Research
Research into the mechanisms and possible therapeutics for retinoblastoma not only benefits retinoblastoma children, but has great potential to serve as a basis for all cancers.
“When the final history of cancer research is one day written, retinoblastoma will deserve its own chapter. Studies of this rare childhood tumor have yielded the first widely accepted tumor progression model, the first cloned tumor suppressor gene, and several other firsts in basic cancer research.” (from Journal of the National Cancer Institute, Vol. 96, No. 15, August 4, 2004)
Prof. Dr. med. Vinodh Kakkassery, Chief Physician at Chemnitz Hospital
“The KAKS is: Good. Exciting. Courageous. Perhaps the latter is what makes it special. I see the courage of KAKS to believe in research, to invest in retinoblastoma research. To invest money, time and love. Research and medicine are often far removed from people, alien, incomprehensible. KAKS goes right into that.”
Mission
of KAKS
In contrast to other cancers, there has been little research into molecular biological therapies for retinoblastoma for a long time.
The reason: retinoblastoma is a rare disease and there is no commercial interest.
The Children’s Eye Cancer Foundation has changed this and enables young doctors in particular to carry out research in this field and has now initiated, supported and completed various research projects – each in search of new therapies, diagnostic methods or aftercare and early detection.
All this in cooperation with university hospitals throughout Europe.
PUBLICATIONS
Here you will find an overview of the publications that have emerged from Kaks research.
Gene analysis of retinal organoids
PDF for download
Tumor marker development – Liquid Biopsy – Early diagnosis / EU project
https://www.cell.com/cancer-cell/fulltext/S1535-6108(15)00349-9
https://www.gesundheitsforschung-bmbf.de/de/nirbtest-8466.php
https://www.gesundheitsforschung-bmbf.de/de/era-net-transcan-3413.php
Diagnostic pathway recognition
Analysis of the diagnostic pathway in children with retinoblastoma to improve early detection
Research
projects
We strive to continuously improve the care of retinoblastoma patients in the areas of early detection, therapy and aftercare.
Since 2010, the Children’s Eye Cancer Foundation has initiated and funded numerous research projects in cooperation with renowned research institutions.
Here we present current projects and the results of previous projects.
Origin of RB: Single-cell RNA sequencing
Prof. Bornfeld, Prof. Lohmann
KAKS is involved in funding this DKS study
The aim is to optimize long-term care and improve the long-term prognosis and quality of life.
To this end, the long-term therapeutic consequences of the various forms of treatment were determined and the individual genetic disposition was also taken into account with regard to the risk of secondary tumors.
In addition, adult patients and parents were informed about preventive measures.
Children and adolescents were included in the first phase and adult patients in the second phase (collective of approx. 570 test subjects).
Status: The studies have been completed and the results are available.
The results of the first phases have since been published (Temming et al. 2015, Pediatric Blood Cancer)
Funded by KAKS and the German Childhood Cancer Foundation.
University Hospital Münster – PD Dr. Kornelius Kerl Group
If retinoblastoma is diagnosed early, the spread of the disease is limited to the eye.
If the retinoblastoma has already spread outside the eye, the children have an unfavorable prognosis despite intensive treatment.
Chemotherapy is regularly carried out in such cases.
However, the cancer can also develop resistance to this therapy, so that certain cells survive.
Last year, Dr. Kornelius Kerl’s group at the University of Münster, with the support of KAKS, used single-cell RNA sequencing to analyze which cancer cells survive chemotherapy.
Such cells are responsible for the development of therapy resistance and relapses of the disease.
In the course of this research project, the group was able to show that only very specific cell populations of these tumors respond to chemotherapy, while other retinoblastoma cells survive these therapies.
This project has thus made it possible to characterize therapy-resistant tumour cell populations in retinoblastomas for the first time.
Based on this knowledge, future projects now aim to find out how therapy resistance can be overcome in order to be able to treat effectively when conventional chemotherapy does not respond.
Funded by the KAKS.
Overview of a typical RNA-Seq workflow Griffith et al.
doi: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004393
Detection of minimal residual disease in patients with retinoblastoma
University Hospital Essen – Prof. Dr. Petra Ketteler et al.
The gold standard for the detection of metastases is the cytological examination of bone marrow aspirates and cerebrospinal fluid, but the sensitivity is limited.
The aim of this study is to establish a more sensitive method for detecting metastatic cells in blood, bone marrow
and cerebrospinal fluid.
To this end, the marker CRX should be measured using real-time PCR (qPCR) and digital droplet PCR (ddPCR).
The photoreceptor gene CRX can serve as a diagnostic marker for retinal tumors.
The bone marrow and cerebrospinal fluid samples were cytologically evaluated by a pediatric hemato-oncologist and these results were correlated with the qPCR, ddPCR and clinical results.
Thus, ddPCR for CRX was identified as a suitable method for the quantification of retinoblastoma cell lines.
This should enable earlier detection of CRX expression in qPCR and ddPCR than in cytology.
This KAKS-funded research project was presented as a poster at the Research Day of the University of Duisburg Essen.
Clinical and morphological images of a child with an extraorbital retino-blastoma.
A: MRI of the head (saggital) shows an intracranial tumor.
B: MRI of the spine shows metastasis.
C: Bone marrow smear shows small blue stained retinoblastoma cells lying together as a cluster.
Combating chemoresistance
University Hospital Lübeck – Group PD Dr. rer.
nat.
Vinodh Kakkassery
This project lays the foundation for the development of a switch for programmed cell death (apoptosis) in chemotherapy-resistant RB tumors by therapeutically inhibiting the switching enzymes (sphingokinases).
The results to date can be found in the literature list on the website.
Further publications are in preparation.
First, the role of sphingosine and sphingosine-1-phosphate as a signal for cell death and survival in the resistant retinoblastoma cells in cell culture was investigated and differences in the extracellular matrix between the resistant and therapy-sensitive cells were identified.
Proteomics analysis was performed to investigate the protein level.
The proteomics analysis revealed further differences between these two cells.
A particular focus of the working group was on the investigation of calcium channels and their response to various channel activators and inhibitors.
Currently, another manuscript on this topic is almost finalized by the research group, which deals with the role of the nerve growth factor in the development of resistance of retinoblastoma in this cell model.
These results were achieved with the support of the KAKS Foundation.
The next step is to link up with the National Center for Tumor Diseases (www.nct-dresden.de) and the Oncoray Research Center (www.oncoray.de) in Dresden in order to achieve the final therapeutic research goal with these structures.
Funded by the KAKS.
Translation of 22,000 genes (genome) into the transcriptome (RNA) and subsequent expression into the proteome (protein), which can then be analyzed to see which genes are converted into proteins and in what form.
Source: https://www.proteomicscenter.nl/background-information/
Zebrafish as an Orthotopic Tumor Model for Retinoblastoma / Mimicking Routes of Human Metastasis
Adjuvant therapy for children treated by enucleation at diagnosis of retinoblastoma
Consortium Universities of Essen, Berlin, Zurich, Münster, Graz, Tübingen
Retinoblastoma is often associated with misdiagnosis of clinical signs and lack of referral to a specialized retinoblastoma center.
Precise molecular genetic biomarkers such as circulating tumor DNA for risk stratification may help to improve risk stratification, reduce side effects and ensure survival.
In this prospective, multicenter observational study, data from 184 patients with retinoblastoma and primary enucleation were collected between 2013 and 2020.
The study showed that primary enucleation alone and additional risk-stratified adjuvant chemotherapy lead to very high cure rates in most retinoblastoma patients.
The study was funded by the German Childhood Cancer Foundation and the Children’s Eye Cancer Foundation.
This project was recently published: EJC Pediatric Oncology 1 (2023) 100004
https://doi.org/10.1016/j.ejcped.2023.100004
Collaborative study to identify Biomarkers to adjust treatment intensity for children with Retinoblastoma (CoBioRB)
University Hospital Essen – Prof. Dr. Petra Ketteler
The first part of this study characterizes the genetic and genomic features of retinoblastoma subtypes in order to identify biomarkers for tumors with high or low metastatic risk.
In the second part, the amount of circulating tumor DNA (ctDNA) in biological fluids of retinoblastoma patients will be quantified.
This is based on the hypothesis that the amount of ctDNA correlates with tumor burden and metastatic risk.
For this purpose, tumor tissue, blood, cerebrospinal fluid and aqueous humor from 35 patients in 6 centers will be collected to analyze the ctDNA and correlate the results with the clinical course.
The biomarkers are intended to help identify patients at risk of metastasis in order to reduce the late effects of treatment in others.
Co-BioRB will form the basis for a pan-European study (“EURBG2”) on biomarkers for risk stratification in the adjuvant treatment of retinoblastoma.
Migration of WERI-RB-1 cells in the first 5 days after injection of
80-100 suspension cells into the vitreous cavity of the left eye of a
2-day-old zebrafish
(A) on day of injection,
(B) on day 1,
(C) on day 2 and
(D) on Day 5 post injection in a lateral view;
(E) on the day of Injection with transmitted light
(F) on day 1,
(G) on day 2 and
(H) on day 5 post injection in dorsal view.
Only the dorsal view shows the initiation of migration of WERI-RB-1 cells in the brain already after one day post injection.
After 5 days post injection, only a few cells are found in the eye and most have migrated ti the brain.
On day 8 post injection no tumor cells were visible (not shown).
Use of CAR-T cells in retinoblastoma
Dr. med Annette Künkele – Charité, Universitätsmedizin Berlin, Prof. Dr. Ulrich Schraermeyer – University Hospital Tübingen
Publication of the results of this project to date: Andersch et al.
BMC Cancer (2019) 19:895(https://doi.org/10.1186/s12885-019-6131-1).
In 2016, KAKS initiated the first retinoblastim project using immune cells to combat tumors.
The aim is for tumor cells to be recognized by certain markers on their surface and destroyed by the body’s own immune cells.
The investigations showed that the contact of the immune cells (CAR-T cells) with the cancer cells led to the generation of functional CAR-T cells that were able to specifically recognize these cancer cells by the surface markers, i.e. CD171-specific as well as GD2-specific CAR-T cells.
The targeted attack of the CAR-T cell via the CD171 or GD2 molecules on the surface of the cancer cells led to the effective killing of all retinoblastoma cells.
Gene analysis of retinal organoids
University Hospital Essen – Dr. rer.
nat.
Deniz Kanber,
Prof. Dr. Laura Steenpaß
Publications: Schipper L, Kanber D, Steenpass L (2018) Generation of heterozygous and homozygous hESC H9 sublines carrying inactivating mutations in RB1.
Stem Cell Res.
PDF for download
Why does retinoblastoma develop in the eye and in young children in particular?
It is not yet clear why the tumor only develops in the eye in young children and which molecular mechanisms contribute to tumor development.
What is clear is that the alleles of the retinoblastoma gene are defective (mutated) or missing in all cells in hereditary retinoblastoma and that each cancer develops from a single initial cell.
But what prevents the development in other cells?
These cells also no longer have a functioning RB protein (tumor guard), which prevents uncontrolled cell growth in healthy cells.
However, research on human retinal cells in their developmental stages is not possible.
Therefore, in order to clarify these and other fundamental questions and to find out more about the initial cells of the tumor in the eye, a laboratory model of eye development with stem cells was developed in which the effects of mutations in the retinoblastoma gene can be investigated.
Previously published results from other groups show that the origin of the tumor appears to be localized in cells of the neural retina and not in the retinal pigment epithelium.
Specifically, a retinal cell model for retinoblastoma has so far been developed with the help of organoid technology.
The model is based on human embryonic stem cells (hESC) being made to differentiate as organoids in the laboratory.
Organoids are organ-like structures that are a few millimetres in size and consist of several cell types of the actual organ.
The retinal organoids generated in the project exhibit all seven retinal cell types that develop in their natural order in the embryo (ganglion cells, horizontal cells, amacrine cells, cones, rods, bipolar cells and Müller cells) and are organized in the known retinal layers, i.e. the anglial cell layer, the inner nuclear layer and the outer nuclear layer.
After successful production of the organoid structures, stem cells were generated that carry a mutation on one or both copies of the retinoblastoma RB1 gene.
The development of the mutated and non-mutated cells was then compared.
Even with simple means, it was possible to see that the retinal organoids and the retinoblastoma organoids form different structures.
So far, the observation has only been based on staining of frozen sections and a few targeted RNA analyses.
For the characterization and for a better understanding of the retinal organoid differentiation with regard to the specific effect of the retinoblastoma mutation (RB1 inactivation), a comprehensive genetic analysis (whole transcriptome analysis) was required in the next step.
The Children’s Eye Cancer Foundation funded this analysis.
The resulting data will provide a fundamental insight into the development and composition of different retinoblastoma cell populations in RB1-mutated organoids and also generate guidelines for sampling and analysis for future work
Supported by the KAKS.
Tumor marker development - Liquid Biopsy - Early diagnosis / EU project
EU-funded liquid biopsy project
Another major liquid biopsy project in which KAKS is involved is the EU Era-net Transcan Tumor Marker Project, which is funded by the European Union with over EUR 1 million.
KAKS was also able to fulfill its self-imposed task of providing start-up funding for this project by financing part of the preliminary work at the Free University of Amsterdam (VumC) and also incorporating results and material from the above KAKS tumor marker project at the UK-Essen.
Prof. Dr. Petra Temming’s group, which carried out a large part of the early marker research, is also involved in the EU project.
The EU-funded tumor marker project started in 2018 with universities from four countries as part of a major research collaboration.
We are proud that, with the help of A Heart for Children, we were able to contribute to the preparatory work for this important project and are now funding the integration of tumor data from the various national tumor centers.
The aim of the project, which is also funded by the BMBF, is also to develop a non-invasive blood test for the early detection of retinoblastoma or secondary tumors via exosomes (cf.
on the preliminary work at http://dx.doi.org/10.1016/j.ccell.2015.09.018).
At the same time, a systematic and comprehensive retinoblastoma database/biobank is being set up in the Netherlands with KAKS support and patient data from the last 13 years is being processed.
The project also focuses on disseminating the results to the specialist public and patients via specialist articles, lectures and regularly updated information.
Last year in Amsterdam, researchers from fifteen centers in Europe, North and South America and Japan founded the IRiSC – International Retinoblastoma and Second Cancer Consortium as part of this project.
By including all newly diagnosed cases of Rb and extending medical care, it will now be possible to assess the risks of second tumors much more accurately and develop personalized prevention/screening.
International cooperation is extremely important here because research into rare diseases is very difficult due to the small amount of data available.
KAKS is involved in the following two exciting subprojects.
Hereditary retinoblastoma: Nine centers from Europe and the USA with 115 families are involved in the hereditary retinoblastoma subproject.
Another project in this context is the previously reported NIRBtest project for the detection of Rb primary and secondary tumors using a blood test, as originally initiated by KAKS in 2012 with the help of Bild e.V..
The blood test, for which samples were collected in the first phase, is now based on three blood components: Free tumor DNA, extracellular vesicles and platelets.
All of these blood elements can provide information about a tumor and are collected centrally, but analyzed in France, the Netherlands or Germany, depending on the type.
Diagnostic path recognition
University Hospital Essen
This study has been completed.
The aim of this study was to analyze the diagnostic pathway in children with retinoblastoma in order to improve early detection.
The results show that the most common initial symptoms are leukocoria and strabismus.
The initial symptoms are mostly noticed by the parents.
However, pediatricians often do not attribute these symptoms to a tumor disease.
And in the first few months, children usually only see the pediatrician, but not an ophthalmologist.
We have launched extensive projects to educate paediatricians so that they can recognize retinoblastoma in the future.
Study details: Data was collected from 1049 Rb patients between 1992-2011 in Essen.
The most common symptoms were leukocoria and strabismus (squint) and it is the parents who usually recognize these symptoms first.
The survey shows that the time to diagnosis has not changed significantly over the last 20 years.
Our aim is to change this too.
We already have initial indications that our work is shortening the time to diagnosis.
Status: Completed
Initiated and supported by KAKS
Rb inhibitors against tumor cells
University Hospital Essen – Dr. Temming
KAKS prefers to fund projects in which substances already known for the treatment of other diseases are used.
Such substances are generally of no interest for commercial research.
However, they have long been tested for their side effects and are usually available on the market at low cost.
One such project is the KAKS-funded search for potential inhibitors that can be used to treat retinoblastoma.
The search was successful because the inhibitors against the cell cycle regulator polo-like kinase 1 (PLK1) and against MYCN regulating factors, such as Aurora A kinase and the BET domain protein BRD4, proved to be extremely effective.
The use of the inhibitors led to a reduction in growth and an increase in cell death (apoptosis) of the retinoblastoma cells in vitro.
This KAKS-funded study now forms the basis for in vivo studies, which should make a major contribution to the development of new therapies for retinoblastoma patients.
Supported by the KAKS.
Late effects study at Essen University Hospital
Prof. Bornfeld, Prof. Lohmann
KAKS is involved in funding this DKS study
The aim is to optimize long-term care and improve the long-term prognosis and quality of life.
To this end, the long-term therapeutic consequences of the various forms of treatment were determined and the individual genetic disposition was also taken into account with regard to the risk of secondary tumors.
In addition, adult patients and parents were informed about preventive measures.
Children and adolescents were included in the first phase and adult patients in the second phase (collective of approx. 570 test subjects).
Status: The studies have been completed and the results are available.
The results of the first phases have since been published (Temming et al. 2015, Pediatric Blood Cancer)
Sponsored by KAKS and the German Childhood Cancer Foundation.
Prevention of radiotherapy induced secondary tumors
Dr. Sylvie Julien, Prof. Dr. Ulrich Schraermeyer, Prof. Dr. H. Peter Rodemann – University Hospital Tübingen
Status: Completed
Published under https://www.ncbi.nlm.nih.gov/pubmed/27694105
Initiated by KAKS and supported by the German Childhood Cancer Foundation.
Abstract
Retinoblastoma (Rb) is the most frequent primary intraocular tumor in children and, if left untreated, can cause death.
Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities.
There are several models described for Rb, but none of them fully meet our requirements.
The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods.
We injected human retinoblastoma Y79 cells intravitreally into both eyes of immune-deficient nude mice.
The incidences of retinoblastoma as well as growth velocity were analyzed 3, 6, 9 and 12 weeks after cell injection in vivo by SLO/OCT as well as ex vivo by electron microscopy (EM) and hematoxylin/eosin (HE) staining.
Furthermore, internal organs were histologically screened for potentially occurring metastases.
Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumor growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumor-development.
After 12 weeks, 67.5% of all analyzed eyes (29 of 42) contained a retinoblastoma.
At early stages of Rb development, the SLO/OCT analysis correlated with the histology results.
If the tumors were too large, only histological investigations were feasible.
The ultrastructural characteristics of the xenograft tumors were very similar to those described for patient’s tumors.
In one mouse, brain metastases were observed.
Our retinoblastoma mouse model closely resembles the human disease.
SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies.
Tumor marker development
Liquid Biospy – Early diagnosis
Charité Berlin and University Hospital Essen
Research into the early detection of cancer through blood analysis (“liquid biopsy”) has made enormous progress in recent years: experts speak of a “revolution” and “breakthrough” in the early detection of cancer.
KAKS is right in the middle of this with two tumor marker projects (Berlin and Amsterdam).
Blood analysis was tackled as a central research objective by the KAKS at a very early stage.
Three approaches were initially tried for the early detection of retinoblastomas and secondary tumors in blood samples:
1. early detection of antibodies in the blood that the body had previously produced against cancer cells
2. identification of cancer cell surface markers,
3. detection of tumor cells circulating in the blood.
These approaches were researched with the support of Bild e.V. Ein Herz für Kinder and the KAKS at the Max Planck Institute in Potsdam in the team of Peter Seeberger and the UK-Essen in the team of Petra Temming.
We were able to identify useful cancer marker proteins on the cell surface of cancer cells in the test tube.
However, these approaches then showed that the antibodies and the circulating tumor cells do not have the necessary sensitivity for detection in the patient’s blood.
The project at the Charité in Berlin was then expanded to include the latest highly sensitive “liquid biopsy” technologies for the analysis of tumor DNA in blood and also a marker protein analysis by one of the most successful marker specialists, Dr. Bergmann.
Again with the support of Bild e.V., we financed a doctoral thesis to establish a standardized protocol for the processing of blood samples, which should ensure that samples obtained can be properly processed independently of the user and in such a way that even small amounts of tumor DNA can be identified in the sample.
Furthermore, a list of candidate genes and genomic alterations that are typical of the most common secondary tumors was compiled by means of literature research and the re-analysis of publicly available sequencing data.
This list, together with precise information on which DNA regions need to be covered in which way when sequencing the patient’s blood, forms the basis for the sequencing-based early detection test.
In addition to the retinoblastoma gene RB1, this test also takes into account those genes that are most frequently altered in secondary tumors.
After processing the blood, special probes are now used to detect the DNA sections of the cancer cells and the candidate genes are specifically examined for characteristic changes using a so-called hybrid capture sequencing assay.
In parallel approaches, the DNA tests are tested on the blood of retinoblastoma patients and various second tumor types as well as on healthy people for comparison.
Most recently, a comparable technique was able to detect 8 different tumors in the blood of 98% of patients.
Initiated by KAKS, sponsored by KAKS and Bild hilft e.V. – Ein Herz für Kinder
PROJECT-
PROMOTION
The Children’s Eye Cancer Foundation primarily supports therapy optimization studies and application-oriented scientific projects.
As a trust foundation of the German Childhood Cancer Foundation (DKS), we, the Children’s Eye Cancer Foundation, are in the fortunate position of being able to draw on the resources of this large foundation.
In order to meet high quality standards, the German Childhood Cancer Foundation has appointed a panel of experts: Seven experts advise the foundation and the Children’s Eye Cancer Foundation on all matters relating to project funding.
Invitation to tender
Project applications undergo a standardized review process.
For larger research projects, we are able to draw on the DKS’s highly developed review process, including a panel of experts, thanks to its generous support.
The expert committee has received a mandate from the German Childhood Cancer Foundation to evaluate all project applications and make decisions in agreement with the Foundation’s Board of Directors.
Composition of the expert committee of the German Childhood Cancer Foundation
Prof. Dr. Dr. med. Michael Frühwald, Augsburg (Chair)
PD Dr. med. Gabriele Escherich, Hamburg (Deputy Chair)
Prof. Dr. med. Uta Dirksen, Essen
PD Dr. med. univ.
PhD Miriam Erlacher, Freiburg
Prof. Dr. med. Jan-Henning Klusmann, Frankfurt
Prof. Dr. med. Stefan Pfister, Heidelberg
Prof. Dr. med. Martin Stanulla, Hanover
The expert committee meets twice a year in a closed meeting.
Information on the procedure
The planned research project must be in the field of all diseases and secondary diseases associated with retinoblastoma and meet the funding criteria of the Children’s Eye Cancer Foundation.
Project applications can be submitted at any time.
(Source: German Childhood Cancer Foundation).
Doing good, donating or endowing
Thank you for your support.
Whether research, early detection or parent-child projects – at KAKS, distances are short and your donation goes directly to the right place.
Research
partner
A heartfelt thank you
- University Hospital Tübingen, Clinic for Ophthalmology
- Max Planck Institute of Colloids and Interfaces, Potsdam
- University Hospital Essen, Clinic for Ophthalmology
- University Hospital Children’s Hospital III Essen
- Essen University Hospital, Institute of Human Genetics
- University Hospital Amsterdam, Holland
- Charite, Children’s Hospital
- University Hospital Münster
- University Medical Center Schleswig-Holstein