EGFR as a stable marker of prostate cancer dissemination to bones

Abstract

Background: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men. Although 5-year survival in patients with localised disease reaches nearly 100%, metastatic disease still remains incurable. Therefore, there is a need for markers indicating metastatic dissemination.

Methods: EGFR overexpression (EGFR^over) was tracked in 1039 primary tumours, circulating tumour cells from 39 d'Amico high-risk patients and metastatic samples from 21 castration-resistant PCa cases. EGFR status was compared to clinical parameters and multiple molecular factors were assessed using immunohistochemistry and gene ontology analysis. The functional aspect of EGFR was evaluated by plating PC-3 cells on soft and rigid matrices.

Results: EGFR^over was found in 14% of primary tumours, where it was associated with shorter metastasis-free survival and was an independent indicator of worse overall survival. EGFR^over correlated with a pro-migratory and pro-metastatic phenotype of tumour cells as well as rich collagen fibre content. All circulating tumour cells (detected in 13% of cases) were positive for EGFR, independent of their EMT-related phenotype. EGFR^over was more prevalent in castration-resistant bone metastases (29% of patients) and supported growth of human PCa cells on rigid matrices mimicking bone stiffness.

Conclusions: EGFR^over is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.

Fig. 1. Clinical significance of EGFR overexpression in primary tumours.

a Representative immunohistochemical staining of EGFR in primary prostate tumours, scale bar 100 μm. b Kaplan–Meier estimates of metastasis-free survival, n = 776. c Univariate and multivariate analysis of overall survival.

Fig. 2. Characteristics of EGFR overexpression in primary tumours.

a Vimentin expression in EGFR^neg-to-mod and EGFR^over cases, n = 415 tumours. b Expression of EMT-related markers in EGFR^neg-to-mod and EGFR^over cases, n = 383 tumours. c EpCAM expression in EGFR^neg-to-mod and EGFR^over cases, n = 501 tumours. d Prevalence of blood and lymphatic vessels in EGFR^neg-to-mod and EGFR^over cases, n = 472 tumours. e Representative images of collagen content quantification (left panel), collagen content distribution in EGFR^neg-to-mod and EGFR^over cases, n = 120 patients. f GO BP terms enriched in genes upregulated in EGFR^positiveCOL1A1^positive tumours; top 20 terms with the lowest p value plotted against fold enrichment and ordered according to −log10(FDR); dot size represents the number of genes associated with the term, dot colour represents −log10(FDR); analysed with Functional Annotation Tool by DAVID Bioinformatics Resources 6.81. g GO BP terms enriched in genes downregulated in EGFR^positiveCOL1A1^positive tumours; top 20 terms with the lowest p value plotted against fold enrichment and ordered according to −log10(FDR); dot size represents the number of genes associated with the term, dot colour represents −log10(FDR); analysed with Functional Annotation Tool by DAVID Bioinformatics Resources 6.81.

Fig. 3. EGFR overexpression in circulating tumour cells from d’Amico high-risk patients.

a Representative CTC cluster composed of two cells positive for EGFR, pan-keratin and EpCAM and negative for CD45; scale bar 10 μm. b Representative single CTC positive for EGFR and pan-keratin and negative for EpCAM and CD45. c Characteristics of CTCs detected in d’Amico high-risk patients. Number of CTCs expressing EGFR, EpCAM and pan-keratin/total detected CTCs.

Fig. 4. Characteristics of EGFR overexpression in metastases.

a Representative immunohistochemical staining of EGFR^over in bone metastasis. b Representative immunohistochemical staining EGFR^over in visceral metastases. c EGFR staining intensity distribution in primary tumours (n = 1841), visceral metastases (n = 36) and bone metastases (n = 39). d EGFR and EpCAM staining intensity distribution in bone and visceral metastases. e Percentage of EGFR-positive cells in bone and visceral metastases. f Collagen content distribution in EGFR^neg-to-mod and EGFR^over metastases (n = 71). g Representative images of EGFR^low and EGFR^high PC-3 cell growth on soft (0.2 kPa) and rigid (25 kPa) matrices. Quantification of PC-3 cells proliferation using Ki-67 marker on soft (0.2 kPa, n = 70 cells) and rigid (25 kPa, n = 91 cells) matrices.