cfDNA is extracted from blood, it is a non-invasive way to detect disease; however, there is some concern that cfDNA does not contain the same biomarkers as tumor tissue. Tumor tissue is typically removed and stored as formalin-fixed, paraffinembedded tissue, a process that preserves the morphological structures well but chemically modifies and degrades the nucleic acids. 

Here we show: 

• Sequencing of cfDNA captures the majority of variants that found in sequenced FFPE DNA 

• More indels are identified using cfDNA than with FFPE DNA, especially with breast tissue 

• Distribution of variants across the genome differs when sequencing FFPE DNA 

• More previously identified clinically relevant variants, as identified by the ClinVar database were found when sequencing FFPE DNA 

This study is small and further work should be done using larger data sets to gain more conclusive information. 



9. Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study. Yang et al. Targeted Oncology. 2019;14:719-728. Published: 06 November 2019. 

(Note: Apostle MiniMax technology is used in this clinical study.)

Background  Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations.  Objective  We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC).  Methods  Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples.  Results  We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression.  Conclusions We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC. Trial Registration Clinical Trials.gov identifier: NCT02804100. 



4. Correlation between mutations found in FFPE tumor tissue and paired cfDNA samples. (n=3) Saunders L and Patel A. American Association for Cancer Research (AACR).  (abstract #2233). Atlanta, GA. April 2, 2019 


Liquid biopsies represent a promising area of facilitating cancer research as taking blood is less invasive than tumor biopsies. The cell free DNA (cfDNA) present in the blood includes DNA derived from cancer cells and cancer biomarkers can be detected in the extracted cfDNA. However, cfDNA is a less direct view of what is happening in the tumor, and can have a different genetic profile than the tumor tissue itself. 

Tumor tissue is typically removed and stored as formalin-fixed, paraffin-embedded tissue, a process that preserves the morphological structures well but chemically modifies and degrades the nucleic acids. This tissue is often used to look for cancer- associated mutations despite these difficulties; however, it does not always correlate with the mutations seen in cfDNA. 

In this poster we present a comparison of matched FFPE and plasma samples to determine how many mutations are seen in both tissues. We also look at where the mutational mismatches appear in the chromosome. Different chromosomal regions can have different mismatch rates, and we use this to draw conclusions about the best chromosomal locations for biomarkers. 

Conclusions: 

Both FFPE and cfDNA detect at least two thirds of the observed indels and at least 90% of the observed SNVs. As SNVs are more likely to be found in both tissue types, they are more suitable for biomarker use if looking across different tissues. This was true for all three cancers tested. 

Different regions of the chromosome have different rates of mismatches in mutation detection between plasma and FFPE tissue. The first 10-30%, 40-50%, 60-80%, and 90-100% of the chromosome have the lowest rates of mismatch and provide the best locations for biomarkers detectible in both tissues. Future work will focus in increasing the sample size to further narrow down the areas of the chromosome with the highest likelihood of good mutation detection in both plasma and FFPE. 



Oncology-related Publications (2018)


1. Competitive evolution of NSCLC tumor clones and the drug resistance mechanism of first-generation EGFR-TKIs in Chinese NSCLC patients. Deng et al. Heliyon. VOLUME 4, ISSUE 12, E01031, DECEMBER 01, 2018

Purpose - Although many studies have reported on the resistance mechanism of first-generation EGFR TKIs (1st EGFR TKIs) treatment, large-scale dynamic ctDNA mutation analysis based on liquid biopsy for non-small cell lung cancer (NSCLC) in the Chinese population is rare. Using in-depth integration and analysis of ctDNA genomic mutation data and clinical data at multiple time points during the treatment of 53 NSCLC patients, we described the resistance mechanisms of 1st EGFR TKIs treatment more comprehensively and dynamically. The resulting profile of the polyclonal competitive evolution of the tumor provides some new insights into the precise treatment of NSCLC.

Experimental design - A prospective study was conducted in patients with advanced NSCLC with acquired resistance to erlotinib, gefitinib or icotinib. By liquid biopsy, we detected mutations in 124 tumor-associated genes in the context of drug resistance. These 124 genes covered all tumor therapeutic targets and related biological pathways. During the entire course of treatment, the interval between two liquid biopsies was two months.

Results - Unlike the common mutations tested in tissue samples, our data showed a higher coverage of tumor heterogeneity (32.65%), more complex patterns of resistance and some new resistance mutation sites, such as EGFR p.V769M and KRAS p.A11V. The major resistance-associated mutations detected were still EGFR p.T790M (45.28%), other point mutations in EGFR (33.9%), and KRAS and NRAS mutations (15.09%). These mutation ratios might be considered as a preliminary summary of the characteristics of Chinese patients. In addition, starting from the two baseline mutations of the EGFR gene (19del vs. L858R), we first described the detailed mutation profile of the EGFR gene. Although there was no significant difference in the number of patients with EGFR p.19del and EGFR p.L858R baseline mutations (24% vs. 16%, P = 0.15), patients from the EGFR p.19del baseline group were much more likely to develop EGFR p.T790M resistance mutations (62.1% vs. 19.3%, P = 0.007). Through careful integration of gene mutation information and clinical phenotype information, an interesting phenomenon was found. Although the variant allele fraction (VAF) of the EGFR p.T790M mutation was significantly linearly correlated with that of the EGFR drug-sensitive mutation (r = 0.68, P = 0.00025), neither VAF was associated with the tumor volume at the advanced stage. It was shown that other tumor clones might contribute more to the resistance to 1st EGFR TKIs treatment than tumor clones carrying the EGFR p.T790M mutation when resistance developed. By further analysis, we found that, in some patients, when the primary tumor clones detected were those carrying EGFR−/− mutations (both types the EGFR p.19del/p.L858R and EGFR p.T790M mutation types were missing), most of them showed a poor prognosis and ineffective late treatment, indicating that EGFR−/− played a more important role than EGFR p.T790M in the process of NSCLC drug resistance in these patients. From the perspective of the clonal evolution of NSCLC tumor cells, these phenomena could be explained by the competitive evolution between different tumor clones. In addition, two new mutations, KRAS p.A11V and EGFR p.V769M, emerged significantly during drug resistance in NSCLC patients and had shown obvious competitive clonal evolution characteristics. Combined with clear clinical drug resistance phenotypic information, we believed that these two new mutations might be related to new drug resistance mechanisms and deserve further study. We have also seen an interesting phenomenon. In some patients undergoing 1st EGFR TKIs treatment, the EGFR p.T790M mutation appeared, disappeared, and reappeared, and this spatial and temporal diversity of the EGFR p.T790M mutation was regulated by targeted drug and chemotherapy and was correlated with the individual tumor mutation profile.

Conclusions - The constitution and competitive evolution of the tumor clones have a decisive influence on treatment and can be regulated by targeted drugs and chemotherapy. Additionally, EGFR p.T790M spatial and temporal diversity during treatment warrants more attention, and this spatial and temporal diversity may be useful for the choice of treatment strategies for certain NSCLC patients. Through longitudinal cfDNA sample analysis, the resistance mechanism and dynamic clinical features of Chinese NSCLC patients are systematically established as reliable and meaningful to understand acquired resistance and make further personalized treatment decisions dynamically. Two new potential drug resistance-associated mutations in EGFR and KRAS have been found and are worthy of further study. Finally, our research shows that the evolutionary process of tumor cloning can be artificially regulated and intervened, possibly providing a new way to treat tumors.

(Methods section) - Cell free DNA was isolated using Apostle MiniMaxTM High Efficiency cfDNA Isolation Kit (Standard Edition) according to the manufacturer's protocol.

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