CAM 204141

Liquid Biopsy

Category:Laboratory   Last Reviewed:October 2019
Department(s):Medical Affairs   Next Review:April 2020
Original Date:July 2016    

National Cancer Institute defines liquid biopsy as a test done on a sample of blood for the detection of cancer cells from a tumor that are circulating in the blood or for the detection of cell free DNA pieces from tumor cells that are in the blood (Domínguez-Vigil, Moreno-Martínez, Wang, Roehrl, & Barrera-Saldaña, 2018). Liquid biopsies are non-invasive blood tests since circulating tumor cells (CTCs) and cell-free tumor DNA (cfDNA) fragments are shed into the bloodstream from existing tumors and can be detected in blood (Curigliano, 2014; Haber & Velculescu, 2014). The presence of CTCs can be indicative of metastatic disease (Alix-Panabieres & Pantel, 2013).. 

Regulatory Status 
At this time there is only one FDA-approved liquid biopsy test, which is a diagnostic for non-small lung cancer (NSCLC) was approved by the FDA in 2016. The test — cobas EGFR Mutation Test v2 from Roche Diagnostics is 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. Additionally, many labs have developed specific tests that they must validate and perform in house.  These laboratory-developed 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.


  1. For patients with Stage IIIB/IV non-­small cell lung cancer (NSCLC), liquid biopsy (plasma genotyping) is considered MEDICALLY NECESSARY in either of the following situations:
    1. At diagnosis: ­When results for EGFR single nucleotide variants (SNV) and insertions and deletions (indels); ALK and ROS1 rearrangements; and PD-L1 expression are not available AND when tissue based comprehensive somatic genomic profiling test (CGP) is infeasible (i.e., quantity not sufficient for tissue based CGP or invasive biopsy is medically contraindicated); OR
    2. At progression: For patients progressing on or after chemotherapy or immunotherapy who have never been tested for EGFR SNVs and indels; and ALK and ROS1 rearrangements, and for whom tissue-­based CGP is infeasible (i.e., quantity not sufficient for tissue-­based CGP); OR For patients progressing on EGFR tyrosine kinase inhibitors (TKIs).
    3. If no genetic alteration is detected by plasma genotyping, or if circulating tumor DNA (ctDNA) is insufficient/not detected, tissue-based genotyping should be considered.
  2. Liquid biopsy test for PIK3CA mutation is considered MEDICALLY NECESSARY for individuals diagnosed with breast cancer that is HR-positive, HER2 negative, and if therapy with alpelisib is being considered.
  3. Liquid biopsy test for screening, detecting and monitoring any other malignancy or tumor is considered investigational and/or unproven and therefore is  considered NOT MEDICALLY NECESSARY. 

The science of noninvasive disease monitoring has advanced greatly since circulating cell-free   DNA (cfDNA) was first reported in body fluids by Mandel and Metais. Since then, the evolution of sensitive cfDNA detection technologies has enabled the development of liquid biopsies with many clinical applications. For example, in oncology, the use of liquid biopsy allows for patient stratification, screening, monitoring treatment response and detection of minimal residual disease after surgery or recurrence. Liquid biopsies have grown in importance because the genetic profile of tumors can affect how well patients respond to a certain treatment. However, this characterization is currently achieved through a biopsy despite the inherent problems in procurement of tissue samples and the limitations of tumor analyses. For example, the invasive nature of a biopsy poses a risk to patients and can have a significant cost (Brock, Castellanos-Rizaldos, Hu, Coticchia, & Skog, 2015).

Tumor sampling from some cancer types also remains difficult resulting in inadequate amount of tissue for genetic testing (Brock et al., 2015). In the case of advanced or metastatic non-small cell lung cancers (NSCLC) as many as 69% of cases do not have accessible tissue (J.-Y. Douillard et al., 2009). Even when tissue can be collected, preservation methods such as formalin fixation can cause false positive results for genetic tests (Quach, Goodman, & Shibata, 2004). Finally, due to tumor heterogeneity, biopsies often suffer from sample bias (Bedard, Hansen, Ratain, & Siu, 2013).  Liquid biopsies are becoming more popular as they provide an opportunity to genotype in a less invasive and expensive manner. However, the low sensitivity (between 60-80%) and higher number of false negative cases compared to traditional tissue biopsy are limitations associated with liquid biopsies (Sequist & Neal, 2017).

Approaches to liquid biopsy analysis
Circulating tumor cells (CTCs)
According to Brock et al, CTCs are cells shed into the vasculature from a primary tumor and may constitute seeds for subsequent growth of additional tumors (metastasis) in distant organs (Brock et al., 2015). They have been detected in various metastatic carcinomas (Mavroudis, 2010) but are extremely rare in healthy subjects and patients with nonmalignant diseases (Brock et al., 2015). Clinical evidence indicates that patients with metastatic lesions are more likely to have CTCs amenable to isolation but their frequency is low, often ~1-10 CTCs per mL of whole blood (Miller, Doyle, & Terstappen, 2010). As 1 mL of blood contains ~7×106 white blood cells and ~5×109 red blood cells, technologies capable of reproducibly isolating a single CTC from the background of all other blood components are fundamental. While such levels of sensitivity are challenging, there are several novel developments in this area, including positive selection, negative selection, physical properties or even enrichment-free assays to efficiently isolate these rare CTCs (Alix-Panabieres & Pantel, 2013). However, Bettegowda et al stated that an advantage of ctDNA is that it can be analyzed from bio-banked biofluids, such as frozen plasma (Bettegowda et al., 2014).

Typically, CTCs are defined as cells with an intact viable nucleus, cytokeratin positive, epithelial cell adhesion molecule (EpCAM) positive and with the absence of CD45 (Brock et al., 2015). Unfortunately EpCAM and other markers are not always expressed on CTCs (Grover, Cummins, Price, Roberts-Thomson, & Hardingham, 2014). In addition, non-tumor epithelial cells are known to circulate in the blood of patients with prostatitis or patients undergoing surgery (Brock et al., 2015; Murray et al., 2013). The heterogeneity of CTCs is a major challenge from a technical standpoint. This has led to alternative strategies of CTC enrichment such as the CTC-iChip which does not rely on tumor antigen expression (Brock et al., 2015; Karabacak et al., 2014).

Sequencing the genetic material from CTCs has demonstrated that the majority are not cancer cells, even when the isolated cell(s) fit the phenotypic criteria of being a CTC. One study by Marchetti A et al (2014) developed a protocol to recover the CTC enriched samples from the cartridge of the Veridex platform and found that from 37 NSCLC patients, the EGFR mutation allele abundance ranged between 0.02% and 24.79% with a mean of 6.34%. Brock et al concluded that the number of CTCs found in the blood is therefore highly dependent on how the platform defines a cell as a CTC (Brock et al., 2015; Marchetti et al., 2014). The CellSearch CTC test, a Food and Drug Administration (FDA) approved actionable CTC test, requires that samples are processed within 96 hours of collection after being drawn into the Cellsave preservative tube. This test does not analyze the molecular genetics of the tumor; rather Cellsave is a platform for CTC numeration. A positive test (more than five detected CTCs for metastatic breast and prostate cancer and more than three CTCs for metastatic colorectal cancer per 7.5 mL of blood) is associated with decreased progression-free survival and decreased overall survival in these patients (Aggarwal et al., 2013).

Overall, although CTCs have produced some promising results in evaluating prognosis of patients with varying cancers, further studies are needed to assess the clinical utility of these biomarkers (Adamczyk et al., 2015; Bidard, Proudhon, & Pierga, 2016; Foukakis & Bergh, 2017; Ignatiadis & Dawson, 2014).

Cell-free DNA (cfDNA)
There is currently an intensive research effort to understand the utility of cfDNA in various clinical fields, such as cancer research, non-invasive prenatal testing and transplant rejection diagnostics (Brock et al., 2015). In a systematic review and meta-analysis of 20 studies and 2012 cases covering assessment of EGFR mutational status in NSCLC, Luo, Shen and Zheng found a sensitivity of 0.674, a specificity of 0.935, and area under the curve of 0.93. The authors concluded that detection of EGFR mutation by cfDNA is of adequate diagnostic accuracy and cfDNA analysis could be a promising screening test for NSCLC (Luo, Shen, & Zheng, 2014).

In a study Jiang P et al (2015) observed that most cfDNA in plasma is reportedly fragmented, around 150-180 bp in length with a higher prevalence of tumor associated mutations in the shorter fragments. Per authors, when analyzing the mutation abundance with massively parallel sequencing, a significant correlation was found between mutations and fragments less than 150 bp.  Notably, the size of the majority of cfDNA fragments overlaps well with the size of histone DNA (Jiang et al., 2015)

A direct comparison of mutation detection on cfDNA vs. CTCs showed a higher abundance of the mutation on the cDNA from the same patient and recent large studies comparing the effectiveness of cfDNA analysis to tissue biopsy in NSCLC showed the clinical value of the liquid biopsy approach (J. Y. Douillard et al., 2014). This positive result led to an approval to use cfDNA analysis for EGFR mutation analysis for IRESSA in Europe (in patients where a tumor sample was not evaluable), making it the first EGFR tyrosine kinase inhibitor for which cfDNA testing is included in the label. Although promising, challenges remain when using cfDNA to characterize the mutation status of a tumor. In addition to the low copy number of mutant alleles, the median half-life of cfDNA in circulation ranges from 15 minutes to a few hours (Brock et al., 2015).

Brock et al in their review observed that the total concentration of cfDNA in the blood of cancer patients varies considerably with tumor specific mutations ranging from undetectable (less than 1 copy per 5 mL of plasma) to patients with over a hundred thousand copies of the mutation per mL of plasma. The authors note that “the challenge of how to maximize the yield of the cfDNA and pair this with a platform sensitive enough to detect rare variants in the background of wild-type DNA remains. Optimally, the ability to detect mutations in plasma should not be limited to a subpopulation of patients with very high mutant copy numbers in circulation (Brock et al., 2015).”

While many analytical platforms report the mutation load with an allelic frequency compared to the wild-type DNA platforms relying solely on the allelic frequency without recording the number of mutations have limitations. This is because the allelic frequency of a gene is affected by the amount of wild-type DNA not related to the tumor. Therefore, it is important to consider the processes that affect the amount of wild-type DNA in circulation (Brock et al., 2015). For example, exercise increases cfDNA levels almost 10-fold (Breitbach, Sterzing, Magallanes, Tug, & Simon, 2014). Other pre-analytical variables such as blood collection, the cellular process leading to its release, and extraction protocols affect the amount and size range of cfDNA fragments in a sample (Devonshire et al., 2014).

In the last few years, the exosome field has grown exponentially impacting various areas of research. Studies demonstrating that exosomes are actively released vesicles (carrying RNA, DNA and protein) and can function as intercellular messengers. Yáñez-Mó M, et al highlights these developments  (2015) in a review outlining the biological properties of exosomes and other extracellular vesicles (EV’s). However, Gould et al (Gould & Raposo, 2013) observed that the exosome field still lags behind as the standardization of  extracellular vesicle (EV) types are not yet firmly established. The majority of exosomes range in size from 30-200 nanometers (nm) in diameter and are isolated from all bio-fluids, including serum, plasma, saliva, urine and cerebrospinal fluid (Brock et al., 2015).

Due to the size of an exosome, on average just over 100 nanometers, the entire transcriptome cannot be packaged inside every vesicle. By way of comparison, retrovirus particles with a similar size can package only around 10 kb, so it is likely that a single vesicle of that size carries only a limited number of transcripts. However, exosomes are extremely abundant (10e11 per mL of plasma) and when isolating the vesicle fraction, most of the transcriptome can be detected(Graham Brock, 2015). Per Huang X et al (2013), and Kahlert C et al (2014), Exosomal RNA can be used for mutation detection as well as global profiling of most types of RNA, and the profile alone (without mutation characterization) can be utilized for diagnostics (Brock et al., 2015). In the study ‘Immune modulation of T-cell and NK (natural killer) cell activities by TEXs (tumour-derived exosomes)’, Whiteside TL et al (2013) observed that exosome investigations have focused on the important physiologic and pathophysiologic functions of these vesicles in micro-metastasis, angiogenesis and immune modulation and as a means for detection of tumor specific mutations in bio-fluids (Whiteside, 2013). Consequently, in 2012, interest in this new field increased when the National Institute of Health (NIH) dedicated the large strategic Common Fund to study these new entities of extracellular RNA. The goal of this effort is to better understand how exosomes can be utilized for biomarkers and therapeutics as well as understanding this new mechanism of intercellular communication (NIH, 2017).

Mutation detection and RNA profiling
Analysis of nucleic acids present in bodily fluids can provide a better understanding of the disease, as summarized in Table below.

Comparison of the analysis capability of CTCs, cfDNA and exosomes from: (Brock et al., 2015)

Analysis capability






Point mutations, InDels, amplifications, deletions, translocations




Epigenetic modifications

Methylation patterns




RNA transcription profiles

Levels/activity of mRNA, microRNA, long non codingRNA, RNA splice variants




Phenotypic studies of cells from the tumor

Cell morphology, protein localization, in vivo studies




Inflammatory response, stromal and other systemic changes

Inflammatory RNA and protein markers




Analysis of RNA as well as DNA and protein profiles from tumor cells

Separate or in combination




Can utilize bio-banked samples

Frozen plasma, urine and other bio-fluids




CTCs, circulating tumor cells; cfDNA, cell-free DNA; InDels, insertions/deletions.

RNA profiling from biofluids is also difficult. However, since exosomes contain RNA, it was possible to separate the fragile RNA from the large amounts of RNases and PCR inhibitors. As cell-free RNA in blood is immediately degraded, RNAs in serum and plasma were either protected inside vesicles, in protein complexes or associated with HDL particles (Brock et al., 2015; Graham Brock, 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).

Clinical Validity and Utility
Seeberg et al conducted a prospective study to assess the prognostic and predictive value of CTCs in 194 patients with colorectal liver metastasis referred to surgery. 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 decision-making before liver resection in these patients (Seeberg et al., 2015)”.

Groot et al 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, Rahbari, Buchler, Koch, & Weitz, 2013).”

Zhang et al 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).”

Oxnard et al 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 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).”

FDA approval of use of the 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. 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 test is Guardant360 by Guardant Health Inc. Guardant360 is a gene panel that sequences 73 genes associated with NSCLC and reports the percentage of cfDNA. The manufacturer purports that this genetic test will allow providers to make better treatment decisions based on the mutations present in the patient (Health, 2017). The gene panel was analytically validated, with 99.8% accuracy on 1000 consecutive samples (Lanman et al., 2015).

Another study evaluating the clinical utility of this test was performed by Kim et al. 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).

A third commercially available test is the Liquid GPS by NantHealth Inc. This test assesses both cfDNA and ctDNA, and measures targeted therapy, chemotherapy, and immunotherapy markers. For example, this test evaluates the biomarker AR-V7, which is considered a predictor of prostate cancer treatments. The targeted therapy biomarkers are as follows: EGFR, HER2, AR (or AR-V7), c-MET, ROS1 fusion, ALK fusion, KRAS, BRAF, and NRAS. The chemotherapy markers are as follows: ERCC1, XRCC1, MGMT1, TUBB3, hENT1, TP, TS, RRM1, TOP1, TOP2A, and TOP2B. The immunotherapy markers are as follows: PD-L1, TIM-3, CTLA-3, and LAG-3 (NantHealth, 2018).

Other proprietary liquid biopsy tests are available to assess genes associated with numerous conditions. Oncobeam has 20 liquid biopsy PCR-based tests for the evaluation of gene mutations, which follow the same principle as other cell-free DNA tests (cells shedding DNA fragments into the circulatory system and into the plasma where it can be easily examined) (Oncobeam). Oncobeam uses a proprietary method in which the DNA is isolated and amplified with PCR. Then, the wild-type and mutant strains are tagged with separate fluorescent probes, and finally quantified with flow cytometry (Diehl et al., 2008; Oncobeam). Oncobeam’s liquid biopsies include assessments for the EGFR, ALK, and ROS1 mutations, and these panels have been observed to detect as low as 0.04% fraction of mutation (Oncobeam). Foundation has also created proprietary tests that examine cell-free DNA. Foundation’s test evaluates features like microsatellite instability, specific types of mutations, and examines 70 commonly altered oncogenes (Foundation, 2018). A prior version of this test (covering 62 genes) was evaluated at 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).

National Comprehensive Cancer Network (NCCN)
NCCN guidelines for non-small cell lung cancer (NSCLC) (NCCN, 2019d) strongly advises “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 “Recent data suggest that plasma genotyping (also known as liquid biopsy or plasma biopsy) may be considered instead of tissue biopsy; however, if the plasma biopsy is negative, then tissue biopsy is recommended if feasible (Oxnard et al., 2016; Sacher et al., 2016)”.

However, the NCCN goes on to state that cell-free or circulating tumor DNA testing should not be used in lieu of tissue diagnosis. The NCCN notes that specificity is generally very high for cell-free tumor testing but is lacking in sensitivity and that standards for testing 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, if there is insufficient material for a molecular analysis (NCCN, 2019d).

NCCN states that “the clinical use of Circulating Tumor Cells (CTC) in metastatic breast cancer is not yet included in the NCCN Guidelines for Breast Cancer (NCCN, 2019d) for disease assessment and monitoring.” However, assessment of the PIK3CA mutation may be performed through liquid biopsy if the tumor is HR-positive, HER2 negative, and if therapy with alpelisib is being considered.

NCCN found that although the presence of androgen receptor splice variant 7 (AR-V7) in CTCs is associated with abiraterone and enzalutamide resistance, it has not been validated and has a low prevalence (3%) in patients before treatment with abiraterone, enzalutamide, and taxanes.  The NCCN panel believes that at this time testing for AR-V7 CTCs would not be useful to inform treatment decisions in prostate cancer (NCCN, 2017, 2019g).

NCCN guidelines for colon cancer and small cell lung cancer do not address use of circulating tumor cells or circulating tumor DNA for patient management (NCCN, 2019a, 2019h).

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, 2019e).

For leptomeningeal metastases, the NCCN notes that assessment of CTCs “increases sensitivity of tumor cell detection and assessment of response to treatment” (NCCN, 2018).

For pancreatic adenocarcinomas, the NCCN acknowledges that circulating cell-free DNA is being investigated as a biomarker for screening (NCCN, 2019f).

For esophageal, esophagogastric junction cancers, and gastric cancers, the NCCN states that the role of liquid biopsy for genomic profiling of esophageal and esophagogastric junction cancers “remains unclear at this point and is a subject of ongoing investigation” (NCCN, 2019b, 2019c).

American Society of Clinical Oncology (ASCO)
In 2007, ASCO published recommendations for the use of tumor markers in the prevention, screening, treatment, and surveillance of breast cancer. ASCO stated that circulating tumor markers has demonstrated insufficient evidence to support routine use in clinical practice and therefore recommend against their use for diagnosis or to guide treatment. ASCO also cannot recommend any FDA-approved tests until further information confirms the validity and utility of these tests (Harris et al., 2007).

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 (Poznak et al., 2016).

National Academy of Clinical Biochemistry (NACB)
In 2008, the NACB issued practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast and ovarian cancers (Sturgeon et al., 2008). The NACB panel’s recommendation on measurement of circulating prostate cancer cells in peripheral blood stated that “although initial results are encouraging, these techniques are not yet sufficiently validated to warrant recommending their application in routine clinical practice.”

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).

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).

American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology (2017)
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).


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Coding Section 

Codes Number Description
  81235  EGFR (epidermal growth factor receptor) (eg, non-small cell lung cancer) gene analysis, common variants
  81404  Molecular pathology procedure, Level 5 (eg, analysis of 2-5 exons by DNA sequence analysis, mutation scanning or duplication/deletion variants of 6-10 exons, or characterization of a dynamic mutation disorder/triplet repeat by Southern blot analysis) 
  81479  Unlisted molecular pathology procedure 
ICD-10-CM diagnosis codes:  C34.00  Malignant Neoplasm Of Unspecified Main Bronchus  
  C34.01   Malignant neoplasm of right main bronchus
  C34.02   Malignant neoplasm of left main bronchus
  C34.10   Malignant Neoplasm Of Upper Lobe, Unspecified Bronchus Or Lung
  C34.11   Malignant neoplasm of upper lobe, right bronchus or lung
  C34.12   Malignant neoplasm of upper lobe, left bronchus or lung
  C34.2   Malignant Neoplasm Of Middle Lobe, Bronchus Or Lung
  C34.30   Malignant Neoplasm Of Lower Lobe, Unspecified Bronchus Or Lung
  C34.31  Malignant neoplasm of lower lobe, right bronchus or lung  
  C34.32  Malignant neoplasm of lower lobe, left bronchus or lung
  C34.80  Malignant Neoplasm Of Overlapping Sites Of Unspecified Bronchus And Lung  
  C34.81  Malignant neoplasm of overlapping sites of right bronchus and lung  
  C34.82  Malignant neoplasm of overlapping sites of left bronchus and lung  
  C34.90  Malignant neoplasm of unspecified part of unspecified bronchus or lung  
  C34.91  Malignant neoplasm of unspecified part of right bronchus or lung  
  C34.92   Malignant neoplasm of unspecified part of left bronchus or lung

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each Policy. They may not be all-inclusive.

This medical policy was developed through consideration of peer-reviewed medical literature generally recognized by the relevant medical community, U.S. FDA approval status, nationally accepted standards of medical practice and accepted standards of medical practice in this community, Blue Cross and Blue Shield Association technology assessment program (TEC) and other non-affiliated technology evaluation centers, reference to federal regulations, other plan medical policies and accredited national guidelines.

"Current Procedural Terminology© American Medical Association.  All Rights Reserved" 

History From 2016 Forward     


Interim review to provide verbiage regarding PIK3CA mutation testing, updating medical necessity criteria for Stage IIIb/IV NSCLC testing. Reformatting policy for clarity. 


Annual review, no change to policy intent. Updating coding. 


Interim review of policy to allow for medical necessity criteria related to Guardant360 testing. Also updating description, references and coding. 


Annual review, policy being rewritten entirely to allow for limited indications being medically necessary.


Annual review, no change to policy intent. 


Update review date. No other changes made. No change to policy intent 



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