Gene expression profiling of circulating tumor cells captured by MicroCavity Array is superior to enumeration in demonstrating therapy response in patients with newly diagnosed advanced and locally advanced non-small cell lung cancer

Abstract

Background: Circulating tumor cells (CTCs) are a promising non-invasive tool for monitoring therapy response. The only Food and Drug Administration (FDA)-approved test is limited to enumeration of epithelial CTC without further characterization and is not approved for the management of non-small cell lung cancer (NSCLC). Here we use a MicroCavity Array (MCA) system to capture CTC agnostic of epithelial markers for further molecular testing in NSCLC.

Methods: CTCs were enumerated by fluorescent microscopy as longitudinal sampling throughout disease management from 213 NSCLC patients. CTC-enriched samples from a subset of 127 patients were interrogated for gene expression by reverse transcription polymerase chain reaction (RT-PCR) using a customized pre-selected panel of 20 genes.

Results: At least 1 CTC was detected by enumeration in 53.8% of samples. Most patients had fewer than 5 CTCs (91%) and the highest observed count was 35 CTCs. Enumeration of single CTCs was not prognostic, although detection of CTC clusters at any time point was associated with increased risk of progression [hazard ratio (HR) 3.00, 95% confidence interval (CI): 1.1-8.2, P=0.0318]. In contrast, 124 (97.6%) patients with samples interrogated for gene expression had at least 1 gene detectable in at least 1 sample, and 101 (79.5%) had at least one elevated epithelial gene in at least one timepoint. High expression of BCL2, CD274 [programmed death-ligand 1 (PD-L1)], CDH1, EPCAM, FGFR1, FN1, KRT18, MET and MUC1 were associated with poor prognosis. Patients with CTCs positive for at least 3 epithelial genes at baseline all progressed within 10 months (HR 8.2, P<0.001, 95% CI: 3.2-21.1). BCL2, CD274 (PD-L1), EPCAM and MUC1 remained significant independent prognostic factors in multivariate, time-dependent analyses of progression and death.

Conclusions: The selective profile of CTC genes and identification of CTC clusters better correlated with prognosis than enumeration of enriched CTC in NSCLC patients in this study.

Keywords: Blood; biomarkers, tumor; liquid biopsy; neoplastic cells, circulating.

Figure 1

Study design. (A) Schematic of CTC sample collection in NSCLC (treatment naïve or recurrent) patients. Samples were collected up to 9 times including at the start of radiation (baseline), at the midpoint of radiation, at the completion of radiation and at the first clinical follow-up. Additional samples were collected at routine visits for up to 2 years. The number of samples collected for CTC enumeration and CTC gene expression are shown for each timepoint. (B) Flow diagram depicting schematic of samples processed for the study. Tx, therapy; CTC, circulating tumor cell; SOC, standard-of-care; HD, healthy donor; NSCLC, non-small cell lung cancer; NA, not available; qPCR, quantitative polymerase chain reaction.

Figure 2

Distribution of CTC counts. (A) CTC counts ranged from 0 to 35 CTC per sample. The distribution for the full study is shown with scaled histograms for each visit as inset. (B) CTC count distribution at each timepoint as the percentage of samples with 0, 1, 2–4, 5–10 or more than 10 CTCs. CTC, circulating tumor cell.

Figure 3

CTC counts lacked prognostic potential. (A) Kaplan-Meier analysis at each timepoint for PFS and OS using a threshold of 5 CTCs. (B) Patients displaying CTC clusters at any timepoint had a higher risk of progression. PFS, progression-free survival; OS, overall survival; CTC, circulating tumor cell.

Figure 4

Distribution of CTC gene expression. (A) Heat map and hierarchical clustering of gene expression. Samples at each time point are split by patients who experienced progression and sorted by CTC count. SNAI2 had weak but significant negative correlation with CTC count. Samples with CTC counts >10 are displayed as 10. (B) Comparison of average gene expression across timepoints for all patients. Red line shows the mean at each timepoint. Comparisons between baseline and each timepoint are shown as P-values using Wilcoxon signed-rank test. (C) The longitudinal analysis was repeated for the cohorts of patients who later progressed (blue) and those who did not (red). (D) Comparison of average gene expression between Progressors and Non-Progressors at each timepoint using Wilcoxon signed-rank test. CTC, circulating tumor cell.

Figure 5

Univariate survival analysis by gene expression of enriched CTC. Univariate gene expression for baseline (left) and first follow-up following completion of radiation therapy (right). Top shows Univariate Cox proportional hazard ratio models for each gene as a continuous variable showing the hazard ratio for each doubling (1-Ct change) in gene expression for PFS (A,E) and OS (B,F); the bottom shows Kaplan-Meier analysis of gene expression stratified using HD blood to define positive expression for PFS (C,G) and OS (D,H). CTC, circulating tumor cell; PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; HD, healthily donor.

Figure 6

Univariate survival analysis by number of CTC genes expressed. Left: from the 9 genes that had significant effects in Cox analysis (BCL2, CD274, CDH1, EPCAM, FGFR1, FN1, KRT18, MET and MUC1), as the number of positive genes goes up, survival times decrease (red line) for PFS (A) and OS (D). Note that median survival is not reached for the low expression group at several thresholds which shows as missing points for the green line. Red line: median survival for patients with baseline sample > threshold; green line: median survival for patients with baseline sample ≤ threshold; blue line hazard ratio for the positive group relative to the negative group; horizontal dashed blue line shows HR =1. Right: 4 epithelial genes: Kaplan-Meier analysis of epithelial genes CDH1, EPCAM, KRT18 and MUC1 at each timepoint for PFS (B,C) and OS (E,F) by number of positive genes (B,E) and any positive expression (C,F). PFS, progression-free survival; OS, overall survival; CTC, circulating tumor cell; HR, hazard ratio; Epi, epithelial.