Objective MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis.
Design We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan–Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression.
Results MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=−0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175).
Conclusions MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.
- COLORECTAL CANCER
- MICROSATELLITE INSTABILITY
- MOLECULAR GENETICS
- RNA EXPRESSION
- LIVER METASTASES
Statistics from Altmetric.com
Significance of this study
What is already known on this subject?
Metastasis is the main cause of cancer-related death in colorectal cancer (CRC) cases.
Reports on miR-224 involvement in CRC metastasis are inconsistent, with some studies supporting prometastatic function of miR-224 and others reporting its antimetastatic function.
A comprehensive and systemic analysis addressing significance of miR-224 in CRC metastasis is absent.
What are the new findings?
Expression levels of miR-141, miR-181b, miR-221 and miR-224 are significantly increased in primary CRC with metastatic dissemination compared with early-stage diseases. Among these, miR-224 shows higher expression in microsatellite stable CRCs compared with those with microsatellite instability.
Ectopic overexpression of miR-224 increases CRC cell motility in vitro and promotes CRC metastasis to lung and liver in two orthotopic CRC mouse models.
SMAD4 mediates miR-224's pro-metastatic effect and shows inverse correlation with miR-224 in CRC tumour samples.
High miR-224 expression in primary CRC tumours is associated with shorter overall and metastasis-free survival of patients in multiple CRC cohorts.
How might it impact on clinical practice in the foreseeable future?
Our data strongly support that miR-224 is an activator for CRC metastasis via targeting SMAD4, and suggest that miR-224, alone or combination with SMAD4, may be an independent prognostic marker for survival of patients with CRC.
Colorectal cancer (CRC) is the second leading cause of cancer death among adults, with over 1.6 million new cancer cases and 580 350 deaths estimated to have occurred in the USA in 2013.1 Metastatic spread of tumour cells remains the ultimate cause of cancer-related death in most CRC cases. While most cases of localised CRC (stages I and II) are curable by surgical excision, only about 70% of stage III CRC cases with regional lymph node metastasis are curable by surgery combined with adjuvant chemotherapy. Advanced metastatic disease (stage IV), despite improved survival due to recent advances in chemotherapy and targeted agents, remains largely incurable.2 ,3 Therefore, it is of critical importance to understand the key molecular switches involved in CRC metastasis and identify biomarkers for CRC malignancies and prognostic markers for patient survival.
MicroRNAs (miRNAs) are 18-nucleotide to 25-nucleotide RNAs that control gene expression at the post-transcriptional level. Based on sequence complementarity, miRNAs bind to targeted protein-coding genes, prevalently at their 3′ untranslated region (UTR), and consequently affect messenger RNA (mRNA) stability or interfere with protein translation.4 The functional importance of miRNAs in physiology and disease has been widely appreciated. MiRNAs are differentially expressed in normal and tumour tissues, and unique miRNA expression patterns have been characterised in many types of cancer including CRC.5 ,6 ,7 However, CRC metastasis-related miRNAs and their biological roles in CRC metastasis remain to be identified. In the invasive regions of primary CRC, organised structure of the tumour is lost: adhesion molecules that maintain cell–cell contact are downregulated, whereas molecules responsible for invasive and migratory behaviour are upregulated.8 These findings suggest that we could decipher the metastatic potential of tumours by analysing miRNA expression in primary CRC specimens.
In the current study, we aimed to identify miRNAs associated with CRC metastasis and explore their biological significance as well as diagnostic and prognostic value.
Six independent CRC cohorts (Italy set 1, Italy set 2, UK set, Romania set, Austria set and The Cancer Genome Atlas (TCGA) set) of 449 tumour samples (table 1) and 172 non-neoplastic mucosal tissues were included in this study.
Samples were obtained from University of Ferrara (Italy set 1), Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) s.r.l., IRCCS (Italy set 2), University of Southampton (UK set), the Oncology Institute Cluj-Napoca (Romania set) and Medical University of Graz (Austria set), respectively. Samples from patients with biopsy proven adenocarcinomas were obtained fresh at the time of surgery and snap frozen prior to being deposited. Exclusion criteria were evidence of a hereditary tumour, presence of multiple or mucinous tumours and tumours with histologically identified extensive necrosis. Tumours were classified according to the WHO pathological classification system. Microsatellite analysis was evaluated with a fluorescence-based PCR method using the five markers of the Bethesda panel (D5S346, D17S250, D2S123, BAT25 and BAT26) plus BAT40. According to the guidelines of the International Workshop of Bethesda,9 tumours were classified as microsatellite instability-high (MSI-H) (instability at two or more loci), MSI-L (instability at single locus) or microsatellite stable (MSS) (no instability). Patient's clinical information was registered and follow-up data were recorded at checkup. All samples were obtained with patient's informed consent. The institutional research and ethics committee approved this study.
Total RNA was isolated using TRIzol reagent (Invitrogen) for Italy set 1, Italy set 2 and Romania set, and using RNAqueous-Micro Kit for UK set. Formalin-fixed paraffin embedded samples were used for RNA extraction in Austria set. We used a custom miRNA microarray containing quadruplicates of 389 human miRNA probes for profiling as previously described.10 Based on the microarray results, we further examined metastasis-related miRNAs using quantitative real-time PCR (qRT-PCR) analysis with TaqMan MiRNA assays (Life Technologies, Grand Island, New York, USA). Biological functions of miRNAs were tested using in vitro motility assays and orthotopic mouse models of CRC. To identify the mRNA targets, we used a prediction algorithm, and the Human Tumor Metastasis RT2 Profiler PCR array (SA Bioscience, Frederick, Maryland, USA) for the screening, and qRT-PCR, western blot, luciferase assay, immunocytochemistry and rescue experiments for validation. Clinical correlation was tested with multiple sets of CRC samples and publically available TCGA data.11 Other methods are shown in online supplementary methods, and primers are listed in online supplementary table S1. An overview of the study design is presented in figure 1.
In vivo study
Six-week-old to 8-week-old severe combined immunodeficiency (SCID) mice (Charles River, UK) were anaesthetised prior to midline laparotomy and exteriorisation of the caecum. A 1:1 suspension of cells and Matrigel was injected submucosally into the caecal wall under magnified vision, raising a bleb on the caecum. For each animal, 0.5×106 cells stably overexpressing green fluorescent protein (GFP) tagged miR-224 or control were implanted orthotopically, with the entire experiment conducted in duplicate. Primary tumours grew in all animals. When showing signs of disease or >10% weight loss, mice were humanely culled, and colon, liver and lungs were harvested. Excised tissue was paraffin embedded, and stained with H&E.
Statistical analysis was carried out in R statistical environment (version 3.0.1) (http:///www.r-project.org/). Differences between groups were analysed using Student t test (two-tailed), assuming equal or unequal variance determined by the F-test of equality of variances. Graphics represent the mean±SD, unless otherwise stated. The Spearman correlation coefficients were computed to assess the correlation between expression level of miR-224 and its target genes in clinical samples. For survival analysis, we divided patients into low/high groups using as cut-off the value that optimally separated the patients, and used the Kaplan–Meier method to estimate the survival curves, and the log-rank test for the comparison. A p value of <0.05 was considered statistically significant.
Identification of metastasis-related miRNAs in CRC
We performed miRNA microarray using primary CRCs from patients with or without metastasis at diagnosis (Italy set 1; n=4 and 8, respectively), and in cell lines derived from a primary CRC lesion (SW480) or its metastatic dissemination to the lymph node (SW620). According to the microarray data (see online supplementary figure S1A and table S2), 11 miRNAs significantly correlated with the presence of metastasis and were tested by qRT-PCR in a larger cohort of samples (Italy set 1, comprising 85 primary CRC tumours and 25 matched adjacent non-neoplastic colon mucosae). Four miRNAs (miR-141, miR-181b, miR-221 and miR-224; table 2) showed higher expression in primary CRCs with metastatic dissemination (stages III–IV) compared with early stages (stages I–II), as well as in the CRC metastasis-derived SW620 compared with primary CRC-derived SW480 cell line. Among these, miR-181b, miR-221 and miR-224 were significantly increased in neoplastic CRC tissue compared with normal mucosa. Higher levels of miR-224 in advanced stages and in tumour versus normal tissue were supported by analysis from multiple cohorts of primary CRC, an independent dataset from TCGA consortium, and cell lines with different metastatic features (see table 1, figure 2A, online supplementary figures S1B and S2).
Association of miR-224 expression with microsatellite status and tumour site
Unlike miR-181b and miR-221, miR-224 levels were significantly higher in MSS samples compared with MSI-H tumours, concordant with the notion that MSI-H CRCs are less aggressive and less prone to metastatic spread than MSS tumors12 (figure 2B). Also, consistent with the reported association of CRC localisation with microsatellite status,13 miR-224 levels were significantly lower in right colon tumours than those occurring in the left colon and rectum (figure 2B). These findings were validated in TCGA dataset comprising 143 samples (figure 2C), and Italy set 2 comprising 67 samples (figure 2D). Concomitantly, in situ hybridisation showed stronger miR-224 staining in epithelial cells of MSS CRCs compared with normal and MSI-H tumour samples (fold change=3.6) (see figure 2E and online supplementary figure S3).
Prometastatic activity of miR-224 in vitro and in vivo
We selected five miRNAs (the four miRNAs from primary screening, miR-141, miR-181b, miR-221 and miR-224, as well as miR-222 that contains identical seed sequences with miR-221) for evaluation with transwell-based assays (migration, haptotaxis and invasion) using HCT116 cells. Cell motility increased consistently only upon overexpression of miR-224 and miR-141 by either transient or stable transfection (see figure 3A, online supplementary figures S4 and S5A). Next, we investigated in vivo activity of miR-224 using orthotopic CRC SCID mouse model. Direct caecal implantation of HCT116 cells stably overexpressing miR-224 (n=4) resulted, at 5 weeks post-procedure, in a greater number and size of metastatic tumour deposits in the liver and lungs compared with control cells (figures 3B, C). Liver replacement was almost complete in some mice implanted with HCT116–miR-224, suggesting a rapid metastatic and growth process. Similarly, stable expression of miR-224 (75-fold induction) with a GFP coexpression plasmid construct in RKO, a CRC cell line with low miR-224 expression (see online supplementary figure S1B), promoted both in vitro cell motility (see online supplementary figure S5B) and in vivo tumour metastasis to the liver in the orthotopic SCID mouse model (figure 3D). Although RKO lung metastases were not as overt as those with HCT116 cells, by immunostaining with anti-GFP antibodies, we observed a significantly greater number of miR-224-RKO cells disseminated to the lung compared with control RKO cells (figure 3E). The metastatic phenotype observed using two cell models supports the prometastatic function of miR-224 in CRC.
SMAD4 and CDH1 as miR-224 targets
We performed a PCR array comprising 84 metastasis-related genes on SW480 cells. Among the 13 genes that were reduced more than half by miR-224 overexpression (see online supplementary table S3), only CDH1 and SMAD4 were predicted miR-224 targets by the miRGEN database (http://diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi) (figure 4A). Overexpression of miR-224 decreased the mRNA and protein expression of CDH1 and SMAD4, both upon transient and stable miRNA transduction (see online supplementary figure S6 and figure 4B, respectively). Inversely, knockdown of miR-224 in HCT116 and AAC1/82 increased SMAD4 protein expression (figure 4C). Next, we generated luciferase constructs containing SMAD4 and CDH1 3′ UTR sequences (pGL3-CDH1, pGL3-SMAD4 constructs A and B, and their respective miR-224 target mutants).14 Cotransfection with synthetic miR-224 precursor reduced the luciferase activity of pGL3-CDH1, but not the mutant construct with the predicted interaction sequence deleted (figure 4D). The reporter activity of pGL3-SMAD4 construct A, which was reduced to half by miR-224 overexpression, reverted after deletion of binding site 1 but not of binding site 2 (figure 4D). On the contrary, pGL3-SMAD4 construct B containing binding site 3 was not affected by miR-224 (figure 4D). Independent experiments with point mutants of the binding sites consistently showed that binding site 1 is the interaction site for miR-224 action (see online supplementary figure S7). As a further proof, in the HCT116 cells transfected with GFP-miR construct, we observed a mutually exclusive expression pattern of GFP (indicating miR-224 expression) and SMAD4 (figure 4E).
Inverse correlation of miR-224 and SMAD4 in clinical samples
We moved on to measure SMAD4 and CDH1 protein levels in stages I and II CRC samples with low miR-224 levels and in stage IV samples with high miR-224 levels selected from the Italian CRC set 1. Consistent with in vitro findings, we detected an inverse correlation between miR-224 levels and SMAD4 protein expression (figure 5A). To exclude the possibility that SMAD4 expression arises from tissues other than the epithelial cells, we performed laser capture microdissection in normal colon and CRC tissues and observed a reciprocal expression of miR-224 and SMAD4 mRNA (figure 5B). However, we did not find an inverse correlation between CDH1 and miR-224 (data not shown). Similarly, in the TCGA consortium data (n=133), we found an inverse correlation between miR-224 and SMAD4 (Spearman Rs=−0.44, p<0.0001), but not with CDH1 (Spearman Rs=0.32, p=0.0002) (see online supplementary figure S8).
SMAD4 as effector of miR-224 in promoting cell motility
We next investigated whether SMAD4 is the mediator of miR-224's prometastatic effect. Similar to what occurred after miR-224 transfection, silencing of SMAD4 increased HCT116 cell motility (figure 5C). Moreover, exogenous SMAD4 expression with a miR-224-resistant SMAD4 construct (SMAD4 without its 3′ UTR containing the putative miR-224 target site) abrogated miR-224's ability to promote cell migration and invasion (figure 5D). Taken together, the phenocopy and the rescue experiment support the hypothesis that SMAD4 is a key effector of miR-224's prometastatic capacity.
Association of miR-224 with survival of patients with CRC
To determine the clinical significance, we performed patient survival analysis in five CRC cohorts with available follow-up information (table 1). In all the cohorts, patients with high miR-224 expression had shorter overall survival compared with those with low miR-224 levels with p=0.0259 in TCGA dataset (n=143), p=0.0137 in Italy set 1 (n=54), p=0.0207 in Italy set 2 (n=68), p=0.0181 in UK set (n=41) and p=0.0331 in Romania set (n=38) (figure 6A–E and table 3). Multivariate analysis showed a trend of correlation but did not reach statistical significance, possibly due to small group size after stage separation. To examine if miR-224 association with survival depends on stage, we used an Austrian sample cohort comprising 74 colon tumours, with majority of them in stages III and IV. In the multivariate analysis, miR-224 showed prognostic value on overall survival (HR 2.36, 95% CI 1.32 to 4.21; p=0.0037) independent of tumour stage (figure 6F and table 3). In addition, in the TCGA dataset, combined analysis of miR-224 with SMAD4 expression increased the separation of the survival curves obtained by either gene alone, and patients with miR-224 (high)/SMAD4 (low) had shorter survival compared with those with miR-224 (low)/SMAD4 (high) (HR 4.12, 95% CI 1.1 to 15.41; p=0.0175) (see figure 6G and online supplementary figure S9). Interestingly, although we did not observe an inverse correlation for miR-224 and CDH expression, and CDH1 alone did not predict patient survival, combined analysis with CDH1 greatly improved the prediction power of miR-224 for patient overall survival (p=0.0009) (see online supplementary figure S10). Furthermore, in the UK set (where complete clinical information was available), patients with high miR-224 expression in primary CRC had shorter metastasis-free survival than those with low miR-224 expression (HR 6.51, 95% CI 1.97 to 21.51; p=0.0008) (figure 6H). Notably, miR-224 showed higher sensitivity and specificity (area under the curve=0.739) for metastasis-free survival than for overall survival in the receiver-operating characteristic analysis (see online supplementary figure S11).
The role of miR-224 during CRC initiation and progression remains controversial:15–21 miR-224 is overexpressed in IBD-associated CRC,21 it promotes CRC tumour growth in mice by repressing PHLPP1 and PHLPP2,19 and ectopic miR-224 expression decreases the chemoradiosensitivity of CRC,22 all of which suggest an oncogenic function. Yet, reports that methotrexate-resistant colon cancer cells express lower miR-22423 and that miR-224 suppresses tumour growth and metastasis in vivo,20 suggest that an opposite role in CRC may be true. The latter study also showed lower miR-224 expression in metastatic CRC cell lines versus non-metastatic cell lines and in metastatic tumours in the lung versus primary CRC tumours.20
In the current study, we analysed miR-224 expression in primary tumour samples, and both our in vitro and in vivo data strongly support a prometastatic rather than antimetastatic function of miR-224 in CRC. This prometastatic influence is also reflected in outcome data from multiple international patient cohorts, which demonstrated that elevated miR-224 expression is associated with advanced disease stages, and impaired survival in CRC in a manner consistent with previous reports.19
Interestingly, among the clinical parameters we have analysed (including tumour stage and tumour site), microsatellite status was the most significant factor associated with miR-224 expression in the 85 Italian CRC samples and the 143 TCGA dataset cases. MSS CRCs, which account for 80% of all CRC cases, are characterised by their unstable chromosomal status, and an aggressive clinical course with early metastasis and poor prognosis.24 Although a previous report identified that miR-224 expression in CRCs with proficient DNA mismatch repair is more than twice that of CRC with defective DNA mismatch repair,25 this association between miR-224 expression and MSS status has not previously been examined in depth. MSS is identified by the absence of multiple MSI marks, and because no direct test for MSS status currently exists, quantitating miR-224 expression in CRC specimen may offer a promising avenue for the development of future diagnostic applications.
SMAD4 protein is a transcription factor required for synergistic transcriptional activity in response to transforming growth factor-β (TGF-β). Loss of SMAD4 in CRC has been shown to switch TGF-β function from tumour suppression to the promotion of tumorigenicity and metastasis.26 Furthermore, loss of SMAD4 function due to 18q genomic deletion, or protein inactivation or suppression, has been associated with advancing tumour stage and metastatic status in CRC.27 ,28 However, CRC tumours with or without 18q21 allelic imbalance showed no difference in SMAD4 levels, suggesting that additional mechanisms of SMAD4 regulation may also apply.29 Furthermore, while 18q21 allelic imbalance and SMAD4 mutations did not perform well as prognostic markers, the absolute level of SMAD4 protein expression was found to have prognostic utility in CRC.29
SMAD4 has been identified as a miR-224 target in several disease models17 ,30 ,31 including CRC.18 Here we provide multiply validated evidence that SMAD4 is a miR-224 target using in-silico target prediction analysis, overexpression and knockdown cell models, luciferase assays and expression correlation in clinical samples. Furthermore, we have established that the prometastatic activity of miR-224 is, in part, mediated by SMAD4 using phenocopy and rescue experiments. We also identified CDH1 as a miR-224 target, by target prediction, qRT-PCR, western blot and luciferase assay; however, CDH1 mRNA shows positive, instead of inverse, correlation with miR-224 levels in clinical samples. Despite this discrepancy, high miR-224 and low CDH1 show a strikingly better prediction for overall survival of patients with CRC than either gene alone, suggesting that miR-224-CDH1 regulation may also play a role in CRC pathogenesis.
We are aware of the limitations of the present study. First, the PCR array we used for screening does not cover all the possible miR-224 targets. It is possible that miR-224 promotes metastasis by mediating multiple targets rather than SMAD4 alone. Second, although we used multiple independent sets of CRC samples, larger cohorts are required to validate the association between miR-224 expression and patient survival, and the prognostic advantage of combining the expression levels of miR-224 and its mRNA targets. Third, survival analysis in the current study may be confounded by the treatment received by patients. In future retrospective and prospective biomarker analysis, the potential impact of therapy on outcome should also be considered.
Despite these limitations, our study has a number of potential implications for clinical practice. First, the association between miR-224 and MSS status suggests that there may be potential for the development of a specific diagnostic marker based on miR-224 expression. Second, miR-224 expression alone or in combination with its target genes (SMAD4, CDH1 or possibly other targets) may serve as prognostic marker in CRC. The recent finding that patients with CRC with MSS status and loss of SMAD4 expression had significantly worse survival32 highlights the promise of this approach. Third, high miR-224 expression in advanced CRCs and its prometastatic consequences suggest that this miRNA could be an ideal candidate for targeted therapeutic interventions, particularly in tumours that express low levels of SMAD4. The recent success of miravirsen (an locked nucleic acid (LNA) anti-miR-122) in a clinical trial for treating HCV infection33 indicates technical plausibility of anti-miR-224 treatment.
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online supplement
HL, KP and CI contributed equally.
AHM, GAC and MSN made equal contributions to this work.
Contributors AM, GAC and MSN: designed the study; MSN, HL, KP, MI, RS, MB, CB, VP, MIA, KV, VS, AH, XZ, KL, KB, JHS, SK, PZ-M and IB-N: performed the wet-lab experiments; JP, GP, RG, IV, MP, GH, FF, MF, AI, CIo, GL, SRH, IB-N and AM: obtained samples and clinical data; CIv, RM, LX, HL and XW: performed statistical analysis; CIv, CIs and EM: performed TCGA analysis; HL, KP, GAC, AM and MSN: performed data analysis and interpretation; HL, KP, MI and MSN: wrote the initial draft; HL, GAC, MSN, AM, MB, C. Isella and EM: revised the report. All authors reviewed this report and approved the final version.
Funding GAC is The Alan M. Gewirtz Leukemia & Lymphoma Society Scholar. Work in GAC laboratory is supported in part by the NIH/NCI grants 1UH2TR00943-01 and 1 R01 CA182905-01, the UT MD Anderson Cancer Center SPORE in Melanoma grant from NCI (P50 CA093459), Aim at Melanoma Foundation and the Miriam and Jim Mulva research funds, the Brain SPORE (2P50CA127001), the Center for radiation Oncology Research Project, the Center for Cancer Epigenetics Pilot project, a 2014 Knowledge GAP MDACC grant, a CLL Moonshot pilot project, the UT MD Anderson Cancer Center Duncan Family Institute for Cancer Prevention and Risk Assessment, an SINF grant in colon cancer, the Laura and John Arnold Foundation, the RGK Foundation and the Estate of C. G. Johnson, Jr,. HL is an Odyssey fellow, and his work is supported in part by the Odyssey Program at The University of Texas MD Anderson Cancer Center. MP is supported by an Erwin-Schroedinger Scholarship of the Austrian Science Funds (project no. J3389-B23). MIA is supported as a fellow by the Foundation for Science and Technology, Portugal (SFRH/BPD/91011/2012). SRH is the recipient of the Frederick F. Becker Distinguished University Chair in Cancer Research from The University of Texas. Work in MF's laboratory is supported in part by award number P30CA014089 from the National Cancer Institute. Work in EM's laboratory is supported in part by funds from AIRC (IG n. 12944 and 5×1000 project n. 9970), FPRC and Regione Piemonte (E-LAB). CI is a University of Torino fellow and is supported in part by Regione Piemonte (E-LAB). KP and AM are supported by grant funding from Wessex Medical Research and Cancer Research UK (C28503/A10013). Work in MSN's laboratory is supported in part by funds from AIRC (MFAG n. 13589), 5×1000 to CRO and Marie Curie Actions (CIG n. 303877).
Competing interests None.
Patient consent Obtained.
Ethics approval University of Ferrara, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) s.r.l., University of Southampton, the Oncology Institute Cluj-Napoca, Medical University of Graz, and MD Anderson Cancer Center.
Provenance and peer review Not commissioned; externally peer reviewed.
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.