NETest Liquid Biopsy Is Diagnostic of Lung Neuroendocrine Tumors and Identifies Progressive Disease

Abstract

Background: There are no effective biomarkers for the management of bronchopulmonary carcinoids (BPC). We examined the utility of a neuroendocrine multigene transcript "liquid biopsy" (NETest) in BPC for diagnosis and monitoring of the disease status.

Aim: To independently validate the utility of the NETest in diagnosis and management of BPC in a multicenter, multinational, blinded study.

Material and methods: The study cohorts assessed were BPC (n = 99), healthy controls (n = 102), other lung neoplasia (n = 101) including adenocarcinomas (ACC) (n = 41), squamous cell carcinomas (SCC) (n = 37), small-cell lung cancer (SCLC) (n = 16), large-cell neuroendocrine carcinoma (LCNEC) (n = 7), and idiopathic pulmonary fibrosis (IPF) (n = 50). BPC were histologically classified as typical (TC) (n = 62) and atypical carcinoids (AC) (n = 37). BPC disease status determination was based on imaging and RECIST 1.1. NETest diagnostic metrics and disease status accuracy were evaluated. The upper limit of normal (NETest) was 20. Twenty matched tissue-blood pairs were also evaluated. Data are means ± SD.

Results: NETest levels were significantly increased in BPC (45 ± 25) versus controls (9 ± 8; p < 0.0001). The area under the ROC curve was 0.96 ± 0.01. Accuracy, sensitivity, and specificity were: 92, 84, and 100%. NETest was also elevated in SCLC (42 ± 32) and LCNEC (28 ± 7). NETest accurately distinguished progressive (61 ± 26) from stable disease (35.5 ± 18; p < 0.0001). In BPC, NETest levels were elevated in metastatic disease irrespective of histology (AC: p < 0.02; TC: p = 0.0006). In nonendocrine lung cancers, ACC (18 ± 21) and SCC (12 ± 11) and benign disease (IPF) (18 ± 25) levels were significantly lower compared to BPC level (p < 0.001). Significant correlations were evident between paired tumor and blood samples for BPC (R: 0.83, p < 0.0001) and SCLC (R: 0.68) but not for SCC and ACC (R: 0.25-0.31).

Conclusions: Elevated -NETest levels are indicative of lung neuroendocrine neoplasia. NETest levels correlate with tumor tissue and imaging and accurately define clinical progression.

Keywords: Biomarker; Blood; Bronchopulmonary carcinoid; Lung cancer; NETest; Neuroendocrine tumor; PCR; Progression; Transcript.

Fig. 1.

a NETest in controls and BPC: Measurements of the NETest identified elevated levels in BPC (red) versus controls (blue) (p < 0.0001). b AUROC for BPC and controls: the AUROC for differentiating BPC from controls was 0.96 (95% CI: 0.92–0.98, p < 0.0001). Mean ± SEM.

Fig. 2.

NETest in BPC – relationship to histological assessment. a NETest in AC and TC with image-detectable disease: measurements of the NETest-identified levels were not significantly elevated in AC (atypical carcinoid) with evidence of disease (red) versus TC (typical carcinoid) with evidence of disease (orange) (p = 0.12). b NETest in AC by stage: NETest levels were significantly elevated in AC with distant metastases (M1) compared to AC with no metastatic disease (N0M0) (p < 0.03). Levels were not significantly elevated compared to those with lymph node disease (N+M0) but no distant metastases (p = 0.18). Group analysis identified NETest was significantly different across all 3 groups (p < 0.02). c NETest in TC by stage: NETest was significantly elevated in TC with distant metastases (M1) compared to those without metastatic disease (N0M0) (p = 0.01) and with lymph node disease (N+M0) but no distant metastases (p < 0.001). Group analysis identified NETest was significantly different across all 3 groups (p = 0.0006). Means ± SEM.

Fig. 3.

NETest in BPC – relationship to clinical status and metastatic disease. a Disease status (RECIST based): measurements of the NETest-identified levels were significantly elevated (p < 0.0005) in stable disease (SD – orange) and progressive disease (PD – red) compared to no evidence of disease (NED) “surgical cures” (white). Levels were significantly elevated (p = 0.0026) in PD compared to SD. b Metastatic disease: NETest levels were significantly elevated in SD with no evidence of distant metastases (SD-M0) compared to NED (p < 0.03). Levels were elevated in SD with distant metastases (SD-M1) versus SD-M0 (p = 0.003). Levels were similarly elevated in PD with (PD-M1) and without metastases (PD-M0). Both PD groups were significantly elevated versus SD-M0 (p = 0.03; p < 0.0003, respectively). Means ± SEM.

Fig. 4.

NETest in IPF and BPC. a NETest in controls, IPF, and BPC: NETest levels were not significantly elevated in IPF versus controls (p = 0.23). Levels were significantly (p < 0.0001) lower than in BPC. b AUROC for IPF and BPC: the AUROC value differentiating BPC from IPF was 0.81 (95% CI: 0.74–0.87, p < 0.0001). Means ± SEM.

Fig. 5.

NETest in other malignant lung diseases (non-BPC). a NETest in controls and different lung neoplasias: NETest levels were elevated in SCLC (p < 0.0001), LCNEC (p < 0.0001), and ACC (p = 0.0067). SCC levels were not elevated versus controls (p = 0.09). Both ACC and SCC had significantly lower expression than BPC (p < 0.0001). No differences were noted between BPC and either SCLC (p = 0.80) or LCNEC (p = 0.12). b AUROC for BPC and ACC/SCC: the AUROC value for differentiating BPC from ACC/SCC (as a group) was 0.84 (95% CI: 0.78–0.90, p < 0.0001). No difference was noted for SCLC and LCNEC versus BPC (AUROC: 0.56, p = 0.31). The difference between SCLC/LCNEC versus ACC/ SCC (AUROC: 0.74, p = 0.0002) was significant. Means ± SEM.

Fig. 6.

NETest gene expression in matched tumor tissue:blood samples from malignant lung diseases. Linear regression (Pearson) analysis of log-transformed normalized values of each of the individual tumor-blood pairs identified R to range from 0.82 (BPC; n = 6, p < 0.0001) to 0.68 (SCLC; n = 4, p < 0.0001) to 0.25 (ACC; n = 5, p = 0.12) to 0.31 (SCC; n = 5, p = 0.06). In the graph plots, the pairs (blood-tissue) were averaged, and error bars indicate standard errors of the mean. The dashed line represents the best linear fit to each dataset.