Circulating Cell-Free miR-375 as Surrogate Marker of Tumor Burden in Merkel Cell Carcinoma

Abstract

Purpose: Merkel cell carcinoma (MCC) is an aggressive skin cancer with neuroendocrine differentiation. There is an unmet need for MCC-specific blood-based surrogate biomarkers of tumor burden; circulating cell-free miRNA may serve this purpose.

Experimental design: Expression of miR-375 was quantified in 24 MCC and 23 non-MCC cell lines, 67 MCC and 58 non-MCC tumor tissues, sera of 2 preclinical MCC models, and sera of 109 patients with MCC and 30 healthy controls by nCounter human-v2-miRNA expression or miR-375-specific real-time PCR assays. The patients' sera consisted of two retrospective (discovery and training) and two prospective (validation) cohorts.

Results: miR-375 expression was high in MCC cell lines and tissues compared with non-MCCs. It was readily detected in MCC-conditioned medium and sera of preclinical models bearing MCC xenografts. miR-375 levels were higher in sera from tumor-bearing patients with MCC than in tumor-free patients or healthy controls (P < 0.0005). Moreover, miR-375 serum levels correlated with tumor stage in tumor-bearing (P = 0.037) but not in tumor-free (P = 0.372) patients with MCC. miR-375 serum level showed high diagnostic accuracy to discriminate tumor-bearing and tumor-free patients with MCC as demonstrated by ROC curve analysis in the retrospective cohorts (AUC = 0.954 and 0.800) as well as in the prospective cohorts (AUC = 0.929 and 0.959). miR-375 serum level reflected dynamic changes in tumor burden of patients with MCC during therapeutic interventions.

Conclusions: Circulating cell-free miR-375 proved as a surrogate marker for tumor burden in MCC without restriction to polyomavirus positivity; it thus appears to be useful for therapy monitoring and the follow-up of patients with MCC.

Conflict of interest statement

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

P.N. has served as a consultant for EMD Serono, Pfizer as well as Merck and has received research support to his institution from Bristol-Myers Squibb and EMD Serono. P.N. activities with EMD Serono, Merck and Pfizer are related to the submitted report (therapy of advanced MCC).

D. Schadendorf has received speaker honoraria from Amgen, Astra Zeneca, MerckSerono, Novartis, BMS, Roche and MSD; he has received advisory board honoraria from Amgen, Array, BMS, Imcore, Incyte, MerckSerono, MSD, Mologen, Novartis, Pfizer, Philogen, Pierre Fabre, Regeneron, Sanofi and 4SC; and he has received research funding from BMS and Novartis. D.S.’s activities with Bristol-Myers Squibb, MerckSerono and Pfizer are related to the submitted report (therapy of advanced MCC).

S.U. declares advisory board and speakers’ honoraria from Bristol-Myers-Squibb, Merck Sharp & Dohme and Roche, as well as grant and travel support from Bristol-Myers Squibb, Merck Sharp & Dohme, Roche und medac.

J.C.B. has received speaker honoraria from Amgen, MerckSerono, and Pfizer; he has received advisory board honoraria from Amgen, CureVac, eTheRNA, Lytix, MerckSerono, Novartis, Rigontec, and Takeda; and he has received research funding from Boehringer Ingelheim, BMS and MerckSerono. J.C.B.’s activities with Bristol-Myers Squibb, MerckSerono and Pfizer are related to the submitted report (therapy of advanced MCC).

N.Ø. has received advisory board honoraria from Zealand Pharma and MiNDERA Inc. and the Institution has a patent or intellectual property in MiNDERA Inc.

A.W. has an immediate family member working at LEO Pharma, and the Institution has a patent or intellectual property in MiNDERA Inc.

All other authors indicated no potential conflicts of interest.

©2018 American Association for Cancer Research.

Figures

Fig. 1:

Study flow diagram of serum analysis for circulating cell-free miR-375, including two retrospective and two prospective MCC patient cohorts.

Fig. 2:

The highly expressed miR-375 in MCC cell lines and tissues is also present as cell-free miRNA in MCC-conditioned media as well as in sera of MCC-bearing preclinical models. A: Heat map depicting the relative expression of the 30 most abundant miRNAs in six MCC cell lines. Data obtained by nCounter® Human v2 miRNA Expression Assay (NanoString Technology). B: miR-375 expression in 24 MCC cell lines (21 classical MCPyV positive [n=14, red] or negative [n=7, gray], and three variant MCC cell lines [blue]; details in S. Tab. 1) as well as 23 non-MCC skin cancer (melanoma, squamous cell carcinoma), lung cancer, kidney, and fibroblast cell lines (details in S. Tab. 2) was quantified by RT-qPCR in triplicates. The relative expression of miR-375 was normalized to U6 and is depicted relative to 293T cells as calculated by the 2-ΔΔCq method. C: miR-375 expression in 58 non-MCC skin cancer tissue samples (48 melanomas and ten basal cell carcinomas) as well as 67 MCC tissue samples (49 MCPyV positive [red] and 18 MCPyV negative [gray]) was determined by RT-qPCR in triplicate. The expression level of miR-375 was normalized to U6 and is depicted relative to one randomly selected melanoma sample as calculated by the 2-ΔΔCq method. D: In situ hybridization (ISH) for miR-375 in a representative MCC tissue. Intense miR-375 ISH signal (right), and background staining for the scrambled control (left). Scale bar, 10 μm. E: The presence of miR-375, miR-200c, miR-182, miR-19b and miR-106b in 200μl of conditioned medium from the MCC cell line WaGa (48 hours culture of 106 cells per ml) and in the cells themselves was determined by RT-qPCR in triplicate. The ratio of respective miRNA calculated by the 2-ΔΔCq method in conditioned medium to the cells is depicted. F: miR-375 presence in conditioned medium from seven different MCC cell lines was determined in triplicate. The expression level of miR-375 was normalized to spiked-in cel-mir-39 and is depicted relative to MCC13-conditioned medium as calculated by the 2-ΔΔCq method. G: Circulating cell-free (cf) miR-375 in sera of chicken embryos bearing 4-day-old xenotransplants of WaGa MCC cells on the chorioallantoic membrane was determined by RT-qPCR in triplicate. The expression level of miR-375 was normalized to spiked-in cel-mir-39 and is depicted relative to the serum of an untreated chicken embryo as calculated by the 2-ΔΔCq method. H: cf miR-375 in sera of NOD.CB17-Prkdcscid/J mice with or without subcutaneous WaGa MCC xenografts was determined by RT-qPCR in triplicate. The expression level of miR-375 was normalized to spiked-in cel-mir-39 and is depicted relative to the sera of tumor-free NOD.CB17-Prkdcscid/J control mice as calculated by the 2-ΔΔCq method. Mann-Whitney U test were performed as described in Statistical analysis; p*<0.05, p**<0.005, p***<0.001.

Fig. 3:

Circulating cell-free miR-375 in serum discriminates MCC patients with and without presence of disease: The retrospective discovery and training cohorts. A, B: cf miR-375 in sera of MCC patients was determined by RT-qPCR in duplicate, and normalized to spiked-in cel-mir-39. Values were calculated relative to the serum of a MCC patient with no evidence of disease (Graz cohort) by the 2-ΔΔCq method. Results are depicted in Cleveland dot plots categorized in patients with no (NED) or with evidence of disease. C, D: Receiver operating characteristic (ROC) curves showing the sensitivity and specificity of miR-375 serum levels to discriminate tumor-bearing versus NED patients. The areas under the curve (AUC), optimal cut-off values and their sensitivity and specificity are given. A, C: Graz cohort; B, D: Seattle cohort. Patients’ characteristics are given in S. Tab. 3 and 4. The horizontal line indicates the median, Mann-Whitney U test and pROC R were performed as described in Statistical analysis; p***<0.001

Fig. 4:

Circulating cell-free miR-375 in serum discriminates MCC patients with and without presence of disease: The prospective validation cohorts. A, B: cf miR-375 in sera of MCC patients was determined by RT-qPCR in duplicate, and normalized to spiked-in cel-mir-39. Values were calculated relative to the serum of an MCC patient with no evidence of disease (Graz cohort) by the 2-ΔΔCq method. Results are depicted in Cleveland dot plots categorized in patients with no (NED) or with evidence of disease. C, D: Receiver operating characteristic (ROC) curves showing the sensitivity and specificity of miR-375 serum levels to discriminate tumor-bearing versus NED patients. The areas under the curve (AUC), optimal cut-off values and their sensitivity and specificity are given. E, F: The mean optimal miR-375 serum level cut-off was calculated from the optimal cut-off values of the retrospective discovery and validation cohorts as 2.42. Proportions of MCC patients of the prospective cohorts with (red) or without (blue) tumor burden below or above this mean optimal cut-off are depicted. Percentages of MCC patients with tumor burden within each group are given. A, C and E: Essen cohort, B, D and F: Melbourne cohort. Patients’ characteristics are given in S. Tab. 3–4. The horizontal line indicates the median, Mann-Whitney U test and pROC R were performed as described in Statistical analysis; p*<0.05, p**<0.005, p***<0.001.

Fig. 5:

Circulating cell-free miR-375 serum levels correlate with disease stage and tumor burden of MCC patients cf miR-375 in sera of MCC patients was determined by RT-qPCR in duplicate, and normalized to spiked-in cel-mir-39. Values were calculated relative to the serum of an MCC patient with no evidence of disease (Graz cohort) by the 2-ΔΔCq method. A, B: Results are depicted in Cleveland dot plots combined for all four cohorts categorized by AJCC stage at the time of blood draw for patients with (A) or without (B) evidence of disease; to discern from which cohorts the samples were derived, the data points were color-coded: Graz - green, Seattle - yellow, Essen - dark red, Melbourne - purple. Correlation analysis between cf miR-375 serum levels and MCC tumor stages was performed in R using the “ggpubr” package, the horizontal line indicates the median. C: Correlation analysis for cf miR-375 serum level and MCC tumor burden as quantified by PET/CT scan for the Melbourne cohort was performed in R using the “ggpubr” package. D, E and F: cf miR-375 serum levels are plotted over the course of disease together with the tumor volume which was calculated from PET/CT scans depicted above the respective graphs for three exemplary patients from the Melbourne cohort (D: p_#16, E: p_#6 and F: p_#10). The numbers circled indicate the different therapies: ①Radiation therapy, ②Chemotherapy and ③Immunotherapy. The clinical course of patient D is described in Results; patient E showed disease progression during chemotherapy; patient F showed a complete response to checkpoint inhibition.