cfDNA and ctDNA as Oncologic Markers: Comparison
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Please note this is a comparison between Version 2 by Lindsay Dong and Version 1 by Jonathan Dao.

The detection of circulating tumor DNA (ctDNA) in liquid biopsy samples as an oncological marker is being used in clinical trials at every step of clinical management. As ctDNA-based liquid biopsy kits are developed and used in clinics, companies work towards increased convenience, accuracy, and cost over solid biopsies and other oncological markers. The technology used to differentiate ctDNA and cell-free DNA (cfDNA) continues to improve with new tests and methodologies being able to detect down to mutant allele frequencies of 0.001% or 1/100,000 copies. 

  • liquid biopsy
  • cfDNA
  • ctDNA
  • cancer screening
  • complementary diagnostic

1. Introduction

Screening and early diagnosis are essential strategies to decrease mortality, treatment cost, and disease burden of cancer [1]. Diagnosis of various cancers at earlier stages increases five-year survival and cure rate, and diagnosis at later stages has dramatically higher medical costs [2,3][2][3]. After diagnosis, determining prognosis and molecular profiling plays a central role in patient management, especially as precision medicine becomes the status quo of treatment selection [4,5][4][5].
As described by the U.S. Preventive Services Task Force (USPSTF), active screening methods, such as mammograms for breast cancer, pap smears for cervical cancer, or colonoscopy for colorectal cancer, have been an effective means of early detection in older patients [6]. Mammograms prevent 21 deaths per 10,000 women screened over 10 years, pap smears prevent up to 8.34 deaths per 1000 women screened to the age of 65, and colonoscopies prevent up to 28 deaths per 1000 adults screened from the age of 45 to 65 [7,8,9][7][8][9]. Additionally, low-dose computed tomography is recommended as a screening method for lung cancer in patients aged 50–80 years who have a 20 pack-year smoking history and currently smoke or have quit within the past 15 years which would prevent 503 deaths per 100,000 adults over a lifetime of screening. Even with these screening methods, limited sensitivity, specificity, and low incidence of cancer keeps them from being recommended to healthy individuals, and each method only screens for one cancer type [6]. 
Liquid biopsy (LB) is a technique that is being studied at every step of cancer management for screening, minimal residual disease (MRD), monitoring for recurrence in the adjuvant setting, and treatment selection for advanced cancer. LB mostly uses peripheral blood, but can also include other body fluids, such as urine, saliva, feces, pleural effusions, ascites, and cerebrospinal fluid (CSF) [14][10]. LB methods have been used to detect circulating tumor DNA (ctDNA), RNA (ctRNA), circulating tumor cells (CTCs), microRNA (miRNA), extracellular vesicles (EVs), and tumor-educated platelets (TEPs). As a method of early detection, LB can be used to screen for multiple cancers at once, becoming more cost-effective than the array of screening methods used currently [15][11]. For use in diagnosis, LB has several advantages over solid tissue biopsy. LBs are non-invasive, samples are easier to obtain, and result in faster turnaround time [16][12]. In addition, LB can be performed repeatedly to monitor for response and progression and achieve better detail of the tumor’s spatial heterogeneity [10,17][13][14]. However, LB does not allow for histological evaluation, which would require a solid biopsy, and most panels have a limited number of genes. While there are some FDA-approved LB tests for prognosis and treatment selection, such as CellSearch, cobas EGFR Mutation Test v2, Guardant360 CDx, and FoundationOne Liquid CDx [18][15], LB tests for screening and diagnosis are still being clinically validated [4,17][4][14].

2. Circulating Free DNA

Circulating cell-free DNA (cfDNA) are extracellular fragments of dsDNA between 120–220 bp long, centered around 167 bp, which is associated with the nucleosome pattern of cfDNA in apoptosis [19][16]. cfDNA has a short half-life that varies from 4 min to 2 h, which lends itself to applications in monitoring. cfDNA can be found in various body fluids, such as blood, urine, or cerebrospinal fluid [20,21,22,23][17][18][19][20]. Under normal conditions, cfDNA can come from apoptosis, neutrophil extracellular traps (NETs), and erythroblast enucleation [24,25,26][21][22][23]. In plasma, cfDNA originates from granulocytes (32%), erythrocyte progenitors (30%), lymphocytes (12%), monocytes (11%), vascular endothelial cells (9%), and hepatocytes (1%) [27][24]. The cfDNA can be increased in normal physiological processes, such as physical exercise, or in pathological processes that increase cell death, such as inflammation, sepsis, or myocardial infarction [28,29,30,31][25][26][27][28]. The ctDNA is the fraction of cfDNA that originates from tumor cells, which comes from three sources: apoptosis, necrosis, and active secretion. While ctDNA can come from apoptosis with fragment lengths similar to healthy patients, ctDNA is more fragmented or shorter than cfDNA [20,32,33][17][29][30]. In necrosis, chromatin does not fragment in a nucleosome pattern but is cleaved at random generating fragments of various sizes, which contributes to both shorter and longer fragments of >10,000 bp [34][31]. While apoptosis and necrosis are straightforward answers to the source of ctDNA, the concentration of cfDNA cannot be fully explained by the death of tumor cells [35][32]. However, the exact mechanisms of the actively secreted DNA are not fully understood with possible mechanisms being exosomes or amphisomes [36,37][33][34].

3. Total cfDNA Concentration

In healthy individuals, the concentration of cfDNA in plasma is between 0–10 ng/mL with the serum concentration of cfDNA being 10 times higher [38,39,40][35][36][37]. Most cfDNA in serum concentration comes from the process of clotting in the collection tube, which makes plasma better for clinical applications [41,42][38][39]. In patients with cancer, the concentration of cfDNA in plasma can be from 0 to over 1000 ng/mL [39,40,43,44][36][37][40][41]. This varies amongst cancer types. For instance, patients with gliomas had less ctDNA than other solid tumors, such as the pancreas, colon, breast, or ovary [45][42]. The concentration of cfDNA also varies with stage, where metastatic cancers have more cfDNA followed by locally advanced and then localized cancers [40][37]. A major challenge in the implementation of cfDNA in clinical use is the variance from the lack of standardization in methodology, such as collecting samples from serum versus plasma, extracting cfDNA with different kits, measuring samples using different techniques, time of collection, and timing for response [36,46,47][33][43][44]. Total cfDNA concentration has been studied extensively for its application as an early detection biomarker. For instance, a meta-analysis showed that using cfDNA concentration for diagnosis of lung cancer yielded a pooled sensitivity of 80% and specificity of 77% [36][33]. A study on differentiating prostate cancer from benign hyperplasia showed a sensitivity of 73.2% and specificity of 72.7% using cfDNA concentration, which aligns with a more recent study stating the low sensitivity and specificity result in poor diagnostic utility [37,48][34][45]. However, total cfDNA concentration could be an effective measure for prognosis determination and patient monitoring. Tumor markers (e.g., PSA, CEA, CA-125, etc.) are used in prognosis determination or monitoring during/after treatment for patients with cancer. In pancreatic cancer, the American Society of Clinical Oncologists (ASCO) guidelines recommend that CA19-9 in serum and CT are used for monitoring during treatment [51][46]. However, CA19-9 is not recommended for prognostic prediction of outcomes, and 5–10% of the population are Lewis antigen negative, with little to no secretion of CA19-9 [52,53][47][48]. In prostate cancer, PSA continues to be a mainstay in screening, prognosis, and monitoring after treatment in conjunction with other modalities [54,55][49][50]. However, the use of PSA may be associated with overdiagnosis and overtreatment for indolent cancers [56][51].  In a study of 74 patients with advanced/metastatic pancreatic cancer, high levels of total cfDNA were associated with new distant metastasis (NDM) with 91% sensitivity and 95% specificity. Researchers also found that total cfDNA concentration was associated with worse progression-free survival (PFS) and overall survival (OS). In two cases, they found that cfDNA was elevated in the first month after treatment of chemotherapy while CA19-9 levels were below detection prior to detection of NDM on CT scans [59][52]. For prostate cancer, in a meta-analysis of 23 studies, total cfDNA concentration was found to be a poor diagnostic tool as previously discussed, but total cfDNA had similar prognostic value to PSA for PFS and OS [61][53]. Interestingly, PSA and cfDNA were independent of each other, and the combination of the two increased the discrimination between indolent vs. lethal prostate cancer [61][53]. In non-small cell lung cancer (NSCLC), a meta-analysis of 22 studies found that patients with elevated cfDNA concentration tend to have shorter PFS and OS [66][54]. A study within that meta-analysis of 218 patients found that individuals in the top third of cfDNA concentration before treatment showed a significantly shorter PFS and OS than patients in the lower two-thirds. However, cfDNA concentration during or after treatment did not have any association with the response to treatment [70][55].  Overall, total cfDNA concentration has promise as a biomarker for prognosis in cancers discussed above, often being as accurate or better than serum protein biomarkers currently used for pancreatic, prostate, and breast cancer [59,61,62][52][53][56]. The meta-analysis for CRC noted that due to a lack of CEA measurements comparative conclusions cannot be made, but cfDNA could be a viable prognostic marker [64][57]. Total cfDNA also showed promise in NSCLC as a prognostic marker [66,67,70][54][55][58]. 

4. ctDNA

While cfDNA can be increased in healthy patients for various reasons, ctDNA detection is more specific to tumors. It has been established that ctDNA matches sequencing from tumor tissue, with the concordance of mutations in ctDNA and tumor tissue greater than 60–80% in various cancers [45,74,75,76,77,78,79][42][59][60][61][62][63][64]. Mutations are sequenced from ctDNA through targeted analysis or whole genome sequencing. The ctDNA can also be differentiated from cfDNA through analysis of aberrant methylation.

4.1. Techniques

Targeted sequencing looks for specific gene mutations or rearrangements that are common in a particular tumor type, which requires prior knowledge of the region of interest to design the proper assay. Generally, targeted sequencing uses PCR-based methods, such as droplet digital PCR (ddPCR), beads, emulsions, amplification, magnetics (BEAMing), and amplification refractory mutation system (ARMS) qPCR. In ddPCR, sample DNA molecules are separated into droplets, which are amplified by end-point PCR. Then, using fluorescent probes, positive and negative reactions are quantified where the copy number of target DNA is quantified by comparing the two [80][65]. The analytical sensitivity of ddPCR was 0.001%, detecting 1 mutant copy in 100,000 wild-type copies [80][65]. With BEAMing, sample DNA is separated onto beads with common primers attached, which are emulsified in oil. The strands attached to primers are then amplified, centrifuged, and collected with a magnet [81][66]. The beads can then be analyzed for mutations with fluorescent probes and flow cytometry, which can detect target DNA with a sensitivity of 0.01% (1/10,000 copies) [81,82,83][66][67][68]. In ARMS qPCR, target DNA is amplified with primers that are complementary to the mutant sequence. If the primer does not anneal properly then extension does not occur, with qPCR detecting the extension of target DNA [84][69]. ARMS qPCR can detect target DNA with a sensitivity of 0.1% (1/1000 copies) [85][70]. There are also methods that apply next-generation sequencing (NGS), which has higher throughput than PCR, to a target region, such as Targeted Error Correction Sequencing (TEC-Seq), Tagged-Amplicon deep sequencing (TAm-Seq), Safe-Sequencing System (Safe-SeqS), CAncer Personalized Profiling by deep sequencing (CAPP-Seq), or Personalized Analysis of Rearranged Ends (PARE). TEC-Seq uses primers with predetermined barcodes for targeted capture of multiple regions and deep sequencing of captured DNA fragments. TEC-Seq was able to detect 100% and 89% of mutations present at 0.2% and 0.1% mutation allele frequency (MAF) respectively [40][37]. TAm-Seq uses specialized primers to amplify regions of interest, identifying mutations of 2% MAF with a sensitivity of over 97% [87][71]. Safe-SeqS adds a unique identifier (UID), which is able to detect target DNA with a sensitivity of 0.05% (1/2000) [88,89][72][73] CAPP-Seq combines optimized library preparation with bioinformatics to create a “selector”, which reflects recurrent mutations. The selector is applied to tumor DNA to identify the patient’s mutations, and then applied to ctDNA for quantification, detecting target DNA down to 0.02% (1/5000 copies) MAF [90][74].  DNA from tumors also have aberrant DNA methylation, which occurs early during tumorigenesis, provides information on the tumor’s origin, and are homogenous across populations [27,92,93][24][75][76]. Almost every tumor type is characterized by progressive CpG-island-specific hypermethylation and global CpG hypomethylation [94][77]. The GRAIL test uses whole genome bisulfite sequencing (WGBS) targeted at over 100,000 methylation regions combined with machine learning to detect cancer and predict tissue of origin (TOO) localization detecting ctDNA down to 0.023% (~1/5000 copies) [95,96][78][79]. The OverC test uses an altered WGBS technique called enhanced linear splinter amplification sequencing (ELSA-seq), which mitigates the damage to DNA caused by bisulfite treatment and is able to detect ctDNA down to 0.02% (1/5000) [97,98][80][81]. 

4.2. Early Detection/Screening

Multiple tests have been developed for the purposes of screening or early detection of cancer in asymptomatic patients. Some tests, such as Epi proColon® and Bluestar Genomics’ 5hmC Assay, are geared towards detecting a single type of cancer, and others, such as CancerSeek, Galleri™, and OverC MCD Assay, are used as multi-cancer early detection tests. Overall, the goal of any screening test is to have high sensitivity in the early stages, specificity to limit false positives and lower costs for patient adherence. Currently, the only FDA premarket-approved liquid biopsy test used for screening is the Epi proColon®, which can be offered to patients who are unwilling or unable to be screened by other recommended methods. It is performed by detecting methylated SEPT9 DNA using real-time PCR and a fluorescent probe [107][82]. Epi proColon® cites three clinical validation trials that led to the FDA’s premarket approval: a multi-center study of 1544 patients across Germany and the U.S. comparing Epi proColon® to colonoscopies for screening of CRC, which achieved a sensitivity of 68% and a specificity of 80% [118][83]; a study of 290 patients comparing Epi proColon® with a fecal immunochemical test (FIT) found the Epi proColon® test was statistically non-inferior to FIT. The sensitivity for CRC detection was 73.3% for Epi proColon versus 68.0% for FIT. Specificity was 81.5% for Epi proColon and 97.4% for FIT respectively [119][84]; and finally, a small two-site randomized controlled trial of 413 patients comparing the adherence to the Epi proColon® blood test, which had significantly higher uptake than FIT (99.5% vs. 88.1%) [128][85]. Other meta-analyses of 8643 and 2271 patients respectively, evaluating the diagnostic Epi proColon® test agree that it has high diagnostic value for CRC, especially with different algorithms, symptomatic patients, and patients with low compliance [129,130][86][87]. While the USPSTF acknowledges that there has been more evidence for the effectiveness of the Epi proColon® test, the USPSTF recommendation does not include serum tests “because of limited available evidence”, and more research is needed to continue evaluating the accuracy and effectiveness of these tests [131,132][88][89].  The Bluestar Genomics’ 5hmC Assay is another test used for single cancer type detection of FDA interest, receiving the Breakthrough Device designation in 2021 [114][90]. In 2020, Bluestar Genomics compared 64 patients with pancreatic ductal adenocarcinoma (PDAC) to 243 patients without cancer and demonstrated the ability to differentiate and classify the methylation patterns of PDAC [134][91]. Since then, a pre-print article from 2021 used 89 patients with PDAC and 596 control patients to generate a methylation library, and the predictive model was validated against 79 patients with PDAC, 506 patients with other types of cancer, and 163 patients without cancer. When used with the validation patients, the assay achieved an overall sensitivity of 51.9% and specificity of 100.0%, and for patients with new-onset diabetes, the assay achieved a sensitivity of 55.2% and specificity of 98.4% [120][92].  The latest ctDNA-based screening test to be announced as receiving the FDA Breakthrough Device designation is Burning Rock’s OverC MCD blood test early in 2023 [117][93]. The OverC assay uses a technique called ELSA-seq, which is a type of WGBS, with increased yield, methylome coverage, and reproducibility [97][80]. Taking this technique, a prospective multicenter study (PROMISE, NCT04972201) compared and combined cfDNA methylation, cfDNA mutation, and microRNA expression assays in the early detection of 9 cancers. Participants were split into training and test sets with 981 and 492 patients respectively. The methylation model performed the best out of the three, with a sensitivity of 72.4% and a sensitivity of 99.2% overall. The combination of three tests correctly predicted the TOO 75.3% of the time, with 90.9% of cases being in the top two predictions [124,147][94][95]. Through the FDA approval of Epi proColon®’s use in screening for CRC in patients who are unwilling to undergo other recommended screening methods, they confirm the niche that liquid biopsies have in the screening space. However, the reluctance of the FDA and USPSTF to approve and recommend other liquid biopsy screening tests shows there is still a long way to go before overcoming challenges in sensitivity, clinical validation, and cost. One challenge that was previously a concern was the lower specificity of detecting ctDNA due to CHIP, which are mutations associated with myeloid cancers and frequently mutated in healthy patients [151][96]. However, DETECT-A was able to exclude CHIP mutations and achieve a specificity of 98.9% [121][97]. Overall, the specificity of ctDNA-based liquid biopsy tests were ~99% with a few exceptions.  Of greater concern for the utility of liquid biopsies is the lack of sensitivity, which could range from 27.1% to 80.6%, and gets lower with earlier-stage cancers. Low ctDNA shedding tumors with low tumor burden or less metastatic spread are likely contributors to lowering the sensitivity of the test through sampling bias [152][98]. While various tests have been analytically validated to detect ctDNA in MAF down to 1/100,000, clinically, once the MAF drops below 0.01% (1/10,000 copies) the use of a 10 mL sample of blood will not contain a single ctDNA fragment to sequence or detect [153][99]. However, newer approaches are working on increasing the sensitivity of the liquid biopsy tests. Multi-omics combines the analysis of ctDNA with other biomarkers, such as fragmentation length or serum proteins. 

4.3. Treatment Selection/Companion Diagnostics

The FDA defines a companion diagnostic device as an in vitro test that provides information that is essential for the safe and effective use of a corresponding therapeutic product [160][100]. These are tested analytically and clinically to ensure that companion diagnostic devices do not lead to withholding appropriate therapy or administering inappropriate therapy [160][100]. In an era of increasing precision medicine, companion diagnostics are an integral part of identifying targetable mutations in the patient’s cancer. Recognizing this, the FDA has given 5 ctDNA-based tests pre-market approval. Some are PCR-based tests, such as Roche’s cobas® EGFR Mutation Test v2, and Qiagen’s therascreen test. Some are NGS-based tests, such as the FoundationOne®Liquid CDx, Guardant360® CDx, and Agilent’s Resolution ctDx FIRST assay. The PredicineCARE cfDNA Assay has received the FDA Breakthrough Device designation and also utilizes NGS.

4.3.1. BRCA

While not detecting ctDNA, the first liquid biopsy-based companion diagnostic test was Myriad’s BRACAnalysis CDx®, which is approved for the identification of deleterious germline BRCA (gBRCA) variants from whole blood products in breast, ovarian, pancreatic, and metastatic castration-resistant prostate cancer (mCRPC), using Sanger sequencing and multiplex PCR. The results provide aid in identifying eligible patients for a PARP inhibitor, such as olaparib, talazoparib, or rucaparib [106][101]. The initial FDA priority review hinged on the completion of a randomized, double-blind, placebo-controlled, phase 2 study of olaparib, which showed a significantly prolonged progression-free survival with BRCA mutation-positive patients most likely to benefit from treatment [179][102]. In a pooled analysis of 300 patients from phase 1 and 2 trials, olaparib elicited durable responses in patients with relapsed gBRCA mutations [180][103]. The FDA analysis noted a limited representation of BRCA1/2 variants in the previous clinical trials and requested data from the ongoing clinical trials [161][104]. In those follow-ups, confirmatory, phase 3 trials (SOLO2, SOLO3), olaparib demonstrated a clinically significant survival benefit in the same patient population [181,182][105][106]. In the initial approval of BRACAnalysis, Myriad provided evidence of the analytical sensitivity and specificity being ~99% for both, and the clinical validity of BRACAnalysis was confirmed through a concordance rate between local test results and BRACAnalysis of 96.7% during clinical trials [161][104]. In the subsequent extension of BRACAnalysis’s indications to other PARP inhibitors and cancer types, some of the clinical trials used BRACAnalysis as part of the inclusion criteria, and that showed the efficacy of drugs being tested on the specific type of cancer [162,163,164][107][108][109]. In the other indications, the clinical trials cited use Foundation Medicine’s NGS-based assay. The study of Rucaparib in ovarian carcinoma (ARIEL2) used the Foundation Medicine T5 NGS assay, and the study of olaparib in mCRPC (PROfound) used the FoundationOne CDx NGS assay. Both of the aforementioned tests use solid tissue biopsy samples and included mutations other than BRCA1/2 [165,166,167,168][110][111][112][113]. While niraparib was not officially on the list of companion diagnostic indications, the final label update for BRACAnalysis notes that in a randomized, double-blind, phase 3 trial testing niraparib on 203 patients with gBRCA mutations, and 350 patients without gBRCA mutations the progression-free survival for the gBRCA+ cohort was significantly longer than the non-gBRCA cohort. Thise study used the myChoice HRD test on solid biopsy samples to identify the BRCA1/2 mutations [183,184][114][115]. NGS from Foundation Medicine was approved by the FDA on 19 December 2016 in the form of the FoundationFocus CDxBRCA Assay for use in detecting BRCA1/2 alterations following the Rucaparib ARIEL1/2 clinical trials [185][116]. The initial study enrolled patients with BRCA mutations detected by local testing. Later, the BRCA mutations were confirmed at a central Foundation Medicine lab using the Foundation Medicine T5a Panel [167,185][112][116]. Similar to BRACAnalaysis, the approval of FoundationOne Liquid CDx was based on a bridging study measuring the concordance compared to the clinical trial tissue assay, as well as the effectiveness of the FoundationOne liquid biopsy test to select patients for treatment with rucaparib [169,174][117][118]. When 217/491 patients from the ARIEL2 patient population were tested, the positive percent agreement was 93.8% (60/64) and the negative percent agreement was 97.4% (149/153) [169][117].

4.3.2. EGFR

Similar to complementary diagnostic tests for detecting BRCA mutations, tests for detecting EGFR mutations required both analytical and clinical validation. Roche’s cobas® EGFR Mutation Test v1 was an RT-PCR test for the detection of exon 19 deletions or exon 21 L858R missense mutations in non-small cell lung cancer (NSCLC), for which erlotinib is indicated [187][119]. The EURTAC study was a phase 3 trial that screened 1044 patients using a clinical trial assay with a combination of methods for comparing erlotinib vs. cisplatin chemotherapy [188][120]. The FDA then approved the tissue-based cobas® EGFR Mutation Test v1 test based on retrospective testing of 487 samples, which had 432 results that could be compared to the clinical trial assay [187,189][119][121]. When compared, the cobas® EGFR Mutation Test v1 had an overall percent agreement of 96.3% (416/432) [189][121]. While retrospective analysis of the progression-free survival from the EURTAC study was not completed, the cobas® EGFR Mutation Test v1 was used to enroll 217 patients for treatment with erlotinib vs. gemcitabine/cisplatin showing a significant increase in PFS using erlotinib in EGFR mutation-positive patients [190][122]. The ASPIRATION and FAST-ACT2 studies also supported the effectiveness of erlotinib in patients with EGFR mutations detected by the cobas® EGFR Mutation Test v1 [191,192][123][124]. In another bridging study, Roche retrospectively tested samples from the ENSURE, ASPIRATION, and FAST-ACT2 studies with the cobas® EGFR Plasma Test v2 [101,171][125][126]. In a pooled analysis of the 897 paired samples available, an imperfect concordance with a positive predictive agreement (PPA) of 72.1% (339/470) and negative predictive agreement (NPA) of 97.9 (418/427) was recorded [171][126]. However, a negative plasma test would lead to patients using a solid tissue biopsy to determine the EGFR status, so a positive predictive value (PPV) of 97.6% was enough to approve the first liquid biopsy test for detecting exon 19 deletion and exon 21 L858R mutation in 2016 - the cobas® EGFR Plasma Test v2. [193][127]. With osimertinib, cobas EGFR Mutation Test v1 was used in the inclusion criteria for the AURA2 phase 2 clinical trial [194][128]. Then, in a bridging study, the cobas EGFR Plasma Test v2 test detected a T790M gatekeeper mutation with a PPA of 56.8% and a NPA of 80.2% [172][129]. Osimertinib was also established as a first-line treatment option for patients with exon 19 deletion and L858R mutation, where both plasma and tissue are now used [195,196,197][130][131][132]. Plasma or tissue was also approved as a companion diagnostic for gefitinib, but no trials are cited in the approval [173][133].

4.3.3. Other

The Guardant360 CDx has also been approved for the detection of KRAS G12C mutations to indicate usage of sotorasib based on concordance studies comparing samples from the CodeBreaK100 study originally analyzed with the Therascreen KRAS RGQ PCR Kit [176,203,204][134][135][136]. With 189 patients compared, the PPA was 70.7% (82/116) and the NPA was 100% (73/73). Importantly, the Guardant360 CDx had no false positives and an overall response rate in the positive patient population of 38% [176,177][134][137]. The newest approval from the FDA of the Agilent Resolution ctDx FIRST assay was also for the detection of the KRAS G12C mutation for use with adagrasib, another KRAS inhibitor [178][138]. Predicine announced that the FDA granted the breakthrough device designation to the PredicineCARE companion diagnostic assay on 20 September 2022 [116][139]. This followed a clinical trial using their NGS-based assay to detect mutations in ctDNA in different types of breast cancer [208][140]. Among 141 patients with advanced breast cancer, 112 (79.4%) had plasma samples with mutations detected. Then, 21 patients had solid biopsies to compare to their plasma samples with the same single nucleotide variant detected in 6 plasma samples out of 10 tissue samples (6/10) in the PIK3CA gene, 5/9 in TP53, and 5/6 in ERBB2 [209][141].

4.4. Minimal Residual Disease

In patients with curative disease, monitoring after treatment generally consists of serial measurements of various serum proteins and serial radiographic imaging [212,213][142][143]. However, there are some gaps in the utility of certain serum proteins, such as in pancreatic and breast cancer, as previously discussed [52,53,57][47][48][144]. Serum proteins also run into issues with limited sensitivity and specificity [212][142]. While imaging improves the detection of recurrence, it can only detect macroscopic disease [214][145]. Also, imaging is associated with a risk of radiation exposure and inconclusive readings due to normal reactions to treatment [212,215][142][146]. Analysis of ctDNA has shown utility for detecting relapse and resistance mutations before clinical progression occurs [216,217,218][147][148][149]. Inivata’s RaDaR™ Assay uses an enhanced version of the TAm-Seq technology, which originally covered 6 genes with a 97% sensitivity and specificity at 2% MAF [87][71]. The InVisionFirst assay used 36 genes targeted for NSCLC and was able to detect target DNA down to 0.25 MAF with a sensitivity of 92.46% [226][150]. Building on that, the RaDaR Assay uses personalized targets based on whole genome sequencing of tumor tissue, which amplifies up to 48 patient-specific tumor variants from cfDNA [102][151]. With 48 variants, the assay had a sensitivity of 100% at 0.002% MAF, and with only 16 variants, the assay had a sensitivity of 95% at 0.004% MAF [102][151]. This approach has since been studied for the detection of MRD in NSCLC, head and neck squamous cell carcinoma (HNSCC), breast cancer, and melanoma. In the “LUng cancer—Circulating tumor DNA” (LUCID) study, 88 patients had their ctDNA levels measured before and after treatment with surgery [227][152]. For the 77 patients undergoing observation >2 weeks after the end of treatment, ctDNA was detected with a clinical specificity of 98.7% (150/152) samples in 64.3% (18/28) patients who later experienced a clinical recurrence of their first tumor, with lead times of ~200 days from detection to disease recurrence [219][153]. For HNSCC, 17 patients with stage 3 or 4 diseases had samples taken before and after surgery with chemoradiotherapy as needed [220][154]. With later-stage diseases, the RaDaR assay was able to detect pre-operative ctDNA in 100% of patients, and ctDNA was detected 108 to 253 days before clinical recurrence occurred in 100% (5/5) of patients with no false positives [220][154].  Natera’s Signatera Assay is also based on a personalized targeted multiplex PCR-based NGS technology, which can track up to 16 clonal variants from cfDNA in plasma [103,223][155][156]. In the initial studies applying the technology to 49 patients with breast cancer, the Signatera Assay was able to detect ctDNA down to MAF of 0.01% and ctDNA was detected in 94% (16/17) patients 0.5–24 months prior to distant metastatic recurrence with detection in 89% (16/18) of overall recurrence. Again, there were no false positives from this study [103][155]. Similar findings were demonstrated in 122 early-stage CRC and 68 urothelial bladder carcinoma patients detecting 87.5% (14/16) of recurrence with an average lead time of 8.7 months and 100% (13/13) of recurrence with a median lead time of 96 days respectively [223,224][156][157]. In the urothelial bladder carcinoma study, the specificity was 98% (48/49) [224][157]. Similar to the use of ctDNA-based liquid biopsies in screening, the detection of ctDNA is highly specific for the presence of cancer, with the detection of MRD having a specificity >90% in all the completed studies [103,219,220,221,222,224,225][153][154][155][157][158][159][160]. The decrease in specificity is likely due to the repetition of the test over a shorter time frame, and possible detection of dormant disease [222][159]. With the detection of MRD, clinicians can obtain information on disease processes weeks and months ahead of current monitoring by serum proteins or imaging [225,231][160][161]. For example, the MRD tests have promising utility in monitoring for response to systemic therapy [224,234,237,240][157][162][163][164]. However, with the high specificity comes a lower sensitivity. The analytical sensitivity of both the RaDaR Assay and Signatera Assay are impressive being 0.002% MAF and 0.01% MAF respectively [102,222][151][159]. However, the clinical sensitivity could range from 64.3% (18/28) to 100% (13/13) [219,221,224][153][157][158]. The sensitivity of the tests increased with the disease burden being better at detecting distant over local recurrence [103,222][155][159]. 

5. Conclusions

The clinical applications of quantitative and qualitative analysis of cfDNA using liquid biopsy can be exciting solutions to weaknesses of solid biopsy, serum proteins, and imaging. While the clinical utility of measuring total cfDNA as a screening tool is low, there are promising studies using it to determine patients’ prognosis, treatment response, and recurrence. However, total cfDNA being increased in normal physiological processes, a lack of consensus for levels of cfDNA during treatment, and an overall lack of standardization in methodology holds it back from being studied in large-scale clinical trials. In contrast, a variety of ctDNA-based companion diagnostic devices have been clinically validated and are used in clinics under pre-market approval from the FDA for treatment selection. An increase in positive percent agreement might increase the clinical utility of these companion diagnostic devices, but the innovation has mostly been in the addition of group approvals, which will make it easier for the industry to create and push new companion diagnostic devices to market. Using ctDNA detection for other parts of clinical management, such as screening and MRD, is promising with clinical trials completed showing high specificity for detecting cancer. However, several factors, such as low tumor burden and sampling bias, contribute to low sensitivity, but there are new innovations in technology, multi-omics, and sampling to find solutions to these issues. With that said, larger clinical trials using ctDNA-based liquid biopsy tests are still ongoing to prove their clinical validity and propel them to mainstream use.

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