We chose to engage with the issue of glioma in our iGEM project. Engagement with serious topics such as cancer requires a high degree of responsibility and intellectual honesty. Since solely doing literature research and working in an isolated fashion is not compatible with these requirements, it was crucially important to us to enter into a mutual dialog with people involved in treating, diagnosing and researching glioma as well as experts in RNA therapeutics, drug development and related topics.
Through our conversations with neurosurgeons, oncologists, pathologists and other experts, we learned many things about the diagnostics, treatment and biology of glioma. Much of what we learned through these discussions confirmed or completely changed our perception on this issue, and helped us keep our project in line with clinical reality.
This page provides an overview of the interesting conversations we had, the invaluable feedback we received and the ways in which our project was influenced by the input of experts and stakeholders.
Prof. Dr. med. Matthias Simon
We met with Prof. Dr. med. Matthias Simon, neurosurgeon and director of the neurosurgery department of the Evangelisches Klinikum Bethel (EvKB) clinic in Bielefeld. Our broad discussion ranged from the importance of tumor subtyping during the resection of gliomas to the challenges and opportunities of administering anti-cancer drugs directly into the brain. Dr. Simon explained to us that the subtype of a glioma determines the extend to which a neurosurgeon has to remove adjacent tissue, since more aggressive and invasive gliomas tend to infiltrate surrounding tissue more than lower grade gliomas. Hence, the most important information to a neurosurgeon is wether or not a given tumor is a glioblastoma, since these are aggressive grade 4 tumors that require extensive tissue removal. We were not aware that the specific type of a brain tumor not only influences the post-operative treatment strategy, but also the surgical procedure. We adopted this knowledge by prioritizing the analysis of point mutations like the IDH1 mutational status (which differentiates glioblastomas from other gliomas) in our diagnostic bioinformatics pipeline, so this information can be relayed to the neurosurgeon as fast as possible.
Dr. Simon also explained that a histopathological examination of tumor tissue during surgery usually does not differentiate between astrocytomas and oligodendrogliomas. However, this would be relevant information to a surgeon since oligodendrogliomas tend to require less aggressive removal of tissue. A key takeaway from this discussion is that an intraoperative diagnostics workflow should be able to distinguish all adult glioma subtypes, which includes a differentiation between astrocytomas and oligodendrogliomas. Regarding our mRNA therapeutics, Dr. Simon encouraged us to go along with this idea and emphasized the importance of discovering new approaches to glioma treatment. He explained that many classical approaches such as tyrosine kinase inhibitors did not show the expected efficacy in clinical studies. We also discussed possible challenges of administering our therapeutics, since tumor cells may be located deep inside the brain tissue. A solution containing lipid nanoparticles loaded with mRNA would have to reach these remaining cancer cells, since they are mainly responsible for tumor recurrence. Another aspect of drug administration to be considered is the diffusion limitation and penetration depth of lipid nanoparticles. We discussed this issue based on the clinical experience with wafers which continously secrete chemotherapeutic substances and are placed into the brain after tumor removal.
Prof. Dr. rer. nat. Sven Thoms
Since our therapeutic approach is based on the conditional translation of a toxic protein inside tumor cells, the safety and specificity of our therapeutics were a great concern to us. A possible issue that we were concerned about is the phenomenon of ribosomal translational readthrough. Given certain conditions, a ribosome may “skip” a stop-codon and simply continue translating an open reading frame. In the context of our therapeutics, this phenomenon might potentially lead to off-target expression of the toxic payload in non-cancerous cells, without the necessary editing of the stop-codon. Therefore, we approached Prof. Dr. Thoms, who researches the translational readthrough phenomenon, and discussed this issue with him. In his estimation, this effect would most likely not be an issue, since certain conserved motifs in the RNA are required to elicit an efficient readthrough. It is unlikely that these motifs would be found in our sensor mRNA constructs, however, we could nevertheless decide to screen sensors for these motifs. Dr. Thoms provided us with the necessary information on how to approach such an analysis. In addition, even if a readthrough occurred, the fraction of mRNA molecules affected by a readthrough would most likely be minimal. We concluded that for our project, the phenomenon of ribosomal readthrough is not an issue, and therefore decided to not investigate it further.
Dr. Roland Coras and Dr. Almut Mentz
We had a joint discussion with Dr. Roland Coras, senior physician at the department of neuropathology at Erlangen University Hospital, and Dr. Almut Mentz, head of the molecular pathology department at the Klinikum Bielefeld hospital. Our conversation focused mainly on the diagnostics and tumor-markers of gliomas.
Dr. Coras told us of the tumor-markers he analyzes during his work with glioma patients and the diagnostic methods employed. He explained that the informative value of classical histopathology strongly depends on the experience of the pathologist and on the quality of tissue samples. Intraoperative histopathological diagnosis can therefore range from an exact classification of the tumor subtype to merely an estimation of tissue malignancy. Especially the distinction between astrocytomas and oligo-dendrogliomas is difficult by a standard frozen section procedure. In addition, it is especially difficult to accurately diagnose diffuse glioma in its early stages, since it may not present with obvious features of neoplasms on tissue examination. Hence, accurate molecular diagnostics is of crucial importance. Dr. Coras explained that the combination of IDH1 mutational status and TERT promoter mutational status would be particularly interesting for intraoperative diagnostics. We also discussed the molecular markers we are trying to detect with our mRNA therapeutics. Dr. Coras gave us the feedback that it is sensible to focus specifially on the IDH1 R132H mutation, since this is by far the most common point mutation found in IDH1-mutated gliomas. Further points of discussion were the prognostic values of tumor-markers for chemotherapy and the analysis of DNA methylation.
Dr. Mentz explained to us which diagnostic methods are routinely used to analyze molecular markers in cancer. These include, among others, next-generation sequencing (NGS), fluorescence / chromogenic in situ hybridization (FISH / CISH), quantitative PCR (qPCR) and DNA methylation analysis. Some of these methods may require several days to produce results and are therefore not applicable for intraoperative tumor diagnostics. She also provided general feedback to us regarding our diagnostics project.
Prof. Dr. med. Michael Weller
We spoke with the scientific spokesperson of the German Glioma network and clinic director for neurology in Zurich, Pr. Dr. med. Michael Weller. We discussed current glioma research and certain aspects of our project. Pr. Dr. med. Weller has been doing glioma research for the past three decades. What motivates him to keep going, is one simple message: "you have to do something, you can’t just wait for someone else to do something." Even though there hasn't yet been the aspired breakthrough, he keeps going. At the moment, his team is focused on CAR-T-cells and cytokines. They also work on RNA therapeutics, and though they do not work on targeted RNA therapeutics, Weller believes that our project is an interesting and viable approach. "If your idea wasn't this innovative, I would not have replied to you", he said.
1st Main problem of glioma treatment: very few clear targets...
Regarding our novel mRNA therapeutics, we asked Weller about possible targets in adult brain cancer for us to go after. About the IDH1 mutation, we learned that it is a driver mutation that occurs in almost all corresponding cancer cells. In addition, while there are other known targets, no successful new treatment options were created. One concern, however, was that it is unclear what would happen if the mutation is lost in certain cells. It may occur that the therapy results in selection for cancer cells that do not carry the IDH1 mutation. This could be dangerous, as it is suspected that such cancer cells are suspected to be more aggressive. However, Pr. Dr. med. Weller stated that it is first of all a good thing if therapeutics are able to kill a lot of the remaining cancer cells after surgical removal of the tumor. Additionally, we discussed the use of EGFR as a target for our sensor. Weller warned us about the heterogeneity of glioma that have EGFR overexpression, which could be an obstacle in our project. The main issue seems to be toxicity, since healthy cells express EGFR, too. However, Weller believes that this is unlikely to be a problem for the mRNA therapy when applied locally. "For a functioning EGFR-targeted therapy, there are a multitude of other cancers that could be treated", he added.
2nd Main problem of glioma research: complicated drug delivery...
We asked Weller about his experience with clinical applications of gene therapies for gliomas. His only experience: "so far, it has never worked...". He says, more often than not, the problem is dysfunctional drug delivery. The issue with many local therapies is reaching cancer cells deep inside the brain. It is important to be aware of limitations like these. From our conversation with him, we concluded that drug delivery using lipid nanoparticles is the best approach for us. He also added that this could potentially be applied using convection. This approach is also advantageous considering the limitations of systemic application caused by the blood brain barrier. "One day, cargo drug delivery will be the standard", he said.
About intraoperative diagnostics...
Many researchers are working on intraoperative diagnostics, all around the globe. Pr. Dr. med. Weller states that Nanopore sequencing provides a promising approach for this. We asked if we should look at point mutations or methylations for our diagnostics. After discussing this topic, we agreed that analyzing methylations is more of a complementary idea, and observing point mutations is the way to go.
Conclusion
Weller stated: "biologics are clearly the future of tumor therapy". He is sure, that our idea is both interesting and new; "the way the toxin is activated is what's innovative about the concept". We were relieved to hear that the problems we are faced with are common amongst those who do research in the field of glioma therapy. On the topic of the iGEM competition, Pr. Dr. Weller is positive that it can help us all to find our way through the multitude of fields of scientific study.