European Collaboration

European Collaboration

The European collaboration we are funding, jointly with the EHE Foundation, includes the Istituto Nazionale dei Tumori (INT) in Milan, Italy; the Institute of Cancer Research (UK); and the Royal Marsden Hospital, London

The overall research program entitled “Evaluation of Cytokines and Hormones as Biomarkers for EHE” is a multi-center collaborative project that comprises a number of key objectives which at a high level include the identification of novel prognostic and predictive markers; to better describe the natural history of the disease and its radiologic features; to collect prospective data on quality of life and the activity of systemic agents administered in EHE; as well as to comparatively assess the activity of drugs in newly generated preclinical models of EHE directly obtained from patients’ tumor specimens.

The different objectives or projects, can be summarised as follows:

  1. The identification and validation of novel circulating and tissue biomarkers to inform patient management (prognosticators and predictors of response to medical agents) as well as potential therapeutic targets (Project 1);
  2. The development of patient-derived xenograft (PDX) models (Project 2);
  3. The assessment of the activity of drugs relevant for the disease (Project 3);
  4. The generation of PDX-derived cell lines to investigate the cellular and molecular determinants of drug activity and to be submitted to a CRISPR whole genome screen to identify new therapeutic targets (Project 4);
  5. The dentification and evaluation of miRNAs (Project 5); and
  6. The description of the radiologic characteristics of EHE and their correlation with the clinical outcome (Project 6).

Updates of these different projects are provided below.

Project 1
: The assessment of circulating cytokines, hormones (and miRNAs), and ERα, Erβ and GPER expression etc and the identification of a novel biomarker for EHE;

Using a protein array able to simultaneously detect the expression of a hundred different cytokines in plasma samples of EHE patients (n=15) and healthy individuals (n=6), a small panel of inflammatory cytokines was found to be differentially expressed. Among them the research team focused on Growth and Differentiation Factor-15 (GDF-15), a member of the TGF-β super-family, which plays multiple roles in a wide variety of cellular processes. Using a specific ELISA assay, they looked at the concentration of circulating GDF-15 in a retrospective series of 23 EHE patients and observed a statistically significant association of GDF-15 levels with EHE aggressiveness. This result was confirmed in a second cohort of 21 EHE patients prospectively collected within the currently ongoing observational study.

The assessment of circulating hormones and tissue expression of ERα, Erβ and GPER is ongoing.

The assessment of miRNAs is undertaken in Project 5.

Project 2
: Development of additional PDX models;

Thus far the team have only one fully established EHE PDX model, whose ability to reproduce the characteristics of the originating clinical tumor has been confirmed in terms of histo-morphology, presence of the WWTR(TAZ)::CAMTA1, overall transcriptomic profile (as detected by RNA-seq) and presence of CDKN2A homozygous deletion. A cell line was established following disaggregation of the EHE PDX.

Project 3
: Assessment of activity of drugs;

The PDX model was used to comparatively assess the activity of doxorubicin and sirolimus (at different doses). Doxorubicin showed almost negligible activity while sirolimus caused a dose-dependent tumor volume inhibition in treated mice and induced the down-regulation of mTOR signaling. The PDX is currently being used to assess the activity of inhibitors of the Hippo pathway, such as the TEAD family of transcription factors.

This PDX and the corresponding cell line were exploited to provide further evidence supporting the value of GDF-15 in EHE. GDF-15 was detected in the medium of the EHE cell line as well as in the blood of EHE PDX but not in healthy mice or in mice carrying another sarcoma type, confirming that the cytokine was released by the EHE. Interestingly, the team also found that sirolimus decreased the abundance of GDF-15 in both their in vitro and in vivo EHE models.

Project 4
: Use of CRISPR in cell lines to help identify genes that confer drug resistance or sensitivity;

The EHE cell lines will soon be provided to Dr. Paul Huang (ICR, London) to start the CRISPR-based experiments.

Project 5
: Identification and evaluation of miRNAs

The expression profiling of plasmatic miRNAs has been initially carried out using the OpenArray Technology (which evaluate the expression of 754 different miRNAs) in the retrospective series of 23 EHE patients.  Six miRNAs were found differentially expressed between patients with indolent and aggressive EHE. In order to evaluate the overall miRNome (global profile of expressed miRNAs), we repeated the analysis on both retrospective and prospective series of patients by miRNA-seq. The results are currently being analyzed by a dedicated bioinformatician.

Project 6
: Establishment and assessment of a radiological data base.

The clinically-annotated radiologic scan collection on the study platform has been started. Response assessment is also collected by REDcap CRFs. The data analysis is planned after the end of the study.

Overall the project is progressing well.

  • The study is currently open to enrolment in Milan and in London. Clinical and radiologic data, blood samples and tumor specimens have all been collected from participants.
  • A circulating cytokine significantly associated to disease aggressiveness has been identified and the results validated in an independent patients’ cohort
  • Using the newly generated preclinical models of EHE the research team have demonstrated the superior antitumor activity of sirolimus compared to doxorubicin.
  • The search for additional prognostic/predictive biomarkers is ongoing on tissue and blood samples.

The research team remain excited and believe that data collected within this study will provide them with: 1) a better picture of the disease to inform the decision on how to treat patients with EHE and to define the prognosis based on disease presentation; and 2) preclinical and clinical data to identify new treatment options and design better prospective clinical studies.

Dr Silvia Stacchiotti and Dr Nadia Zaffaroni are joint PI’s of the research taking place at INT, while Dr Paul Huang is PI for the ICR, London. Dr Sandro Pasquali is also a key member of the INT research team.

Dr S. Stacchiotti
Nadia Zaffaroni
Dr N. Zaffaroni
Sandro Pasquali
Dr S. Pasquali
Dr P. Huang