Non‐invasive epigenomic molecular phenotyping of the human brain via liquid biopsy of cerebrospinal fluid and next generation sequencing

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can non-invasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and five presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to non-invasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In five of the nine cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the three patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for non-invasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for non-invasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity.

Download Full-text

A pilot study of next generation sequencing–liquid biopsy on cell-free DNA as a novel non-invasive diagnostic tool for Klippel–Trenaunay syndrome

Vascular ◽

10.1177/1708538120936421 ◽

2020 ◽

pp. 170853812093642

Author(s):

Maria Palmieri ◽

Anna Maria Pinto ◽

Laura di Blasio ◽

Aurora Currò ◽

Valentina Monica ◽

...

Keyword(s):

Next Generation Sequencing ◽

Liquid Biopsy ◽

Somatic Mutations ◽

Causative Role ◽

Next Generation ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Klippel Trenaunay Syndrome ◽

Generation Sequencing

Objectives Somatic mosaicism of PIK3CA gene is currently recognized as the molecular driver of Klippel–Trenaunay syndrome. However, given the limitation of the current technologies, PIK3CA somatic mutations are detected only in a limited proportion of Klippel–Trenaunay syndrome cases and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next generation sequencing liquid biopsy using cell-free DNA has emerged as an innovative non-invasive approach for early detection and monitoring of cancer. This approach, overcoming the space-time profile constraint of tissue biopsies, opens a new scenario also for others diseases caused by somatic mutations. Methods In the present study, we performed a comprehensive analysis of seven patients (four females and three males) with Klippel–Trenaunay syndrome. Blood samples from both peripheral and efferent vein from malformation were collected and cell-free DNA was extracted from plasma. Tissue biopsies from vascular lesions were also collected when available. Cell-free DNA libraries were performed using Oncomine™ Pan-Cancer Cell-Free Assay. Ion Proton for sequencing and Ion Reporter Software for analysis were used (Life Technologies, Carlsbad, CA, USA). Results Cell-free circulating DNA analysis revealed pathogenic mutations in PIK3CA gene in all patients. The mutational load was higher in plasma obtained from the efferent vein at lesional site (0.81%) than in the peripheral vein (0.64%) leading to conclude for a causative role of the identified variants. Tissue analysis, available for one amputated patient, confirmed the presence of the mutation at the malformation site at a high molecular frequency (14–25%), confirming its causative role. Conclusions Our data prove for the first time that the cell-free DNA-next generation sequencing–liquid biopsy, which is currently used exclusively in an oncologic setting, is indeed the most effective tool for Klippel–Trenaunay syndrome diagnosis and tailored personalized treatment.

Download Full-text

A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

Current Bioinformatics ◽

10.2174/1574893615666200324133451 ◽

2020 ◽

Vol 15 ◽

Author(s):

Zheng Jiang ◽

Hui Liu ◽

Siwen Zhang ◽

Jia Liu ◽

Weitao Wang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Microsatellite Instability ◽

Liquid Biopsy ◽

Tumor Tissue ◽

High Sensitivity ◽

Next Generation ◽

Tissue Samples ◽

Next Generation Sequencing Technology ◽

Medication Selection ◽

Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

Download Full-text

670. Rapid, Non-invasive Detection of Invasive Mycoplasma hominis Infection using the Karius Test, A Next-Generation Sequencing Test for Microbial Cell-free DNA in Plasma

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.863 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S390-S390

Author(s):

Priya Edward ◽

William V La Via ◽

Mehreen Arshad ◽

Kiran Gajurel

Keyword(s):

Next Generation Sequencing ◽

Case Series ◽

Mycoplasma Hominis ◽

Microbial Cell ◽

Next Generation ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Hominis Infection ◽

Generation Sequencing

Abstract Background Mycoplasma hominis is typically associated with genital infections in women and is a rare cause of musculoskeletal infections often in immunocompromised hosts. Diagnosis of invasive Mycoplasma hominis infections are difficult due to challenges in culturing these organisms. Molecular diagnostics require an index of suspicion which may not be present at the time of tissue sampling. Accurate, rapid diagnosis of Mycoplasma hominis infections are important for antibiotic management. Methods Two cases of invasive Mycoplasma hominis infections are presented in which the Karius test (KT) was used to make the diagnosis. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell-free DNA (mcfDNA). After mcfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of > 1400 organisms. Organisms present above a statistical threshold are reported. Case review was performed for clinical correlation. Results A young woman with lupus nephritis status post renal transplant developed persistent fever with progressive multifocal culture-negative osteoarticular infection despite empiric ceftriaxone. An adolescent female presented with an ascending pelvic infection progressing to purulent polymicrobial peritonitis (see table) requiring surgical debridement and cefipime, metronidazole and micafungin therapy; her course was complicated by progressive peritonitis/abscesses. Karius testing detected high-levels of Mycoplasma hominis mcfDNA in both cases – at 3251 molecules/microliter (MPM) in the first case and 3914 MPM in the second case. The normal range of Mycoplasma hominis mcfDNA in a cohort of 684 normal adults is 0 MPM. The patients rapidly improved with atypical coverage with doxycycline and levofloxaxin. Clinical findings in 2 patients with M. hominis infection detected by the Karius Test Conclusion Open-ended, plasma-based NGS for mcfDNA provides a rapid, non-invasive method to diagnose invasive Mycoplasma hominis infection. This case series highlights the potential to diagnose infections caused by fastidious pathogens to better inform antimicrobial therapy and achieve favorable outcomes. Disclosures William V. La Via, MD, Karius (Employee)

Download Full-text

Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma

Scientific Reports ◽

10.1038/s41598-021-82266-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Imteyaz Ahmad Khan ◽

Safoora Rashid ◽

Nidhi Singh ◽

Sumaira Rashid ◽

Vishwajeet Singh ◽

...

Keyword(s):

Next Generation Sequencing ◽

Pancreatic Ductal Adenocarcinoma ◽

Early Stage ◽

Ductal Adenocarcinoma ◽

Next Generation ◽

Serum Samples ◽

Tissue Samples ◽

Non Invasive ◽

Specific Symptoms ◽

Generation Sequencing

AbstractEarly-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

Download Full-text