Refining management of lung cancer: ctDNA testing comes of age


Lung cancer is the leading cause of cancer death, accounting for 18.4% of the total cancer deaths worldwide. Despite major advances in cancer care, lung cancer is associated with a high mortality rate, in part because of late-stage diagnosis, for which the 5-year survival rate is approximately 4% (CA Cancer J Clin. 2018;68:394-424; CA Cancer J Clin. 2017;67:7-30).

Across the spectrum of lung cancer care, classification of disease subtype, selection of treatment, and detection of resistance mechanisms increasingly relies on molecular and genetic information. With breakthroughs in research, the lung cancer treatment paradigm is at the forefront of personalized medicine and treatment approaches are shifting away from conventional chemotherapy toward targeted therapies (Cancer Treat Rev. 2019;78:31-41).

Currently, the classification and management of lung cancer is increasingly guided by molecular and genetic testing. However, obtaining tumor tissue for testing is often challenging. Acquiring sufficient tumor tissue from biopsy samples is a common barrier given that a smaller percentage of tumor cells are often present. In addition, for patients progressing on a particular therapy, multiple biopsies are not always feasible as the quality of the biopsy may be insufficient to allow for molecular testing. As a result, a significant need remains for alternative forms of molecular and genetic testing.

Circulating tumor DNA is found in serum and plasma fractions from blood.
credit: Rachel June Wong/Wikimedia/ CC BY-SA 4.0

Applying ctDNA in cancer care

Liquid biopsy tools, such as circulating tumor DNA (ctDNA), offer the potential to transform cancer care through noninvasive tumor monitoring, earlier disease detection, therapeutic response prediction, and treatment personalization. ctDNA is a component of cell-free DNA that is shed by malignant tumors into the bloodstream. Cell-free DNA fragments contain valuable information for decoding the tumor’s genetic characteristics. However, levels of ctDNA are generally low, especially in patients with nonmetastatic cancer, which warrants highly sensitive methods for detection and quantitation. (J Clin Oncol. 2018;36[16]:1631-41; J Thorac Dis. 2019;11[Suppl 1]:S113-26). 

Recent advances in sequencing technology have enabled ctDNA testing to be technically feasible, which has allowed for integration of the technology into clinical practice. Despite sensitivity-related challenges, significant potential for ctDNA analysis remains.

Upon detection and evaluation, ctDNA reflects the genetic makeup of the tumor and provides similar molecular findings, compared with tissue biopsy, which reflects the genetic composition of a single biopsied or resected tumor. ctDNA provides a “snapshot” of the DNA from all locoregional and metastatic tumors in the body. Therefore, ctDNA testing can complement a single biopsy and help elucidate tumor heterogeneity, which, in turn, may optimize targets for treatment, monitor response, and detect resistance to therapy (Cancer Treat Rev. 2019;78:31-41).

As ctDNA is a blood-based test, repeat testing is possible, which may help monitor tumor dynamics over time. Furthermore, if carefully optimized for certain clinical scenarios, ctDNA testing may also be a cost-effective option over radiographic examinations and invasive procedures.

ctDNA is “currently being used to identify targetable and resistance mutations in NSCLC when tissue is not obtainable or insufficient, and that ctDNA analysis has improved our ability to manage advanced-stage disease in a more personalized fashion.”


In non–small cell lung cancer (NSCLC), targeted therapies directed against mutations in EGFR, ALK, ROS1, and BRAF have improved clinical outcomes for patients presenting with these genetic alterations. For patients with these mutations, targeted therapy is the preferred treatment option (JAMA Oncol. 2019;5[2]:173-80).

The National Comprehensive Cancer Network guidelines recommend screening for actionable mutations as the standard of care in NSCLC. However, tumor tissue is often difficult to obtain, particularly for patients with metastatic disease. In addition, there are cases where the clinically actionable mutations present change over the course of treatment, and these genetic transformations can be difficult to monitor using tissue biopsy alone.

Because of spatial and temporal tumor heterogeneity, the use of biopsy from a single metastatic site makes clinical evaluation of resistance mutations challenging. Accordingly, ctDNA analysis represents a minimally invasive testing technique, with the potential for detecting molecular alterations in NSCLC patients (J Thorac Dis. 2019;11[Suppl 1]:S113-26; JAMA Oncol. 2019;5[2]:173-180; NCCN Clinical Practice Guidelines in Oncology: Non–Small Cell Lung Cancer. Version 6.2020.).

Aadel Chaudhuri, MD, PhD, of Washington University, St. Louis, explained that ctDNA is “currently being used to identify targetable and resistance mutations in NSCLC when tissue is not obtainable or insufficient, and that ctDNA analysis has improved our ability to manage advanced-stage disease in a more personalized fashion.”

The NCCN Clinical Practice Guidelines in Oncology for NSCLC state that ctDNA testing may be considered when a patient is medically unfit for invasive tissue sampling or if, after pathologic confirmation of an NSCLC diagnosis, there is insufficient material for molecular analysis.

Currently approved clinical use of cell-free DNA in NSCLC include the Cobas EGFR Mutation test v.2 CE-IVD (Roche) and Therascreen mutation kits (Qiagen). These tests are recommended for patients with NSCLC who are unable to undergo a tissue biopsy, or with acquired resistance to EGFR tyrosine kinase inhibitors (Eur Respir Rev. 2020;29[155]:190052).

Dr. Aadel Chaudhuri

credit: ELLA MARU STUDIO / Science Source


Small cell lung cancer accounts for approximately 15% of all lung cancers. Characterized by rapid growth and early metastases, SCLC is extremely aggressive and patients are quick to relapse. SCLC is also the deadliest form of lung cancer, with a 5-year survival of about 5%. Difficulties in early diagnosis and inadequacy of current therapeutic strategies contribute significantly to the poor outcomes observed in SCLC (Exp Mol Med. 2019;51[12]:1-13).

As SCLC is usually detected in later stages, therapeutic options are limited to cytotoxic chemotherapy, which fail to improve overall survival (OS) significantly. These chemotherapy regimens usually include a combination of platinum-based alkylating agents, such as carboplatin or cisplatin, and topoisomerase inhibitors, such as irinotecan or etoposide. Though SCLC tumors are initially responsive to chemotherapy, most will relapse given the development of chemoresistance. Hence, a better understanding of the mechanisms of tumor initiation, early-stage progression, and treatment resistance are needed for the development of novel therapeutic strategies in the settings of early diagnosis and prevention.

In recent years, there has been substantial advancement in understanding of the genomic landscapes for many cancer types, which has led to the identification of novel biomarkers and therapeutic targets. Better understanding the genomic intricacies of SCLC may help in identifying the somatic alterations in the SCLC genome, which is key to elucidating the underlying tumor mechanisms. Unraveling tumor genetics could also optimize existing treatments and help select patients most likely to benefit from targeted therapies (Nat Commun. 2018;9[1]:3114).

Although the benefits of genomic profiling have been well characterized, compared with other solid tumors, few studies have investigated the genomic landscape of SCLC. At least in part, this is because of lack of adequate tumor tissue, as surgical resection of SCLC is rare. Another reason is the rapid progression of recurrent disease. Patients with relapsed SCLC often require prompt treatment initiation and rarely undergo biopsies for molecular testing. There remains an urgent need for novel studies characterizing the genomic landscape of SCLC, particularly for patients already on treatment.

Unlike in NSCLC, mutations in therapeutic targets are rare in SCLC. TP53 and RB1 alterations play an essential role in SCLC tumorigenesis. Other frequently mutated genes in SCLC include NOTCH1-4, CREBBP, and EP300. Copy number alterations of MYC, MYCL1, and MYCN have also been reported in SCLC. ctDNA testing enables the detection of genomic alterations in relapsed disease in a clinically meaningful time frame and may also help elucidate the mechanisms of treatment resistance in SCLC and aid in developing novel treatment strategies (EBioMedicine. 2016;10:117-23).

SCLC is highly metastatic, with early hematogenous spread. As a result, ctDNA may be readily detectable in SCLC patients. ctDNA sequencing can also be leveraged to detect somatic mutations in SCLC patients. Apart from somatic mutations, ctDNA may be utilized to study the subclonal architecture of SCLC. Looking at patterns of initial response to chemotherapy/radiotherapy, and seeing invariable patterns of relapse, it has been postulated that treatment-naive SCLC may harbor subclones of inherently refractory cancer cells that give rise to relapse. Better understanding the subclonal architecture of SCLC and its molecular evolution during treatment may provide new insight into the mechanisms underlying recurrence and help guide the development of novel therapeutic approaches.

Besides being a noninvasive test, ctDNA analysis is suitable for providing real-time analysis by evaluating genomic alterations over time, especially during the course of treatment. Studies have demonstrated that ctDNA level correlates with tumor changes on CT imaging. Several studies are ongoing assessing whether ctDNA can detect disease recurrence/progression earlier than conventional imaging studies and whether early detection will improve clinical outcomes. Mutations from ctDNA are usually easier to identify in late-stage malignancies. However, as SCLC is a rapidly proliferating malignancy, with early hematogenous spread, ctDNA is readily detectable in SCLC patients, regardless of disease stage.

“The biggest advancement [of ctDNA technology in SCLC/NSCLC] has been data showing that ctDNA technology has promise for early lung cancer detection and could serve as an adjunct to low-dose CT screening.”

Early lung cancer detection

Although ctDNA has many attractive features, its use in detecting early-stage lung cancer has not been well established. ctDNA may only represent a small fraction of cell-free DNA and may not be representative of the localized tumor. In addition, the concentration of ctDNA is lower in early-stage disease, compared with later stages (Cancer Treat Rev. 2019;78:31-41).

However, recent studies in patients with early-stage NSCLC demonstrate that significant progress is being made in this area. Dr. Chaudhuri noted: “The biggest advancement [of ctDNA technology in SCLC/NSCLC] has been data showing that ctDNA technology has promise for early lung cancer detection and could serve as an adjunct to low-dose CT screening.”

Radiologic screening with low-dose CT is recommended for adults with high-risk lung cancer; however, the implementation of radiologic screening is impaired by high rates of false positives and low compliance with follow-up imaging.

To develop a noninvasive screening method for NSCLC, researchers determined ctDNA detection rates in patients with early-stage NSCLC using a tumor-informed approach (Nature. 2020;580[7802]:245-51). The study demonstrated that despite the extremely low levels of ctDNA in early-stage lung cancers, ctDNA remains present prior to treatment initiation in most patients, and is strongly prognostic. The study also showed that most somatic mutations in the cell-free DNA of lung cancer patients, and of risk-matched controls, reflect clonal hematopoiesis, and are nonrecurrent.

Based on these findings, the researchers developed an approach for noninvasive NSCLC screening that integrates improved molecular techniques with machine learning to detect the presence of NSCLC-derived cell-free DNA in a blood sample. The machine-learning method termed “lung cancer likelihood in plasma (Lung-CLiP),” can discriminate early-stage lung cancer patients from risk-matched controls. The availability of blood-based tests, such as ctDNA, have the potential to increase screening uptake in high-risk adults and reduce mortality for patients with lung cancer.

credit: Centers for Disease Control and Prevention / Science Source

Detecting MRD and predicting PFS, OS

Cancer recurrence is the leading cause of postoperative NSCLC mortality. As a result, there has been considerable research aimed at developing markers predictive of postoperative disease recurrence. As ctDNA in plasma indicates the presence of residual tumor tissue, researchers are exploring the use of ctDNA to evaluate the minimal residual disease (MRD) after surgical resection in NSCLC. Investigating MRD would facilitate the use of adjuvant therapies and postoperative follow-up in patients at risk of recurrence, and limit exposure to cytotoxic agents and ionizing radiations in low-risk patients (J Thorac Dis. 2019;11[Suppl 1]:S113-26).

The TRACERx study investigated phylogenetic tracking and MRD detection using ctDNA profiling following resection in patients with stage I-III NSCLC. The researchers generated patient-specific anchored-multiplex polymerase chain reaction enrichment panels for 78 patients with stage I-III disease who had undergone surgery. The researchers found that 45 patients had a relapse of their primary NSCLC, and ctDNA was detected at or before clinical relapse in 37 of these patients. Among these patients, time from ctDNA detection to clinical relapse was 151 days, and the median time to relapse from surgery was 413 days. The researchers concluded that ctDNA is an adjuvant biomarker capable of detecting MRD following surgery and defining the clonality of relapsing disease (2020 AACR Virtual Meeting II, Abstract CT023.).

Some evidence suggests ctDNA levels are also associated with progression-free survival (PFS) and OS, with the capacity to provide vital information on prognosis and treatment response. An analysis of the phase 1 TATTON study showed that ctDNA clearance of EGFR mutation – that is, detectable mutation at baseline that became undetectable or dropped below a predetermined threshold – is predictive of extended PFS for patients with EGFR-mutant, MET-amplified NSCLC with detectable ctDNA at baseline. The study also demonstrated that most ctDNA-evaluable patients had a molecular response to the combination of osimertinib (EGFR inhibitor) and savolitinib (MET inhibitor). For patients with and without ctDNA clearance, the median PFS was 9.1 months versus 3.9 months, respectively – a 66% improvement for those with ctDNA clearance (P = .0146). (2020 AACR Virtual Meeting II, Abstract CT303.).

Challenges and the way forward

The proportion of ctDNA in cell-free DNA varies considerably. It can range from 0.01% to over 90% and is correlated with disease burden. The sensitivity of ctDNA detection depends on ctDNA levels; thus, highly sensitive assays are required in patients with earlier stage disease (Cancer Treat Rev. 2019;78:31-41).

Clonal hematopoiesis is a well-known cause of genotyping discordance between plasma and tumor samples. Clonal mutations are derived from the hematopoietic system and may be mistaken for tumor mutations since similar genetic alterations may be present in both samples. In the measurement of ctDNA, clonal hematopoiesis can prompt false-positive results owing to the detection of nonreference variants in the plasma (Thorac Surg Clin. 2020;30[2]:165-77).

The presence of ctDNA fragments among individuals without any diagnosed cancer is common and constitutes a serious challenge for the development of ctDNA tests for the early detection of cancer.

Further, there can be various analytical and/or technical issues that pose a challenge to ctDNA analysis. The short half-life of ctDNA and the probability of contamination of plasma cell-free DNA with DNA from healthy leukocytes is a matter of concern. Therefore, it is essential that blood is collected in specialized tubes or processed and stored quickly after collection. Finally, the integration of ctDNA testing in standard clinical practice would require cross-validation and standardization between assays and tissue testing.

Genetic profiling is the first step in targeted treatment, and ctDNA analysis can greatly simplify the whole process, making it a more practical and feasible option than conventional tissue biopsy.

ctDNA is already used in clinical practice for the detection of genomic alterations. Although significant progress has been made in determining treatment efficacy and predicting prognosis, additional studies are required to explore additional applications in lung cancer.

“I see the technology improving significantly such that it is being used reliably in the clinic for posttreatment MRD detection and early lung cancer detection. I still see it being used most widely in the metastatic setting to identify targetable and resistance mutations to optimize treatment regimens and patient survival outcomes,” Dr. Chaudhari said.

While studies are deciphering the role of ctDNA in early lung cancer, there is much interest in analyzing whether a therapeutic strategy guided by ctDNA analysis can improve PFS, OS, and patients’ quality of life, compared with radiologic/clinical monitoring.

In the coming years, as ctDNA technology continues to advance, uptake is expected to increase as continued improvement and further optimization occurs. While monitoring ctDNA over time is not yet routinely used in practice, it is expected that one future application of the technology will involve monitoring the most optimal treatment choice and disease relapse.

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