Clinical Utility of Cell-Free DNA for the Detection of ALK Fusions and Genomic Mechanisms of ALK Inhibitor Resistance in Non-Small Cell Lung Cancer

Abstract

Purpose: Patients with advanced non-small cell lung cancer (NSCLC) whose tumors harbor anaplastic lymphoma kinase (ALK) gene fusions benefit from treatment with ALK inhibitors (ALKi). Analysis of cell-free circulating tumor DNA (cfDNA) may provide a noninvasive way to identify ALK fusions and actionable resistance mechanisms without an invasive biopsy.Patients and Methods: The Guardant360 (G360; Guardant Health) deidentified database of NSCLC cases was queried to identify 88 consecutive patients with 96 plasma-detected ALK fusions. G360 is a clinical cfDNA next-generation sequencing (NGS) test that detects point mutations, select copy number gains, fusions, insertions, and deletions in plasma.Results: Identified fusion partners included EML4 (85.4%), STRN (6%), and KCNQ, KLC1, KIF5B, PPM1B, and TGF (totaling 8.3%). Forty-two ALK-positive patients had no history of targeted therapy (cohort 1), with tissue ALK molecular testing attempted in 21 (5 negative, 5 positive, and 11 tissue insufficient). Follow-up of 3 of the 5 tissue-negative patients showed responses to ALKi. Thirty-one patients were tested at known or presumed ALKi progression (cohort 2); 16 samples (53%) contained 1 to 3 ALK resistance mutations. In 13 patients, clinical status was unknown (cohort 3), and no resistance mutations or bypass pathways were identified. In 6 patients with known EGFR-activating mutations, an ALK fusion was identified on progression (cohort 4; 4 STRN, 1 EML4; one both STRN and EML4); five harbored EGFR T790M.Conclusions: In this cohort of cfDNA-detected ALK fusions, we demonstrate that comprehensive cfDNA NGS provides a noninvasive means of detecting targetable alterations and characterizing resistance mechanisms on progression. Clin Cancer Res; 24(12); 2758-70. ©2018 AACR.

Figure 1.

Consort Diagram

Figure 2.

Cohort 1 (Newly identified ALK fusion) Genomic Landscape. Individual patient results and cfDNA identified alterations for Cohort 1, newly identified ALK fusions. Tumor tissue ALK status was known for 21 (50%) of cohort 1 cases: 5 negative (NEG), 5 positive cases (POS), 11 insufficient tissue to perform analysis or unable to obtain tissue for analysis (QNS). The remainder of the samples had tissue status that was unknown (Unk). Alterations identified are in the following columns and denoted by color, blue shades = fusion, green = RAS / RAF / EGFR / MET variant; red = amplification; light gray = tumor suppressor / other pathway gene mutations. Asterisk (*) indicates instances in which only the reciprocal ALK-EML4 fusion was detected in ctDNA. If known, the prior systemic therapy is listed, as is the days between diagnosis and blood draw for Guardant 360 (Median 21 days; range 1 – 1056 days).

Figure 3.

Cohort 2 Genomic Landscape. Individual patient results are shown in rows and ctDNA identified alterations are shown in each column. Patients C2–1, C2–2, and C2–3, had multiple progression samples collected, denoted as 1 and 2. The most recent treatment is noted in the following column, if known. The mutational allele frequency (MAF) is shown in each box for fusions, resistance mutations, and somatic mutations in alternative oncogenes. The maximum somatic alteration allele frequency for each sample in shown in the far right column. T = fusion previously detected in tissue but not detected in ctDNA at progression. Asterisk (*) indicates instances in which the reciprocal ALK-EML4 fusion was also detected in ctDNA. Color legend: blue/ purple shades=fusion, orange=on-target resistance mutation; green=RAS/RAF/EGFR/MET mutation; red=amplification. ^Sample C2–17 is the progression sample from patient C1–26 in cohort 1

Figure 4.

NSCLC Case with Multiple ctDNA Timepoints Across Disease Trajectory. Patient C2–3: At initial diagnosis, EML4-ALK fusion was detected in ctDNA (and tissue). Crizotinib was initiated with 32% reduction in target lesion in 3 months. Treatment was switched to ceritinib due to side effects. G360 was drawn again when progressing on ceritinib and the original fusion was detected along with ALK F1174V, a mutation conferring resistance to crizotinib and ceritinib. Alectinib was then initiated and after initial response, progression was noted and G360 was drawn again: F1174V was no longer present in circulation but G1202R was identified, a mutation conferring resistance to all FDA-approved ALKi but predicted to be sensitive to lorlatinib and brigatinib. Lorlatinib is currently only available by clinical trial.

Figure 5.

Genomic Landscape of Cohort 3 Cohort 3, genomic landscape for patients with unknown clinical status. Individual patient results are shown in rows and ctDNA identified alterations are shown in each column. The second column lists the tissue status; POS- FISH positive, NEG – FISH negative, UNK- unknown. Color legend: blue shades = fusion, green=RAS/RAF/EGFR/MET mutation; red=amplification; light gray=tumor suppressor/other pathway gene mutations. Asterisk (*) indicates instances in which only the reciprocal ALK-EML4 fusion was detected in ctDNA.

Figure 6.

Cohort 4 Genomic Landscape. A, Individual patient results are shown in rows and ctDNA identified alterations are shown in each column. The EGFR mutation subtype is shown in parentheses. The most recent treatment is noted in the far-right column. The mutational allele frequency (MAF) is shown in each box for fusions, resistance mutations, and somatic mutations in alternative oncogenes. Color legend: blue shades=fusion, orange=on-target resistance mutation; green=RAS/RAF/EGFR/MET mutation; red=amplification; light gray=tumor suppressor/ other pathway gene mutations Eleven ALK fusions (6 STRN-ALK, 5 EML4-ALK) were identified in six patients drawn when progressing on an EGFR TKI (of 1450 NSCLC cases with EGFR driver mutations in the Guardant database.) EGFR T790M was also present in 5 of 6 pts (4 by ctDNA in 7 tests, 1 by tissue [T]) along with multiple amplification events. In three pts (*) tissue testing results from initial diagnosis were available and were EGFR positive, ALK fusion negative. In patient C4–3, progression tissue biopsy, the presence of the STRN-ALK fusion was confirmed by an RNA based NGS assay. B, Relative to the highest mutant allele fraction (MAF) variant in circulation, the EGFR driver mutations appear to be clonal while both T790M and the fusions appear to be subclonal. This information, combined with available treatment-naïve tissue testing results, suggest that the ALK fusion events are emergent alterations.