Genomic Analysis Reveals Secondary Glioblastoma After Radiotherapy in a Subset of Recurrent Medulloblastomas
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Acta Neuropathologica https://doi.org/10.1007/s00401-018-1845-8 ORIGINAL PAPER Genomic analysis reveals secondary glioblastoma after radiotherapy in a subset of recurrent medulloblastomas Ji Hoon Phi1,2 · Ae Kyung Park3 · Semin Lee4,5 · Seung Ah Choi1,2 · In‑Pyo Baek6 · Pora Kim7 · Eun‑Hye Kim8 · Hee Chul Park9 · Byung Chul Kim9 · Jong Bhak4,5 · Sung‑Hye Park10 · Ji Yeoun Lee1,2,11 · Kyu‑Chang Wang1,2 · Dong‑Seok Kim12 · Kyu Won Shim12 · Se Hoon Kim13 · Chae‑Yong Kim14 · Seung‑Ki Kim1,2 Received: 15 December 2017 / Revised: 2 April 2018 / Accepted: 2 April 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Despite great advances in understanding of molecular pathogenesis and achievement of a high cure rate in medulloblastoma, recurrent medulloblastomas are still dismal. Additionally, misidentifcation of secondary malignancies due to histological ambiguity leads to misdiagnosis and eventually to inappropriate treatment. Nevertheless, the genomic characteristics of recurrent medulloblastomas are poorly understood, largely due to a lack of matched primary and recurrent tumor tissues. We performed a genomic analysis of recurrent tumors from 17 pediatric medulloblastoma patients. Whole transcriptome sequencing revealed that a subset of recurrent tumors initially diagnosed as locally recurrent medulloblastomas are secondary glioblastomas after radiotherapy, showing high similarity to the non-G-CIMP proneural subtype of glioblastoma. Further analysis, including whole exome sequencing, revealed missense mutations or complex gene fusion events in PDGFRA with augmented expression in the secondary glioblastomas after radiotherapy, implicating PDGFRA as a putative driver in the development of secondary glioblastomas after treatment exposure. This result provides insight into the possible application of PDGFRA-targeted therapy in these second malignancies. Furthermore, genomic alterations of TP53 including 17p loss or germline/somatic mutations were also found in most of the secondary glioblastomas after radiotherapy, indicating a crucial role of TP53 alteration in the process. On the other hand, analysis of recurrent medulloblastomas revealed that the most prevalent alterations are the loss of 17p region including TP53 and gain of 7q region containing EZH2 which already exist in primary tumors. The 7q gain events are frequently accompanied by high expression levels of EZH2 in both primary and recurrent medulloblastomas, which provides a clue to a new therapeutic target to prevent recurrence. Considering the fact that it is often challenging to diferentiate between recurrent medulloblastomas and secondary glioblastomas after radiotherapy, our fndings have major clinical implications both for correct diagnosis and for potential therapeutic interventions in these devastating diseases. Keywords Medulloblastoma · Recurrence · Secondary glioblastoma after radiotherapy · Genomic analysis Introduction Medulloblastoma (MB) is the most common malignant brain tumor in children. Decades of advances in surgery, adjuvant therapies, and patient care have led to a high cure rate of MB Ji Hoon Phi, Ae Kyung Park and Semin Lee contributed equally to which was invariably fatal in Harvey Cushing’s time. Clini- this article. cal trials are ongoing for reducing radiation dose to decrease Electronic supplementary material The online version of this late toxic efects in survivors. Molecular subgrouping of MB article (https://doi.org/10.1007/s00401-018-1845-8) contains has been well-established by a series of genomic studies supplementary material, which is available to authorized users. [27]. There are four main subgroups of MB, namely, WNT, SHH, Group 3, and Group 4, which exhibit distinct gene * Seung‑Ki Kim [email protected] expression patterns, ages of onset, clinical behavior, and prognoses. More recently, genome-wide DNA methylation Extended author information available on the last page of the article Vol.:(0123456789)1 3 Acta Neuropathologica profling has been recognized as a more robust and accurate revealed a misdiagnosis of secondary GBM after RT. PDG- method for not only molecular subgrouping of MB but also FRA is a frequently altered gene in GBM [4, 36]. CNS tumor classifcation [17]. Patients with WNT MB have This study demonstrates the power of genomic analyses a > 90% chance of survival [8, 37]. In contrast, patients with for diagnosis of brain tumors, especially during recurrence. Group 3 tumors and MYC amplifcation face a far worse Integrated diagnosis, combining histology and molecular prognosis (< 50% survival) [39]. Molecular targeted therapy characteristics, is recommended for recurrent MBs. Fur- is also attempted for the treatment of refractory MBs with thermore, altered PDGFRA signaling pathways can be tar- altered SHH signaling pathways [42, 43]. geted by receptor tyrosine kinase inhibitors, and outcomes Notwithstanding the advancements in MB treatment and of patients with secondary GBM after RT with PDGFRA research, the situation regarding recurrent MB looks difer- mutations can be improved with appropriate diagnosis and ent. Currently, at least a quarter of all MB patients sufer therapies. from tumor recurrence, and the prognosis is dismal. There is no efective therapy for recurrent MB if standard treatments have already been delivered [21]. The genomic and molecu- Materials and methods lar characteristics of recurrent MB are poorly understood, largely because of the lack of tumor tissues. It is known Patients and samples that recurrent MBs frequently acquire increased histological anaplasia and chromosomal imbalance [16]. A recent study Specimens were collected from 17 patients from three cent- has shown that the emergence of combined MYC and p53 ers, namely, Seoul National Children’s Hospital (SNUCH), pathway defects during recurrence leads to extremely poor Severance Hospital, and Seoul National University Bundang outcomes [13]. Another study has shown that recurrent MBs Hospital (SNUBH), with appropriate consent from institu- are derived through clonal selection from existing clones tional review boards. The primary tumor in all the patients in primary tumors and harbor a remarkable number of new was MB. Details of patient characteristics and samples are genetic events [24]. Interestingly, despite the accumulation described in Table S1. The biospecimens were composed of of genetic variations, the molecular subgroup of a MB is 14 tumors paired with patient-matched primary MB samples highly stable during recurrence [40]. Therefore, progressing (11 pairs of primary–recurrent MB and 3 pairs of primary genomic changes coexist with stable molecular subgroups MB–secondary GBM after RT); one pair of locally recur- during MB recurrence. To overcome the treatment resistance rent-distantly metastasized MB without a primary tumor of recurrent MBs, it would be desirable to fnd and target sample; one pair of secondary GBM after RT, from two emergent genomic aberrations in recurrent tumors. independent operations, without a primary MB sample; and A confounding problem in the management of MB recur- one unpaired secondary GBM after RT without a primary rence is the presence of secondary malignancies [15]. Whole MB sample. Normal DNA from peripheral whole blood cells neuro-axis radiation is the mainstay of treatment for MBs. (WBCs) was available for 12 patients. For DNA and RNA Therefore, secondary malignancy, especially malignant extraction from frozen tissues, DNeasy Blood and Tissue gliomas, can develop in a small percentage of survivors [5]. Kit (Qiagen) and mirVana miRNA Isolation Kit (Invitro- Clinically, it is often challenging to diferentiate between the gen) were used, respectively. Extraction of DNA and RNA two entities based solely on imaging results. Furthermore, from FFPE tissues was carried out using RecoverAll Total matched primary and recurrent tumor tissues are uncommon Nucleic Acid Isolation Kit (Thermo Fisher Scientifc). because surgery is usually not indicated for recurrent MBs. Even pathological examinations often provide misleading RNA sequencing and analysis information due to histological ambiguity, such as high degrees of anaplasia during recurrence. However, misdiag- mRNA sequencing was performed using a TruSeq RNA nosis of secondary glioblastoma (GBM) after radiotherapy Library Preparation Kit and TruSeq RNA Access Kit (Illu- (RT) as recurrent MB can result in grim outcomes because mina) and sequenced on an Illumina HiSeq 2000 or 2500 the treatment protocols and regimens are diferent. platform. We used an in-house script to trim low-quality Genomic and epigenomic analyses have shown great reads [proportion of ambiguous nucleotide (N ratio) > 0.1 potential not only in stratifying diseases but also in correct- or proportion of nucleotide with low Phred quality score ing misdiagnoses [49]. In the genomic analyses of primary (< 20) > 0.4]. The filtered sequence data were analyzed and recurrent MBs, we found that a subset of recurrent using STAR [7] for alignment, Cufinks v2.2.1 1 [47] for tumors contains mutations or gene fusion events in PDG- assembly, and a known set of reference transcripts from FRA, which is not characteristic of MB. The gene expression Ensembl v72 for expression estimation. The details of RNA pattern of these recurrent tumors co-segregated with that of sequencing and alignments are summarized in Table S2. GBM rather than MB. Re-evaluation of histological images Gene fusions were identifed by searching for the spanning 1 3 Acta Neuropathologica reads and split reads by ChimeraScan