Abstract 930: Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors
Authors: De Mattos-Arruda Leticia , Mayor Regina , Ng Charlotte K. Y. , Weigelt Britta , Martínez-Ricarte Francisco , Torrejon Davis , Oliveira Mafalda , Raventos Carolina , Arias Alexandra , Guerini-Rocco Elena , Martínez-Sáez Elena , Lois Sergio , Marín Oscar , de la Cruz Xavier , Piscuoglio Salvatore , Towers Russell , Vivancos Ana , Peg Vicente , Ramon y Cajal Santiago , Rodon Jordi , Felip Enriqueta , Sahuquillo Joan , Tabernero Josep , Cortes Javier , Reis-Filho Jorge S. , Seoane Joan 
Published: August 2015

Abstract
Background: Genetic characterization and monitoring of brain tumors is challenging given the restricted sampling of tumors and the limited abundance of brain tumor-derived circulating tumor DNA (ctDNA) in the plasma. Here, we sought to define whether cerebrospinal fluid (CSF) could serve as a ‘liquid biopsy’ for the genetic characterization of tumor DNA originated from the central nervous system (CNS) of patients (pts) with brain tumors.
Methods: CSF, plasma and tumor tissue were obtained from pts with glioblastoma (GBM), brain metastases from breast (BMBC) and lung cancers (BMLC) and leptomeningeal carcinomatosis (LC). Whole exome sequencing was done in CSF and tumor (n = 3) and was coupled with digital PCR for monitoring CSF and plasma ctDNA (n = 6). Targeted capture massively parallel sequencing of 39 samples from 4 BMBC pts, including samples from CSF, plasma and matched metastatic sites obtained at autopsy was performed using two independent platforms (MSK-IMPACT (341 genes) and BC panel (254 genes most frequently mutated in BC)) comprising 488 unique genes. Sequencing was performed on an Illumina HiSeq2000. Single nucleotide variants were defined using MuTect, and indels using Varscan. Copy number alterations were assessed using Varscan2 and GISTIC.
Results: Whole exome sequencing of tumor and CSF DNA revealed a high proportion of tumor-derived cell-free DNA in the CSF of 3 pts (1 GBM, 2 BMBC). We identified actionable somatic mutations (EGFR L858R, IDH1 R132H) and also IDH1 mutations associated with molecular diagnosis of proneural GBM. In CSF and plasma ctDNA of 6 pts with GBM, BMBC, BMLC, we observed that the mutations present in the CSF ctDNA, but not plasma ctDNA, and their mutant allele fractions (MAFs) correlated with brain tumor burden. In 3 pts with suspected LC, we compared the results of cytopathologic analysis and CSF ctDNA obtained from samples used for cytopathologic diagnosis, and observed that CSF ctDNA was more robust and sensitive for the diagnosis of LC. The analysis of synchronous CSF ctDNA, plasma ctDNA and intra- and extra-cranial metastases from 4 autopsied BMBC pts showed that CSF ctDNA recapitulated the somatic genetic alterations present in the intra-cranial lesions. We detected similar MAFs for the truncal mutations (RB1, KMT2D, AHNAK2) in both CSF and plasma DNA of BMBC3, a pt with Li-Fraumeni syndrome and a diagnosis of concurrent BMBC and esthesioneuroblastoma; however, mutations in PIK3CB, PAK7, MSH5 found only in the CNS implant of each disease were only detected in the CSF but not in the plasma DNA.
Conclusions: Brain tumor-derived ctDNA is abundantly present in the CSF of brain cancer pts and compared to plasma ctDNA, CSF ctDNA is more representative of the brain lesions. Our results demonstrate that massively parallel sequencing can be performed using CSF DNA, allowing for the non-invasive genomic characterization and monitoring of brain lesions.
Citation Format: Leticia De Mattos-Arruda, Regina Mayor, Charlotte K. Y. Ng, Britta Weigelt, Francisco Martínez-Ricarte, Davis Torrejon, Mafalda Oliveira, Carolina Raventos, Alexandra Arias, Elena Guerini-Rocco, Elena Martínez-Sáez, Sergio Lois, Oscar Marín, Xavier de la Cruz, Salvatore Piscuoglio, Russell Towers, Ana Vivancos, Vicente Peg, Santiago Ramon y Cajal, Jordi Rodon, Enriqueta Felip, Joan Sahuquillo, Josep Tabernero, Javier Cortes, Jorge S. Reis-Filho, Joan Seoane. Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 930. doi:10.1158/1538-7445.AM2015-930