Establishment of a molecular classification platform for medulloblastoma by qPCR in real time

Abstract

Medulloblastoma is the most common malignant brain tumor in childhood and according to World Health Organization it is subdivided in 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. SHH is divided in SHH TP53 WT and Mutated. These subgroups have distinct molecular characteristics and clinical associations, and differ in the prognosis of patients impacting the risk stratification and the type of treatment to be used. For example, the WNT subgroup has the best prognosis compared to the others and has an 95% overall survival in 5 years and a frequency of 1 in 3 patients. Phase II clinical studies in the USA aim to de-escalate radiotherapy and / or chemotherapy in patients with WNT medulloblastoma, reducing long-term sequelae caused mainly by radiation. This protocol, when used in Brazil, would positively impact the quality of life of patients and also reduce the budget of public hospitals. For the SHH subgroup, an inhibitor of this pathway, the Vismodegib, is being tested as a new subgroup-specific treatment agent. Hospitals that do not use the molecular classification, in a certain way, establish the same toxic and nonspecific treatment protocol for all patients. This factor highlights the great importance of the molecular classification approach for precision medicine that aims to establish the "ideal" protocol for different tumor subtypes. In Brazil and also in Latin America, there is no commercialization of any molecular test for medulloblastoma, we would be the first company to commercially implement this molecular test. There are some private or public / private hospitals that use NanoString to classify medulloblastoma, a methodology developed internationally and at a high cost, which still requires a minimum set of 6 samples for processing and analysis, which generates technical, cost and result release time disadvantages. In addition, these hospitals do not sell the exam, being only for internal use and research, and may even become future customers. In view of the current scenario, the objective of this project is to establish and validate a medulloblastoma molecular classification platform by an accurate and sustainable cost-benefit technique: real-time PCR (RT-qPCR). The basic principle is to measure the level of expression of a reduced set of genes that will be able to classify the medulloblastoma molecularly using samples derived from paraffin tissue (FFPE). These genes will be selected through bioinformatics analysis using public databases of medulloblastoma transcriptome available online (approximately 1000 cases). Using this wealth of information and the artificial intelligence by decision tree methodology, a minimum set of highly sensitive and specific genes will be determined to classify medulloblastoma into 3 molecular subgroups, WNT, SHH and non-WNT / SHH. Subsequently, this set of genes will be validated by RT-qPCR and the determined SHH samples will be sequenced for the TP53 gene in frozen and FFPE tissues of medulloblastoma in collaboration with Hospital das Clínicas de Ribeirão Preto-HCFMRP-USP. To develop this proposal, NEOGENYS Molecular Diagnosis has the support of associated researchers and collaborators, and infrastructure of the Molecular Biology Laboratory of Pediatric Oncology at HCFMRP-USP, in an agreement already formalized with the University of São Paulo. With this, the expertise of our team added to the team of clinicians and collaborators positions NEOGENYS as a company that aims to democratize the access to molecular tests in the oncology field and of international potential to impact on public health and quality of life of cancer patients. (AU)