EGFR as a stable marker of prostate cancer dissemination to bones

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 (EGFRover) 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 EGFRover 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. EGFRover 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. EGFRover 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 EGFRover is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.


INTRODUCTION
Prostate cancer (PCa) is the second most frequent malignancy in men worldwide. 1 Although 5-year survival in patients with localised PCa is nearly 100%, metastatic disease still remains incurable. 2,3 Therefore, there is an urgent need for markers that could help to detect initial stages of tumour dissemination, probability of recurrence and predict preferred sites of metastasis in order to personalise patients' treatment.
Epithelial-mesenchymal transition (EMT) and plasticity are involved in metastatic progression of PCa. [4][5][6] In addition, significant roles for the epidermal growth factor receptor (EGFR) have been suggested in prostate tumorigenesis and progression. 7,8 EGFR expression was previously shown to be associated with high grade, advanced stage and high risk for prostate-specific antigen (PSA) recurrence 9 and bone metastases. 10 In addition, EGFR was also shown to control bone development. 11 Indeed, as one of the regulators of EMT, (de)differentiation, proliferation and angiogenesis, EGFR might initiate and/or promote tumour dissemination and metastasis and thus may be considered as a surrogate marker of high metastatic potential. 10 However, there is a lack of a complex study evaluating EGFR expression in PCa in the context of tumour characteristics and at various stages of PCa dissemination.
Thus, in the current study, the expression of EGFR protein was assessed in the dissemination cascade-throughout the disease process from primary tumours to disseminated circulating tumour cells (CTCs) and metastatic samples obtained from castrateresistant PCa (CRPC) patients at the time of death. It was also compared to clinical parameters and multiple molecular factors (including EMT-related proteins, collagen fibre content, vascular and lymphatic vessels numbers) to evaluate its feasibility as a stable marker in the PCa dissemination process.

METHODS
PCa patients of cohort I to study primary tumours One-thousand two hundred PCa patients were included in this study (Supplementary Table 1) based on their signed informed consent, after the approval of the local Ethics Committee (i.e. Ethik Kommission www.nature.com/bjc der Aerztekammer Westfalen-Lippe und der Medizinischen Fakultaet der Westfaelischen Wilhelms-Universitaet Muenster, Germany, no. 2007-467-f-S). The patients underwent radical prostatectomy at the Department of Urology in the Prostate Centre University Clinic Münster (Germany) between 1993 and 2004. The variable clinicopathological and molecular parameters were documented as described. 12,13 Time to biochemical recurrence was defined as time between prostatectomy and the time point of first serum PSA increase >0.1 ng/mL followed by another value >0.1 ng/mL after surgery. Metastasis-free survival was defined as the time between prostatectomy and occurrence of clinically defined metastases. Overall survival was defined as the time between the prostatectomy and patient death. Last follow-up was completed in June 2019. The median follow-up was 76 months (range 0.1-273 months).
PCa patients of cohort II to study CTCs Fifty-nine d'Amico high-risk PCa patients treated in the Martini-Clinic at the University Medical Center Hamburg-Eppendorf (Hamburg, Germany) between 2012 and 2013 were enrolled in this study after informed consent based on the approval of the local ethical review board number PV3779 as presented in Supplementary Table 2. Blood samples (at mean volume of 7.5 mL, range 5-12 mL; first 2 mL of collected blood discarded to avoid contamination by skin cells) were collected into EDTA tubes. Last follow-up was completed in September 2015. The median observation time was 13 months (range 1-25 months).
PCa patients of cohort III to study metastases Visceral and bone metastases were obtained from 21 PCa patients who died of metastatic CRPC and who signed written informed consent for a rapid autopsy performed within 6 h of death, under the aegis of the Prostate Cancer Donor Program at the University of Washington and approved by the Institutional Review Board of the University of Washington (Supplementary Table 3).
Tissue microarrays (TMAs) TMAs with primary or metastatic PCa samples were prepared as previously described. 13,14 The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) data set RNA-seq data (RNASeqV2, RSEM normalised) covering normalised counts of sequences aligning to 20,531 genes were obtained for 497 PRAD patients from TCGA portal (data status of 28 January 2016). The methods of biospecimen procurement, RNA isolation and RNA sequencing were previously described by TCGA Research Network. 15 Isolation of CTCs Peripheral blood was processed within 24 h of collection. Peripheral blood mononuclear cell (PBMC) fraction, preferably containing CTCs, was enriched using Ficoll density gradient centrifugation, resuspended in 5 mL of 1× phosphate-buffered saline (PBS) and centrifuged to prepare microscopic slides, each containing 500,000 cells. The slides were left overnight to air-dry at room temperature and used within 24 h for further CTC analysis.
Immunohistochemical (IHC) detection and evaluation of EGFR To detect EGFR, TMA sections were deparaffinised and treated with Proteinase K Ready-to-Use (Dako) for 6 min and Perioxidase-Blocking Solution (Dako) for 5 min. TMAs were incubated overnight at 4°C with mouse monoclonal anti-EGFR in vitro diagnostic antibody (E30, Dako) diluted 1:20, envisioned by EnVision Kit, Rabbit/Mouse (Dako) and counterstained with haematoxylin (Merck, Germany). The intensity (negative, weak, moderate or strong), subcellular localisation of the staining (membranous, cytoplasmic, nuclear) and the percentage of positive tumour cells were documented. Two tumour samples (TMA tissue cores) from each patient were assessed individually by two independent observers, experienced in IHC analysis. The EGFR intensity (negative, weak, moderate, strong) was evaluated according to the analogical recommendations for HER2 testing in breast cancer proposed by American Society of Clinical Oncology. 16 To evaluate overall score corresponding to one patient, maximal intensity of EGFR was chosen from two analysed tumour samples. If one tissue core was uninformative, the overall score corresponded to the remaining one.
IHC detection and evaluation of other proteins IHC for vimentin, epithelial cell adhesion molecule (EpCAM) and keratins K8/18 and K19 was performed, evaluated and categorised as negative vs. positive staining as described. 13,14 The number of vascular or lymphatic vessels was examined in each tumour sample as the number of vessels with visible lumen, positive for CD34 and podoplanin staining, respectively. 12 EGFR/EpCAM/pan-keratin/CD45 immunocytochemical staining on CTCs Immunocytochemical staining identifying CTCs was performed for each patient on 3 slides containing 500,000 PBMCs each. The slides were fixed for 10 min with the Fixation Solution B for Epithelial Cell Detection Kit (Micromet AG; 135 μL diluted in 10 mL of 1× PBS), incubated for 5 min with Peroxidase-Blocking Solution (Dako) and subsequently for 20 min with AB blocking serum (Bio-Rad Medical Diagnostics) diluted 1:10 in 1× PBS. CD45 was detected by incubation with a mouse antibody (NCL-LCA-RP, Novocastra, diluted 1:100, 45 min) and secondary rabbit polyclonal anti-mouse antibody labelled with horseradish peroxidase (Dako, diluted 1:100, 30 min) followed by addition of DAB substrate (Dako, diluted 1:50, 10 min). EGFR was detected by incubation with rabbit polyclonal antibody (sc-03, SantaCruz, diluted 1:100, overnight at +4°C) and secondary antirabbit antibody labelled with Alexa 555 (Life Technologies, diluted 1:200, 45 min). EpCAM was detected by incubation with mouse antibody (NCL-ESA, Novocastra, diluted 1:100, 45 min) and secondary anti-mouse Alexa 350-conjugated antibody (Life Technologies, diluted 1:200, 45 min). Subsequently, cells were incubated for 45 min with anti-pan-keratin antibody AE1/AE3 (eBioscience, diluted 1:700) and C11 (Cell Signalling Technology, diluted 1:300) both directly labelled with Alexa 488. Nuclei of the cells were counterstained with Red-Dot (Biotum, diluted 1:200, 30 min) and covered with coverslips with one drop of Moviol (Sigma Aldrich). Three slides per patient were screened and evaluated under the fluorescence microscope (Axioplan2) in five fluorescent channels and brightfield for putative CTCs under magnification ×400 and ×600. A cell was classified as a CTC based on its cellular and nuclear morphology (inclusion criteria: intact and non-apoptotic cell morphology, intact non-leucocyte-like nucleus, non-granulocyte-like morphology, cell diameter of minimum 5 µm) and absence of CD45 staining. Keratins, EGFR and EpCAM expression was evaluated in such cells and documented. If no CTCs were found, a subsequent three slides were stained and analysed to confirm CTC status in such patients.
With the gene ontology analysis, multiple genes involved in cell migration, adhesion and proliferation, as well as angiogenesis regulation, were significantly upregulated in tumours expressing EGFR and COL1A1 (Fig. 2f and Supplementary Data 1). On the contrary, multiple genes involved in translation, transcription and mitochondrial metabolism were downregulated in this subgroup of tumours ( Fig. 2g and Supplementary Data 2).
EGFR is an EMT-independent marker of CTCs in d'Amico high-risk PCa patients Single CTCs (n = 11) and CTC clusters (n = 2) were isolated from 5 (12.8%) of 39 analysed d'Amico high-risk patients. The CTC yield varied between 1 and 5 CTCs/1,500,000 PBMCs per patient. All detected CTCs showed strong EGFR and were negative for CD45 (Fig. 3a-c and Supplementary Fig. 2a). Other common CTC markers including pan-keratins and EpCAM were also evaluated. Only three patients had CTCs positive for EpCAM, including all cells from both clusters (Fig. 3a, c). Pan-keratin status of the detected CTCs was heterogonous, varying from negative (five cells) through weak (six cells) to moderate expression-five cells in two clusters (Fig. 3a-c). CTCs' yield and phenotype were not associated with any tumour characteristics (Supplementary Table 4). Despite the short observation time after surgery (≤25 months) and limited follow-up cohort, patients with EGFR over 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 positive COL1A1 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 positive COL1A1 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.

a b c
Pan-cytokeratin Pan-cytokeratin EGFR as a stable marker of prostate cancer dissemination to bones P Nastały et al.
CTCs had significantly shorter time to biochemical recurrence and time to metastasis than patients negative for CTCs (n = 25, Kaplan-Meier log-rank analysis, p = 0.002 and p < 0.001, respectively, Supplementary Fig. 2b, c). In addition, 3 of those 5 patients with EGFR over CTCs developed distant metastases to lymph nodes (patient with CTCs negative for EpCAM) or bones (patient with weakly positive CTCs for EpCAM), whereas none of the patients developed metastasis in the cohort negative for CTCs (n = 23, Fisher's exact test, p = 0.005).
EGFR overexpression is most frequent in CRPC metastases to bones Membranous and membranous/cytoplasmic EGFR expression was also evaluated in 75 tissue cores from castration-resistant metastases of 21 patients (Fig. 4a, b). EGFR expression was significantly more frequent in castration-resistant bone metastases, when compared to its distribution in primary tumours (n = 39 vs. n = 1841, Chi 2 = 11.543, p = 0.009, Fig. 4c, d). Interestingly, also EpCAM strong intensity reached 76% in bone metastases from CRPC patients (Fig. 4d). In bone metastases, the percentage of EGFR-positive tumour cells frequently reached 100% per tumour sample (Fig. 4e) and its mean value was significantly higher than in the cohort of EGFR-positive primary tumours (92%, n = 13 vs. 58%, n = 1258; two-tailed Mann-Whitney test, p < 0.0001). There was no correlation between EGFR over and EpCAM, K8/18/19 and vimentin expression in castration-resistant bone and/or visceral metastases (data not shown). In addition, there was a borderline correlation between higher prevalence of collagen fibres (>50%) and the EGFR over phenotype of tumour cells in metastases (n = 71, Chi 2 = 5.934, p = 0.051, Fig. 4f).
High expression of EGFR improves PC-3 proliferation on rigid matrices On collagen-coated rigid matrices (25 kPa), mimicking bone tissues rigidity, PC-3 cells sorted according to their high EGFR expression, adhered and grew more efficiently than sub-lines with low EGFR expression (two-tailed Mann-Whitney test, p < 0.0001, Fig. 4g and Supplementary Fig. 3a, b). On the other hand, such increase in adhesion and growth was not observed on soft substrate (0.  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.
EGFR as a stable marker of prostate cancer dissemination to bones P Nastały et al.
by K19/vimentin ratio assessed by real-time PCR (data not shown).

DISCUSSION
Our data indicate that EGFR over might be a candidate biomarker of stable marker of PCa dissemination cascade. EGFR overexpression, found both in primary tumour and CTCs, was an indicator of poor prognosis. In primary tumours, it was associated with shorter metastasis-free survival, which has been previously shown to be related with a significant risk of death from PCa. 25 Our observation is similar to the ones from other groups, 9 performed, however, mainly in CRPC. 26,27 The expression of EGFR was reported to be associated with EMT, 28,29 which promotes pro-migratory and pro-survival behaviour of tumour cells, generating their aggressive phenotype. 4 Based on our data, EGFR overexpression correlated with promigratory and pro-metastatic phenotype of PCa tumour cells. In EGFR over tumours, we also found a higher collagen fibre content that can influence cell migration, invasiveness and proliferation and indicate worse prognosis, 30,31 Gene ontology analysis further confirmed that cancers with high expression of both EGFR and gene-encoding type I collagen have upregulated genes involved in cell migration and adhesion. Tumours with strong EGFR expression had a larger number of blood and/or lymphatic vessels that could facilitate haematogenous and lymphatic dissemination of cancer cells. 32 Of note, EMT is a phenomenon hindering detection of CTCs in bloodstream. [33][34][35] Our results suggest that EGFR seems to be a stable signature of PCa progression, which might serve as surrogate marker of CTCs undergoing EMT. Importantly, based on our collected data, in a d'Amico high-risk patient cohort, inclusion of EGFR can improve both CTC detection and stratification of patients. However, this result needs further confirmation in a larger cohort of patients as the absolute number of CTC-positive patients and actual number of CTCs in the present study are too low to allow for strong conclusions.
In our study, EGFR over was enriched in bone metastases, suggesting that organ-specific factors such as its stiffness or tumour microenvironment might result in regulation of harbouring and/or nesting of EGFR-positive tumour cells. PCa cells that disseminate show an exquisite tropism for the bone. 36 In an autopsy study, 90% of the men who had died with haematogenous metastases of PCa were diagnosed with bone metastases. 37 However, the possible molecular mechanisms involved in governing bone metastases tropism is still poorly understood. 36 Bone is among tissues characterised by elastic moduli with the greatest stiffness. 38 It was also reported that EGFR can be involved in rigidity sensing after associating with nascent adhesions under rigidity-dependent tension. 39 Moreover, human squamous cell carcinoma cells, in response to matrix stiffening, increased EGFR expression and invasiveness. 40 In our study, EGFR positively correlated with collagen fibre content that can indicate tumour stiffness. 40,41 Together with our data showing improved proliferation of EGFR-overexpressing PCa cells on rigid matrices, it can be speculated that EGFR can promote growth of cells on rigid substratum and bone metastases.
To sum up, the data collected within this study suggest that EGFR is a marker of PCa dissemination, independent of EMT.