Liquid Biopsy AHS - G2054

RNA profiling from biofluids also poses numerous challenges. However, the discovery that exosomes contained RNA made it possible to separate the fragile RNA from the large amounts of RNases and PCR inhibitors that are present in most biofluids. As cell-free RNA in blood is immediately degraded, RNAs in serum and plasma are either protected inside vesicles like an exosome, in protein complexes with the Ago2 protein or associated with HDL particles (Brock et al., 2015) . The levels of these microRNAs are tightly regulated in normal cells, and dysregulation has been implicated in a number of human diseases, e.g., cardiovascular (Thum & Condorelli, 2015) and neurological, and is strongly linked to cancer development and progression. However, microRNAs represent only a minor fraction of the transcriptome. By contrast, the nucleic acids in exosomes can be isolated and the entire transcriptome examined (Brock et al., 2015).

The most significant hurdle for all forms of liquid biopsy remains the relative rarity of nucleic acid derived from a tumor against the background of normal material found in most patient samples. In fact, the majority of cell, cell-free nucleic acids, microRNAs and exosomes in a liquid biopsy will have originated from normal cells with numbers fluctuating as a consequence of biological variations (Brock et al., 2015).

Furthermore, although liquid biopsy was first introduced with serum, other liquid media, such as urine and cerebrospinal fluid (CSF), have been used to evaluate other conditions. Cell-free DNA is not necessarily confined to blood, and other media have been proposed.

Urine

Urine’s primary advantage over blood is that it is non-invasive, allowing for more convenient testing. Urinary cell-free DNA (UcfDNA) has been proposed as a biomarker for the detection and diagnosis of certain cancers, particularly bladder and prostate cancer (Lu & Li, 2017). An example of this is SelectMDX. SelectMDX evaluates two mRNA cancer-related biomarkers (HOXC6 and DLX1 with KLK3 as a reference gene) to assist a clinician in deciding to continue routine screening or to order a prostate biopsy. This test is considered a “non-invasive urine test” (a liquid biopsy) and reports a binary result of “increased risk” or “very low risk” (MDx, 2023). Van Neste et al. evaluated this test at a 0.90 area under curve in a validation cohort. The authors concluded that the mRNA signature was one of the most significant components of the validation results (Van Neste et al., 2016). Shore et al assessed the effect of SelectMDX results on clinical decision making and found that out of 253 patients SelectMDX evaluated as “negative”, only 12% underwent a biopsy (Shore et al., 2019).

Xu et al. (2021) assessed the diagnostic value of urinary exosomes for urological tumors. The authors performed a systematic review and meta-analysis of 16 studies with a total of 3224 patients. Diagnostic value was calculated based on the number of true positives, false positives, true negatives, and false negatives. The sensitivity of using urinary exosomes for the diagnosis of urological tumors was 83% and the specificity was 88%. Sensitivity and specificity results were similar regardless of urinary exosome content type and tumor type. The authors conclude that “urinary exosomes may serve as novel non-invasive biomarkers for urological cancer detection” (Xu et al., 2021).

Cerebrospinal Fluid (CSF)

Cerebrospinal Fluid (CSF) is a colorless, clear liquid produced by the choroid plexus. CSF acts to control flow of molecules to the central nervous system (CNS). Due to the tight control of the CSF, it may play a significant role in assessing several conditions. CSF is traditionally used to evaluate conditions such as meningitis, but it has also been used to assess central nervous system cancers, such as leptomeningeal metastases (Demopoulos, 2022; Johnson & Sexton, 2023). In addition to widely-known measures of pathology in CSF (opening pressure, total protein, glucose, cell count with differential), circulating tumor cells in CSF have also been proposed as markers for epithelial tumors (Demopoulos, 2022).

Lin et al. (2017) evaluated the diagnostic accuracy of circulating tumor cells in CSF (CSF-CTC) in patients with leptomeningeal metastasis (LM). There were 30 of 95 total patients diagnosed with LM based on a combination of CSF cytology and MRI. CSF-CTCs were detected in 43 patients (median 19.3 CSF-CTC/mL). Based on receiver operating curve analysis, the optimal cutoff was found to be 1 CSF-CTC/mL, identifying patients at a rate of 93% sensitivity, 95% specificity, positive predictive value 90%, and negative predictive value 97% (Lin et al., 2017). Diaz et al. (2022) studied the clinical utility of CSF-CTC by evaluating how CSF-CTC quantification was able to predict the outcome of LM. The authors performed a single institution retrospective study of 101 LM patients with solid tumors. The CSF-CTC count significantly predicted survival continuously (p=0.0027). The authors conclude that “CSF-CTCs quantification predicts survival in newly diagnosed LM, and outperforms neuroimaging” and suggest CSF-CTC can be used for LM prognosis and to assess disease burden (Diaz et al., 2022).

Mathios and Phallen (2022) published a review paper noting “significant strides” towards understanding the molecular mechanisms of brain cancer. Research advances in the field include a focus on the “tumor microenvironment” and identifying molecular biomarkers with liquid-based analyses (such as CSF in liquid biopsy). While it is a rapidly advancing area of research, clinical utility is currently limited, that is, there are currently “no approved noninvasive tests that are clinically useful” for gliomas. The authors point to Cristiano et al. (2019) as an example of a study that analyzed genome-wide cfDNA fragment features (in a variety of cancers); the authors were able to distinguish patients with cancer from non-cancer patients (as well as isolate the tissue of origin). In another glioma-specific study, Mouliere et al. (2018) detected five of 13 patients’ brain tumors (38%) using a cfDNA fragmentation-based approach to analyze cfDNA fragments and copy number alterations in CSF. In conclusion, the authors note that, despite recent excitement over promising studies, liquid biopsy approaches to brain cancer are still “in their infancy” (Mathios & Phallen, 2022).

Proprietary Testing

The FDA approval of use of Roche Cobas EGFR Mutation Test in plasma was based on evaluation of plasma samples from the ENSURE study (Wu et al., 2015), a multicenter, open-label, randomised, Phase III study of stage IIIB/IV NSCLC patients. A total of 98.6% of the patients enrolled (214/217) had a plasma sample available for testing. The agreement between the cobas EGFR Mutation Test in plasma and tissue was evaluated for detection of EGFR mutations. In 76.7% of tissue-positive specimens, plasma was also positive for an EGFR mutation. Plasma was negative for EGFR mutation in 98.2% (95.4%, 99.3%) of tissue-negative cases. The patients whose plasma results were positive for exon 19 deletion and/or an L858R mutations treated with erlotinib had improved progression-free survival (PFS) compared to those treated with chemotherapy (FDA, 2016).

Another commercially available, FDA-approved test is Guardant360 by Guardant Health Inc. Guardant360 is a gene panel that sequences 74 genes (including 18 amplifications and six fusions) associated with NSCLC and reports the percentage of cfDNA (Guardant, 2023). The manufacturer purports that this genetic test will allow providers to make better treatment decisions based on the mutations present in the patient (Health, 2023). The gene panel was analytically validated, with 99.8% accuracy on 1000 consecutive samples (Lanman et al., 2015).

FoundationOne has also created proprietary FDA-approved test that examines cell-free DNA. Foundation One’s liquid CDx test evaluates 324 genes using circulating cell-free DNA and is FDA-approved to report short variants in 311 genes (FoundationOne, 2022, 2024). A prior version of this test (covering 62 genes) was evaluated based on 2666 reference samples. The assay reached >99% sensitivity of short variants of allele frequencies of >0.5%, >95% sensitivity of allele frequencies 0.25%-0.5%, and >70% sensitivity of allele frequencies 0.125%-0.25%. Out of 62 healthy volunteers, no false positives were detected (Clark et al., 2018).

Biodesix is another laboratory that offers a liquid biopsy panel. Biodesix offers two tests; one called GeneStrat, tests EGFR, ALK, ROS1, RET, BRAF, and KRAS (Biodesix, 2023). Sensitivities of 78%- 100% for EGFR, ALK, and KRAS with the GenStrat test were shown in multiple validation studies (Mellert et al., 2017). GeneStrat also detected over 88% of RET or ROS1-positive patients (Mellert et al., 2018). Biodesix also offers GeneStrat NGS, a broad 52 gene panel also evaluated through blood-based liquid biopsy technology.

Other firms that offer liquid biopsy testing include ResolutionBio (now part of Agilent) which offers Agilent Resolution ctDx FIRST(“companion diagnostic to KRAZATI™ adagrasib) for the detection of KRAS G12C in non-small cell lung cancer [NSCLC]”) and Agilent Resolution ctDx LUNG, which focuses on actionable genes for lung cancer such as EGFR and ALK; Circulogene (tests BRAF, EGFR, KRAS, ALK, ROS1, PD-L1, and MSI), Neogenomics (InvisionFirst, 37-gene panel including 10 actionable genes), and Biocept (CNSide™). As liquid biopsy is a rapidly emerging field, it is possible that many more tests will find their way into the clinical setting (Biocept, 2023; Circulogene, 2023; Neogenomics, 2023; ResolutionBio, 2024).

In addition to panels designed to target cancer or cancer type specific mutations, tests are beginning to emerge that perform the role of a genetic screen in asymptomatic individuals. One such test includes the GRAILGalleri multi-cancer early detection (MCED) test, which claims to look for a signal shared by at least 50 types of cancer with a single blood test (Grail, 2024).

According to Turnbull C. (2024) the Galleri-MCED test has an overall sensitivity of only 27.5% for earlystage cancer. The test sensitivity improves to 52.8% by restricting analysis to 12 cancers that the Galleri authors specified as being of high unmet need. However, for several of these 12 cancers, including pancreatic, oesophageal, biliary, and liver, the mortality gains from population screening may be low because these cancers are mainly diagnosed in individuals older than 70 years and prognosis is poor regardless of stage (Turnbull C., 2024).

Klein et al. (2021) held a Circulating Cell-free Genome Atlas substudy including 4077 participants in an independent validation set of Galleri (cancer: n=2823; non-cancer: n=1254, non-cancer status confirmed at year-one follow-up). Specificity for cancer signal detection was 99.5%. Overall sensitivity for cancer signal detection was 51.5% with the sensitivity increasing with stage. Stage I-III sensitivity was 67.6% in the 12 prespecified cancers and was 40.7% in all cancers. Cancer signals were detected across more than 50 cancer types. Overall accuracy of cancer signal origin prediction in true positives was 88.7%. The study concluded that “The MCED test demonstrated high specificity and accuracy of cancer signal origin prediction and detected cancer signals across a wide diversity of cancers. These results support the feasibility of this blood-based MCED test as a complement to existing single-cancer screening tests” (Klein et al., 2021).

Clinical Validity and Utility

Seeberg et al. (2015) conducted a prospective study to assess the prognostic and predictive value of CTCs in 194 patients with colorectal liver metastasis referred to surgery. A total of 153 patients underwent a resection (41 patients had an unresectable tumor), and CTCs were detected in 19.6% of patients. Patients with unresectable tumors had a 46% CTC positivity rate compared to 11.7% for resectable tumors. Patients with two or more CTCs experienced reduced time to relapse/progression. Two or more CTCs was a strong predictor of progression and mortality in all subgroups of patients. The authors concluded that “CTCs predict nonresectability and impaired survival. CTC analysis should be considered as a tool for decisionmaking before liver resection in these patients” (Seeberg et al., 2015).

Groot Koerkamp et al. (2013) performed systematic review and meta-analysis to investigate the prognostic value of CTCs in patients with resectable colorectal liver metastases or widespread metastatic colorectal cancer (CRC). The results of 12 studies representing 1,329 patients were suitable for pooled analysis. The overall survival and progression-free survival were worse in patients with CTCs, with hazard ratios of 2.47 for overall survival rate and 2.07 for progression-free survival. The authors concluded that “the detection of CTCs in peripheral blood of patients with resectable colorectal liver metastases or widespread metastatic CRC is associated with disease progression and poor survival (Groot Koerkamp et al., 2013).”

Zhang et al. (2012) conducted a meta-analysis of published literature on the prognostic value of CTC in breast cancer. Forty-nine eligible studies enrolling 6,825 patients were identified. The presence of CTC was significantly associated with shorter survival in the total population and the prognostic value of CTC was significant in both early and metastatic breast cancer. The authors concluded that “the detection of CTC is a stable prognosticator in patients with early-stage and metastatic breast cancer. Further studies are required to explore the clinical utility of CTC in breast cancer (Zhang et al., 2012).”

Pinzani et al. (2021) assessed that the clinical validity of CTCs has been demonstrated in cancer screening, prognosis, and monitoring treatment responses. In the original article by Cabel et al. (2017), using the Cellsearch® technique in early non-metastatic cancer has reported low CTC detection rates (5-30% depending on cancer type), with limited specificity since “some circulating epithelial cells can be found in individuals with inflammatory disease or even in some healthy individuals.” However, in the preliminary report of another study, it was found that a CTC count >25 could “distinguish lung cancer from benign lesions in patients with abnormal lung imaging. CTC count was also shown to be an “independent prognostic factor in non-small cell lung cancer and small cell lung cancer;” despite this, CTCs are rare in the non-metastatic setting, and thus cannot be completely utilized as an independent prognostic factor in the localized setting. With respect to the independent cancers, Cabel et al. (2017) summarizes the clinical validity of CTC detection in Figure 1.

On the clinical utility of CTC, Cabel et al. (2017) initially stated “the clinical utility of CTC detection (i.e. does it improve patient outcome) has yet to be demonstrated before it can be implemented in routine clinical practice.” In recent time, it was seen that specific CTC features may have clinical utility in “[predicting] the sensitivity to specific immunotherapies,” and in the case of ER+ MBCs, ER-CTCs can develop and reflect “acquisition of therapy resistance by the primary tumor” (Pinzani et al., 2021).

Figure 1. Clinical validity of circulating tumor cells (CTC): level of evidence according to clinical settings (Cabel et al., 2017).

Oxnard et al. (2016) found that: “Sensitivity of plasma genotyping for detection of T790M was 70%. Of 58 patients with T790M-negative tumors, T790M was detected in plasma of 18 (31%). ORR and median PFS were similar in patients with T790M-positive plasma (Objective response rate [ORR], 63%; progression-free survival [PFS], 9.7 months) or T790M-positive tumor (ORR, 62%; PFS, 9.7 months) results. Although patients with T790M-negative plasma had overall favorable outcomes (ORR, 46%; median PFS, 8.2 months), tumor genotyping distinguished a subset of patients positive for T790M who had better outcomes (ORR, 69%; PFS, 16.5 months) as well as a subset of patients negative for T790M with poor outcomes (ORR, 25%; PFS, 2.8 months) (Oxnard et al., 2016).” The authors concluded that “upon availability of validated plasma T790M assays, some patients could avoid a tumor biopsy for T790M genotyping (Oxnard et al., 2016).”

A review by Sacher et al. (2016) genotyped 180 patients with NSCLC using plasma droplet PCR (plasma ddPCR). This was done to validate the plasma droplet PCR technique, and the study identified 115 EGFR mutations and 25 KRAS mutations. The plasma ddPCR was measured to have 82% sensitivity for the EGFR 19 del, 74% for L858R, 77% for T790M, and 64% for KRAS. The positive predictive value was 100% for every mutation apart from T790M at 79%. The authors concluded that the technique “detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies”. The authors also noted that this assay “may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease (Sacher et al., 2016).”

Kim et al. (2017) evaluated the clinical utility of Guardant360. This study used the Guardant360 panel to detect mutations in patients with metastatic NSCLC and other cancers. Somatic mutations were detected in 59 patients, 25 of which had actionable mutations. Out of the 73-patient NSCLC cohort, 62 were found to have somatic mutations and 34 had actionable mutations. After these genetic findings were identified, molecularly matched therapy was provided to 10 patients with gastric cancer (GC) and 17 with NSCLC. Response rate was 67% in GC and 87% in patients with NSCLC, while disease control rate was 100% for both types (Kim et al., 2017).

Odegaard et al. (2018) validated the Guardant360 cell-free DNA sequencing test and aimed to “demonstrate its clinical feasibility”. The authors found that the test could detect variants down to “0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies”. Clinical validation in a cohort of over 750 patients demonstrated high accuracy and specificity, with positive percent agreement (with PCR) of 92%-100% and negative percent agreement of over 99%. In terms of feasibility, the authors performed the test in 10593 patients and found the technical success rate to be over 99.6% and the clinical sensitivity to be 85.9%. The authors also noted that 16.7% of these mutations were targetable with FDA-approved treatments (with 72% with “treatment or trial recommendations”) with as many as 34.5% of non-small cell cancer samples having a targetable mutation (Odegaard et al., 2018).

Aggarwal et al. (2019) evaluated the utility of plasma-based sequencing in improving mutation detection in patients with non-small cell lung cancer. The authors first performed next-generation sequencing (NGS) on tissue, then plasma-based sequencing. A total of 229 patients had concurrent sequencing, and NGS alone detected 47 targetable mutations. Addition of plasma sequencing brought that number to 82 targetable mutations. Furthermore, 36 of 42 patients that received “plasma next-generation sequencing–indicated therapy” achieved a “complete or a partial response or stable disease”. The authors concluded that “adding plasma next-generation sequencing testing to the routine management of metastatic non–small cell lung cancer appears to increase targetable mutation detection and improve delivery of targeted therapy” (Charu Aggarwal et al., 2019).

Leighl et al. (2019) evaluated the utility of “comprehensive cell-free DNA analysis” to identify genomic biomarkers in patients with newly diagnosed metastatic non-small cell lung cancer (NSCLC). 282 patients were included. Tissue genotyping (current standard of care) identified a guideline-recommended biomarker in 60 patients, whereas cell-free DNA identified a relevant biomarker in 77 patients. Concordance between the two methods was 80% (48 biomarkers detected in both methods). For FDA-approved targets (EGFR, ALK, ROS1, BRAF), concordance was >98.2% with 100% positive predictive value for cell-free DNA. Cell-free DNA was also found to have a faster median turnaround time (9 days compared to 15 for tissue genotyping), and “guideline-complete” (assessment of all eight guideline-recommended biomarkers [EGFR, ALK, ROS1, BRAF, RET, MET amplification and exon 14 skipping, and HER2]), was significantly more likely (268 patients vs 51) (Leighl et al., 2019).

Dudley et al. (2019) have developed a novel high-throughput sequencing method that uses urine-derived tumor DNA (utDNA) known as utDNA CAPP-Seq (uCAPP-Seq) to detect bladder cancer. This technique was used to analyze samples from 118 patients with early stage bladder cancer and 67 healthy adults. “We detected utDNA pretreatment in 93% of cases using a tumor mutation-informed approach and in 84% when blinded to tumor mutation status, with 96% to 100% specificity (Dudley et al., 2019).” These results show that utDNA can be used to diagnose early-stage bladder cancer with high sensitivity and specificity.

Wang et al. (2018) performed a meta-analysis to determine the diagnostic performance of cell-free DNA (both blood and urine) assays in bladder cancer. 11 studies encompassing 802 patients were included. The authors evaluated cell-free DNA assays at the following statistics: “sensitivity 0.71, specificity 0.78 positive likelihood ratio 3.3, negative likelihood ratio 0.37, diagnostic odds ratio 9, and area under curve 0.80. No publication bias was identified. The authors concluded that “cell-free DNA has a high diagnostic value in bladder cancer” (Wang et al., 2018).

Hopefully, cfDNA can be used to indicate prognoses of personalized peptide vaccine therapy in patients with NSCLC. Waki et al. (2021) identified that cfDNA integrity “decreased after the first cycle of vaccination” and that those with “high prevaccination cfDNA integrity survived longer than those with low prevaccination integrity (median survival time (MST): 17.9 versus 9.0 months, respectively; hazard ratio (HR): 0.58, p= .0049),” showing that monitoring cfDNA levels could contribute to quantifying treatment success and predicting patient lifespans.

For exosome-based liquid biopsy, Yu et al. (2021) have proposed a synergistic alternative of combining cfDNA and exosomal RNA to “increase the sensivity of mutation detection… the exosome component enables a combination of exosomal RNA, cfDNA, and disease specific proteins… the unique composition of the exosome compartment makes these vesicles particularly amenable for multi-analyte testing, since they carry cancer-informative DNA, RNA, proteins, lipids, oligosaccharides, and metabolites. In one study, a high sensitivity (92%) for EGFR mutations was found for utilizing exosomal RNA and ctDNA together and remained high in a subpopulation that’s been difficult for ctDNA assays to detect (88% sensitivity). ExoRNA and ctDNA combined analyses on BRAF, KRAS, and EGFR mutations in exosomes and respective ctDNA have also better correlated the biomarkers with treatment outcomes when compared to ctDNA alone (Yu et al., 2021).

Lee et al. (2021) analyzed the clinical utility of ctDNA to reliably detect EGFR in ctDNA. The authors compared EGFR analysis results between tissueDNA (tDNA) and ctDNA from 554 NSCLC cases. ctDNA analysis detected EGFR mutation in 57.3% of cases. ctDNA detection correlated with metastatic stage and disease progression (p<0.001). The authors followed up after an average of 41.09 month and found that, “survival analysis revealed ctDNA status and M stage (p < 0.001) to be independent predictors of overall survival in the multivariate analysis.” The authors conclude that ctDNS is clinically useful for EGFR analysis, but note the possibility of false negatives and recommend using tDNA to confirm ctDNA results in some situations (Lee et al., 2021). Syeda et al. (2021) evaluated the use of ctDNA as a biomarker for melanoma. The authors measured changes in ctDNA and survival following “BRAF, MEK, or BRAF plus MEK inhibitor therapy” in patients participating in two clinical trials. The BRAFV600-mutant was measured in ctDNA before and during treatment. “Elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome.” The authors conclude that BRAFV600-mutation ctDNA analysis can be used as a biomarker to predict clinical outcomes (Syeda et al., 2021).

Dang and Park (2022) completed a systematic review on the use of ctDNA for liquid biopsy and the potential challenges as a primary cancer screening marker for minimal residual disease. They cite that more studies need to be performed to evaluate the positive and negative predictive values of existing tests utilizing ctDNA for this purpose. Despite this, the figure below details their understanding of positive benefits in the potential utility of ctDNA across the cancer spectrum (Dang & Park, 2022):

Guidelines and Recommendations

National Comprehensive Cancer Network (NCCN)

The NCCN guidelines for non-small cell lung cancer (NSCLC) strongly advise “broader molecular profiling with the goal of identifying rare driver mutations for which effective drugs may already be available, or to appropriately counsel patients regarding the availability of clinical trials. Broad molecular profiling is a key component of the improvement of care of patients with NSCLC”. Furthermore, the NCCN states that “data suggest that plasma genotyping (also known as plasma ctDNA testing or liquid biopsy) may be considered at progression instead of tissue biopsy to detect whether patients have T790M; however, if the plasma biopsy is negative, then tissue biopsy is recommended ” (NCCN, 2023k).

However, the NCCN goes on to state that cell-free/circulating tumor DNA testing should not be used in lieu of histologic tissue diagnosis. The NCCN notes that specificity is generally very high for cell-free tumor testing but is lacking in sensitivity (up to 30% false-negative rate) and that standards for analytic performance characteristics of cell-free tumor DNA have not been well established. The use of cell-free or circulating tumor DNA may be considered in specific clinical situations, such as if a patient is medically unfit for an invasive tissue sampling or if there is insufficient material for a molecular analysis following pathologic confirmation of an NSSCLC diagnosis in the initial diagnostic setting (but “follow-up tissue-based analysis for all patients in which an oncogenic driver is not identified should be planned).” The NCCN notes that “data suggest that plasma ctDNA testing can be used to identify ALK, BRAF, EGFR, HER2, MET exon 14 skipping, RET, ROS1 and other oncogenic biomarkers that would otherwise not be identified in patients with metastatic NSCLC” (NCCN, 2023k).

For NSCLC, the NCCN provides the following specific recommendations for liquid biopsy:

“The use of cell-free/circulating tumor DNA testing can be considered in specific clinical circumstances, most notably:

• If a patient is medically unfit for invasive tissue sampling
• In the initial diagnostic setting, if following pathologic confirmation of a NSCLC diagnosis there is insufficient material for molecular analysis, cell-free/circulating tumor DNA can be used; however, follow-up tissue-based analysis for all patients in which an oncogenic driver is not identified should be planned (see NSCL-18 for oncogenic drivers with available targeted therapy options).
• In the initial diagnostic setting, if tissue-based testing does not completely assess all recommended biomarkers owing to tissue quantity or testing methodologies available, consider repeat biopsy and/or cell-free/circulating tumor DNA testing.
• In the initial diagnostic setting, if the feasibility of timely tissue-based testing is uncertain, concurrent cfDNA testing may aid in biomarker evaluation for treatment selection, provided negative results are considered per above limitations” (NCCN, 2023k).

The NCCN lists “comprehensive germline and somatic profiling to identify candidates for additional targeted therapies” as part of the workup for recurrent stage IV (M1) breast cancer.” They go on to specifically note that “tissue or plasma-based circulating tumor DNA (ctDNA) assays may be used. Tissue-based assays have greater sensitivity, but ctDNA may reflect tumor heterogeneity more accurately.” The NCCN also states that assessment of the PIK3CA mutation may be performed through liquid biopsy if the tumor is HR-positive, HER2 negative, and if therapy with alpelisib plus fulvestrant is being considered. Finally, for the management of breast cancer with liquid biopsy techniques, the NCCN states that “the clinical use of Circulating Tumor Cells (CTC) or circulating DNA (ctDNA) in metastatic breast cancer is not yet included in the NCCN Guidelines for Breast Cancer for disease assessment and monitoring”, though the sentence that follows would indicate that this statement refers to a count of CTCs, not their use for genotyping: “Patients with persistently increased CTC after three weeks of first-line chemotherapy have a poor PFS and OS” (NCCN, 2023e).

The NCCN states that AR-V7 testing in CTCs “can be considered to help guide selection of therapy in the post-abiraterone/enzulamide metastatic CRPC [castration-resistant prostate cancer] setting”. The NCCN does not comment on any particular liquid medium over another (e.g. urine, CSF, serum). However, the NCCN does specify the use of circulating DNA for rucaparib treatment, stating that “the preferred method of selecting patients for rucaparib treatment is somatic analysis of BRCA1 and BRCA2 using a circulating tumor DNA sample” (NCCN,2023a). SelectMDx is also acknowledged by the NCCN; “the panel believes that SelectMDx score is potentially informative in patients who have never undergone biopsy, and it can therefore be considered in such individuals” (NCCN, 2023m).

With regards to circulating tumor DNA (ctDNA) in colon cancer, the NCCN “panel believes that there are insufficient data to recommend the use of multigene assays, Immunoscore, or post-surgical ctDNA to estimate risk of recurrence or determine adjuvant therapy” (NCCN, 2023g).

For neuroendocrine tumors, NCCN notes that CTCs have been studied as prognostic markers, but state that more research is required. There is no single biomarker available that is satisfactory as a diagnostic, prognostic, or predictive marker (NCCN, 2023j).

For a primary CNS lymphoma, the NCCN remarks that cerebrospinal fluid analysis may “possibly” include gene rearrangement evaluation. For leptomeningeal metastases, the NCCN notes that assessment of ctDNA in CSF “increases sensitivity of tumor cell detection and assessment of response to treatment” (NCCN, 2023f).

For pancreatic adenocarcinomas, the NCCN acknowledges that circulating cell-free DNA is being investigated as a biomarker for screening. The NCCN also notes that if tumor tissue is not available, cell-free DNA testing may be considered (NCCN, 2023l).

For esophageal, esophagogastric junction cancers, and gastric cancers, the NCCN states “testing using a validated NGS-based [next generation sequencing] genomic profiling assay performed in a CLIA-approved laboratory may be considered. A negative result should be interpreted with caution, as this does not exclude the presence of tumor mutations or amplifications (NCCN, 2023h).

For acute myeloid leukemia, the NCCN notes that “morphologically detectable,” circulating leukemic blasts from peripheral blood may be used to detect molecular abnormalities (NCCN, 2023b).

For bladder cancer, the NCCN mentions RT-PCR testing for FGFR2/3 gene alterations, but does not specify whether this can be done through a liquid biopsy or cell-free DNA. The only comment made is that the laboratory should be CLIA-approved (NCCN, 2023d).

The NCCN guidelines for small cell lung cancer and hepatocellular carcinoma do not address use of CTCs, ctDNA, or liquid biopsy for patient management (NCCN, 2023i, 2023n). In biliary tract cancers, the NCCN states that “a cell-free DNA (cfDNA) test may also be considered for identifying gene mutations. This technique may not reliably identify gene fusions or rearrangements depending on the panel used and the specific partner gene” (NCCN, 2023c).

American Society of Clinical Oncology (ASCO)

In 2016, ASCO published updated recommendations for the use of tumor markers in treatment of metastatic breast cancer. ASCO found that although CTCs may be prognostic, they are not predictive for clinical benefit when used to guide or influence decisions on systemic therapy for metastatic breast cancer. ASCO recommends clinicians to not use these markers as adjunctive assessments ( Van Poznak et al., 2015). Similarly, ASCO recommended against use of CTCs to guide decisions about adjuvant systemic therapy for individuals with early stage invasive breast cancer (Andre et al., 2019).

In 2019, ASCO stated that clinicians “should not use circulating biomarkers as a surveillance strategy for detection of recurrence in patients who have undergone curative-intent treatment of stage I-III NSCLC or SCLC”. ASCO states that further data is required to validate this approach (Schneider et al., 2019).

In 2018, ASCO and the College of American Pathologists (CAP) released a joint review on “circulating tumor DNA analysis in patients with cancer”. In it, they note that apart from the assays that have received “regulatory appeal”, most assays have “insufficient evidence” for both clinical validity and clinical utility. They note discordant results between circulating DNA assays and tissue genotyping. Furthermore, they remark on the lack of evidence for use in monitoring therapy effectiveness, diagnosing early-stage cancer, or cancer screening.

However, they point to evidence that well-validated assays may support initiation of targeted therapy (Merker et al., 2018).

National Academy of Clinical Biochemistry (NACB) now known as the American Association for Clinical Chemistry (AACC)

In 2010, the NACB issued practice guidelines for the use of tumor markers in liver, bladder, cervical, and gastric cancers. It found that CTCs had “questionable” clinical utility in the assessment of liver cancer and did not recommend their use (Sturgeon et al., 2010).

The NACB published an updated guideline in 2020. For liver cancer, they note circulating cell-free serum DNA as “undergoing evaluation” for “predictive marker for distant metastasis of hepatitis C virus–related HCC.” The plasma proteasome is also undergoing evaluation for “assessment of early HCC in patients with chronic viral chronic hepatitis; assessment of metastatic potential of HCC.” Finally, circulating methylated DNA is undergoing evaluation for HCC screening, detection, and prognosis. No other circulating tumor markers for bladder, cervical, and gastric cancers were mentioned (Sturgeon et al., 2020).

College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP)

An expert panel was convened to review and update the CAP-IASLC-AMP Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors. This panel consists of practicing pathologists, oncologists, and a methodologist.

The panel states there is “insufficient evidence to support the use of circulating cell-free plasma DNA (cfDNA) molecular methods for the diagnosis of primary lung adenocarcinoma”. According to the panel, there is also “insufficient evidence to support the use of circulating tumor cell (CTC) molecular analysis for the diagnosis of primary lung adenocarcinoma, the identification of EGFR or other mutations, or the identification of EGFR T790M mutations at the time of EGFR TKI-resistance” (College of American Pathologists, 2018; Lindeman et al., 2018).

However, the panel acknowledges that “In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cell-free plasma DNA (cfDNA) assay to identify EGFR mutations” (Lindeman et al., 2018).

In 2021, the IASLC published an updated consensus statement on liquid biopsy testing. They note that liquid biopsy “includes a variety of methodologies for circulating analytes. From a clinical point of view, plasma circulating tumor DNA is the most extensively studied and widely adopted alternative to tissue tumor genotyping in solid tumors, including NSCLC” (Rolfo et al., 2021).

The following recommendations were presented in a consensus statement:

1. In clinical practice, ctDNA collection, sample handling, and automated processing should be performed using standardized and clinically validated procedures to reduce operator variability and false-negative results.
2. Because of the growing number of guideline-recommended oncogene targets to be assessed in advanced NSCLC, testing of plasma ctDNA should be performed by a clinically validated NGS platform rather than single-gene, PCR-based approaches, both in treatment-naive patients and those associated with multiple mechanisms of acquired resistance (MOR) to targeted agents. Where plasma NGS is not available owing to technical and economic constraints, single-gene or low multiplex-based approaches may represent appropriate alternatives. Use of limited PCR analysis for EGFR mutations as the initial step in molecular assessment, for example, remains highly relevant in areas of the world where the EGFR mutation rate is high. Nevertheless, single-gene testing should not be considered complete, and if negative, serial testing for additional actionable biomarkers must be pursued.
3. The benefit of tissue and plasma NGS is now established in several clinical practice settings. It is anticipated, owing to broad-based coverage of requisite oncogenes, decreased turnaround times, and emerging data on cost effectiveness, that in the near future, NGS will become increasingly available worldwide. Implementation of a multidisciplinary MTB to assist clinicians in treatment decision-making is advisable, as described previously.
4.  In patients with oncogene-addicted NSCLC, liquid biopsy is emerging as not only complementary to tissue-based analysis but also acceptable as the initial approach (“plasma first”) for biomarker evaluation at the time of diagnosis and for monitoring the efficacy of targeted therapies. Finally, a plasma-first approach is appropriate for identification of MOR to targeted therapies in many clinical settings.
5. Indications for liquid biopsy in patients with nononcogene-addicted NSCLC are less well defined at this time, although there are several promising areas of investigation. As noted previously, bTMB is an emerging biomarker, pending completion of ongoing prospective randomized trials and refinement of methodology.

American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology

These joint guidelines from these societies were published regarding molecular biomarkers for colorectal cancer. Despite the potential of liquid biopsy for assessment of tumor recurrence and treatment resistance, the technique “awaits robust validation and further studies to determine their clinical utility” (Sepulveda et al., 2017).

European Society for Medical Oncology (ESMO) and Chinese Society of Clinical Oncology (CSCO)

These guidelines state that liquid biopsy can be used as “the initial test for the detection of a T790M mutation [for EGFR in NSCLC], and if tests are negative, a re-biopsy should be attempted if feasible” (Wu et al., 2018).

The European Association of Urology (EAU), European Society for Radiotherapy and Oncology (ESTRO), European Society of Urogenital Radiology (ESUR), and the International Society of Geriatric Oncology (SIOG)

The joint guidelines on prostate cancer state that “In asymptomatic individuals with a prostate-specific antigen (PSA) level between three to ten ng/mL and a normal digital rectal examination, use one of the following tools:

• risk-calculator- provided it is correctly calibrated to the population prevalence; magnetic resonance imaging of the prostate- Strong
• an additional serum, urine, or tissue-based test- Weak”.

These joint guidelines acknowledged SelectMDX as a test to isolate urine biomarkers, but the guidelines noted “the clinically added value of SelectMDX in the era of upfront MRI and targeted biopsies remains unclear” (Mottet et al., 2023).

American Society of Colon and Rectal Surgeons (ASCRS)

The ASCRS released clinical practice guidelines for the management of colon cancer. The guidelines state that “the use of multigene assays, CDX2 expression analysis, and ctDNA may be used to complement multidisciplinary decision-making for patients with stage II or III colon cancer” (Vogel et al., 2022).

State and Federal Regulations, as applicable

Food and Drug Administration (FDA)

There are four FDA-approved liquid biopsy tests as of January 10, 2023. The Cobas EGFR Mutation Test v2 from Roche Diagnostics is an assay purported to detect epidermal growth factor receptor (EGFR) gene mutations in NSCLC patients. The test is intended as a companion diagnostic test for the cancer drug Tarceva (FDA, 2016), and a similar test for the T790M mutation has been produced by the same company. A second test is the Cell Search® Ciculating Tumor (CTC) Test, which is used to predict and analyze outcomes for individuals with metastatic breast, prostate, or colon cancer (CellSearch, 2024). A third test is Guardant360® CDx, which detects ctDNA and other common genetic errors in order to help in the choice of a therapeutic or treatment (Health, 2023). Lastly, FoundationOne® Liquid CDx is an FDA-approved liquid biopsy test that detects ctDNA and may be able to assist a provider in determining the type of treatment that will be most effective (FoundationOne, 2022).

Many labs have developed specific tests that they must validate and perform in house. These laboratorydeveloped tests (LDTs) are regulated by the Centers for Medicare and Medicaid (CMS) as high-complexity tests under the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88). As an LDT, the U. S. Food and Drug Administration has not approved or cleared this test; however, FDA clearance or approval is not currently required for clinical use. 

Billing/Coding/Physician Documentation Information

This policy may apply to the following codes. Inclusion of a code in this section does not guarantee that it will be reimbursed. For further information on reimbursement guidelines, please see Administrative Policies on the Blue Cross Blue Shield of North Carolina web site at www.bcbsnc.com. They are listed in the Category Search on the Medical Policy search page.

Applicable service codes: 81162, 81163, 81164, 81194, 81210, 81235, 81275, 81276, 81309, 81405, 81406, 81462 81479, 86152, 86153, 0011M, 0091U, 0155U, 0177U, 0179U, 0229U, 0317U, 0332U, 0333U, 0337U, 0338U, 0343U, 0356U, 0368U, 0388U, 0395U, 0410U, 0453U, 0490U, 0491U, 0492U, 0496U, 0499U, 0500U, 0501U, 0507U

BCBSNC may request medical records for determination of medical necessity. When medical records are requested, letters of support and/or explanation are often useful, but are not sufficient documentation unless all specific information needed to make a medical necessity determination is included.

Scientific Background and Reference Sources

For Policy Titled: Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management

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Medical Director review 5/2019

Medical Director review 7/2019

For Policy Titled: Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management (Liquid Biopsy)

Medical Director review 11/2019

Specialty Matched Consultant Advisory Panel 3/2020

Medical Director review 3/2020

Medical Director review 4/2020

Medical Director review 2/2021

Specialty Matched Consultant Advisory Panel 3/2021

Medical Director review 3/2021

Medical Director review 4/2021

Medical Director review 6/2021

For Policy Titled: Liquid Biopsy

Medical Director review 4/2022

Medical Director review 4/2023

Medical Director review 4/2024

Policy Implementation/Update Information

For Policy Titled: Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management

1/1/2019 New policy developed. BCBSNC will provide coverage for detection of circulating tumor cells and cell free DNA in cancer management when it is determined to be medically necessary and criteria are met. Medical Director review 1/1/2019. Policy noticed 1/1/2019 for effective date 4/1/2019. (lpr)

6/11/19 Reviewed by Avalon 1st Quarter 2019 CAB. Added CPT 81479 to Billing/Coding section. Deleted CPT codes 86152 and 86153 from Billing/Coding section. No change to policy statement. Medical Director review 5/2019. (lpr)

7/30/19 Under “When Covered” section: removed item B. “Testing is performed using the Cobas EGFR Mutation Test, Guardant360 test, or OncoBEAM test.” Medical Director review 7/2019. (lpr)

11/12/19 Deleted coding table from Billing/Coding section. Wording in the Policy, When Covered, and/or Not Covered section(s) changed from Medical Necessity to Reimbursement language, where needed. Minor reformatting of policy statements; no change to policy statement intent. (hb)

For Policy Titled: Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management (Liquid Biopsy)

12/31/19 Reviewed by Avalon 3rd Quarter 2019 CAB. Under “When Not Covered” section added the statement “Testing to predict treatment response using circulating tumor DNA in all other cancer types is investigational. Added “Liquid Biopsy” to the title of the policy. Policy Title changed from “Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management” to Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management (Liquid Biopsy).” Added CPT codes 81277, 81404, 86152, 86153 to Billing/Coding section. Medical Director review 11/2019. (lpr)

4/14/20 Specialty Matched Consultant Advisory Panel review 3/18/2020. No change to policy statement. (lpr)

5/12/20 Reviewed by Avalon 1st Quarter 2020 CAB. Medical Director review 4/2020. Added CPT codes 81301, 0091U to Billing/Coding section. Updated When Covered section statement. Added CellSearch is investigational to When Not Covered section. References added. (lpr)

2/23/21 Off cycle review. Under “When Covered” section: Added coverage criteria for additional mutations for NSCLC as well as use for breast cancer. Medical Director review 2/2021. (lpr)

4/6/21 Specialty Matched Consultant Advisory Panel review 3/17/2021. No change to policy statement. (lpr)

7/1/21 Reviewed by Avalon 1st Quarter 2021 CAB. Medical Director review 4/2021, 6/2021. Added CPT codes 81162, 81163, 81164, 0011M, 0155U, 0177U, 0179U, 0229U, 0239U, 0242U to Billing/Coding section; removed CPT 81277. Updated Description and Policy Guidelines. Added Related Policies section under Description. Under “When Covered” section, added “Note: If the above criteria for medical necessity have been met (i.e., when tissue biopsy is contraindicated or quantity of tissue available is insufficient), panel testing using NGS for up to 50 genes may be performed.” Added references. No change to policy intent. (lpr)

For Policy Titled: Liquid Biopsy

5/31/22 Reviewed by Avalon 1st Quarter 2022 CAB. “When Covered” section reformatted and removed 50 gene limit sentences. Moved Note 1 & 2 and reformatted “When Not Covered” section. Updated policy guidelines, references. Added policy AHS- M2178 to related policies section and removed AHS-M2109 due to archival. Added CPT 0137U to Billing/Coding section. Medical Director review 4/2022. Policy title changed from “Detection of Circulating Tumor Cells and Cell Free DNA in Cancer Management (Liquid Biopsy)” to “Liquid Biopsy.” (lpr)

9/30/22 Added the following CPT codes to Billing/Coding section: 0332U, 0333U, 0337U, 0338U, 0343U, 0346U. (lpr)

12/30/22 Added PLA code 0356U to Billing/Coding section for effective date 1/1/2023. (lpr)

3/31/23 Added PLA code 0368U to Billing/Coding section. (lpr)

5/16/23 Reviewed by Avalon 1st Quarter 2023 CAB. Medical Director review 4/2023. Expanded medical necessity criteria under “When Covered” section. Updated policy guidelines, description, and references. Removed Notes 1, 2. Deleted related policies section. Under Billing/Coding section, added CPT codes: 81194, 81210, 81275, 81276, 81405, 81406, 0332U, 0333U, 0337U, 0338U, 0343U, 0356U, 0368U; deleted CPT codes: 81301, 81404, 0239U, 0242U, 0346U. (lpr)

9/29/23 Added PLA code 0410U to Billing/Coding section for 10/1/23 code update. (lpr)

12/29/23 Added CPT code 81462 to Billing/Coding section for 1/1/2024 code update. (lpr)

5/15/24 Reviewed by Avalon Q1 2024 CAB. Medical Director review 4/2024. Added related policies section. Updated policy guidelines and references. Under Billing/Coding section: added CPT codes 0388U and 0395U. No change to policy intent. (lpr)

9/4/24 Added PLA code 0453U to Billing/Coding section. (lpr)

10/1/24 Added PLA codes: 0490U, 0491U, 0492U, 0496U, 0499U, 0500U, 0501U, 0507U to Billing/Coding section for 10/1/24 code update. (lpr)