Liquid biopsy in non-small cell lung cancer: A minimally invasive solution for molecular testing and real-time monitoring

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Although tissue biopsy is currently the standard of care for non-small cell lung cancer, liquid biopsy is a promising, minimally invasive technique for diagnosing and monitoring patients, with this technology quickly becoming a clinical reality.

NSCLC requires tailored therapy because of its many different forms. With the increasing number of targeted therapies, molecular testing is essential for producing the best outcomes. After initial diagnosis of disease and selection of therapy, patients must be repeatedly tested for resistance and treatment response. Complicating matters, approximately 70% of patients with NSCLC present with advanced disease. Formalin-fixed, paraffin-embedded tissue specimens are generally used for testing these patients, but tissue sampling is often not performed because of patient discomfort, difficulty in obtaining enough tissue, and other challenges.

Problems with tissue biopsies are increased in patients who have developed resistance to tyrosine kinase inhibitors (TKIs), which commonly occurs within 12 months of starting TKI therapy. Among these patients, approximately 25% are ineligible for a second biopsy because of metastatic disease or poor health status. Even when patients are eligible, up to 20% of samples obtained through re-biopsy are uninformative because of a lack of genetic material or the absence of tumor within the sample.

Advantages of liquid biopsy

Liquid biopsy, which involves analysis of body fluids, circumvents many of the obstacles of tissue biopsy with its minimally invasive approach (Table). Cell-free DNA (cfDNA), circulating tumor cells (CTCs), exosomes, microRNAs or platelets can be evaluated from fluids. These fluids include:

• blood;
• plasma;
• serum;
• pleural effusions;
• ascites;
• cerebrospinal fluid;
• urine; and
• saliva.

Notably, urine and saliva testing are completely noninvasive and amenable to home collection, so serial sampling is not limited by the patient’s performance status.

At the time of diagnosis, liquid biopsy can be used for rapid molecular profiling to select targeted agents. Blood samples can provide genetic information for all lesions, both primary and metastatic, and show a more complete picture of tumor heterogeneity, even in light of tumor fibrosis or necrosis. CTC counts can be used as a prognostic marker after surgery, chemotherapy and radiotherapy. Also, liquid biopsy can be easily repeated for monitoring disease, helping to predict drug response and resistance.

Additional benefits of liquid biopsy include savings in time and money. Liquid biopsy offers a rapid turnaround time for results of 1 to 2 weeks with compared with 3 to 6 weeks typical for tissue biopsy. One example of savings comes from a recent study of urine-based vs. tissue-based testing, demonstrating up to $1,680 in savings per patient, which was the result of avoiding biopsy procedures, associated complications and tissue-based molecular testing in 56% of patients.

Table. Advantages of liquid biopsy vs. tissue biopsy

Abbreviations: NR, not reported; ORR, objective response rate; OS, overall survival; PFS, progression-free survival.

Molecular testing guidelines

Approved targeted therapies are available for three genetic alterations in NSCLC:

• epidermal growth factor receptor (EGFR) mutations;
• c-ros oncogene 1 (ROS1) rearrangements; and
• anaplastic lymphoma kinase (ALK) rearrangements.

These alterations should be tested for at the time of diagnosis in all patients with adenocarcinoma or mixed histology including adenocarcinoma. Current guidelines also recommend that EGFR mutation status be evaluated in squamous cell carcinoma where an oncogenic driver may be involved.

If EGFR, ALK or ROS1 tests are negative or unknown, then PD-L1 expression testing should be performed before first-line treatment in all patients with metastatic NSCLC. High PD-L1 expression, which is defined by a tumor proportion score of at least 50%, warrants PD-L1 inhibitor immunotherapy.

The most recent guidelines also describe analysis of several other oncogenes at the time of diagnosis, including RET, HER-2, BRAF, RAS and MET, although associated target therapies are off-label. After diagnosis, molecular profiling is needed upon disease progression and for therapeutic monitoring, especially when resistance mutations are present.

Impact and accuracy of liquid biopsy techniques

EGFR mutations are associated with reduced survival and have been reported in 43% to 89% of adenocarcinomas, about 48% of patients of East Asian origin and 10% to 20% of white patients. EGFR TKI therapy is the most effective targeted therapy for NSCLC. Although TKIs are effective, most patients will go on to develop resistance mutations. The EGFR T790M resistance mutation is the most common, and is found in approximately 50% to 60% of NSCLC patients with acquired resistance. This secondary mutation conveys resistance to first- and second-generation TKIs such as:

• erlotinib (Tarceva; Genentech, Astellas);
• gefitinib (Iressa, AstraZeneca); and
• afatinib (Gilotrif, Boehringer Ingelheim).

However, the mutation remains sensitive to osimertinib (Tagrisso, AstraZeneca), a third-generation TKI.

In a recent study comparing urine-based liquid biopsy with tissue biopsy, the sensitivity of EGFR T790M detection was 72%. In samples that met recommended urine volumes of 90 mL to 100 mL, the sensitivity reached 93%, which was comparable to plasma-based testing. Prolonged progression-free and overall survival rates were reported with urine-based testing, based on a 7% increased detection of EGFR T790M and timely treatment initiation with a third-generation EGFR TKI. The combination of urine and plasma testing yielded the identification of more EGFR T790M–positive cases compared with tissue biopsy alone (95% vs. 83%, respectively). Importantly, the concordance between urine and tissue EGFR T790M results has been reported at 81%.

Along with differences in liquid biopsy methods, patient age and metastasis may also influence EGFR mutation detection. The ASSESS study found that in patients with EGFR-mutant NSCLC tumors, plasma detection of EGFR was higher in patients younger than 65 years of age, and increased sensitivity was seen in patients exhibiting higher metastatic grades and more metastatic sites.

Current and emerging technologies

Because of the heterogeneous nature of pooled CTCs, technological challenges for isolating and enriching tumor-specific CTCs, and the lack of standardized methods, liquid biopsy studies have mostly focused on cfDNA. In 2016, the FDA approved the cobas EGFR Mutation Test, v2 (Roche Molecular Systems), for plasma testing using cfDNA. This was the first liquid biopsy test approved by the FDA for clinical decisions, and it demonstrates a concordance of over 91% between tumor tissue and cfDNA.

Although it is not yet FDA-approved, the therascreen EGFR Plasma RGQ PCR Kit (Qiagen) has a reported concordance of 97%, with a sensitivity of 62% to 68% and a specificity of nearly 100%. On the horizon, LungSelect (Personal Genome Diagnostics) is a digital next-generation sequencing-based method that detects point mutations, deletions, insertions and translocations, and has demonstrated specificity of more than 99.9%.

Other investigated liquid biopsy tests include:

• mass spectrometry genotyping;
• denaturing high-performance liquid chromatography;
• peptide nucleic acid (PNA) clamping-assisted polymerase chain reaction (PCR);
• droplet digital PCR; and
• BEAMing (beads, emulsion, amplification and magnetics).

Some of these techniques show promise. Of note, PNA clamping-assisted PCR has exhibited up to 97% concordance with tissue testing, and droplet digital PCR can offer a turnaround time of 1 week or less with up to 100% specificity and 82% sensitivity.

A transformative period

The molecular testing landscape for NSCLC is rapidly evolving, and the advancement of new biomarker-based therapies will undoubtedly influence future treatments. Existing and emerging liquid biopsy techniques provide safe, accurate and minimally invasive collection of samples that can facilitate regular monitoring of molecular tumor characteristics.

Although the data encourage incorporation of plasma-based EGFR mutation detection into current molecular diagnostics, it is not time to abandon tissue-based biopsies just yet; they remain the gold standard. However, liquid biopsies should be considered when a tumor biopsy is unsuccessful or unsafe and in patients with a high tumor burden. Developments in the years to come are highly anticipated, as truly noninvasive NSCLC monitoring through urine or saliva samples is becoming a clinical reality.

References

Duréndez-Sáez E, Azkárate A, Meri M, et al. New insights in non-small-cell lung cancer: circulating tumor cells and cell-free DNA. J Thorac Dis. 2017;9(Suppl 13):S1332-S1345.

Yoon HJ, Lee HY, Lee KS, et al. Repeat biopsy for mutational analysis of non-small cell lung cancers resistant to previous chemotherapy: adequacy and complications. Radiology. 2012;265:939-948.

Zhang Z, Xiao Y, Zhao J, et al. Relationship between circulating tumour cell count and prognosis following chemotherapy in patients with advanced non-small-cell lung cancer. Respirology. 2016;21:519-525.

Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17:223-238.

Sands J, Hornberger J. P3.02b-010 Urine detection of EGFR T790M mutation in non-small-cell lung cancer: an outcomes and total cost of care analysis. J Thorac Oncol. 2017;12:S1191.

Reck M, Rabe KF. Precision diagnosis and treatment for advanced non-small-cell lung cancer. N Engl J Med. 2017;377(9):849-861.

Reckamp KL, Melnikova VO, Karlovich C, et al. A highly sensitive and quantitative test platform for detection of NSCLC EGFR mutations in urine and plasma. J Thorac Oncol. 2016;11:1690-1700.