Funded Grants

Use of cerebrospinal fluid genetics for the diagnosis, subgrouping, and minimal residual disease monitoring in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children, and despite recent improvements in treatment strategies, still approximately one third of children die from their disease and at least another third of patients can only be cured at high cost, namely severe neurological, mental, psychosocial, endocrine, cardiac, and renal deficits. In the past, diagnosis was solely derived from histopathological assessment of the tumor tissue. Recent evidence from genome‐wide screening projects (including DNA copy‐number analysis, gene expression profiling, and DNA¬methylation analyses) clearly suggests that medulloblastoma in fact is a group of molecularly, epidemiologically, and prognostically distinct diseases comprising at least four major variants. Across all of these molecular variants, patients with microscopic or macroscopic dissemination of their tumor (throughout the CNS, occasionally even throughout the body) have a much worse probability of being cured.

One major reason, why brain tumors as a group have replaced leukemias (although the latter being much more common) as the leading cause of cancer death in children, is that our knowledge of the genetic repertoire of leukemias is much more comprehensive. These scientific insights have been successfully exploited for molecular diagnostics, treatment monitoring, tailoring treatment intensity to disease risk, and molecular targeted therapies in selected cases for more than a decade. This body of knowledge is only starting to grow for medulloblastoma, the deadliest brain tumor in children. A clinically challenging decision on whether to regard patients as clinical high risk is the definition of “microscopic metastases”. To justify this diagnosis, “united cell structures” of malignant cells have to be present in the cerebrospinal fluid (CSF) of patients at the time of primary diagnosis according to a widely used classification system proposed by Chang and colleagues in the late 1960s. It is anticipated, however, that almost all patients have some kind of CSF dissemination and that medulloblastoma is in fact a disease that involves the entire CNS rather than only the cerebellum, where the primary tumor occurs. This might be illustrated by the fact, that many patients who are considered non‐metastatic at the time of diagnosis, will develop a relapse at a distant site, e.g., the spine. Patients with recurrent disease have a fatal prognosis. Most of them die within one year after tumor recurrence was diagnosed. As a result of this, a refined detection of CSF dissemination is urgently warranted and a very promising avenue to achieve this is by tracking and quantifying tumor‐specific genetic aberrations in this bodily liquid. Identifying tumor‐specific mutations in cellular and non‐cellular DNA floating in the CSF could not only be used for the diagnosis of CSF dissemination, but also for molecular subgrouping, treatment monitoring (i.e., clearance of the CSF in the course of treatment), and surveillance of patients after completing treatment (i.e., detection of early microscopic relapse). The latter two aspects are again in analogy to a very successful concept that has been applied in leukemia patients for many years, namely “minimal residual disease monitoring”. In leukemia patients, this is achieved by detecting characteristic genetic aberrations in the leukemia cell clone in the bone marrow down to a level of 1 tumor cell in 100.000 normal cells. This information is used all over the world to stratify patients into therapy groups with different treatment intensity, to monitor treatment response, and for early detection of leukemia recurrence. If it was possible to use CSF genetics for the same purpose in brain tumor patients, this would revolutionize current clinical practice.

Our approach to test the feasibility of using CSF genetics in the diagnosis and treatment monitoring of medulloblastoma patients involves three important steps. In a first phase, we will investigate 30 medulloblastoma patients, from whom we have collected frozen tumor samples, CSF at primary diagnosis, and blood (germline control). By sequencing all coding exons in the tumor DNA using next‐generation sequencing techniques and compare these with the germline of the same patient, we will determine the repertoire of somatic (i.e., tumor specific) mutations in these 30 tumor samples. In a second step, we will quantify the abundance of 5‐10 tumor‐specific mutations in the CSF of the respective patient down to a resolution of 1 copy of the mutant allele in 100 copies of the wildtype (germline) allele. Finally, in the third phase of the project we will be able to compare serial CSF samples from ten patients over the course of their disease to assess, whether it was possible to quantify molecular CSF clearance after chemotherapy.

Preliminary data obtained from a pilot patient who´s tumor harbored somatic mutations in the two cancer genes TP53 and CTNNB1, demonstrates that it is feasible to extract sufficient amounts of high quality DNA from 1 ml of CSF for at least 30‐50 PCR reactions and to detect tumor‐specific mutations in a semi‐quantitative fashion. This pilot project intriguingly demonstrates the principal feasibility of our approach, which now has to be proven on a larger basis in a reasonably large cohort of patients (independent of the presence of microscopic dissemination by cytological definition).

In summary, we strongly believe that genetic tests in the CSF of medulloblastoma patients has the potential to significantly influence clinical decision making in terms of diagnosis, assessment of therapy response, selection of therapeutic agents, and patient surveillance. Therefore, we think our proposal exactly serves the major goals of the JSM foundation call on brain cancer research, namely the generation of novel insights leading to increased cure rates and improve functional outcome for brain tumor patients in a truly translational sense.