MERMAID newsletter – summer 2019

 

At the MERMAID Project we would like to wish you a pleasant summer and provide you an update of the development of the project.

MERMAID III is the MERMAID Project’s largest research project to date with a budget of DKK 40 mio. which DKK 35 mio. has been received in funds and commitments.

 

Evaluation

The research in MERMAID III is every year evaluated by a independent scientific group, The Independent Audit Committee (IAC). The members of the group are

Dr Ranjit Manchanda MD, MRCOG, PhD, Clinical Senior Lecturer, Consultant Gynaecological Oncologist, Barts Cancer Institute, Queen Mary University of London, UK
Prof Ian Jacobs MD, FRCOG, Vice Chancellor, University of New South Wales, Sydney, Australia
Dr Christer Borgfeldt MD, Phd, Professor Department of Obstetrics and Gynecology, Ska?ne University Hospital, Lund University, Sweden.

IAC write in their summary for the research in 2018:

This is to certify that following review of outputs from the three Work-streams the committee is happy to recommend continued funding of the Mermaid Programme.
While there have been challenges along the way, the productivity of the overall programme remains good. We expect investigators address some of the issues highlighted and hope outputs will increase over the next year as they continue with planned implementation of ongoing projects.

(On behalf of the Audit Committee)

Dr Ranjit Manchanda MD, MRCOG, PhD 

 

The research 
The goal of the research is to identify one or more methods of diagnosing ovarian cancer at an early stage.
Only around 40% of women diagnosed survive. However, 90% of women diagnosed at an early stage survive, whereas a mere 5-10% survives when diagnosed at late stage. Hypothetically, via identifying all women at the earliest stage, the survival rate could increase to 90%.

The research in MERMAID III is divided into three sub-projects.

It is  coordinated by professor Bent Ottesen, Project director Rigshospitalet, Copenhagen University Hospital and is carried out by three professors: Susanne Krüger Kjær, Claus Høgdall and Jan Blaakær, all very dedicated and with great competence within gynecological research.

In the following the professors of the projects describe the development in the research.

Early detection and long-term survival
Head of research: Professor Susanne Krüger Kjær, The Dansih Cancer Society (Kræftens Bekæmpelse)/Copenhagen University Hospital, DK.

Risk of ovarian cancer among women diagnosed with ovarian borderline tumor
Whether subsequent serous carcinoma in women with a prior serous borderline tumor (SBT) represents malignant progression or an independent primary tumor is unclear, and the combined clinicopathologic and molecular features of SBTs and their subsequent carcinomas have not been fully characterized. This is important to know, as it will potentially affect the follow-up strategy of women with SBT. We have previously identified all women with an ovarian serous borderline tumor diagnosis in the nationwide Danish Pathology Data Bank and/or the national Danish Cancer Registry during 1978–2002.

In this study, we analyzed a cohort of 42 women initially diagnosed with SBT who subsequently developed serous carcinoma, out of a total of 1,025 cases of ovarian SBT from a nationwide population-based cohort. Review of the diagnostic slides was performed from this subset of SBTs and matched metachronous invasive serous carcinomas. DNA was extracted from tissue blocks available for both SBT and carcinoma. We found that more than 90% of the paired samples had identical mutation profiles. These findings point to a tumor progression relationship between the borderline tumor and their subsequent carcinomas. As part of a more in-depth molecular study, whole exome sequencing (a genomic technique for sequencing all of the protein-coding region of genes in a genome) will be performed to further address this clonality issue. This will be performed in collaboration with Johns Hopkins Hospital og State Key Lab of Molecular Oncology, National Clinical Research Center for Cancer, Chinese Academy of Medical Sciences and Peking Union Medical College; Collaborative Innovation Center for Cancer Medicine, Beijing.

The results have been presented among others at a seminar at Johns Hopkins Medical Center:
(Kjaer SK, Hannibal CG, Frederiksen K). Serous borderline tumors. Seminar, Johns Hopkins Medical Center, Baltimore, USA, Sept. 2018, and below are elisted some examples of publications from this project.
https://www.ncbi.nlm.nih.gov/pubmed/28873354

https://www.ncbi.nlm.nih.gov/pubmed/28248817

https://www.ncbi.nlm.nih.gov/pubmed/25188864

 

Early detection of ovarian cancer
In a previous part of the study funded by MERMAID III, the researchers have shown that it was possible to identify up to 63% of women with ovarian cancer in an early stage by testing for mutations in cervical samples obtained at routine screening against cervical cancer.

These promising results were obtained in a population of 150 women with ovarian cancer. Since then, we have worked on improving the test together with our collaborators at Johns Hopkins Hospital in Baltimore. It is important that we now test whether these results are also applicable to other study populations in order to determine whether this test can be used in a screening setting.

Along these lines, the researchers have been working to establish a biobank containing cervical cytology samples. The biobank is established at Dept. of Clinical Pathology, Vejle Hospital, which covers the entire region of Southern Denmark. The researchers have currently collected cervical samples from more than 110,000 women. When the examination for cervical cytological abnormalities has been performed, the residual of the cell material is transferred to the biobank using a biobank robot developed especially for this project. Using the nationwide Danish registers, the researchers can identify women who subsequently develop ovarian cancer, and the cell samples from these women can then be retrieved from the biobank and tested for the selected mutations.

Currently, the results have been presented at both national and international meetings and conferences ( e.g.

  • Ørnskov D, Waldstrøm M, Thomsen LT, Munk C, Kjaer SK. A Danish Clinical Cervical Cytology Biobank. Pilot study of samples processing and quality. 32nd International Papillomavirus Conference in Sydney, Australia, Oktober 2018. 
  • Kjær SK. Early detection of ovarian cancer. DCRC Clinical Seminar. Gynecologic cancers. Copenhagen, Denmark, October 31, 2018.
  • Kjaer SK. Gynecologic cancer – is there light at the end of the tunnel ? TeLinde Seminar, Johns Hopkins Medical Center, Baltimore, USA, Sept. 2018

and in the scientific publication below

https://www.ncbi.nlm.nih.gov/pubmed/29563323

 

The project Biomarkers and / or prognostic markers 
Head of research:  Professor Claus Høgdall, Copenhagen University Hospital, Rigshospitalet,DK

For this part of the MERMAID research, blood and tissue from women with ovarian cancer are collected in order to identify biochemical and molecular biologic markers that characterize the disease.

A biomarker or a combination of biomarkers may detect cancer in the early stage so that the patient can be cured, or, even better, identify patients at high risk of developing ovarian cancer and thus prevent the development of the cancer by prophylactic treatments. Biomarkers can also contribute to predicting treatment effects, and thus provide the basis for patients to be offered a personalized and more effective treatment such as biological treatment.

 

The project is divided into 3 sub-studies (microRNA and messengerRNA, methylation and sequencing) including a large number of planned analyses. All studies progress according to the schedule.
The first sub-study investigates microRNAs, which are small molecules that play a very important role in regulating the genes in the cell. This work has resulted in 4 scientific articles published (2016, two articles in 2017, 2018) and 1 article submitted. In addition, during Spring 2019 the results were presented at two annual meetings, as an oral presentation at Dansk Gynækologisk Cancer Gruppe (DGCG) and as a poster at Dansk Patologisk Selskab (DPAS).Laboratory analysis of all known messengerRNAs (mRNA), which is a type of RNA that gets translated into protein, was completed in 2018 and the first scientific article is under preparation. 3 additional scientific articles about mRNA will be written.

The microRNA studies have formed the basis of a Ph.D. dissertation by doctor Kira Prahm, who has been a MERMAID Ph.D. student for the last 3.5 years. The Ph.D. thesis was successfully defended on May 24th, 2019. The microRNA studies in blood and mRNA studies in tissue are now continued by molecular biologist postdoc Douglas Oliveira, employed in November 2018. The analyses have been carried out and the first article (microRNA in blood) was submitted for publication in March 2019. Additionally, the project was presented at the DPAS annual meeting and were awarded “Årets forskerpris” (Researcher of the Year).

Sub-study 2 investigates DNA methylation, also known as epigenetics, that is important for the regulation of genes. The project aims to find DNA methylation markers both for the prediction of chemotherapy resistance as well as for diagnostics and screening. Molecular biologist postdoc Julie Lilith Hentze is responsible for this sub-study. Julie has already published the first MERMAID article (2017) and has recently published a second article about methylation (March 2019). Julie is currently registering patient material and prepares DNA for the planned methylation analyzes.  The methylation study comprises three work packages that will contribute significantly to the knowledge of epigenetics in ovarian cancer. Preliminary results were presented at DGCG’s annual meetings in 2018 and 2019. The sub-study thus proceeds positively according to schedule.

 

 

For the third sub-study, sequencing, the overall goal is to find changes (variants/mutations) in the DNA genetic code, that can explain etiology and/or prognosis, and potentially form a basis for personalized treatment decisions. Molecular biologist postdoc Mikael Kronborg was appointed in April 2018 to lead this sub-study. The patient cohort for these comprehensive studies has been formed and biological material acquired from Bio- and Genome Bank Denmark. DNA sequencing was completed in February 2019. The samples are thoroughly investigated, as both known and unknown variants/mutations need to be described, and the newest techniques in the field, so-called Next-Generation Sequencing, are employed. Data analyses are ongoing. All discovered variants will be validated in a larger setup to ensure that the results can be incorporated into a potential national screening program. The project was presented as a poster at two annual meetings in the Spring 2019, DPAS and DGCG, and at the latter, it was awarded Best Poster.

In conjunction with planning the project, Mikael reviewed the literature for every known genetic mutation influencing ovarian cancer, which resulted in a systematic review article, which has recently been submitted for publication. This sub-study also proceeds positively according to schedule, and will contribute with large datasets, that can easily be used in the other sub-studies.

In addition to the established sub-studies, this MERMAID III project has resulted in a large number of positive spin-off projects. Briefly mentioned:  A) Annexin and S100, which results from a collaboration with a research group in Adelaide, Australia. An article was recently published (May 2019). B) PD-L1, which is an important cancer biomarker and a target for immunotherapy. The results have been presented at scientific congresses and an article has been written, which is currently under review by co-authors, and will be submitted for publication soon. It will be one of the biggest studies of PD-L1 in ovarian cancer. C) P53 autoantibodies and p53 mutations in tumors; a collaboration with Professor R. Bast, from MD Anderson, USA, one of the world’s leading researchers in ovarian cancer markers. The analyses are done and presented, and an article is in preparation. As a direct result three additional antibodies have been optimized and interpretation of the results are ongoing. An article is being prepared covering all findings. D) Organoids; where the project seeks to develop 3-D cell cultures (organoids) from individual patients. The organoids grow as the primary cancerous nodes (thus also called tumoroids), leading to the possibility of predicting the individual effect of both traditional chemotherapy and the newer biologically-based treatments. Currently there is no such test. The first tumoroids have been so promising that the first publication is already under preparation, further studies are planned, and the appointment of a Ph.D. student settled for the Summer of 2019. NGS analyses will be included in the assessment of organoid tumor heterogeneous growth patterns and could have a large impact on planning the posterior treatment of women with ovarian cancer. E) We have investigated RNA stability for some time, as analyses using RNA as starting material can be important in personalized medicine initiatives. Thus, investigations of the stability of RNA in a clinical setting have been performed, and the article detailing this project have just been accepted for publication (May 2019).

 

Publications 2017 – May 2019
In the above period, a total of 29 international articles related to ovarian cancer and with a primary author from this MERMAID sub-study group have been published. During the same period, 12 articles have been published by our international collaboration group Ovarian Cancer Association Consortium (OCAC) with co-authors from this MERMAID sub-study group. Furthermore, with authors from the Mermaid group 6 articles has been published.

 

The Mermaid III research study  ”The Infection Theory” 
Head of research:  Professor Jan Blaakær, University of Southern Denmark (SDU) and the University Hospital, Odense, DK

Our first publication dealing with the infection theory ”High-risk HPV is not associated with epithelial ovarian cancer in a Caucasian population” was published in Infectious Agents and Cancer (2016) 11:39 DOI 10.1186/s13027-016-0087-4.

Actually, we didn’t expect to demonstrate an etiological correlation between HPV and ovarian cancer, but knowing that HPV vira are causing cancers in the cervix, vagina, vulva, anus, tonsils, and tongue base, we simply had to be sure that no correlation existed between ovarian cancer and HPV infection.

The following paper dealing with the infection theory has been published:
“The potential role of infectious agents and pelvic inflammatory disease in ovarian Carcinogenesis” in Infectious Agents and Cancer (2017) 12:25 DOI 10.1186/s13027-017-0134-9.In this publication we reviewed the literature and became convinced that we had to continue our search for a possible infectious agent in the etiology of ovarian cancer.

Epstein-Barr Virus 

In the next study, which has been in Infectious Agents and Cancer, we examined the possible role of Cytomegalo-virus (CMV) and Epstein-Barr Virus (EBV) in ovarian carcinogenesis . We didn’t demonstrate a correlation to CMV but we found EBV in 5% of the ovarian cancer cases. We

compared this observation to a healthy control group (women with a benign tumor in the pelvis) and found a significant difference, indicating that EBV might influence the development of some ovarian cancer cases. This is a very interesting observation as this is the first study to demonstrate EBV in ovarian cancer tissue samples. Moreover, EBV infects epithelial cells and is an established etiological factor in the development of nasopharyngeal and gastric carcinomas. It is also implicated in a range of B- and T- celllymphoproliferative disorders including Burkitt’s lymphoma and angioimmuno-blastic T-cell lymphoma.
Ovarian cancer is made up of different histological subtypes and EBV might be an etiological factor in one or more of these subtypes

Next generation sequencing 

The next step in our research project in the infection theory project was to apply next generation sequencing (NGS) technique to ovarian cancer. We have now finished the 16S ribosomal RNA (rRNA) sequencing of all the ovarian cancer specimens and have generated an enormous quantity of data. These data have to be processed, but the most important thing is to co-operate with a data manager expert (bacterial taxonomy) being familiar with processing this type of dataset.  This data processing will be performed as the next step in the NGS study.

Professor Robert Kurman, John Hopkins Hospital, Baltimore published a possible change in the Fallopian tubes named as ‘carcinoma in situ’ (STIC) and corresponding to the changes preceding cervical cancer (Kurman  & Shih ’The Origin and Pathogenesis of Epithelial Ovarian Cancer: A Proposed Unifying Theory’, Am J Surg Pathol 2010;34:433–443).

In the light of these observations we have decided to examine Danish women with STIC lesions applying our results from the infection theory so far. We have identified the ‘STIC-women’ in the national patient registry and permissions from relevant authorities  have been obtained. We await the results from the NGS study before initiating the STIC study in order to qualify our search for microbiological DNA.

 

Publications and evaluation

During the year a number of research articles has been published by all sub-projects.
Access to the publications and to the eavaluation report is available upon request to Birgitte Blix Treschow, project coordinator, (blix@cancer.dk).