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Circulating Tumor DNA Allele Fraction

A Candidate Biological Signal for Multicancer Early Detection Tests to Assess the Clinical Significance of Cancers
Open AccessPublished:August 07, 2022DOI:https://doi.org/10.1016/j.ajpath.2022.07.007
      Current imaging-based cancer screening approaches provide useful but limited prognostic information. Complementary to existing screening tests, cell-free DNA–based multicancer early detection (MCED) tests account for cancer biology [manifested through circulating tumor allele fraction (cTAF)], which could inform prognosis and help assess the cancer's clinical significance. This review discusses the factors affecting circulating tumor DNA (ctDNA) levels and cTAF, and their correlation with the cancer's clinical significance. Furthermore, it discusses the influence of cTAF on MCED test performance, which could help inform prognosis. Clinically significant cancers show higher ctDNA levels quantified by cTAF than indolent phenotype cancers within each stage because of more frequent mitosis and cell death combined with increased trafficking of cell-free DNA into circulation because of greater vascularization and depth of tumor invasion. cTAF has been correlated with biomarkers for cancer aggressiveness and overall survival; cancers with lower cTAF had better survival when compared with cancers with higher cTAF and with the Surveillance, Epidemiology, and End Results–based survival for that cancer type at each stage. MCED-detected cancers in case-control studies had comparable survival to Surveillance, Epidemiology, and End Results–based survival at each stage. Because many MCED tests use ctDNA as an analyte, cTAF could provide a common metric to compare performance. The prognostic value of cTAF may allow MCED tests to preferentially detect clinically significant cancers at early stages when outcomes are favorable and avoid overdiagnosis.
      Current approaches to cancer screening include imaging-based tests, such as mammography for breast cancer or low-dose computed tomography for lung cancer, and tissue visualization-based tests, such as colonoscopy for colon cancer. These approaches provide useful but limited information on the prognosis of the disease, and they potentially contribute to overdiagnosis of cancer.
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      Just as use of molecular markers has revolutionized cancer treatment decisions, cancer screening technologies could also benefit from the use of molecular biomarkers to inform disease prognosis. As novel biomarkers emerge to select treatment and determine the prognosis of individuals already diagnosed with cancer, the question remains of whether there are biomarkers with prognostic capabilities for early detection tests and how these biomarkers can aid in avoiding overdiagnosis.
      A screening or early detection test should preferably detect cancers in need of a timely treatment decision while avoiding burdening the health care system with the workup of indolent neoplasms. A new paradigm through which this may be achieved is multicancer early detection (MCED) tests, which evaluate analytes, such as circulating tumor DNA (ctDNA), a direct marker for the presence of an invasive malignant process in the body. This review aims to assess whether ctDNA as a biomarker in the screening setting possesses capabilities to characterize cancer similar to biomarkers employed for patient management after disease confirmation, and thus could enhance the utility of cancer screening as we know it. Furthermore, circulating tumor allele fraction (cTAF) is proposed as a metric to compare MCED tests because of its superior prognostic potential than cancer type and/or clinical stage.

      Cancer Biology of ctDNA Release

      Focusing on the biological signals that tumors emit in the body can help design better tests for screening or early detection of cancer. ctDNA is cell-free DNA (cfDNA) that is shed (ie, released) from tumors during cellular apoptosis and necrosis
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      and reaches the circulation. This genetic material in the circulation is packaged into short fragments of on average 165 nucleotides. Not accounting for DNA fragmentation has led to vast underestimation of the ctDNA in cfDNA samples that is available for detection by MCED tests in some studies.
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      and has been found to vary by histologic or molecular tumor subtype, which may be partially explained by the mitotic activities of different subtypes. In addition, trafficking of cfDNA into the circulation depends on the anatomic location of the tumor.
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      This is in part due to barriers present in certain locations, such as the blood-brain barrier for release of cfDNA from brain tumors,
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      and due to potential shedding into systems other than lymph or blood vessels, like airways, intestinal lumen, urinary system, or cerebrospinal fluid. Notably, ctDNA levels do not depend only on the release rate of cfDNA from tumor cells into the tumor microenvironment (TME), but also on presence or absence of transport pathways from the TME into the circulation. Thus, trafficking of cfDNA into the circulation is a distinct process that affects circulating tumor fraction and the detectability of ctDNA in a screening test.
      Circulating tumor fraction represents the fraction of cfDNA in the circulation that originates from a tumor (ie, ctDNA proportion of all cfDNA). ctDNA carries the genomic and epigenetic state of the primary tumor and metastases if present, including fragments that contain tumor-specific somatic small variant and/or abnormal methylation and whose count reflects copy number alterations. Herein, we use the term allele to refer to genetic and epi alleles. Because not all tumor-derived DNA at a mutant locus harbors the tumor allele because of heterozygosity and/or intratumor and intertumor heterogeneity, cTAF is used as a measure of ctDNA signal availability for many cancer detection tests. cTAF is a modeled value that estimates the expected fraction of cfDNA in the circulation that originates from a tumor and contains a tumor-specific allele. Observations of allele frequencies at multiple loci of the tumor genome are combined into a single cTAF estimate. cTAF is therefore an attractive metric to evaluate the performance of an MCED test as it estimates the expected abundance of tumor-specific alleles in a sample. Consequently, the number of tumor-specific features for MCED tests is calculated as the total cfDNA fragments multiplied by cTAF. For example, with an MCED test that scans 105 regions to a depth of 100× per region, a sample with a cTAF of 10−4 would have an expected yield of 1000 tumor-specific alleles. As ctDNA has a short half-life (from 16 minutes to 2.5 hours),
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      tests based on ctDNA capture the status and activity of a tumor close to the time of a blood draw. Figure 1 visualizes the underlying biology that drives ctDNA levels in an individual with cancer and how the resulting set of frequencies of alleles at variant loci drives the estimation of cTAF.
      Figure thumbnail gr1
      Figure 1Origin and fates of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) and their influence on circulating tumor allele fraction (cTAF). Both normal cells (light green) and tumor cells (light purple) shed DNA during cell death (eg, by apoptosis or necrosis). In cancer, tumor cell mitosis increases the amount of DNA that is shed into the tumor microenvironment (TME). In the TME, cfDNA has several fates: it may be digested; phagocytosed; lost into the lumen of the gastrointestinal (GI), pulmonary, or genitourinary tract; or trafficked into the circulation, where it is pooled with cfDNA from other cells in the body. After entering the circulation, cfDNA is subject to further digestion or clearance in the liver, kidney, or spleen. As a result, cfDNA in a blood sample is composed of ctDNA and cfDNA from normal cells that is shed by dying cells and has not been removed by various clearance mechanisms. The most probable cTAF of a blood sample can be estimated by modeling observed allele frequencies (of methylation and/or single-nucleotide variants) at variant loci across the genome, with a high or low cTAF distinguishing high- and low-shedding tumors, respectively.
      Several factors related to tumor biology and the associated TME provide insight into what influences ctDNA levels in blood. First, fast-growing, clinically significant tumors have more cell divisions and therefore generate new cancer DNA at a higher rate. These tumors have cell growth rates that often exceed resource limitations (ie, outgrow vascular supply and further support functions of tumor stroma), which, in turn, limits tumor growth rates.
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      supports that ctDNA shedding can increase with tumor blood flow, perfusion, and angiogenesis, and this provides another mechanistic explanation as to why clinically significant cancers have higher ctDNA levels. As a potential pathophysiological link, increased perfusion and increased vascular permeability increase the flow of interstitial fluid in the TME. Cellular debris from cell death, including cfDNA fragments, can be transported together with such interstitial fluid back into postcapillary venules, terminal ends of lymphatic capillaries, or openings in the tumor neovasculature under development.
      All of these factors together suggest that higher ctDNA levels, and consequently higher cTAF, are associated with clinical significance. Prioritizing such cancers for detection and early treatment may have a positive impact on outcomes in a screened population.

      Multicancer Early Detection Tests and Their Ability to Preferentially Detect Clinically Significant Cancers

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      One such MCED test is already commercially available (https://grail.com/press-releases/grail-presents-interventional-pathfinder-study-data-at-2021-asco-annual-meeting-and-introduces-galleri-a-groundbreaking-multi-cancer-early-detection-blood-test, last accessed July 13, 2022). These MCED tests are intended to be used as a complement to current single-cancer screening tests to detect more cancers and potentially further reduce cancer-related mortality. Because these MCED tests detect abnormal cfDNA presumed to be derived from a tumor, their performance is primarily determined by the availability of ctDNA in a plasma sample, which depends on shedding and trafficking patterns and subsequent cTAF across cancer types and stages. Detection by MCED tests is therefore impacted by the underlying biology of cancer, which drives preferential detection of clinically significant cancers and could potentially inform prognosis.

      Evidence for cTAF as a Biomarker of Clinically Significant Cancers

      A nonlinear relationship between ctDNA detection level and tumor aggressiveness has been suggested in individual cancers thus far.
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      However, additional research is needed to fully understand the differences between different cancer types and to inform clinical use for both single-cancer screening and MCED tests. In breast cancer, there were different levels of ctDNA detected, depending on the molecular tumor subtype,
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      with significantly higher ctDNA in hormone-receptor–negative breast cancers versus hormone-receptor–positive breast cancers at stage II (P < 0.001),
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      and patients with HER2+/estrogen-receptor–negative tumors had higher ctDNA levels than other patients (HER2/estrogen receptor negative, HER2/estrogen receptor positive, HER2+/estrogen receptor positive; P = 0.02).
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      Circulating breast-derived DNA allows universal detection and monitoring of localized breast cancer.
      In multivariate analyses for breast cancer, clinical stage (stage III versus I and IV versus I) and hormone-receptor status were the factors most significantly associated with ctDNA levels and detection of tumor-derived mutations in the blood (P < 0.001 each).
      • Zhou Y.
      • Xu Y.
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      • Zhang Y.
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      • Li P.
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      • Wang J.
      • Xia X.
      • Yang L.
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      • Sun Q.
      Clinical factors associated with circulating tumor DNA in primary breast cancer.
      ,
      • Liu M.C.
      • Carter J.M.
      • Visscher D.W.
      • Kopp K.
      • Shaknovich R.
      • Chen X.
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      • Dong Z.
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      • Fung E.T.
      • Hartman A.-R.
      Blood-based cancer detection in plasma cell-free DNA (cfDNA): evaluating clinical and pathologic tumor characteristics in participants with breast cancer.
      Tumor burden and ctDNA levels were correlated for both localized and metastasized pancreatic ductal adenocarcinoma; pretherapeutic ctDNA levels were associated with shorter disease-free survival in localized cancers, whereas in metastatic pancreatic ductal adenocarcinoma, ctDNA levels were associated with worse overall survival (OS) in contrast to disease-free survival.
      • Kirchweger P.
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      • Wundsam H.
      Circulating tumor DNA correlates with tumor burden and predicts outcome in pancreatic cancer irrespective of tumor stage.
       cTAF has also been associated with OS and/or progression-free survival in various individual cancer types, such as resectable colon cancer, non–small-cell lung cancer, head and neck squamous cell carcinoma, and advanced biliary cancers.
      • Burgener J.M.
      • Zou J.
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      • Bratman S.V.
      Tumor-naïve multimodal profiling of circulating tumor DNA in head and neck squamous cell carcinoma.
      • Hsiehchen D.
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      • Beg M.S.
      Clinical and biological determinants of circulating tumor DNA detection and prognostication using a next-generation sequencing panel assay.
      • Phallen J.
      • Sausen M.
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      • Leal A.
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      • White J.
      • et al.
      Direct detection of early-stage cancers using circulating tumor DNA.
      • Winther-Larsen A.
      • Demuth C.
      • Fledelius J.
      • Madsen A.T.
      • Hjorthaug K.
      • Meldgaard P.
      • Sorensen B.S.
      Correlation between circulating mutant DNA and metabolic tumour burden in advanced non-small cell lung cancer patients.
      • Uson Junior P.L.S.
      • Majeed U.
      • Yin J.
      • Botrus G.
      • Sonbol M.B.
      • Ahn D.H.
      • Starr J.S.
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      • Wylie N.
      • Boyle A.W.
      • Bekaii-Saab T.S.
      • Gores G.J.
      • Smoot R.
      • Barrett M.
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      • Meurice N.
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      • Zhou Y.
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      • Baker A.
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      • Mansfield A.
      • Mody K.
      • Borad M.J.
      Cell-free tumor DNA dominant clone allele frequency (DCAF) is associated with poor outcomes in advanced biliary cancers treated with platinum-based chemotherapy.
      Although the evidence for the relationship between cTAF and cancer aggressiveness in individual cancers is strong, data are mostly limited to specific cancer types. In the context of MCED tests, evidence has been collected to demonstrate how cTAF is related to cancer aggressiveness and OS across cancer types, as well as MCED test performance.
      The Circulating Cell-Free Genome Atlas (CCGA) study, a prospective, case-control, observational study, was conducted to develop and validate an MCED test. CCGA was divided into three sub-studies, each with different objectives. Sub-study 1 identified DNA methylation as the most promising genomic feature for an MCED test.
      • Klein E.
      • Hubbell E.
      • Maddala T.
      • Aravanis A.
      • Beausang J.
      • Filippova D.
      • Gross S.
      • Jamshidi A.
      • Kurtzman K.
      • Shen L.
      • Valouev A.
      • Venn O.
      • Zhang N.
      • Smith D.
      • Yeatman T.
      • Tibshirani R.
      • Williams R.
      • Hartman A.-R.
      • Seiden M.
      • Liu M.
      Development of a comprehensive cell-free DNA (cfDNA) assay for early detection of multiple tumor types: the circulating cell-free genome atlas (CCGA) study.
      Sub-study 2 focused on the development of a targeted methylation assay and training and independent validation of a machine-learning classifier to differentiate cancer/noncancer and predict tissue of origin of the cancer signal.
      • Liu M.C.
      • Oxnard G.R.
      • Klein E.A.
      • Swanton C.
      • Seiden M.V.
      CCGA Consortium
      Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA.
      Sub-study 3 focused on independent validation of a further refined assay and machine learning classifier in a larger population.
      • Klein E.
      • Richards D.
      • Cohn A.
      • Tummala M.
      • Lapham R.
      • Cosgrove D.
      • Chung G.
      • Clement J.
      • Gao J.
      • Hunkapiller N.
      Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set.
      Because CCGA is a case-control study, enrolled participants had a known newly diagnosed cancer status or noncancer status confirmed in multiyear follow-up. However, it still allowed for comparison of cTAF between participants with and without various types of cancer and different clinical presentations and outcomes. Results from different analyses of CCGA are included below.

      cTAF Increases with Aggressiveness and Varies by Orders of Magnitude within Each Cancer Type and Stage

      Samples from CCGA were analyzed for cTAF across stages in multiple cancer types. The results showed that median cTAF increased with stage across cancer types. However, cTAF varied by orders of magnitude within a given cancer type and stage (Figure 2A) (unpublished data). Strong differences in cTAF were observed between cancer types. High-mortality cancers, including esophageal, gastric, hepatobiliary, lung, and pancreatic [bottom 10th percentile of 5-year cancer-specific survival in Surveillance, Epidemiology, and End Results (SEER)], were observed to have higher cTAF than low-mortality cancers, including breast, prostate, and thyroid (top 90th percentile of 5-year cancer-specific survival in SEER) within each stage (Figure 2B) (unpublished data). High/low mortality was defined on the basis of histologic information for lung and esophageal cancers so that cancer subtypes that did not meet the mortality criteria were omitted. Thus, developing an understanding of the range of cTAF in different clinical presentations and outcomes is helpful for establishing and characterizing adequately sensitive screening tests with reference to the underlying cancer biology.
      Figure thumbnail gr2
      Figure 2Distribution of estimated circulating tumor allele fraction (cTAF) by cancer type, stage, and mortality (unpublished data). A: Estimated cTAF varies over orders of magnitude by stage and differs between cancer types. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles, respectively). Each dot represents the cTAF estimate from one participant. Text inserts above each box indicate participants with tumor tissue available and single-nucleotide variants detected in both tissue and cell-free DNA (388 participants)/total participants (644 participants). B: Within stage, deadlier cancers have higher estimated cTAF; using incidence-weighted 5-year survival from Surveillance, Epidemiology, and End Results, cancer types were categorized into high mortality (blue; bottom 10th percentile: esophageal, gastric, hepatobiliary, lung, and pancreas) and low mortality (green; top 90th percentile: breast, prostate, and thyroid). Distribution of estimated cTAF for samples with signal detected within stage are summarized by box plots; each participant was recorded as a dot. The fraction of samples with no signal detected for tumor variants is recorded in boxes at the top. Detection sensitivity of a whole genome methylation–based multicancer early detection (MCED) test at 98% specificity with 95% CIs is reported at the bottom. High-mortality cancers showed higher estimated cTAF than low-mortality cancers within stages II to IV (Kolmogorov-Smirnov test, P < 0.05) and higher MCED test sensitivity within stages I to IV.

      cTAF Influences Performance of Blood-Based cfDNA MCED Tests

      An objective of the CCGA study was to determine the factors that influence MCED test performance. Any classifier whose features are derived from ctDNA-specific attributes should have better performance if more ctDNA with cancer signal is available to analyze (ie, higher cTAF). Indeed, a positive association was observed between detection and ctDNA level among cancer cases. Notably, once cTAF was included as a predictor in a multivariate analysis, cancer type and stage were no longer significant influences on test performance (unpublished data). Because high-mortality cancers have higher median cTAF than low-mortality cancers within each stage, the MCED test developed in the CCGA study preferentially detected high-mortality cancers at each stage (Figure 2B) (unpublished data). This may provide an opportunity to detect high-mortality or clinically significant cancer earlier, when patients can still have favorable outcomes, while avoiding overdiagnosis of indolent disease. Given these characteristics, cTAF is an attractive metric to compare MCED performance between approaches and studies.

      Prognostic Value of cTAF

      Participants from the second CCGA sub-study were followed up for up to 3 years to assess OS and explore how a cancer signal from cfDNA analysis was associated with cancer prognosis across cancer types.
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      Results from this study corroborated findings from unpublished data that higher cTAF was observed in later stages with higher tumor burden, and cancer detection rate increased with cTAF (Figure 3A). Cancers with lower cTAF had better survival compared with cancers with higher cTAF (Figure 3B), which suggests that tumor DNA shedding is a biological factor that is strongly associated with prognosis. Therefore, cTAF could be a useful surrogate biomarker for aggressiveness or clinical significance of cancer across multiple cancer types in addition to clinical stage.
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      In agreement with this inference, cancers not detected by the MCED test (due to low cTAF) had better survival than cancers that were detected; in addition, these nondetected cancers had better survival than expected based on SEER at each stage (Figure 3, C–F). More important, cancers detected by the MCED test had OS comparable to that expected on the basis of SEER at the same stage (Figure 3, C–F), indicating that these cancers could potentially benefit from early detection and thus have favorable outcomes.
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      In a multivariate analysis that included covariates of cancer mortality group (high versus low), method of diagnosis (clinical presentation versus screening), clinical stage (III/IV versus I/II), and age, cancer detection by the MCED test remained a significant predictor of OS (P < 0.0001), suggesting its ability to inform prognosis while avoiding overdiagnosis of indolent cancers.
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      Figure thumbnail gr3
      Figure 3A–F: Association of circulating tumor allele fraction and cancer aggressiveness (A and B) and overall survival by clinical stage in multicancer early detection (MCED) test detected or not detected cancers relative to Surveillance, Epidemiology, and End Results (SEER; C–F). B: Tumor fraction is divided into four quantiles, with 75% to 100% being the highest quantile. C–F: Comparison of Circulating Cell-Free Genome Atlas (CCGA) observed survival versus SEER-based expected survival. Survival curve depicting overall survival for cancer participants of stages I to IV, detected (red) versus not detected (blue) by the MCED test, and CCGA observed (solid) versus SEER-based expected (dashed) survival. (Re-used from Chen et al,
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      2021.)
      For cancers that have existing screening options (eg, breast, lung, and prostate), the MCED test performance was consistent with that observed for all cancers: increased detection in aggressive cancer subtypes, such as hormone-receptor–negative breast cancer and small-cell lung cancer, as well as prostate cancers with high Gleason scores compared with less aggressive cancers with low Gleason scores (P < 0.0001).
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      Thus, an MCED test whose performance is influenced by underlying cancer biology could potentially overcome the issue of overdiagnosis by not detecting cancers with indolent phenotypes.

      Correlation of cTAF with Clinical Biomarkers for Cancer Aggressiveness

      A separate analysis of clinical features of tumor biology in addition to clinical stage (eg, tumor volume and mitotic or metabolic activity and accessibility of tumor DNA to the circulation) was conducted to further understand how cTAF varies and affects cfDNA-based cancer detection.
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      Breast, lung, and colorectal cancers were used in the analysis with the goal of identifying correlates that can generally characterize solid cancers.
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      Clinical correlates of circulating tumor fraction [tumor size, mitotic activity, as reported by %Ki-67–positive (breast cancer), metabolic activity, as reported by positron emission tomography FDG standardized uptake value (lung), and depth of microinvasion (colorectal)], which are clinically established indicators of aggressive tumors, were associated with more frequent detection by the MCED test in the CCGA study.
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      These clinical correlates indicate that faster-growing and deeper-invading tumors have higher cTAF.
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      Consequently, the MCED test was more sensitive for tumors that are associated with higher mortality. The consistent results across lung, breast, and colon cancers may reflect an underlying tumor biology that applies to cancers without current available screening paradigms.
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      In summary, cTAF can be associated with the following: i) presence/absence of clinical biomarkers for cancer aggressiveness,
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      ii) cancer prognosis,
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      and iii) MCED test performance (unpublished data).

      Areas for Future Use of cTAF by MCED Tests

      Several cfDNA-based MCED tests using various genomic approaches are currently in development, with some close to clinical use.
      • Liu M.C.
      • Oxnard G.R.
      • Klein E.A.
      • Swanton C.
      • Seiden M.V.
      CCGA Consortium
      Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA.
      • Klein E.
      • Richards D.
      • Cohn A.
      • Tummala M.
      • Lapham R.
      • Cosgrove D.
      • Chung G.
      • Clement J.
      • Gao J.
      • Hunkapiller N.
      Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set.
      • Cohen J.D.
      • Li L.
      • Wang Y.
      • Thoburn C.
      • Afsari B.
      • Danilova L.
      • et al.
      Detection and localization of surgically resectable cancers with a multi-analyte blood test.
      • Cristiano S.
      • Leal A.
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      • et al.
      Genome-wide cell-free DNA fragmentation in patients with cancer.
      One of them is in fact commercially available (https://grail.com/press-releases/grail-presents-interventional-pathfinder-study-data-at-2021-asco-annual-meeting-and-introduces-galleri-a-groundbreaking-multi-cancer-early-detection-blood-test, last accessed July 13, 2022). Therefore, having a common metric on which to evaluate test performance could be useful for future comparisons of MCED tests. cTAF has been used to define a clinical limit of detection (cLOD) metric that can be used to compare performance across MCED tests (unpublished data).
      • Liu M.C.
      • Jamshidi A.
      • Klein E.A.
      • Venn O.
      • Hubbell E.
      • Beausang J.F.
      • Zhang N.
      • Kurtzman K.N.
      • Hou C.
      • Richards D.A.
      • Yeatman T.
      • Cohn A.L.
      • Thiel D.D.
      • Tummala M.
      • Mcintyre K.
      • Sekeres M.A.
      • Bryce A.H.
      • Seiden M.V.
      • Swanton C.
      Evaluation of cell-free DNA approaches for multi-cancer early detection.
      In the first CCGA sub-study, cLOD of an MCED test was defined as the cTAF at which the probability of detecting a cancer signal was at least 50% while maintaining 98% specificity (unpublished data).
      • Liu M.C.
      • Jamshidi A.
      • Klein E.A.
      • Venn O.
      • Hubbell E.
      • Beausang J.F.
      • Zhang N.
      • Kurtzman K.N.
      • Hou C.
      • Richards D.A.
      • Yeatman T.
      • Cohn A.L.
      • Thiel D.D.
      • Tummala M.
      • Mcintyre K.
      • Sekeres M.A.
      • Bryce A.H.
      • Seiden M.V.
      • Swanton C.
      Evaluation of cell-free DNA approaches for multi-cancer early detection.
      As such, across cancer types and stages, cancers with cTAF above this cLOD will be detected at least half of the time.
      The target cLOD for an MCED test that best balances overdiagnosis versus early cancer detection is not known, and may vary between cancer types. If the cLOD of a test is too low, there exists a risk of overdiagnosis or detecting conditions with low cTAF (ie, indolent neoplasms). Conversely, if the cLOD of a test is not low enough, there exists a risk of underdiagnosis or not detecting cancers that would have benefitted from appropriate treatment and management. Defining the optimal level of detection that is relevant to clinicians and patients without overdiagnosis (hypercancer detection) and underdiagnosis (missing cancer detection) will be useful in preferentially detecting clinically significant cancers that require treatment. In the CCGA study, it was observed that cancers not detected by the MCED test had a better prognosis at each stage than the prognosis expected on the basis of SEER, suggesting that such cfDNA-based MCED tests could potentially overcome the issue of overdiagnosis.
      • Chen X.
      • Dong Z.
      • Hubbell E.
      • Kurtzman K.N.
      • Oxnard G.R.
      • Venn O.
      • Melton C.
      • Clarke C.A.
      • Shaknovich R.
      • Ma T.
      • Meixiong G.
      • Seiden M.V.
      • Klein E.A.
      • Fung E.T.
      • Liu M.C.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      It does not mean that all cancers that were detected by the MCED test have high cancer-specific mortality. In fact, on the basis of modeling studies, early detection of cancers using this same MCED test has predicted stage shift,
      • Hubbell E.
      • Clarke C.A.
      • Aravanis A.M.
      • Berg C.D.
      Modeled reductions in late-stage cancer with a multi-cancer early detection test.
      which can subsequently lead to reduced mortality. Notably, the cancers that were detected followed the mortality rates observed in SEER, whereas the cancers that were not detected had an exceptionally good prognosis.
      The distribution of cTAF in the intended-use population for screening is not yet known and will require assessment in future clinical studies. cTAF may be lower in individuals who are being screened for cancer than in individuals who are symptomatic for cancer or have a known diagnosis of cancer (even if that diagnosis is recent), such as participants of the case-control CCGA study. In the first CCGA sub-study, the cLODs of several different approaches that analyzed whole genome methylation, single-nucleotide variants, or somatic copy number alteration were compared, with whole genome methylation having the lowest cLOD (1.2 × 10−3) in comparison to whole genome somatic copy number alterations, fragmentation profiles, and targeted ultradeep sequencing of short somatic variants (unpublished data).
      • Liu M.C.
      • Jamshidi A.
      • Klein E.A.
      • Venn O.
      • Hubbell E.
      • Beausang J.F.
      • Zhang N.
      • Kurtzman K.N.
      • Hou C.
      • Richards D.A.
      • Yeatman T.
      • Cohn A.L.
      • Thiel D.D.
      • Tummala M.
      • Mcintyre K.
      • Sekeres M.A.
      • Bryce A.H.
      • Seiden M.V.
      • Swanton C.
      Evaluation of cell-free DNA approaches for multi-cancer early detection.
      In the second CCGA sub-study, the whole genome methylation assay was refined to a targeted methylation assay targeting only the most cancer-informative regions of the genome. The associated targeted methylation classifier had an even lower cLOD (1.3 × 10−4) than the whole genome methylation classifier. It is expected that cTAF is lower in the intended-use population (screening population) based on differences reported between participants already diagnosed with cancer and participants prediagnosis in the CCGA study (unpublished data). However, the cLOD of the targeted methylation–based MCED test was about an order of magnitude lower than the whole genome methylation approach, which could allow for cancer detection in the intended-use population. The ability to detect cancer before clinical presentation using this targeted methylation-based MCED test has been demonstrated in the interventional PATHFINDER study.
      • Beer T.M.
      • McDonnell C.H.
      • Nadauld L.
      • Liu M.C.
      • Klein E.A.
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      • Chung K.
      • Lopatin M.
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      • Schrag D.
      Interim results of PATHFINDER, a clinical use study using a methylation-based multi-cancer early detection test.
      In addition, in the exploratory prospective, interventional study, Detecting Cancers Earlier through Elective Mutation-Based Blood Collection and Testing, 26 preclinical cancers in ≈10,000 women, aged 65 to 75 years, with no personal history of cancer and with high adherence to standard-of-care screening were detected using a cfDNA-based MCED test and protein biomarkers.
      • Lennon A.M.
      • Buchanan A.H.
      • Kinde I.
      • Warren A.
      • Honushefsky A.
      • Cohain A.T.
      • et al.
      Feasibility of blood testing combined with PET-CT to screen for cancer and guide intervention.
      Notably, the ability to detect low cTAF cancer cases overcomes issues associated with low ctDNA yield in blood samples for early-stage cancers. Future development of systemized methods for measuring cTAF in different settings for many cancer types will also help standardize comparisons of patient cases and improve biological understanding to reduce overdiagnosis.
      In addition, because cTAF has been shown to correlate with clinical biomarkers of aggressiveness for the most common cancers,
      • Bredno J.
      • Lipson J.
      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      it may be possible to use cTAF to both complement and substitute (when unavailable) the diverse, heterogeneous biomarkers in standard use to inform therapy decisions.
      Competing models exist that differ in their characterization of which cancers are detectable by ctDNA and the impact of MCED on a screening population. One author suggests that detection by cfDNA-based MCED tests depends on the presence of at least micrometastases (unpublished data). However, although the presence of additional tumor cells in a metastasis contributes more ctDNA fragments, a local or regional primary lesion can also be detected before any metastases appear. In addition, a metastasis that is too small to be manifested on imaging is limited in the amount of ctDNA that it can shed. Mitotic activity, lymphovascular invasion, and the histologic type of lung cancer are well known as significant contributors to tumor fraction.
      • Abbosh C.
      • Birkbak N.J.
      • Wilson G.A.
      • Jamal-Hanjani M.
      • Constantin T.
      • Salari R.
      • et al.
      Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.
      When modeling cTAF in lung cancers, only the size and metabolic activity of the primary cancer, but not the presence of tumor-involved lymph nodes, were identified as significant correlates in multivariable regression.
      • Bredno J.
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      • Venn O.
      • Aravanis A.M.
      • Jamshidi A.
      Clinical correlates of circulating cell-free DNA tumor fraction.
      The interventional PATHFINDER study has shown the ability of a targeted methylation-based MCED test to detect cancer before clinical presentation.
      • Beer T.M.
      • McDonnell C.H.
      • Nadauld L.
      • Liu M.C.
      • Klein E.A.
      • Reid R.L.
      • Marinac C.
      • Chung K.
      • Lopatin M.
      • Fung E.T.
      • Schrag D.
      Interim results of PATHFINDER, a clinical use study using a methylation-based multi-cancer early detection test.
      This evidence also disproves models that estimate the feasibility of cancer detection from tumor volume alone,
      • Pons-Belda O.D.
      • Fernandez-Uriarte A.
      • Ren A.
      • Diamandis E.P.
      Prognostic significance of blood-based multi-cancer detection in plasma cell-free DNA.
      as these cannot sufficiently reflect the heterogeneity, varying detectability, and varying clinical significance of neoplasms of the same volume. Such models do not agree with empirical evidence, including prospective interventional studies. Large-scale interventional studies, such as NHS Galleri (N ≈ 140,000; https://www.nhs-galleri.org, last accessed July 13, 2022), are ongoing to better understand the clinical utility of an MCED test and its relationship to cTAF in large screening populations.
      More research is needed on the relationship of cTAF and tumor burden (number of tumor cells), tumor growth and metabolism, metastatic potential, and tumor type, as there are likely cancer types where ctDNA levels depend on anatomic site, histologic type, and further tumor characteristics. In addition, much of the current cancer biology presented herein is representative of solid tumors; the role of cTAF in liquid cancers, like circulating lymphomas and leukemias, may differ because of the predominant hematopoietic origin of cfDNA as well as the access and DNA fragment trafficking conditions from lymph nodes and bone marrow to the circulation.

      Use of cTAF in Other Settings by cfDNA-Based Tests

      cTAF is also the common factor that allows the use of cfDNA tests in the minimal residual disease setting, where detection of putative ctDNA can indicate residual tumor cells after surgery or other interventions, which may lead to cancer recurrence and thus poor outcomes.
      • Abbosh C.
      • Birkbak N.J.
      • Wilson G.A.
      • Jamal-Hanjani M.
      • Constantin T.
      • Salari R.
      • et al.
      Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.
      ,
      • Coombes R.C.
      • Page K.
      • Salari R.
      • Hastings R.K.
      • Armstrong A.
      • Ahmed S.
      • et al.
      Personalized detection of circulating tumor DNA antedates breast cancer metastatic recurrence.
      ,
      • Moding E.J.
      • Nabet B.Y.
      • Alizadeh A.A.
      • Diehn M.
      Detecting liquid remnants of solid tumors: circulating tumor DNA minimal residual disease.
      Caution should be exercised in extrapolating post-treatment minimal residual disease cTAF prognostication to screening populations; ctDNA positivity in a minimal residual disease setting indicates failure of treatment that is not generally appropriate for a screened population, where the individuals do not have a known prior cancer diagnosis and might subsequently have lower cTAF. Nevertheless, there are likely parallels between MCED tests preferentially detecting more clinically significant cancers with higher cTAF and minimal residual disease surveillance tests, which have increasingly higher sensitivity months after surgery or radiation that is consistent with progressive increase in the amount of residual disease over time.
      • Moding E.J.
      • Nabet B.Y.
      • Alizadeh A.A.
      • Diehn M.
      Detecting liquid remnants of solid tumors: circulating tumor DNA minimal residual disease.

      Conclusions

      In this review, we have shown that cTAF is a clinically meaningful biomarker that drives MCED test performance and informs prognosis of cancer. Because detection of cancer by MCED tests is influenced by cTAF, there is the potential for MCED tests to not only detect cancers earlier, but to inform prognosis, reduce overdiagnosis, and inform treatment decisions. Furthermore, cLOD computed against cTAF is an attractive biologically motivated metric to assess and compare MCED test performance.

      Acknowledgments

      We thank Mia DeFino, MS, ELS (DeFino Consulting, LLC, Chicago, IL), and Ruhi Ubale, PhD, CMPP (employee of GRAIL, LLC), for providing medical writing support; Kristi Whitfield (PosterDocs, Oakland, CA) and John Beausang, PhD (employee of GRAIL, LLC), for providing support for figure development; and Erin Spohr (ENGAGE Labs, LLC, Oak Ridge, NJ) for providing copyediting, all paid for by GRAIL, LLC, a subsidiary of Illumina, Inc.

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