Amplicon-Based Liquid Biopsy Detects ALK/ROS1 Fusion and Resistance Mutations in NSCLC

Laura Mezquita, MD, PhD

Amplicon-based liquid biopsy can detect ALK/ROS1 fusions in TKI-naïve patients with non—small cell lung cancer (NSCLC) and allows for the identification of resistance mutations in those treated with TKIs, according to results from a study published in the Journal of Clinical Oncology.

“Liquid biopsy specimens from patients treated with TKIs may affect clinical outcomes and capture heterogeneity of TKI resistance, supporting their role in selecting sequential therapy,” Laura Mezquita, MD, PhD, of Hospital Clínic de Barcelona, and co-authors wrote in their paper.

A total of 128 patients were enrolled in the study; of those, 101 were positive for ALK alterations and 27 were positive for ROS1 fusions. Investigators collected blood samples (n = 405) from 29 patients who were TKI naïve or from 375 patients who were under treatment, including 105 samples collected at disease progression.

Sensitivity for ALK/ROS1 fusion detection was 67%. Investigators noted a higher detection for ALK fusions at TKI failure (n = 33/74; 46%) compared with patients who had therapeutic response (n = 12/109; 11%). Overall, ALK-resistance mutations were detected in 22% of patients (n = 16/74) and 43% of the total ALK-resistance mutations identified occurred after next-generation TKI therapy.

ALK G1202R was the most common mutation detected (7 of 16) and ROS1 G2032R resistance was detected in 30% of patients (3 of 10). The absence of circulating tumor (ct) DNA mutations at TKI failure was linked with a prolonged median overall survival (OS; 105.7 months). Furthermore, complex ALK-resistance mutations were associated with a poor OS at a median of 26.9 months versus not reached (NR) for a single mutation (P =.003) and progression-free survival (PFS) to subsequent therapy was at a median of 1.7 months versus 6.3 months (P =.003).

“Our clinical experience of an amplicon-based, NGS liquid biopsy in a large, real-world, prospective cohort of ALK/ROS1-positive patients with NSCLC provide evidence of the clinical utility of this approach at the time of diagnosis as well as at the time of [disease progression] for detection of resistance mutations,” wrote Mezquita et al. “Liquid biopsy specimens in TKI-treated patients capture heterogeneity of TKI resistance, supporting the role of liquid biopsy in selecting sequential therapy.”

Patients ≥18 years with ALK- and ROS1-fusion—positive NSCLC were prospectively enrolled in the study between October 2015 and August 2018 at 8 French institutions. Samples were collected at any time point at diagnosis and/or at each radiologic evaluation. In blood samples, plasma was isolated and ctDNA analysis was centralized using InVisionFirst-Lung, which detects single nucleotide variants, insertions, and deletions, copy number variations, and fusions, with whole-gene and gene hotspots across a 36-gene panel. The samples were correlated with clinical outcomes.

A median of 2 samples were collected per patient (range, 1-13) and 27 of those samples were eligible for analysis in the treatment-naïve cohort. In 18 patients, the fusion was detected in blood (n = 16 patients with ALK; n = 2 with ROS1), with a sensitivity of 67% for ALK and ROS1 fusion. In the ALK cohort, eight variant 1, two variant 2, and six variant 3 fusions were detected. In the ROS1 cohort, one CD74-ROS1 and one SLC34A2-ROS1 fusion were detected.

“Fusion detection in blood was associated with a high number of metastatic sites and visceral involvement,” the investigators wrote. “The metastatic pattern was not related to ALK variants or ROS1 partner gene.” Plasma next-generation sequencing was done on 25 samples; 44% of these samples were found to have concurrent gene aberrations.

Regarding radiologic response, 143 samples collected at the time of confirmed objective response by RECIST criteria were determined to be evaluable for fusion analysis (ALK, n = 109; ROS1, n = 34). Fusions were detected in 10% of samples with 11% of samples showing ALK mutations and 6% of samples showing ROS1 mutations. Additionally, 121 samples were collected at the time of disease progression on systemic therapies (ALK, n = 96; ROS1, n = 15). Of those samples, 74 were collected at TKI failure, and the detection rate for ALK fusions was 45%; for ROS1 fusions, the detection rate was 30%. According to the investigators, the detection rate was higher in those with visceral and bone metastases.

Additionally, in 22% of samples collected at TKI failure, ALK mutations were detected. These mutations were more frequently detected in patients with bone or liver progression, at 75% to 80%, versus those with exclusive central nervous system (CNS) or thoracic progression, at 10%. At isolated CNS relapse, the detection of ALK mutations was 10%. The detection rate of these mutations after crizotinib (Xalkori) was 0% and after therapy with a next-generation TKI, it was 18%. Notably, in 55% of patients, no mutations were detected. At disease progression, ALK mutations with third-generation TKIs were detected in 43% of samples compared with 29% in samples from patients who were treated with second-generation TKIs and 11% in samples from those treated with crizotinib. ALK G1202R was the most common resistance mutation observed.

Ten samples were collected at the time of disease progression in the ROS1 cohort. Of those samples, 30% had the ROS1 G2032R-resistance mutation and all cases had concurrent mutations. Three patients had sequential samples to assess the emergence of mutations and 1 case showed the emergence of a ROS1 mutation following crizotinib failure.

The absence of mutations in ctDNA was linked with improved OS (74 samples; n = 55 patients). Specifically, the median OS was 58.5 months (95% CI, 26.9-NR) if ≥1 ALK mutations were detected compared with 44.1 months (95% CI, 21.7-NR) if non-ALK mutations were identified versus 105 months (95% CI, 105.7-NR) in patients negative for ctDNA (P =.001).

“This effect was observed regardless of the number of lines of TKI received,” noted the investigators. “This observation also held true when we assessed this outcome in the population exclusively treated with TKI as first-line therapy.”

Additionally, the presence of complex ALK mutations was linked with poor OS, at a median of 26.9 months (95% CI, 13.19-NR) versus NR with single ALK mutation (95% CI, 57.0-NR; P =.003). Investigators also noted this affect in the subgroup of patients who received up front treatment with a TKI (P =.038). In the 4 patients who had ALK G1202R mutations, the median OS was 59.5 months (95% CI, 26.9-NR).

The group with absence of ctDNA mutations had a median PFS of 14.8 months (95% CI, 8.1-23.1) versus 9.6 months (95% CI, 6.6-19.9) if there was ≥1 ALK mutation or 7.8 months (95% CI, 4.5-11.7) if there were non-ALK mutations at TKI failure (P =.31). The median PFS of the 4 cases with emergence of ALK G1202R was 2.7 months (95% CI, 2.03-NR) versus 8.6 months (95% CI, 5.6-10.6) in the remaining population (P =.05).

Additionally, investigators then evaluated the PFS to the subsequent therapy per the ctDNA mutations detected in 56 samples. In the ctDNA-negative group, they found that the median PFS was 20.7 months (95% CI, 6.3-NR) versus 8 months (95% CI, 2.8-NR) in the group with non-ALK mutations versus 2.8 months (95% CI, 1.2-NR) in the group with ≥1 ALK mutations detected (P =.03).

After further investigating PFS in the subgroup of patients who harbored ALK mutations (n = 16), investigators found that the ALK complex mutations were linked with poor efficacy, with a median PFS of 1.7 months (95% CI, 0.9-NR). Conversely, ALK single mutation was associated with longer PFS, with a median PFS of 6.3 months (95% CI, 1.8-NR). Regarding the 4 cases with the emergence of ALK G1202R, the median PFS to the sequential therapy was 3.7 months (95% CI, 1.2-NR) compared with 8.3 months (95% CI, 4.9-NR) in the overall patient population (P =.15).

The authors of the study acknowledged that the study had some limitations. For one, the amplicon-based NGS approach is highly sensitive for mutations, but it is limited to the detection of known fusion partners. Second, the sample size included in the analysis was limited the heterogeneity of the samples collected in different time points is high. Moreover, only 51% of samples were collected after next-generation TKI in the ALK population. Lastly, patients were included as they were diagnosed or at any time of treatment, which could be linked with a bias in recruitment of patients with long survival.

Mezquita L, Swalduz A, Jovelet C, et al. Clinical relevance of an amplicon-based liquid biopsy for aetecting ALK and ROS1 fusion and resistance mutations in patients with non—small-cell lung cancer. J Clin Oncol. 2020:272-282. doi: 10.1200/P0.19.00281