Liquid Biopsy Use in NSCLC Clinical Practice on the Horizon

The capture of whole, circulating tumor cells (CTCs) was the initial focus of liquid biopsies. However, 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 cancer patients.

Compared with conventional tissue biopsy of tumors, liquid biopsy offers advantages in the diagnosis and treatment of non-small cell lung cancer (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.

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 to the detection of mutations in exon 20 of theEGFRgene, where 50% to 60% of acquired resistance to inhibitors ofEGFR’s tyrosine kinase activity is associated with point mutationT790M.¹Other key mutations consist of rearrangements of theALKgene andKRASmutations.

Circulating Tumor Cells

However, 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.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 progression-free survival (PFS) and overall survival (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.

Circulating Tumor DNA

There is only one technology approved by the US Food and Drug Administration (FDA) for CTC detection (in metastatic cancers of the breast, prostate, and colon, but not in metastatic lung cancer). This technique was 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 ofEGFRmutation 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.

Exosomes

Outlook

ctDNA can be also used for mutational analysis. For example, examination ofEGFRstatus in ctDNA from patients with NSCLC has demonstrated the occurrence of the resistance mutationT790Mat progression, but not at the time of treatment initiation. Evaluation of theKRASmutation in ctDNA from patients with NSCLC showed that those with aKRASmutation detectable in plasma had shorter OS and PFS compared with patients with wild-typeKRAS. Thus, detection ofKRASmutations in plasma may have a prognostic as well as a predictive value. Use of ctDNA to detect somatic mutations, particularly ofEGFR, by next generation sequencing (NGS), shows promise.²However, 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.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, IRCAN team leader at INSERM, Nice, France. The utility of liquid biopsy in diagnosis and treatment of NSCLC will increase with discovery of additional biomarkers to differentiate and stratify various forms of the disease, supporting the global trend toward personalized medicine.

Although hurdles remain before liquid biopsy is seen in routine clinical practice for diagnosis and treatment of NSCLC, its considerable potential and the energy being applied to its development mean that every day clinical use of liquid biopsy is inevitable and relatively imminent.

Companies Developing Liquid Biopsy for NSCLC and Other Cancers

Qiagen

Guardant

Sequenom/Illumina

Trovagene

Foundation Medicine

Cynvenio

Biocept

Exosome Diagnostics

Although liquid biopsy testing has not yet entered daily clinical practice in oncology, numerous companies are vying for the opportunity to make the technology commonplace. Here are some of the players:Received CE-IVD marking in Europe on January 21, 2015, for its ctDNA-based companion diagnostic to gefitinib for NSCLC. Test is quantitative polymerase chain reactions (QPCR)-based analysis checkingEGFRstatus across 21 mutations in serum. Announced codevelopment of liquid biopsy assays with Exosome a week earlier.Next generation sequencing (NGS)-based mutation analysis of ctDNA using 68-gene panel with complete exon coverage ofEGFR,BRAF,KRAS, andMET, and detection of rearrangements inALKandROS1. Received additional $50 million in funding February 2015.The companies settled their IP differences December 3, 2014, and have pooled their patent portfolios, announcing plans to enter the cancer market. RUO kits to be released in 2015 while Illumina seeks FDA approval for tests. Strong position in noninvasive prenatal testing (NIPT) market using nearly identical technology.Next generation sequencing of ctDNA forBRAF V600EandKRASmutations in urine as well as in blood. Pipeline includes tests forEGFRandNRASmutations.Roche announced purchase of a majority stake in Foundation Medicine on January 12, 2015, for more than $1 billion. The company currently offers a 315-gene oncology-based test for solid tumors, and another that checks 405 genes for hematologic tumors. Roche says it will convert tests to liquid biopsy.Thermo Fisher Scientific announced the deal on January 13, 2015, to distribute Cynvenio’s LiquidBiopsy platform. Processes CTCs, uses NGS to check mutations in 50 genes important in breast, CRC, and prostate cancers. Mutations of additional cancers on the way, as well as analysis of ctDNA. Samples processed currently in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory but will launch RUO assay kits this year.Uses CTCs and ctDNA from blood for CLIA-validated tests of lung, breast, and gastric cancer. Biocept announced January 7, 2015, that its tests will be offered to a 19-million member preferred provider network. Two weeks later, Biocept announced the launch of a blood test forROS1rearrangements in NSCLC using CTCs.Focuses on advantages of exosomes to derive additional information on lung cancer beyond what is available from ctDNA. In addition to currentALKandEGFR,T790Mtests for NSCLC, company plans to launch a plasma-based cancer panel and a urine-based prostate cancer diagnostic in 2015. Announced codevelopment of liquid biopsy assays with Qiagen on January 13, 2015.

References

1. Rolfo C, Giovannetti E, Hong DS, et al. Novel therapeutic strategies for patients with NSCLC that do not respond to treatment with EGFR inhibitors.Cancer Treat Rev. 2014;40(8):990-1004.
2. Couraud S, Vaca-Paniagua F, Villar S, et al. Non-invasive diagnosis of actionable mutations by deep sequencing of circulating-free DNA in non-small cell lung cancer: Findings from BioCAST/IFCT-1002.Clin Cancer Res. 2014;20:4613-4624.