High Circulating Sonic Hedgehog Protein Is Associated With Poor Outcome in EGFR-Mutated Advanced NSCLC Treated With Tyrosine Kinase Inhibitors

Abstract

Introduction: Growing preclinical evidence has suggested that the Sonic hedgehog (Shh) pathway is involved in resistance to tyrosine kinase inhibitor (TKI) therapy for EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC). However, little is known concerning the prognostic value of this pathway in this context.

Materials and methods: We investigated the relationship between plasma levels of Shh and EGFRm NSCLC patients' outcome with EGFR TKIs. We included 74 consecutive patients from two institutions with EGFRm advanced NSCLC treated by EGFR TKI as first-line therapy. Plasma samples were collected longitudinally for each patient and were analyzed for the expression of Shh using an ELISA assay. The activation of the Shh-Gli1 pathway was assessed through immunohistochemistry (IHC) of Gli1 and RT-qPCR analysis of the transcripts of Gli1 target genes in 14 available tumor biopsies collected at diagnosis (baseline).

Results: Among the 74 patients, only 61 had baseline (diagnosis) plasma samples, while only 49 patients had plasma samples at the first evaluation. Shh protein was detectable in all samples at diagnosis (n = 61, mean = 1,041.2 ± 252.5 pg/ml). Among the 14 available tumor biopsies, nuclear expression of Gli1 was observed in 57.1% (8/14) of patients' biopsies. Shh was significantly (p < 0.05) enriched in youth (age < 68), male, nonsmokers, patients with a PS > 1, and patients presenting more than 2 metastatic sites and L858R mutation. Higher levels of Shh correlated with poor objective response to TKI, shorter progression-free survival (PFS), and T790M-independent mechanism of resistance. In addition, the rise of plasma Shh levels along the treatment was associated with the emergence of drug resistance in patients presenting an initial good therapy response.

Conclusion: These data support that higher levels of plasma Shh at diagnosis and increased levels of Shh along the course of the disease are related to the emergence of TKI resistance and poor outcome for EGFR-TKI therapy, suggesting that Shh levels could stand both as a prognostic and as a resistance biomarker for the management of EGFR-mutated NSCLC patients treated with EGFR-TKI.

Keywords: Sonic Hedgehog (Shh); biomarker; epidermal growth factor receptor (EGFR); non-small cell lung cancer (NSCLC); tyrosine kinase inhibitor (TKI).

Figure 1

Shh signaling expression and activation in EGFR-mutated NSCLC patients: (A) Shh concentration in plasma from patients at diagnostic (n = 61) as assessed by ELISA assays. (B) mRNA expression of Gli1 target genes in NSCLC; data are expressed as mean ± SEM of 14 patients analyzed in duplicate. (C) Representative, IHC of Gli1 in NSCLC (n = 14). (C, D) Expression of Shh in plasma from patients (14) according to Gli1 expression. *p < 0.05.

Figure 2

Shh concentrations in plasma from patients at diagnosis according to patients’ characteristics: Patient samples analyzed for Shh expression were classified according to age, gender, smoking status, performance status at diagnosis, number of metastatic sites, central nervous system (CNS) metastasis, and EGFR mutation type. A Mann–Whitney test was used to analyze the differences between means. *p < 0.05.

Figure 3

Pattern of plasmatic Shh concentration along the course of the disease: (A) Shh was analyzed in plasma collected from patients and classified according to (A) treatment response (responders vs. non-responders); (B, C) and treatment steps (diagnosis or base line, the first evaluation and the progression). A Mann–Whitney test was used to analyze the differences between means. *p < 0.05, ***p < 0.001.

Figure 4

Patient’s survival according to Shh levels: Patient survivals were classified according to (A) Shh levels at diagnosis (Shh^low vs. Shh^High) and (B) Shh levels at the first evaluation (Shh^low vs. Shh^High).

Figure 5

Shh concentrations in plasma from patients at diagnosis according to the presence of secondary mutations. (A) Patient samples analyzed for Shh expression at baseline were classified according to the progression mechanism observed at the time of the progression. (B) Association between Shh concentrations and the presence of T790M mutation at the time to the progression. (C) Shh concentration in plasma from patients according to the presence of a resistance mechanism. A Mann–Whitney test was used to analyze the differences between means. **p < 0.01; ***p < 0.001.