. 2021 Jun 1;13(1):98.

doi: 10.1186/s13073-021-00912-z.

Detection of cryptogenic malignancies from metagenomic whole genome sequencing of body fluids

Wei Gu^{1

2

3

4}, Eric Talevich⁵, Elaine Hsu⁶, Zhongxia Qi⁶, Anatoly Urisman⁷, Scot Federman^{6

8}, Allan Gopez^{6

8}, Shaun Arevalo^{6

8}, Marc Gottschall⁶, Linda Liao⁹, Jack Tung¹⁰, Lei Chen⁹, Harumi Lim⁹, Chandler Ho⁹, Maya Kasowski¹⁰, Jean Oak^{10

9}, Brittany J Holmes^{10

9}, Iwei Yeh⁷, Jingwei Yu⁶, Linlin Wang⁶, Steve Miller^{6

8}, Joseph L DeRisi^{11

12}, Sonam Prakash⁶, Jeff Simko^#⁷, Charles Y Chiu^#^{13

14

15}

Affiliations

¹ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, 94107, USA. weigu@stanford.edu.
² UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA. weigu@stanford.edu.
³ Department of Pathology, Stanford University, Stanford, CA, 94305, USA. weigu@stanford.edu.
⁴ Stanford Health Care, Stanford University, Stanford, CA, 94305, USA. weigu@stanford.edu.
⁵ DNANexus, Mountain View, CA, 94040, USA.
⁶ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, 94107, USA.
⁷ Department of Pathology, University of California San Francisco, San Francisco, CA, 94107, USA.
⁸ UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA.
⁹ Stanford Health Care, Stanford University, Stanford, CA, 94305, USA.
¹⁰ Department of Pathology, Stanford University, Stanford, CA, 94305, USA.
¹¹ Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, 94107, USA.
¹² Chan Zuckerberg Biohub, San Francisco, CA, 94107, USA.
¹³ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, 94107, USA. charles.chiu@ucsf.edu.
¹⁴ UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA. charles.chiu@ucsf.edu.
¹⁵ Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, 94107, USA. charles.chiu@ucsf.edu.

^# Contributed equally.

PMID: 34074327
PMCID: PMC8167833
DOI: 10.1186/s13073-021-00912-z

Detection of cryptogenic malignancies from metagenomic whole genome sequencing of body fluids

Wei Gu et al. Genome Med. 2021.

. 2021 Jun 1;13(1):98.

doi: 10.1186/s13073-021-00912-z.

Authors

Affiliations

¹ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, 94107, USA. weigu@stanford.edu.
² UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA. weigu@stanford.edu.
³ Department of Pathology, Stanford University, Stanford, CA, 94305, USA. weigu@stanford.edu.
⁴ Stanford Health Care, Stanford University, Stanford, CA, 94305, USA. weigu@stanford.edu.
⁵ DNANexus, Mountain View, CA, 94040, USA.
⁶ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, 94107, USA.
⁷ Department of Pathology, University of California San Francisco, San Francisco, CA, 94107, USA.
⁸ UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA.
⁹ Stanford Health Care, Stanford University, Stanford, CA, 94305, USA.
¹⁰ Department of Pathology, Stanford University, Stanford, CA, 94305, USA.
¹¹ Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, 94107, USA.
¹² Chan Zuckerberg Biohub, San Francisco, CA, 94107, USA.
¹³ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, 94107, USA. charles.chiu@ucsf.edu.
¹⁴ UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, 91407, USA. charles.chiu@ucsf.edu.
¹⁵ Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, 94107, USA. charles.chiu@ucsf.edu.

^# Contributed equally.

PMID: 34074327
PMCID: PMC8167833
DOI: 10.1186/s13073-021-00912-z

Abstract

Background: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens.

Methods: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31).

Results: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid.

Conclusions: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.

PubMed Disclaimer

Conflict of interest statement

ET was employed by DNAnexus, Inc. during the duration of the study and by Karius, Inc. prior to publication. The remaining authors declare that they have no competing interests.

Figures

**Fig. 1**
A Schematic of the bioinformatics pipeline. After whole genome sequencing of cell-free DNA from body fluids, adapter sequences are trimmed and aligned to the human genome. The cancer pipeline aligns human reads and counts reads over moving windows across the human genome [12, 17]. The microbial pipeline aligns non-human reads to a microbial database, taxonomically classifies the microbial aligned reads, and identifies pathogens [2, 18]. B Sample type composition of the 205 body fluid samples. C Contingency table comparing conventional cancer detection to sequencing in patients with malignancy. Negative controls did not have a history of cancer and were explained by infections with positive microbiological testing (top). Patients with cancer detected by positive cytology and/or flow cytometry testing of body fluid (bottom). Patients diagnosed with cancer but with negative or ambiguous detection based on conventional clinical testing in the same fluid by cytology or cytometry. D Detection accuracy and tumor fractions. Detection of malignancies through CNV detection in 2 cancer-positive case categories described in C. The “New” category refers to samples collected from patients with a new diagnosis who have no previous cancer history and have not been treated. Tumor fractions were estimated through the magnitude of copy changes detected (see online Methods, “Equation 1”)

**Fig. 2**
Patient PC63 showing biopsies, mNGS pathogen/CNV results, and orthogonal confirmation. A Schematic of the biopsies performed. B–D Histology of the bone marrow, paratracheal lymph node, and lung wedge biopsy show increased eosinophils (arrowheads) that were morphologically normal and indistinguishable from reactive and benign eosinophils. E Bacterial profile from mNGS testing. No viral, fungal, or parasitic pathogens were detected. F Copy number plotting across the human genome derived from metagenomic sequencing data. Six chromosomal scale deletions and duplications are identified, 3 of which accounted for > 90% of the human DNA content. G FISH (fluorescence in situ hybridization) of wedge biopsy, confirming presence of matching clonal complex cytogenetics to BAL fluid in F. Scale bar, 5 μm. H Bone marrow biopsy, confirming presence of matching clonal complex cytogenetics to BAL fluid in F

See this image and copyright information in PMC

References

1. Wilson MR, Sample HA, Zorn KC, Arevalo S, Yu G, Neuhaus J, Federman S, Stryke D, Briggs B, Langelier C, Berger A, Douglas V, Josephson SA, Chow FC, Fulton BD, DeRisi JL, Gelfand JM, Naccache SN, Bender J, Dien Bard J, Murkey J, Carlson M, Vespa PM, Vijayan T, Allyn PR, Campeau S, Humphries RM, Klausner JD, Ganzon CD, Memar F, Ocampo NA, Zimmermann LL, Cohen SH, Polage CR, DeBiasi RL, Haller B, Dallas R, Maron G, Hayden R, Messacar K, Dominguez SR, Miller S, Chiu CY. Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. N Engl J Med. 2019;380(24):2327–2340. doi: 10.1056/NEJMoa1803396. - DOI - PMC - PubMed
1. Miller S, Naccache SN, Samayoa E, Messacar K, Arevalo S, Federman S, et al. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Genome Res. 2019; Available from: http://genome.cshlp.org/content/early/2019/03/07/gr.238170.118. [cited 2019 May 31]. - PMC - PubMed
1. Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, Kawli T, Christians FC, Venkatasubrahmanyam S, Wall GD, Cheung A, Rogers ZN, Meshulam-Simon G, Huijse L, Balakrishnan S, Quinn JV, Hollemon D, Hong DK, Vaughn ML, Kertesz M, Bercovici S, Wilber JC, Yang S. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. 2019;4(4):663–674. doi: 10.1038/s41564-018-0349-6. - DOI - PubMed
1. Goggin KP, Gonzalez-Pena V, Inaba Y, Allison KJ, Hong DK, Ahmed AA, et al. Evaluation of plasma microbial cell-free DNA sequencing to predict bloodstream infection in pediatric patients with relapsed or refractory cancer. JAMA Oncol. 2019; Available from: https://jamanetwork.com/journals/jamaoncology/fullarticle/2757390. [cited 2020 Jan 4] - PMC - PubMed
1. Thoendel MJ, Jeraldo PR, Greenwood-Quaintance KE, Yao JZ, Chia N, Hanssen AD, Abdel MP, Patel R. Identification of prosthetic joint infection pathogens using a shotgun metagenomics approach. Clin Infect Dis. 2018;67(9):1333–1338. doi: 10.1093/cid/ciy303. - DOI - PMC - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Medical
- MedlinePlus Health Information

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Detection of cryptogenic malignancies from metagenomic whole genome sequencing of body fluids

Affiliations

Detection of cryptogenic malignancies from metagenomic whole genome sequencing of body fluids

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Medical