Mesothelin overexpression is a marker of tumor aggressiveness and is associated with reduced recurrence-free and overall survival in early-stage lung adenocarcinoma

Abstract

Purpose: In an effort to identify molecular markers of tumor aggressiveness and therapeutic targets in lung adenocarcinoma (ADC), we investigated the expression of mesothelin (MSLN) in lung ADC, as well as its biologic and clinical relevance.

Experimental design: In a training and validation set of patients with early-stage (I-III) lung ADC (n = 1,209), a tissue microarray consisting of tumors and normal lung tissue was used to examine the association between MSLN expression and recurrence-free survival (RFS) and overall survival (OS). The influence of MSLN overexpression on lung ADC was investigated in vitro and in vivo by use of clinically relevant orthotopic and metastatic xenogeneic and syngeneic mouse models.

Results: MSLN was expressed in 69% of lung ADC tumors, with one in five patients strongly expressing MSLN and no expression in normal lung tissue. Increased MSLN expression was associated with reduced OS [HR = 1.78; 95% confidence interval (CI), 1.26-2.50; P < 0.01] and RFS (HR = 1.67; 95% CI, 1.21-2.27; P < 0.01) in multivariate analyses, even after adjustment for currently known markers of tumor aggressiveness in lung ADC: male sex, smoking history, increasing stage, morphologic pattern, visceral pleural invasion, lymphatic or vascular invasion, and mutation status. In vitro, lung ADC cells overexpressing MSLN demonstrated increased cell proliferation, migration, and invasion; in vivo, mice with MSLN(+) tumors demonstrated decreased survival (P = 0.001).

Conclusions: MSLN expression in patients with early-stage lung ADC is associated with increased risk of recurrence and reduced OS, indicating that MSLN expression is a molecular marker of tumor aggressiveness and a potential target for therapy.

Fig. 1. Mesothelin (MSLN) expression and clinicopathologic characteristics

(A) The sum of MSLN staining intensity and distribution grades was used to determine the total MSLN score, ranging from 0 to 5, at the core level; all cores per patient were averaged to yield an MSLN score at the patient level. (B) Overall, 69% of patients had detectable levels of MSLN in their tumor cores; among MSLN(+) patients, 53%, 28%, and 19% of cores had MSLN staining intensity of 1 (weak), 2 (moderate), and 3 (strong), respectively. Overall survival (C) (P< 0.001) and recurrence-free survival (D) (P=0.002) of patients with stage I to III lung adenocarcinoma were reduced in those with high-level MSLN expression.

Fig. 2. Mesothelin (MSLN) expression and patient survival

The optimal cutoff for comparison of overall survival (OS) was identified in a randomly split cohort. In the training set (A), patients with an MSLN score of 5 (MSLN high score) had reduced OS (P=0.021), compared with patients with an MSLN score of 0 to 4 (MSLN low score), which was confirmed in the validation set (B) (P=0.001). In patients with stage I lung adenocarcinoma, OS (C) (P=0.003) and RFS (D) (P=0.005) remained significantly reduced in patients with high-level MSLN expression.

Fig. 3. Mesothelin (MSLN) expression promotes lung adenocarcinoma (ADC) aggressiveness in vitro

Fluorescence-activated cell sorting (FACS) was performed, after retroviral transduction, using FACSAria (BD Biosciences, San Jose, CA) to selectively obtain adequately transduced cells. FACS for green fluorescent protein (GFP) positivity demonstrated high-level expression of GFP–firefly luciferase. Human MSLN cell-surface expression was detected using allophycocyanin-conjugated antihuman MSLN rat IgG2A (R&D Systems, Minneapolis, MN). Subsequent flow cytometry for analysis of GFP and MSLN expression was performed using either FACSCaliber or LSRII flow cytometers (BD Biosciences, San Jose, CA). Resulting FACS data were analyzed using FlowJo (Tree Star, Ashland, OR) analysis software. (A) A549 and H1299 cells transduced to overexpress MSLN are designated “A549M” and “H1299M,” respectively. Human lung ADC cell lines transduced to overexpress MSLN were compared with cells transduced with control vector. For invasion assays, Matrigel-coated polyethylene terephthalate inserts with 8-um pores were used in 24-well plates. For migration assays, inserts without Matrigel coating were used. (B) MSLN overexpression in H1299 lung ADC cells (H1299M) increased invasion at 24 hours (44 vs 99 cells [2.21-fold increase]; P = 0.0002) and (C) migration at 12 hours (48 vs 96 cells [1.98-fold increase]; P = 0.0004).

Fig. 4. Mesothelin (MSLN) expression promotes lung adenocarcinoma (ADC) tumor progression and decreased survival in vivo

All control tumors were developed from cells transduced with control vector. An orthotopic mouse model of lung ADC was established in the left lung of mice by a single intrapulmonary injection of H1299 cells. (A) Mice were serially imaged using bioluminescence imaging (BLI) to noninvasively assess tumor progression over time. (B) Mice bearing MSLN(+) H1299M tumors accumulated a greater BLI signal over time (P<0.0001), suggesting increased tumor progression with MSLN overexpression. (C) This correlated with the decreased survival of mice bearing MSLN(+) H1299M orthotopic tumors (44 vs 56 days; P=0.0012). (D) To evaluate the relative in vivo proliferation of H1299 tumor cells with and without MSLN expression, we orthotopically injected mice with a heterogeneous population of MSLN-expressing H1299 cells. The initial tumor inoculum was 22% MSLN(+) H1299M on flow cytometry. Tumor cells, identified by green fluorescent protein expression by use of flow cytometry, were then gated for MSLN positivity. H1299M became the dominant tumor cell population over time; these results were statistically significant at days 24 (66% vs 29%; P< 0.01) and 31 (87% vs 12%; P<0.001).