Newsletter march 2021

Dear donors and friends of MERMAID

The research in MERMAID III, which began in mid-2015, has entered its final year. In recent years, researchers have achieved important sub-goals in their work on finding a method or methods for diagnosing ovarian cancer at an early stage. According to the researchers, the research has largely gone according to plan, despite the fact that the challenges due to the COVID 19 pandemic in 2020 which has partially affected the research work.

It was most gratifying when one of the leading researchers in the MERMAID Project, Professor Susanne Krüger Kjær, in the autumn received the prestigious award, the KFJ Award, one of Denmark’s largest medical awards, for her important and groundbreaking research into HPV and uterine cancer, which was the research she spearheaded in the MERMAID II project. 

The research of MERMAID III was initially divided into three sub-projects that has been researched from different angles to find a way to early diagnosis of ovarian cancer.

In two of the research projects, led by Susanne Krüger Kjær (MIIIS) and Claus and Estrid Høgdall (MIIIC), respectively, a good and constructive synergy has been shown in the research process. 

It has therefore been decided to start a collaboration. The focus of the thesis is that a simple cell scrape from the uterus can be used for early detection of ovarian cancer.

The starting point is that the first research results have shown that so-called DNA methylation

markers have potential as biomarkers for ovarian cancer. DNA methylation markers have been shown to distinguish between benign tumors and ovarian cancer. The purpose of the research is to enable early diagnosis with DNA methylation analysis and thereby contribute to increased survival for patients with ovarian cancer.

Based on figures from the Danish Gynecological Cancer Database, early detection can increase the average survival from 40-45% to be up to 90% in respect of patients with ovarian cancer.

The combined research in the two research projects is described in more detail in the following text, where the research leaders explain their respective research, which is expected to be completed by the end of 2021.

The third sub-project, “The infection theory”, led by Jan Blaakær, was completed at the turn of the year. It was not possible to prove that ovarian cancer could be caused by a bacteria or virus as the thesis in the project was. A report on the results of Jan Blaakær’s research will be published when the overall research is completed, and a scientific report on all research results will be published.

Yours sincerely

Birgitte Blix Treschow
Adm. Project coordinator

The MERMAID Project

Executive Summary – MERMAID IIIS

Ovarian cancer – Causes, Early diagnosis, and Long-term survival 

Ovarian cancer is the fourth most common cause of death due to cancer among women in Denmark. Each year around 500 new cases of ovarian cancer are diagnosed and currently more than 4,500 Danish women are living with the disease. Ovarian cancer is the most lethal among the gynecological cancers. This is due to the fact that ovarian cancer is often diagnosed in an advanced stage where the disease has spread beyond the ovaries and because recurrence is common. The prognosis is highly dependent on the stage of disease at diagnosis. Ovarian cancer is a heterogeneous group of cancers with variations in etiology and prognosis. 

Causes of ovarian cancer

On this background, the aim of this part of the Mermaid IIIS project is to identify modifiable causes of ovarian cancer in order to decrease the incidence of the disease. For this part of the project, Danish data will be used in collaboration with international top researchers and their data in order to gain as much data and statistical power as possible and thereby a greater opportunity to identify important associations. It is well known that the number of childbirths decreases the risk of ovarian cancer. Based on one of the largest datasets in the world within this research area, including more than 10,000 women with ovarian cancer and 16,000 healthy women, we have recently found and reported that even pregnancies lasting for a shorter time period cause a statistically significantly decreased risk of ovarian cancer of 16%. That the risk decreased further with increasing number of incomplete pregnancies. Another interesting finding is that breast-feeding is also associated with a statistically significantly decreased risk of ovarian cancer, particularly the type of ovarian cancer with the worst prognosis (high-grade serous ovarian cancer). It could also be mentioned that we have recently found that some types of anti-depressive medication decrease the risk of ovarian cancer. The abovementioned examples are important for our understanding of the mechanisms of this cancer. 

Early diagnosis

In another part of the Mermaid IIIS project, we are collecting cellular material from the cervix from women who are participating in the routine screening program against cervical cancer in Denmark. This is done in order to obtain biological material as a source of DNA. Subsequently, this material will be analyzed for biomarkers among women who develop ovarian cancer to create the possibility to diagnose the disease as early as possible. By the end of 2020, we reached our goal of collecting cervical samples from 150,000 Danish women. This forms the basis of a potential collaboration with researchers in the Mermaid IIIC project.

Finally, we are working together with researchers from Johns Hopkins University, USA, and Odense University Hospital where we are collecting both cervical cell samples and blood samples from women who are suspected to have ovarian cancer in order to test whether we can identify mutations in both types of samples that can increase the sensitivity of a specific test.

Long-term survival after ovarian cancer 

Another aim is to examine factors influencing survival after a diagnosis of ovarian cancer, i.e. are there factors that can help us identify those women with long-term survival after ovarian cancer, and can this be used in our choice of treatment or can survival be prolonged by modifying these factors? The study is based on nationwide Danish data and collection of tissue from women with ovarian cancer. The factors that will be examined are, among others, reproductive factors (e.g. number of births, age at first/last childbirth), socioeconomic factors (e.g. education, disposable income), comorbidity (e.g. infertility, heart disease, diabetes), and use of different kinds of daily medication. 

Publications:

Lee A, Rosenzweig S, Wiensch A, Ramus SJ, Menon U, Gentry-Maharaj A, Ziogas A, Anton-Culver H, Whittemore A, Sieh W, Rothstein J, McGuire V, Wentzensen N, Bandera E, Qin B, Terry KL, Cramer D, Titus L, Schildkraut J, Berchuck A, Goode E, Kjaer SK, Jensen A, Jordan S, Ness R, Modugno F, Moysich K, Thompson P, Goodman M, Carney M, Chang-Claude J, Rossing M, Harris H, Doherty J, Risch H, Pharoah P, Wu A, Pike M, Webb P, Pearce C. Natl Cancer Inst. 2020 Aug 

Babic A, Sasamoto N, Rosner BA, Tworoger SS, Jordan SJ, Risch HA, Harris HR, Rossing MA, Doherty JA, Fortner RT, Chang-Claude J, Goodman MT, Thompson PJ, Moysich KB, Ness RB, Kjaer SK, Jensen A, Schildkraut JM, Titus LJ, Cramer DW, Bandera EV, Qin B, Sieh W, McGuire V, Sutphen R, Pearce CL, Wu AH, Pike M, Webb PM, Modugno F, Terry KL. JAMA Oncol. 2020 Jun 

Bennetsen AKK, Baandrup L, Aalborg GL, Kjaer SK. Gynecol Oncol. 2020 Jun;157(3):693-699

In preparation:

  • Reproductive factors and the risk of non-epithelial ovarian cancer.
    Hemmingsen C, Kjaer SK, Bennetsen AKK, Dehlendorff C, Baandrup L. Gynecol Oncol. (submitted)
  • Prognostic impact of socioeconomic status on long-term survival of non-localized epithelial ovarian cancer ? the Extreme study.
    Baandrup L, Dehlendorff C, Hertzum-Larsen R, Hannibal CG, Kjaer SK. Gynecol Oncol. (submitted)
  • Cancer and precancer following a diagnosis of non-epithelial ovarian cancer.
    Baandrup L, Faber MT, Dehlendorff C, Hemmingsen C, Hertzum-Larsen R, Kjaer SK. (in preparation)
  • Is the improvement in ovarian cancer survival independent of socio-economic status ?
    Faber MT, Horsbøl TA, Baandrup L, Dalton SO, Kjaer SK. (in preparation)

       

Executive Summary – MERMAD IIIC

Biomarkers and/or prognostic markers

The underlying cause of ovarian cancer is unknown. The mortality rate is very high, since symptoms are unspecific or absent until an advanced disease stage. A decisive breakthrough to improve survival would be early detection and diagnosis. The survival rate of late stage patients is less than 20%, whereas that of patients in the early stages is higher than 90%. In Denmark alone, this would save the lives of more than 200 women annually. Technological progress, especially within the field of molecular biology, provides hope that we can achieve our goal of better survival for these women. 

The MERMAID IIIC project gives the opportunity to address the problem from different angles as specified below. The research is based on clinical data registered in the Danish Gynecologic Cancer Database and material collected in Pelvic Mass and GOVEC studies through Danish Cancer Biobank. 

Figure 1. Understanding the nature of ovarian cancer with powerful molecular biology tools. Main research projects in the MERMAID IIIC-project.

 

Our studies – Analyzes of tissues, cell swabs, blood and peritoneal fluid

Biomarkers are playing an increasingly important role in the clinical treatment of cancer patients and are a prerequisite for the selection of patients for individualized treatment (personalized medicine). Biomarkers can be measured in biological materials, such as tissues, cell swabs, blood and peritoneal fluid. The latest biomarkers are based on our genetic material called DNA. Single- or multi-gene “signatures” (DNA-based biomarkers) have shown promising results that can determine which treatment is optimal for the patient. The exact mapping of the sequence of nucleotides A, G, C and T (the building blocks of DNA) has made it possible to study the genetic arrangement of our cells’ DNA (genomics) on a whole new level. This mapping is better known as sequencing. The sequencing has helped to identify changes (mutations) in DNA in e.g. patients with cancer, which may help to better understand cancer mechanisms and therefore target patient treatment. In addition, there are modifications (epigenomics, methylation) that do not change DNA sequences, but they can change how human cells read a DNA sequence. These modifications are equally informative in providing specific disease signatures. In the MERMAID IIIC project, we examine both types of changes (mutations and methylation) to search for the answers to the following questions:

Methylation: a biological process that controls how DNA is read in human cells and which genes are active. 

Can DNA methylations in cervical cell swabs be used for early detection of ovarian cancer?

Ovarian cancer is characterized by cellular changes. One of these changes is the addition of a small chemical group – called a methyl group (-CH3) to DNA, which can result in the gene being deactivated. This chemical group can be removed through a process called demethylation. Research has shown that DNA methylation has potential as biomarkers for ovarian cancer, as it regulates the genes activity. The aim of the project is to enable early diagnosis with DNA methylation analysis, which may potentially be included in the existing national screening of cervical cell swabs from all Danish women.

 

The goal will be fulfilled by:

1) To perform complete methylation studies in tumor tissue to differentiate patients with ovarian cancer from patients with benign conditions, early in the patient’s disease course.

2) To investigate whether methylation markers, found in tissues, can be found in the cervical cell swabs and used in early diagnosis and potential future screening.

3) To test identified DNA methylation markers in cervical cell swabs in a larger cohort of women, referred to hospital for ovarian cancer surgery. This part of the project will be in collaboration with the researchers in Mermaid IIIS.

Once these goals are reached, we will validate findings in a large number of the cervical cell swabs, collected in conjunction with the National Cervical Cancer Screening Program, to determine a potential use in existing routine ovarian cancer diagnostics. We use a new method to examine all known methylation sites on the DNA strand (approximately 850,000). Our study is the first to perform this analysis on ovarian cancer patients. The current data are based on results from the research in MERMAID III. These promising results show that DNA methylation can distinguish ovarian cancer from benign tumors. 

The candidate biomarkers discovered in this study could potentially be included in the national cervical cancer screening program. The focus of the project is earlier and better diagnosis of ovarian cancer using DNA methylation. Based on the data from the Danish Gynecological Cancer Database, early detection can improve the average survival from approx. 45% to be up to 90% in ovarian cancer patients. In fact, the study has promising results – our preliminary results show 15 potential methylation biomarkers specific for ovarian cancer. We are now in the process of collecting and validating a larger collection of samples.

Sequencing: methods that are used to read the genetic information from a DNA segment

How can we find mutations in the genes that are involved in ovarian cancer?

DNA damage repair genes and oncogenes: In this part of the project, we mainly focus on the mutations of DNA damage repair genes. These mutations cause that cells are not able to properly repair their damaged DNA. Until now, the research has been focused mainly on the changes in BRCA-genes that have been shown to have a significant impact on the treatment of ovarian cancer patients. However, there is a growing evidence that mutations in other genes might also be very relevant for optimal treatment. Therefore, we will investigate all the DNA damage repair genes to understand their significance for ovarian cancer. Moreover, in our project “Molecular markers for individualized ovarian cancer treatment”, we will study the well-known genes associated with cancer (oncogenes) to identify changes that might determine the optimal treatment.

In another project, Identifying biomarkers based on genetic changes in ovarian cancer patients, we will use tumor and ascites samples (fluid from the abdominal cavity) to investigate whether the genetic information from the tumor can be reflected in ascites and blood from the same patients (Figure 2) . We are the first group to study this area, and preliminary results show that we were able to isolate DNA from ascites.

Why does immunotherapy work for some but not all sick women? There are different subtypes of ovarian cancer that have a different genetic profile. One of these subtypes is described as clear cells. Unfortunately, chemotherapy has very little effect on clear cells. We have found that this subtype of ovarian cancer also has different sub-profiles, indicating the possibility of more personal treatment. Different sub-profiles of clear cell ovarian cancer show a very different survival. We are now the first to investigate whether identified genetic profiles can explain these differences in survival, as well as whether we can use them to select patients for effective immunotherapy or other treatment.

miRNA/mRNA

Proteins (molecules that perform key functions in the body) are created based on information from messenger RNA (mRNA), which is formed from a DNA code. Another type of RNA is microRNA (miRNA) that has a regulatory effect on mRNA and thereby the production of proteins. MiRNAs have been shown to have important regulatory functions in connection with cancer development, including ovarian cancer. We investigate the potential of miRNA /mRNA correlations as biomarkers for patient survival, in order to find relationships that are functionally significant. The study of tissues from 197 patients has already shown that several miRNA/mRNA signatures are associated with overall survival, and that some are specific for specific subtypes and the prognosis of ovarian cancer. The results are being published. Validation in a larger cohort will be done subsequently with publication and will provide a basis for specific biological treatment options for patients with ovarian cancer.

Publications

Overview of latest publications (2020/2021)

Published

  • Oliveira DNP, Prahm KP, Christensen IJ, Hansen A, Hogdall C. Noncoding RNA (ncRNA) Profile Association with Patient Outcome in Epithelial Ovarian Cancer Cases. Reproductive Sciences. 2020; Oct. DOI: 10.1007/s43032-020-00372-7. 
  • Estrid HøgdallClaus HøgdallThao VoWei ZhouLingkang HuangMatthew MartonStephen M KeefeMichael Busch-SørensenSarah M SørensenJeanette GeorgsenElse MejlgaardLotte NedergaardTorben Steiniche Impact of PD-L1 and T-cell inflamed gene expression profile on survival in advanced ovarian cancer. Int J Gynecol Cancer. 2020 Jul;30(7):1034-1042. DOI: 10.1136/ijgc-2019-001109.  

Submitted manuscript

  • Oliveira DNP, Prahm KP, Christensen IJ, Hansen A, Hogdall C. Gene expression profile association with poor prognosis in epithelial ovarian cancer patients. 2nd Revision from Scientific Reports.
  • Prahm KP, Høgdall C, Karlsen MA, Christensen IJ, Novotny GW, Høgdall E. MicroRNA characteristics in epithelial ovarian cancer and their predictive value for surgical outcome. Scientific Reports. 
  • Oliveira DNP, Hentze JL, O’Rourke CJ, Andersen JB, Hogdall C, Hogdall C. DNA methylation in ovarian tumors – a comparison between fresh tissue and FFPE samples. 1st Revision from Reproductive Sciences

In preparation

  • Lopacinska-Jørgensen J, Oliveira DNP, Novotny GW, Hogdall C, Hogdall E. Integrated microRNA and mRNA signatures associated with overall survival in epithelial ovarian cancer. 
  • Oliveira DNP, Schnack T, Hogdall C, Hogdall E. Identification of ovarian clear cell carcinoma subgroups by mutational signature might predict patient overall survival.
  • Prahm KP, Hogdall C, Karlsen MA, Christensen IJ, Novotny GW, Knudsen S, et al. Molecular prediction of platinium resistance in patients with epithelial ovarian cancer.
  • Oliveira DNP, Prahm KP, Christensen IJ, Hansen A, Hogdall C, Hogdall E. Predicitive association of mRNA expression for surgical outcome in patient with epithelial ovarian cancer.
  • Hentze JL, Lopacinska-Jørgensen J, Oliveira DNP, O’Rourke CJ, Andersen JB, Hogdall C, Oliveira DNP, Hogdall E. DNA methylation patterns in fresh tumor tissue can differentiate early ovarian cancer from benign tumors.
  • Hentze JL, Lopacinska-Jørgensen J, Oliveira DNP, O’Rourke CJ, Andersen JB, Bonde J, Hogdall C, Hogdall E. Diagnostic potential of DNA Methylation profilling of cervical swabs from ovarian cancer patients.
  • Christophersen MK, Poulsen TS, Oliveira DNP, Hogdall C, Hogdall E. Whole Exome Sequencing of epithelial high-grade serous ovarian cancer and ovarian clear cell carcinoma patients.
  • Christophersen MK, Poulsen TS, Oliveira DNP, Hogdall C, Hogdall E. Investigating genetic variants in DNA damage repair genes in epithelial high-grade serous ovarian cancer and ovarian clear cell carcinoma patients.
  • Christophersen MK, Oliveira DNP, Poulsen TS, Hogdall C, Hogdall E. A comparison study between Ion Torrent Suite and GATK Best Practice Guidelines – similarities and differences between proprietary software and publicly available algorithms.
  • Christophersen MK, Poulsen TS, Oliveira DNP, Hogdall C, Hogdall E. Exome sequencing vs targeted NGS panels – in-depth analysis or focused efforts? 
  • Sisman Y, Schnack T, Antonsen SL, Juhler-Nøttrup T, Harling H, Thastrup O, Høgdall E, Høgdall, C. Establishment of ovarian cancer 3D cultures for individualized treatment.