Systemic LRG1 Expression in Melanoma is Associated with Disease Progression and Recurrence

Abstract

The response rates upon neoadjuvant immune checkpoint blockade (ICB) in stage III melanoma are higher as compared with stage IV disease. Given that successful ICB depends on systemic immune response, we hypothesized that systemic immune suppression might be a mechanism responsible for lower response rates in late-stage disease, and also potentially with disease recurrence in early-stage disease. Plasma and serum samples of cohorts of patients with melanoma were analyzed for circulating proteins using mass spectrometry proteomic profiling and Olink proteomic assay. A cohort of paired samples of patients with stage III that progressed to stage IV disease (n = 64) was used to identify markers associated with higher tumor burden. Baseline patient samples from the OpACIN-neo study (n = 83) and PRADO study (n = 49; NCT02977052) were used as two independent cohorts to analyze whether the potential identified markers are also associated with disease recurrence after neoadjuvant ICB therapy. When comparing baseline proteins overlapping between patients with progressive disease and patients with recurrent disease, we found leucine-rich alpha-2-glycoprotein 1 (LRG1) to be associated with worse prognosis. Especially nonresponder patients to neoadjuvant ICB (OpACIN-neo) with high LRG1 expression had a poor outcome with an estimated 36-month event-free survival of 14% as compared with 83% for nonresponders with a low LRG1 expression (P = 0.014). This finding was validated in an independent cohort (P = 0.0021). LRG1 can be used as a biomarker to identify patients with high risk for disease progression and recurrence, and might be a target to be combined with neoadjuvant ICB.

Significance: LRG1 could serve as a potential target and as a biomarker to identify patients with high risk for disease recurrence, and consequently benefit from additional therapies and intensive follow-up.

FIGURE 1

Flowchart of patients with stage III and stage IV melanoma selected for serum/plasma analyses. Number of patients included for the different analysis. A total of 117 patients with paired plasma/serum samples were available at stage III disease and stage IV disease (paired analysis). Because of plasma/serum mismatch and heterogeneity between samples from European centers and MIA, the sera of 65 patients were used for analysis. Of these patients, 33 patients were analyzed by Olink and 64 patients were analyzed by MS (left). Plasma of 108 patients with stage III or stage IV was analyzed by Olink (right).

FIGURE 2

MS analysis of protein change upon disease progression and recurrence. A, Plasma and serum samples of different cohorts of patient with melanoma analyzed for circulating proteins using MS proteomic profiling and Olink analysis. B, Volcano plot showing differential protein expression of serum analysis using MS, comparing protein expression between matched patients with stage III and stage IV disease (n = 64). Proteins higher expressed at stage III disease are displayed on the left, and proteins higher expressed at stage IV are displayed on the right. The protein fold change on a log₂ scale is shown on the x-axis, with the significance indicated by the −log₁₀ scale on the y-axis. The significance cutoff (P = 0.05) is indicated with the black dotted line, showing significant increased proteins for stage III in blue and for stage IV in purple. A two-tailed paired Student t test was used to determine statistical significance between stage III and stage IV samples. C, A Venn diagram for overlapping significant proteins of patients with stage IV melanoma and baseline samples of nonresponding stage III patients with recurrent disease. D, Volcano plot showing differential expression of plasma markers using MS, comparing protein expression of nonresponder patients of the OpACIN-neo study (n = 21) with or without a recurrence. Proteins higher expressed by patients without a recurrence are displayed on the left, and proteins higher expressed by patients with a recurrence are displayed right. The protein fold change on a log₂ scale is shown on the x-axis, with the significance indicated by the −log₁₀ scale on the y-axis. The significance cutoff (P value = 0.05) is indicated with the black dotted line, showing significant increased proteins for patients without a recurrence in green and for patients with a recurrence in red. A two-tailed unpaired Welch t test was used to determine statistical significance between samples of patients with and without a recurrence.

FIGURE 3

LRG1 expression is associated with melanoma progression and recurrence. A–D, Normalized protein expression (log₂) values of LRG1, measured by MS for matched stage III and IV patients (n = 64; A), non-responsive patients at baseline of the OpACIN-neo study (n = 20; B), and at week 6 after neoadjuvant ICB treatment of the OpACIN-neo study (n = 20; C) and all patients at baseline of the OpACIN-neo study (n = 82; D). A two-tailed paired Student t test was used to determine statistical significance between stage III and stage IV samples. A two-tailed unpaired Student t test was used to compare patients with and without a recurrence or response of the OpACIN-neo study. E, sROC curves to define the optimal cutoff (marked by the blue dot) for all patients (left) and nonresponding patients (right) of the OpACIN-neo study for baseline LRG1 expression. The AUC for all patients was 0.598 and for nonresponder patients 0.780, with an optimal cutoff of 24.5504. A Kaplan–Meier curve showing EFS for all patients (F) and nonresponder patients (G) of the OpACIN-neo study with either a high (red) or low (blue) expression of LRG1. The asterisk denotes a patient (in LRG1 low group) who died because of irAEs. H, A Kaplan–Meier curve showing overall survival for nonresponder patients of the OpACIN-neo study with either a high (red) or low (blue) expression of LRG1. P value was calculated using the log-rank test (two-sided) and significance is indicated. *, P < 0.05; ***, P <0.001.

FIGURE 4

LRG1 expression is associated with melanoma recurrence in a second independent cohort. A, sROC curves to define the optimal cutoff (marked by the blue dot) for all patients (left) and nonresponding patients (right) of the PRADO study for baseline LRG1 expression in patient plasma. The AUC for all patients was 0.588 and for nonresponder patients 0.714, with an optimal cutoff of 24.9485. A Kaplan–Meier curve showing EFS for all patients (n = 49; B) and nonresponder patients (n = 11; C) of the PRADO study with either a high (red) or low (blue) expression LRG1. P value was calculated using the log-rank test (two-sided) and significance is indicated.

FIGURE 5

High LRG1 expression is associated with distant metastasis in patients that do not respond to neoadjuvant ipilimumab and nivolumab. LRG1 expression measured in patient plasma by MS for patients without a response upon neoadjuvant ICB of the OpACIN-neo study (n = 20; A) and PRADO study (n = 11; B). Comparing patients without a recurrence (green), patients with a local recurrence (pink) and patients with a distant recurrence (red). A two-tailed unpaired Student t test for comparing patient groups. *, P < 0.05.