Longitudinal therapy monitoring of ALK-positive lung cancer by combined copy number and targeted mutation profiling of cell-free DNA

Abstract

Background: Targeted therapies (TKI) have improved the prognosis of ALK-rearranged lung cancer (ALK⁺ NSCLC), but clinical courses vary widely. Early identification and molecular characterisation of treatment failure have key importance for subsequent therapies. We performed copy number variation (CNV) profiling and targeted panel sequencing from cell-free DNA (cfDNA) to monitor ALK⁺ NSCLC.

Methods: 271 longitudinal plasma DNA samples from 73 patients with TKI-treated metastatic ALK⁺ NSCLC were analysed by capture-based targeted (average coverage 4,100x), and shallow whole genome sequencing (sWGS, 0.5x). Mutations were called using standard algorithms. CNVs were quantified using the trimmed median absolute deviation from copy number neutrality (t-MAD).

Findings: cfDNA mutations were identified in 58% of patients. They included several potentially actionable alterations, e.g. in the genes BRAF, ERBB2, and KIT. sWGS detected CNVs in 18% of samples, compared to 6% using targeted sequencing. Several of the CNVs included potentially druggable targets, such as regions harboring EGFR, ERBB2, and MET. Circulating tumour DNA (ctDNA) mutations and t-MAD scores increased during treatment, correlated with markers of higher molecular risk, such as the EML4-ALK variant 3 and/or TP53 mutations, and were associated with shorter patient survival. Importantly, t-MAD scores reflected the tumour remission status in serial samples similar to mutant ctDNA allele frequencies, and increased with disease progression in 79% (34/43) of cases, including those without detectable single nucleotide variant (SNV).

Interpretation: Combined copy number and targeted mutation profiling could improve monitoring of ALK⁺ NSCLC. Potential advantages include the identification of treatment failure, in particular for patients without detectable mutations, and broader detection of genomic changes acquired during therapy, especially in later treatment lines and in high-risk patients.

Funding: This work was supported by the German Center for Lung Research (DZL), by the German Cancer Consortium (DKTK), by the Heidelberg Center for Personalized Oncology at the German Cancer Research Center (DKFZ-HIPO), and by Roche Sequencing Solutions (Pleasanton, CA, USA).

Keywords: ALK; Liquid biopsy; Non-small cell lung cancer; Therapy monitoring.

Fig. 1

Oncoprint of molecular alterations detected by targeted and shallow whole genome sequencing of cfDNA in the investigated cohort. For t-MAD scores, a patient was graded as high if one cfDNA sample exceeds the first quartile cohort t-MAD value (0.01030), and low otherwise. Similarly, a patient was evaluated positive for CNV by sWGS if the computed tumor fraction of one cfDNA sample exceeded 1%, and negative otherwise.

Fig. 2

Genomic alterations detected in cfDNA show an increased trend across therapy lines. (a) Detection rates of all identified mutations, as well as mutations in ALK, TP53, and other co-mutations by targeted sequencing of cfDNA across treatment lines. Comma-separated significance levels indicate P values for Chi-square test and Chi-square test for trend, respectively. (b) t-MAD scores across treatment lines inferred from copy number profiling of cfDNA by sWGS. * P = 0.01-0.05; ** P = 0.001-0.01; *** P < 0.001; n.s. = not significant. ALK: anaplastic lymphoma kinase; TP53: tumor protein 53; SNVs: somatic nucleotide variants; t-MAD: trimmed median absolute deviation from copy number neutrality.

Fig. 3

Shallow whole genome sequencing (sWGS) informs global copy number number changes in cfDNA. (a) Exemplary genome-wide copy number profiles inferred from sWGS of plasma cfDNA (patient P44). For (b-d), the Mann-Whitney U test was used to compare differences between groups. (b) Comparison of t-MAD scores estimated from shallow whole-genome sequencing (sWGS) data of the first vs. the last available plasma sample from each patient during follow-up. (c) Comparison of t-MAD scores from samples of patients with EML4-ALK fusion variants E13:A20 (V1) and E20:A20 (V2) vs. patients with EML4-ALK fusion variant 3 (V3). (d) Comparison of t-MAD scores from samples of patients without TP53 (TP53⁻) vs. with TP53 (TP53⁺) mutations. (e) Multigroup comparison of t-MAD scores from samples of patients of the distinct variant subtypes and TP53 co-mutation status. *P = 0.01-0.05; ** P = 0.001-0.01; *** P < 0.001; n.s. = not significant.

Fig. 4

Detection of circulating tumor DNA (ctDNA) by either targeted next-generation sequencing (tNGS) or sWGS is associated with shorter overall survival (OS). (a) OS for metastatic ALK⁺ NSCLC from the time-point of liquid biopsy according to the detectability of ctDNA with tNGS. The median OS was 40 months from the time of liquid biopsies without detectable mutations in plasma ctDNA vs. 14 months from the time of liquid biopsies with detectable alterations in targeted NGS (log-rank P < 0.001). Only mutations included in both AVENIO kits were considered. (b) OS from the time-point of liquid biopsy according to the t-MAD scores as global copy number variation (CNV) measure after dichotomizing the samples at the median t-MAD score (0.0078). The median OS was 33 months from the time of liquid biopsies with a t-MAD score below the median vs. 25 months from the time of liquid biopsies with a t-MAD score above the median (log rank P = 0.0125).

Fig. 5

Representative cases exemplifying the utility of tNGS and sWGS of cfDNA in monitoring ALK+ NSCLC tumour response across therapy regimens. (a) Clonal evolution of anaplastic lymphoma kinase (ALK) and tumour protein 53 (TP53) mutations in plasma of patient P28 during sequential TKI and chemotherapy (CTx) treatment. (b-e) Mutation kinetics identified by targeted sequencing and corresponding t-MAD levels inferred from shallow whole-genome sequencing (sWGS) of plasma cfDNA during sequential treatment of patient P28 (b), patient P13 (c), patient P3 (d), and patient P23 (e). ERBB2: Erb-B2 Receptor Tyrosine Kinase 2; PD: progressive disease; bPD: brain-only progressive disease; pPD: pleural progressive disease; SD: stable disease.