Trends in Cancer
Volume 7, Issue 11, November 2021, Pages 995-1009
Journal home page for Trends in Cancer

Review
Technical and biological constraints on ctDNA-based genotyping

https://doi.org/10.1016/j.trecan.2021.06.001Get rights and content

Highlights

  • Circulating tumor DNA (ctDNA) is an emerging source of relevant tumor information across a variety of clinical contexts. In prostate cancer, existing sensitive strategies for early cancer detection and disease monitoring place emphasis on the use of ctDNA to detect treatment-predictive genomic biomarkers in the advanced setting.

  • Multiple technical and biological variables can confound ctDNA-based metastatic cancer genotyping, complicating the integration of ctDNA into routine clinical management strategies.

  • ctDNA fraction (ctDNA%) strongly influences assay detection sensitivity–specificity for different genomic events and is an underappreciated but critical variable in the interpretation of patient results.

  • Copy-number variants (CNVs) are challenging to detect in samples with low ctDNA%, regardless of sequencing approach. Orthogonal methods can modestly improve detection but only to a finite lower limit.

  • Sequencing sample-matched white-blood-cell DNA can improve the characterization of CNVs, assist with estimating ctDNA%, and remove interference from clonal hematopoiesis.

Circulating tumor DNA (ctDNA) enables real-time genomic profiling of cancer without the need for tissue biopsy. ctDNA-based technology is seeing rapid uptake in clinical practice due to the potential to inform patient management from diagnosis to advanced disease. In metastatic disease, ctDNA can identify somatic mutations, copy-number variants (CNVs), and structural rearrangements that are predictive of therapy response. However, the ctDNA fraction (ctDNA%) is unpredictable and confounds variant detection strategies, undermining confidence in liquid biopsy results. Assay design also influences which types of genomic alterations are identifiable. Here, we describe the relationships between ctDNA%, methodology, and sensitivity–specificity for major classes of genomic alterations in prostate cancer. We provide recommendations to navigate the technical complexities that constrain the detection of clinically relevant genomic alterations in ctDNA.

Section snippets

The emerging clinical need for ctDNA profiling in metastatic prostate cancer

Cell-free ctDNA in peripheral blood is an important new biomarker source in oncology. Early detection of plasma ctDNA promises to improve cancer diagnosis, while quantification of ctDNA in people with cancer can prognosticate and monitor the response to therapeutic interventions (Figure 1A,B, Key figure) [1., 2., 3., 4., 5., 6., 7., 8., 9., 10.]. Characterization of genomic alterations in ctDNA is enabling a greater understanding of metastatic disease biology and beginning to help predict drug

Assay design: a tradeoff between breadth and cost

The methodology for cell-free DNA (cfDNA) profiling must match the intended clinical application. Early diagnosis of cancer, the detection of minimal residual disease or treatment response, and the characterization of genomic alteration status are each associated with different optimal approaches that can also differ by cancer type and patient population.

DNA-sequencing approaches can be described by their depth and breadth (Figure 1C). Depth refers to the average redundant read support per

ctDNA% influences results interpretation

Independent of panel design, per-sample ctDNA% is a determinant of sensitivity, specificity, and limits of detection, which may differ for different types of genomic alterations (Figure 2A,B). Accurate ctDNA% estimates are particularly important for distinguishing true from false negatives. True negatives (i.e., the absence of a given alteration despite sufficient ctDNA% to detect the alteration) do not require further confirmatory testing. However, potential false negatives due to low ctDNA%

cfDNA sequencing without a matched normal

Unlike most research tools, clinical assays rarely incorporate patient germline samples into analysis [37,66,67]. Without a matched normal, germline and somatic variants are less easily distinguished [37,66]. Failure to discriminate germline from somatic status is not a major issue for individual variants (e.g., pathogenic BRCA2 mutations), since the clinical relevance of any single-lesion biomarker usually stems from its functional effect. Rather, this failure is liable to reduce the accuracy

Signature approaches

As described above, results of testing for individual mutations or CNVs may be unreliable. However, alterations to some classes of genes are associated with patterns of genome-wide ‘scarring’ (i.e., mutation signatures), creating orthogonal opportunities for detection [83,84]. For example, signatures of HRR deficiency may indicate vulnerability to PARP inhibitors, while signatures of defective DNA mismatch repair are a putative predictive biomarker for immune-checkpoint inhibitor response [40,47

Concluding remarks

In metastatic cancers with expanding therapeutic options, ctDNA screening offers a practical strategy to determine genomic biomarker status. Despite the excitement, the integration of ctDNA testing into routine clinical care for people with metastatic prostate or other cancers is encumbered by technical and biological complexities (Box 3). In particular, low ctDNA% is common and magnifies the known technical limitations of sequencing and bioinformatic methods, which are usually less relevant in

Declaration of interests

A.W.W. has served on advisory boards and/or received honoraria from AstraZeneca, Astellas, Janssen, and Merck. A.W.W.’s laboratory has contract research agreements with Janssen and ESSA Pharma. C.H. has no interests to declare.

References (83)

  • A. Jayaram

    Plasma tumor gene conversions after one cycle abiraterone acetate for metastatic castration-resistant prostate cancer: a biomarker analysis of a multi-center international trial

    Ann. Oncol.

    (2021)
  • F. Favero

    Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data

    Ann. Oncol.

    (2015)
  • M.I. Hosen

    Urinary TERT promoter mutations are detectable up to 10 years prior to clinical diagnosis of bladder cancer: evidence from the Golestan Cohort Study

    EBioMedicine

    (2020)
  • C. Herberts

    Activating AKT1 and PIK3CA mutations in metastatic castration-resistant prostate cancer

    Eur. Urol.

    (2020)
  • C.I. Müller

    Rare mutations of the PIK3CA gene in malignancies of the hematopoietic system as well as endometrium, ovary, prostate and osteosarcomas, and discovery of a PIK3CA pseudogene

    Leuk. Res.

    (2007)
  • A.H.M. Reid

    Novel, gross chromosomal alterations involving PTEN cooperate with allelic loss in prostate cancer

    Mod. Pathol.

    (2012)
  • D.A. Quigley

    Genomic hallmarks and structural variation in metastatic prostate cancer

    Cell

    (2018)
  • R.L. Bowman

    Clonal hematopoiesis and evolution to hematopoietic malignancies

    Cell Stem Cell

    (2018)
  • I. Garcia-Murillas

    Assessment of molecular relapse detection in early-stage breast cancer

    JAMA Oncol.

    (2019)
  • M. Annala

    Circulating tumor DNA genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer

    Cancer Discov.

    (2018)
  • J.J. Chabon

    Integrating genomic features for non-invasive early lung cancer detection

    Nature

    (2020)
  • X. Chen

    Non-invasive early detection of cancer four years before conventional diagnosis using a blood test

    Nat. Commun.

    (2020)
  • E. Christensen

    Early Detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma

    J. Clin. Oncol.

    (2019)
  • T. Reinert

    Analysis of plasma cell-free DNA by ultradeep sequencing in patients with stages I to III colorectal cancer

    JAMA Oncol.

    (2019)
  • R.C. Coombes

    Personalized detection of circulating tumor DNA antedates breast cancer metastatic recurrence

    Clin. Cancer Res.

    (2019)
  • H. Luo

    Circulating tumor DNA methylation profiles enable early diagnosis, prognosis prediction, and screening for colorectal cancer

    Sci. Transl. Med.

    (2020)
  • T. Sumiyoshi

    Clinical implications of genomic alterations in metastatic prostate cancer

    Prostate Cancer Prostatic Dis.

    (2021)
  • J. de Bono

    Olaparib for metastatic castration-resistant prostate cancer

    N. Engl. J. Med.

    (2020)
  • W. Abida

    Rucaparib in men with metastatic castration-resistant prostate cancer harboring a BRCA1 or BRCA2 gene alteration

    J. Clin. Oncol.

    (2020)
  • W.T. Lowrance

    Advanced prostate cancer: AUA/ASTRO/SUO guideline part I

    J. Urol.

    (2021)
  • J.L. Mohler

    Prostate cancer, version 2.2019, NCCN clinical practice guidelines in oncology

    J. Natl Compr. Canc. Netw.

    (2019)
  • J. Armenia

    The long tail of oncogenic drivers in prostate cancer

    Nat. Genet.

    (2018)
  • D. Gonzalez

    Practical considerations for optimising homologous recombination repair mutation testing in patients with metastatic prostate cancer

    J. Pathol. Clin. Res.

    (2021)
  • A.W. Wyatt

    Concordance of circulating tumor DNA and matched metastatic tissue biopsy in prostate cancer

    J. Natl Cancer Inst.

    (2017)
  • B. De Laere

    TP53 outperforms other androgen receptor biomarkers to predict abiraterone or enzalutamide outcome in metastatic castration-resistant prostate cancer

    Clin. Cancer Res.

    (2019)
  • J.S. de Bono

    Randomized Phase II study evaluating Akt blockade with ipatasertib, in combination with abiraterone, in patients with metastatic prostate cancer with and without PTEN loss

    Clin. Cancer Res.

    (2019)
  • R.B. Corcoran et al.

    Application of cell-free DNA analysis to cancer treatment

    N. Engl. J. Med.

    (2018)
  • R.J. Leary

    Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing

    Sci. Transl. Med

    (2012)
  • C. Bettegowda

    Detection of circulating tumor DNA in early- and late-stage human malignancies

    Sci. Transl. Med.

    (2014)
  • A.D. Choudhury

    Tumor fraction in cell-free DNA as a biomarker in prostate cancer

    JCI Insight

    (2018)
  • A. Romanel

    Plasma AR and abiraterone-resistant prostate cancer

    Sci. Transl. Med.

    (2015)
  • Cited by (0)

    View full text