Novel Insights on ctDNA Dynamics During Targeted Therapy and Immune Checkpoint Blockage in NSCLC

Dr. Christian Rolfo

By Christian Rolfo, MD, PhD, MBA, Dr.h.c.
Posted: December 11, 2019

IN REFERENCE TO: Phallen J, Leal A, Woodward BD, et al. Early Noninvasive Detection of Response to Targeted Therapy in Non-Small Cell Lung Cancer. Cancer Res. 2019;79(6):1204-1213.

Anagnostou V, Forde PM, White JR, et al. Dynamics of Tumor and Immune Responses during Immune Checkpoint Blockade in Non-Small Cell Lung Cancer. Cancer Res. 2019;79(6):1214-1225.

Liquid biopsy (LBx) is a new, powerful tool for the molecular profiling of patients with NSCLC that can help oncologists with appropriate treatment selection in oncogene-addicted NSCLCs. Circulating tumor DNA (ctDNA) assays for detection of both EGFR sensitizing and resistance mutations have already entered clinical practice in NSCLC,1 and recently, the NILE study provided evidence that a 73-gene next-generation sequencing (NGS) panel can detect biomarkers (including EGFR, ALK, ROS1, BRAF, RET, MET, HER2, and KRAS) at a rate similar to standard-of-care tissue genotyping tests, with a faster turnaround time; it can also provide the opportunity to rescue patients who were incompletely genotyped or whose initial tissue analysis proved negative for “actionable” biomarkers.2

In addition to tumor genotyping, LBx may potentially allow real-time monitoring of response in patients with cancer. This application may be particularly useful in patients treated with targeted agents favoring early identification of mechanisms of acquired resistance that inevitably occur after initial response, as well as in those treated with immune checkpoint inhibitors (ICIs) in which radiographic interpretation of response might be challenging, thereby overcoming the limits of conventional radiologic assessment methods. Recently, the American Association for Cancer Research published two interesting papers addressing this issue.

Phallen et al. evaluated the role of serial ultrasensitive LBx with targeted error correction sequencing (TEC-Seq) as an early non-invasive detection tool of response in patients with metastatic NSCLC with EGFR or HER2 mutations during treatment with different classes of TKIs. They collected serial blood draws from 28 patients with metastatic NSCLC at baseline and over the course of treatment until disease progression, evaluating the changes of a new metric, cell free tumor load (cfTL), which was defined as the contribution of the most abundant alterations in ctDNA at any particular time point. Changes in cfTL were compared with tumor burden assessed in patients with detectable sequence clones (24 patients) or the qualitative assessment of change from aneuploidy to normal ploidy in those without detectable sequence clones (4 patients). They reported that both ctDNA levels and clonal heterogeneity dramatically reduced in responding patients due to the selective pressure of targeted therapy with a significant reduction of cfTL compared to baseline levels (average of 10.8% at baseline vs. 0.18% at a median time of 19 days after treatment start; p < 0.001); they also noted a decrease of plasma aneuploidy scores (average decrease of 92%; p = 0.002), and a reduction of average number of observed alterations (from 3.6 to 1.1 mutations per patient; p < 0.01). In contrast, patients with stable disease (SD) and progressive disease exhibited a less pronounced (average of 2.24% at baseline vs. 1.04% after treatment; p = 0.03) or limited variation of cfTL (average of 14.23% at baseline vs. 11.84% after treatment; p = 0.6), respectively, and no significant change in the number of mutations observed. Despite the limited sample number, this study further confirmed the findings of previous reports suggesting a potential role for LBx as a non-invasive drug-monitoring method,3,4 allowing an earlier identification of mechanisms of acquired resistance compared with conventional radiologic methodologies. However, to date, it is unclear whether this might be associated with changes in the treatment strategy before radiographic progression or not. The randomized phase II EORTC APPLE trial (NCT02856893) will likely provide further evidence on the utility of this strategy.

Interestingly, cfTL reduction at a median of 19 days was a more accurate predictor of clinical outcome compared with initial CT imaging performed after an average of 47 days (p < 0.0001), allowing a more precise evaluation of patients with nonmeasurable disease or with radiographic SD. This, in turn, may allow a better characterization of these patients and, in turn, overcome the limits of conventional radiographic methodologies. Finally, the authors reported that, in a subset of patients, the effect of the first dose of treatment after 4 to 12 hours showed a 110-fold increase in the rate of emerging mutations, with a relative stability of ctDNA amounts. This finding may potentially affect future combinatorial strategies, allowing us to add different inhibitors to EGFR blockade based on emerging mechanisms of resistance.

In a companion study, Anagnostou et al. evaluated the role of noninvasive monitoring of ctDNA using the TEC-Seq approach in a longitudinal study of T-cell receptor (TCR) repertoire during immune checkpoint inhibition. The study included 24 patients with metastatic NSCLC treated with ICIs and 14 patients with resectable NSCLC (stage I-IIIA) receiving nivolumab as neoadjuvant treatment. At least two serial samples (range 2-8) were collected for all patients. To avoid the potential effect of clonal hematopoiesis,5 ctDNA analysis was focused only on genetic alterations identified through NGS in paired-matched tissue samples. In the metastatic cohort, 19 of 24 patients had ctDNA detectable levels (median mutant allele fraction of 1.87%) either at baseline or other time points, whereas 7 of 14 patients in the early-stage cohort had detectable ctDNA (median allele fraction of 0.34%). They identified three patterns of molecular response in ctDNA: molecular response, corresponding to a dramatic reduction of ctDNA to undetectable levels; molecular resistance, associated with limited fluctuations or a rise of ctDNA levels; and molecular acquired resistance, where tumor-specific variants were undetectable at the time of response followed by increase in mutant allele fraction at the time of acquired resistance. Reduction of ctDNA to undetectable levels was associated with longer PFS (p = 0.001) and OS (p = 0.008) compared with no evidence of ctDNA elimination. Once again, in patients with radiographic SD (12 patients), the molecular response pattern correlated with clinical benefit from immune checkpoint blockade and better predicted the magnitude of therapeutic response than CT imaging. Furthermore, molecular response was associated with major or partial pathologic response in the neoadjuvant cohort, whereas molecular resistance was associated with no pathologic response.

Moreover, 24 patients with metastatic disease had available samples from both tumor infiltrating lymphocytes and peripheral blood lymphocytes for analysis of TCR clonal dynamics. Consistent with the cDNA analysis, distinct patterns in TCR clonotype dynamics were observed with a significant oligoclonal expansion in peripheral blood of pre-existing intratumoral T-cell clones, followed by a significant decrease after acquired resistance. In contrast, in patients with primary resistance, no evidence of TCR clonal expansion of intratumoral TCR repertoire was observed.

The results of this study have several potential clinical implications. First, ctDNA dynamics might complement standard imaging approaches in the therapeutic management of patients with NSCLC treated with ICIs,6,7 allowing a better characterization of pseudo-progression or mixed/dissociated responses. In addition, the clearance of ctDNA, if validated in large prospective studies, might represent a valid tool that would allow a better selection of patients who can benefit from elective discontinuation strategies after selected treatment duration and might help to identify patients who can benefit from combinatorial approaches instead of single-agent ICI therapy. Finally, this study further confirms that more clonal T cell repertoire is predictive of response ICIs targeting PD-1/PD-L18 and TCR clonal dynamics might guide treatment management.

Further prospective studies in large patient population should validate these preliminary data and may support the incorporation of dynamic ctDNA analysis in clinical trials evaluating targeted therapies and immunotherapy in NSCLC. ✦

About the Author: Dr. Rolfo is professor of Medicine and director of Thoracic Medical Oncology and Early Clinical Trials at University of Maryland Greenebaum Comprehensive Cancer Center and University of Maryland School of Medicine. He is vice-president of the International Society of Liquid Biopsy.

1. Rolfo C, Mack PC, Scagliotti GV, et al. Liquid Biopsy for Advanced Non-Small Cell Lung Cancer (NSCLC): A Statement Paper from the IASLC. J Thorac Oncol. 2018;13(9):1248-1268.

2. Leighl NB, Page RD, Raymond VM, et al. Clinical Utility of Comprehensive Cell-free DNA Analysis to Identify Genomic Biomarkers in Patients with Newly Diagnosed Metastatic Non-small Cell Lung Cancer. Clin Cancer Res. 2019 Apr 15. [Epub ahead of print].

3. Oxnard GR, Paweletz CP, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res. 2014;20(6):1698-1705.

4. Chia PL, Do H, Morey A, et al. Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment. Lung Cancer. 2016;98:29-32.

5. Hu Y, Ulrich BC, Supplee J, et al. False-Positive Plasma Genotyping Due to Clonal Hematopoiesis. Clin Cancer Res. 2018;24(18):4437-4443.

6. Cabel L, Proudhon C, Romano E, et al. Clinical potential of circulating tumour DNA in patients receiving anticancer immunotherapy. Nat Rev Clin Oncol. 2018;15(10):639-650.

7. Goldberg SB, Narayan A, Kole AJ, et al. Early Assessment of Lung Cancer Immunotherapy Response via Circulating Tumor DNA. Clin Cancer Res. 2018;24(8):1872-1880.

8. Roh W, Chen PL, Reuben A, et al. Integrated molecular analysis of tumor biopsies on sequential CTLA-4 and PD-1 blockade reveals markers of response and resistance. Sci Transl Med. 2017;9(379): pii: eaah3560.