 The capture of whole, circulating tumor cells (CTCs) was the initial focus of liquid biopsies. Now, the definition of liquid biopsy has expanded to include the collection of cell-free DNA (cfDNA), along with various species of cell-free RNA and exosomes, all of which are capable of providing information on the disease status of patients with cancer.

This expansion in scientific and technological approaches is expected to become particularly meaningful in the diagnosis and treatment of non—small cell lung cancer (NSCLC). Although hurdles remain before liquid biopsy is seen in routine clinical practice for patients with NSCLC, its considerable potential and the energy being applied to its development suggest that everyday clinical use of liquid biopsy is inevitable.

Compared with conventional tissue biopsy of tumors, liquid biopsy offers advantages in NSCLC. A blood draw is minimally invasive for the patient, can be done at the point-of-care, and can be performed serially and inexpensively to monitor disease progression, an approach not possible with tissue biopsies in the lungs.

Liquid biopsy for NSCLC has potential applications in both screening and in monitoring. Survival rates for NSCLC are low, despite advances in treatment of the disease, because patients often present with advanced disease. The availability of screens to detect validated biomarkers of the disease from CTCs, cfDNA, or other elements in blood may make it practical to one day perform routine screening for lung cancer, particularly for smokers.

Additionally, a key benefit to liquid biopsies is the potential for overall characterization of disease progression and genetic variation. Intratumoral heterogeneity is a common feature of primary tumors, but the particular sample collected in a tissue biopsy may not be representative of the tumor as a whole. Heterogeneity also exists between the primary tumor and metastases. Liquid biopsy can address this issue by detecting DNA that is shed by cancer cells in all parts of a tumor, as well as by secondary, metastatic tumors.

The major reason for studying heterogeneity is detection of mutations for the assignment of targeted therapies. In this way, liquid biopsy is a crucial component of the shift to personalized medicine. For NSCLC, this relates in particular to the detection of mutations in exon 20 of the EGFR gene, where 50% to 60% of acquired resistance to inhibitors of EGFR’s tyrosine kinase activity is associated with point mutation T790M.1 Other key mutations in NSCLC have now been identified 

Despite the widely acknowledged potential of liquid biopsy, its integration into everyday clinical practice in oncology has yet to occur.

Alberto Bardelli, PhD, associate professor, Turin University School of Medicine, Italy, expressed the need for further data when summarizing a session on liquid biopsy that he chaired at the 2014 European Society for Medical Oncology(ESMO) Congress. “I have no doubt liquid biopsy will become routine, but right now the evidence is lacking,” he said.

Standardization of analytical processes for liquid biopsy and the development of robust commercial platforms for the analysis of biomarkers in body fluids are being actively pursued.

“There are biomarkers with the potential to be introduced into daily clinical practice in the near future, but success depends on robust validation in sufficiently large, independent, prospectively designed studies,” said Paul Hofman, MD, PhD, a team leader at the Institute for Research on Cancer and Aging in Nice, France.

The signals from a growing body of research are promising. In February, investigators reported that cfDNA was successfully used to assess EGFR mutations in plasma and serum samples from patients with NSCLC collected as part of the EURTAC trial.1 They were able to correlate the type of EGFR mutation, particularly L858R, with overall survival (OS), progression-free survival (PFS), and response to therapy outcomes.

In April, researchers presenting at the AmericanAssociation for Cancer Research (AACR) Annual Meeting reported that liquid biopsies in NSCLC treatment could provide key data on disease progression earlier than other methods available.2

Jonas A. Nilsson, PhD, a researcher in the Department of Radiation Sciences at UmeÃ¥ University in Sweden, and colleagues analyzed the efficacy of monitoring EML4-ALK fusion gene rearrangements in the blood platelets and plasma of 77 patients with NSCLC.

They determined that reverse transcription-polymerase chain reaction testing allowed real-time monitoring of the disease and earlier identification of patients who have developed resistance to treatment with crizotinib, an ALK inhibitor.

“We showed that if we detected EML4-ALK in the platelet fraction before therapy starts and it does not disappear during treatment, it indicates that the patient is not responding to the therapy, which is associated with a shorter time to recurrence and, therefore, other therapies could be tried,” Nilsson said in a statement.

CTCs are intact cancer cells that have been released into the bloodstream and have been associated with worse prognosis in several major cancers, including lung cancer, with an increase in CTCs predicting tumor progression and aggressiveness.

In patients with NSCLC, the CTC count has been correlated with both PFS and OS. Although CTCs are potentially more informative than cfDNA by their very nature as intact cells, CTCs are scarcer and therefore more difficult to obtain from a blood sample.

Thus far, the CellSearch system is the only in vitro CTC diagnostic to gain the FDA’s approval, although other assays are available commercially as laboratory-developed tests. Initially approved in 2004, CellSearch is indicated for detecting the presence of CTCs and monitoring disease progression through CTC levels in patients with metastatic breast, colorectal, and prostate cancer.

The CellSearch technique has been used in clinical trials to establish thresholds for patient CTC counts that could be used as prognostic markers and predictors of patient outcomes. However, the technique relies on the presence of the epithelial cell adhesion molecule (EpCAM) on the surface of the CTC. Because metastatic cells that have undergone an epithelial-mesenchymal transition (EMT) no longer express epithelial biomarkers, the EpCAM system might not detect CTCs of interest in patients with lung cancer.

There are scientific publications demonstrating CTC detection in patients with lung cancer by other means, but these new methods have not yet become part of routine clinical practice. The abundance of methods available for CTC detection may be a contributing factor, because selecting the optimal method can be challenging.

Proper treatment of NSCLC requires evaluation of specific predictive biomarkers, with molecular characterization of EGFR mutation status in patients with NSCLC via tissue biopsy firmly established in current clinical practice. However, the fine-needle aspiration procedures used to collect tumor tissue can make molecular profiling difficult because of the tiny amount of material obtained.

More sensitive techniques have been developed to deal with the small sample sizes characteristic of such tissue biopsies, but analysis of circulating tumor (ct)DNA may be a better solution. Although cfDNA is released by both healthy and tumor cells, the DNA released from normal cells undergoing apoptosis is trimmed into relatively uniform fragments of 185 to 200 base pairs, while the DNA from tumor cells undergoing necrosis varies in size.

Healthy individuals display only cfDNA from apoptotic cells, meaning that prevalence of longer DNA fragments could be used a marker of malignancy, and higher ctDNA levels compared with healthy controls have been demonstrated in patients with cancer.

ctDNA can be also used for mutational analysis.

For example, examination of EGFR status in ctDNA from patients with NSCLC has demonstrated the occurrence of the resistance mutation T790M at progression, but not at the time of treatment initiation.

Evaluation of the KRAS mutation in ctDNA from patients with NSCLC showed that those with a KRAS mutation detectable in plasma had shorter OS and PFS compared with patients with wildtype KRAS.

Thus, detection of KRAS mutations in plasma may have a prognostic as well as a predictive value. Use of ctDNA to detect somatic mutations, particularly of EGFR, by next generation sequencing (NGS), shows promise.3 However, there is still a dearth of widely accepted and approved methods for ctDNA analysis, and detection of ctDNA in patients with lung cancer is not used by physicians in daily practice.

Exosomes represent another potential avenue for revealing mutational heterogeneity and tracking disease progression. Exosomes are small (40 nm to 100 nm diameter), membrane-derived vesicles that are released from normal and malignant cells to blood and other body fluids. Exosomes contain DNA fragments, such as signal proteins and/or peptides, micro RNAs, (mRNAs), and lipids, and they can promote cancer progression when they originate from tumor cells.

Although the normal cellular function of exosomes appears to include cell-to-cell communication via direct activation of surface-expressed ligands or by transfer of molecules between cells, their diagnostic potential is clear. Not only can exosomes be subjected to NGS but also to various RNA and proteomic analyses.

		Rolfo C, Giovannetti E, Hong DS, et al. Novel therapeutic strategies for patients with NSCLC that do not respond to treatment with EGFR inhibitors [published online June 6, 2014]. Cancer Treat Rev. 2014;40(8):990-1004.

		Karachaliou N, Mayo-de las Casas C, Queralt C, et al. Association of EGFR L858R mutation in circulating free DNA with survival in the EURTAC trial [published online February 26, 2015]. 

		Nilsson JA, Karachaliou N, Schellen P, et al. Monitoring rearrangement of EML4-ALK in blood platelets predicts outcome to crizotinib treatment in non-small-cell lung cancer patients. Presented at: 2015 AACR Annual Meeting; April 18-22, 2015; Philadelphia, PA. Abstract 8728.

		Couraud S, Vaca-Paniagua F, Villar S, et al. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in non-small cell lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002 [published online July 10, 2014]. 

Articles

The definition of liquid biopsy has expanded to include the collection of cell-free DNA (cfDNA), along with various species of cell-free RNA and exosomes, all of which are capable of providing information on the disease status of patients with cancer.

Liquid Biopsy Technology Grows More Varied and Potentially More Useful in NSCLC

Toxicities among patients with metastatic colorectal cancer (mCRC) who responded best to regorafenib were“broadly similar” to the overall study population, according to a posthoc analysis of the pivotal phase III CORRECT trial presented at the 2015 Gastrointestinal (GI) Cancers Symposium.

“The results of this exploratory subgroup analysis support the clinical benefit and tolerability of regorafenib as a treatment option for patients with metastatic colorectal cancer,” said lead investigator Axel Grothey, MD, a professor in the Division of Medical Oncology at Mayo Clinic in Rochester, Minnesota.

CORRECT randomized 760 patients with mCRC in a 2:1 ratio to the oral multikinase inhibitor regorafenib (n = 505) or placebo (n = 255). Overall, treatment with regorafenib improved overall survival (OS) by 1.4 months (6.4 vs 5.0 months; HR = 0.77; 

 = .0052) and progression-free survival (PFS) by 0.2 months (1.9 vs 1.7 months; HR = 0.49; 

 <.000001). Based on these data, the FDA approved regorafenib in September 2012 for use in patients with previously treated mCRC.

The subgroup analysis presented at the GI Symposium included 98 patients (19.4%) treated with regorafenib who had PFS >4 months. Forty-seven percent of these patients were 

Patients in this best responder subgroup received a median of 6 cycles of regorafenib, with 92% receiving ≥5 cycles and 20% receiving >8 cycles. Overall, 34% of patients had their regorafenib dose reduced and 87% had dose interruptions. The resulting mean daily dose was 139 mg, or 81% of the planned dose.

The review of toxicities in the subgroup found that these patients mostly experienced adverse events (AEs) at comparable rates to those in the overall trial population, despite exposure to regorafenib for over twice as long as the overall group.

All patients with longer PFS experienced AEs; the most common grade ≥3 AEs were hand-foot skin reaction (20%), hypertension (17%), diarrhea (17%), and fatigue (16%).

Although the AE profile of regorafenib was similar in both populations, some AEs did occur more frequently in patients treated with regorafenib for >4 months. Rates of all-grade diarrhea, hand-foot skin reaction, and weight loss were ≥15% higher in the subgroup with longer PFS than in the overall CORRECT population.

Rates of grade ≥3 diarrhea and hypertension (17% each) in the longer PFS population were twice as high as in the overall population.

According to Grothey, prospective validation of these AE findings among the best responders is needed, in conjunction with biomarker analysis from real-life clinical experience.

"While adverse events across both populations were broadly similar, some did occur more frequently in patients with longer exposure, an observation that is possibly related to the longer duration in this subgroup. Analyses to identify clinical and molecular markers in these patients are ongoing.”

CORRECT was an international trial involving 114 centers across 16 countries in North America, Europe, Asia, and Australia. Patients had to have an ECOG PS of 0 or 1 and had to have progressed within 3 months of receiving standard treatments, including chemotherapy, bevacizumab (Avastin), cetuximab (Erbitux), and panitumumab (Vectibix).

Patients received either 160 mg of oral regorafenib or placebo once daily on a 3-weeks-on/1-week off basis. All patients in the trial also received best supportive care. Radiologic evaluation was done every 8 weeks, with treatment continuing in the absence of progression, unacceptable toxicity, or death.

The primary endpoint was OS, defined as the time from randomization to death from any cause. Secondary endpoint was PFS, defined as the time to disease progression (radiological or clinical) or death from any cause. Patients were permitted dose interruptions or dose reductions for management of treatment-related AEs.

Regorafenib blocks the activity of multiple protein kinases involved in oncogenesis (KIT, RET, RAF-1, BRAF, and BRAF V600E), tumor angiogenesis, (VEGFR 1-3 and TIE2), and the tumor microenvironment (PDGFR and FGFR).

Approximately 50% to 60% of patients diagnosed with CRC develop metastases, and the majority of these patients have unresectable disease.The standard of care for unresectable mCRC is chemotherapy with or without the VEGF inhibitor bevacizumab, but few options are available for patients whose disease progresses after standard therapies.

	Grothey A, Falcone A, Humblet Y, et al. Subgroup analysis of patients with metastatic colorectal cancer (mCRC) treated with regorafenib (REG) in the CORRECT trial who had progression-free survival (PFS) longer than 4 months. 

Adverse events among patients with metastatic colorectal cancer who responded best to regorafenib in the phase III CORRECT trial were "broadly similar" to the overall study population.

Dennis Bittner, PhD

Articles

Liquid Biopsy Technology Grows More Varied and Potentially More Useful in NSCLC

Analysis Confirms Safety of Regorafenib in mCRC Among Best Responders