Over the last 30 years, widespread efforts to characterize

Transcription

1 GALANIS ET AL Integrating Genomics Into Neuro-Oncology Clinical Trials and Practice Evanthia Galanis, MD, Farhad Nassiri, MD, Shannon Coy, MD, Romina Nejad, HBSc, MSc (C), Gelareh Zadeh, MD, PhD, and Sandro Santagata, MD, PhD OVERVIEW Important advances in our understanding of the molecular biology of brain tumors have resulted in a rapid evolution in the taxonomy of central nervous system (CNS) tumors, which culminated in the revised 2016 World Health Organization classification of CNS tumors that incorporates an integrated molecular/histologic diagnostic approach. Our expanding understanding of brain tumor genomics and molecular evolution during the disease course has started to impact clinical management. Furthermore, incorporation of genomic information in ongoing and planned neuro-oncology clinical trials is expected to lead to improved outcomes and result in personalized treatment options for patients with CNS malignancies. Over the last 30 years, widespread efforts to characterize chromosomal abnormalities, genomic mutations, epigenetic alterations, and proteomic changes in cancer cells have rapidly increased our understanding of the molecular biology of neoplasia. 1-6 These advances have challenged the histology-centric paradigm of tumor classification, demanding a reassessment of current diagnostic algorithms and categories and development of novel strategies for incorporating molecular and genetic data into the nosology of neoplasia. 7 A new paradigm is emerging, highlighted by the publication of the 2016 World Health Organization Classification of Tumors of the Central Nervous System, 8 which adopts, for the first time, diagnoses that integrate both histologic and molecular parameters. Application of these recent changes in the criteria and taxonomy of CNS tumors and in clinical practice results in both challenges and opportunities that are many and varied. HISTOLOGIC CLASSIFICATION OF CENTRAL NERVOUS SYSTEM TUMORS Systematic attempts to establish a universal taxonomy for CNS tumors began in the 1950s with the groundbreaking Armed Forces Institute of Pathology and Union for International Cancer Control texts. These efforts were, however, not met with widespread international acceptance. 9,10 Subsequently, World Health Organization (WHO) working groups were established to define international diagnostic standards. The first meetings of a CNS-focused group convened in 1970, culminating in the first WHO blue book for CNS tumors that was published in Similar to classification schemes in other organ systems, the WHO classification of CNS tumors has typically been organized by the presumed histogenesis of tumors ascertained through morphology and immunohistochemistry (e.g., astrocytic, oligodendroglial, or ependymal tumors). Notably, in contrast to the staging of other solid tumors, which relies on the TNM classification system, typically based on tumor size and extent of spread, 12 prognostic information for CNS tumors is conveyed using a WHO grade designation. Grading is based on histologic characteristics such as mitotic rate or vascular proliferation, which have more prognostic value for CNS tumors than does tumor size or extent of invasion. Numerous clinicopathologic studies have driven the iterative and continual refinement of WHO diagnostic categories and these changes have been codified in new editions and revisions of the WHO CNS manual in 1993, 2000, 2007, and ,13-15 INTEGRATED MOLECULAR CLASSIFICATION OF CENTRAL NERVOUS SYSTEM TUMORS In addition to numerous advances in the histologic classification of CNS tumors in each subsequent edition of the WHO manual, investigators across the field of neurooncology have uncovered distinct patterns of recurrent genomic alterations in many CNS neoplasms. Such molecular and genetic findings were included in the 2000 and 2007 From the Division of Medical Oncology, Department of Oncology, Mayo Clinic, Rochester, MN; Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, ON, Canada; Department of Pathology, Brigham and Women s Hospital, Harvard Medical School, Boston, MA; MacFeeters Hamilton Centre for Neuro-Oncology Research, University of Toronto, Toronto, ON, Canada; Ludwig Center at Harvard, Department of Pathology, Boston Children s Hospital, and Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA. Disclosures of potential conflicts of interest provided by the authors are available with the online article at asco.org/edbook. Corresponding author: Evanthia Galanis, MD, Division of Medical Oncology, Department of Oncology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905; galanis.evanthia@ mayo.edu American Society of Clinical Oncology ASCO EDUCATIONAL BOOK asco.org/edbook

2 EVOLVING ROLE OF GENOMICS IN NEURO-ONCOLOGY WHO classifications, but at the time, these were considered as supplemental to the histologic diagnoses. However, as the number of findings has increased and clinicopathologic analyses have matured, the International Society of Neuropathology and WHO working group surveys supported inclusion of molecular criteria in tumor classification with the aim of increasing diagnostic reproducibility and improving patient care. These insights were incorporated into the 2016 revision of the WHO classification. The most substantial changes were in the classification of diffuse gliomas and embryonal tumors, which provide illustrative examples of the new paradigm of integrated molecular classification. Diffuse Gliomas Since their first classification by Bailey and Cushing, 16 diffuse gliomas have been classified by their presumed lineage differentiation (astrocytic or oligodendroglial) based on morphologic features. Oligodendrogliomas were among the first CNS tumors in which specific molecular genetic alterations were recognized, with the identification in 1994 of the codeletion of chromosomal arms 1p and 19q. 17 Further studies showed improved outcome and response to chemotherapy in tumors with these alterations, providing tantalizing proof-of-principle evidence that specific molecular alterations may be simultaneously diagnostic, prognostic, and predictive of treatment response. 18 In 2008, integrated genomic analysis identified recurrent mutations in the isocitrate dehydrogenase genes (IDH1/2), most commonly the IDH1 R132H mutation, in nearly all secondary glioblastomas. Subsequent studies showed that these mutations were also present in the majority of low-grade astrocytomas and oligodendrogliomas. Clinical correlation showed that these mutations are highly predictive of long-term prognosis. 19,20 PRACTICAL APPLICATIONS The 2016 World Health Organization classification of CNS tumors resulted in a major restructuring of some of the most common CNS tumors, including glioma and medulloblastoma. Characterization for IDH mutations is now required as part of the histologic characterization of diffuse gliomas. The presence of both 1p/19q codeletion and IDH mutations is necessary for the diagnosis of oligodendroglioma to be established, and it is impacting the standard-of-care treatment of these patients. The recognition of the molecular evolution and transformation of gliomas during the disease course highlights the importance of repeat biopsies at recurrence. A number of ongoing clinical trials for tumors such as glioma, medulloblastoma, meningioma, and craniopharyngioma are using biomarker-based and adaptive designs that are expected to result in the development of personalized treatment approaches for patients with primary CNS tumors. Additional work identified alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutations in astrocytomas and secondary glioblastomas, which frequently co-occur with mutations in tumor protein 53 (TP53). 21 Such advances suggested that the integration of genotypic findings may provide more biologically relevant diagnostic categories, such as the presence of 1p/19q codeletion and IDH1/2 mutations for oligodendroglioma; of TP53, ATRX, and IDH1/2 mutations for astrocytomas and secondary glioblastomas; and of epidermal growth factor receptor (EGFR) amplification and CDKN2A/2B deletion among other aberrations for primary glioblastomas. 22 Clinicopathologic and molecular studies to explore this possibility showed that integrated molecular data provided superior prognostic significance than traditional histologic classification alone. 23,24 For example, detection of the 1p/19q codeletion in morphologically defined oligoastrocytomas or oligodendrogliomas was associated with clinical outcomes more concordant with genotype rather than histologic phenotype or grade. Thus, targeted gene sequencing and copy-number analysis is becoming increasingly essential for the proper classification of most brain tumors. For gliomas, including the most common malignant glioma diagnosis (i.e., glioblastoma), a complete workup requires IDH mutation assessment by immunohistochemistry using antibodies that recognize the IDH1 R132H mutant protein (Fig. 1) and/or mutation hotspot sequencing that can identify alternative IDH1 mutations such as R132C or IDH2 mutations. Glioblastomas are divided in the 2016 WHO classification of CNS tumors into (1) glioblastoma, IDH-wild type (approximately 95% of cases), which corresponds most frequently with the clinically defined primary or de novo glioblastoma and predominates in patients older than age ; (2) glioblastoma, IDH-mutant (approximately 5% of cases), which frequently corresponds to secondary glioblastoma with a history of prior lowergrade diffuse glioma and preferentially arises in younger patients 25 ; and (3) glioblastoma, not otherwise specified, a diagnosis that is reserved for those tumors for which full IDH evaluation cannot be performed. The definition of full IDH evaluation can differ for older patients with glioblastoma relative to younger adults with glioblastoma and relative to WHO grade II and grade III diffuse gliomas. In the latter situations, IDH sequencing is highly recommended following negative R132H IDH1 immunohistochemistry, whereas the near absence of non-r132h IDH1 and IDH2 mutations in glioblastomas from patients older than approximately age suggests that sequencing may not be needed in the setting of negative R132H IDH1 immunohistochemistry among such patients. 27 In the case of oligodendroglioma and oligoastrocytomas, in addition to IDH mutation, testing for 1p/19q deletion by fluorescence in situ hybridization, array comparative genomic hybridization, or next-generation sequencing is also needed. Accordingly, institutions that do not offer such molecular testing will frequently be required to request testing from outside laboratories. Assays that characterize both mutations and copy-number alterations, such as targeted next-generation sequencing, asco.org/edbook 2018 ASCO EDUCATIONAL BOOK 149

3 GALANIS ET AL provide an efficient approach for capturing the data needed for glioma classification as well as for identifying a wide range of other aberrations that occur at low frequency but that may inform tumor classification, patient care, and clinical trial decisions such as BRAF V600E mutations, histone gene mutations, FGFR fusions, and a range of focal amplifications or deletions. For instance, recurrent mutations in histone H3 genes define an infrequent entity codified in the 2016 WHO classification as H3K27M-mutant diffuse midline glioma. This mutation is thought to result in dysregulation of polycomb repressor complex 2 (PRC2) with resultant genome-wide epigenetic alterations. These tumors may exhibit histologic features of a high-grade (Fig. 2A) or low-grade (Fig. 2C) glioma and may be identified in most cases by immunohistochemistry specific for mutated histone H3F3A when suspected FIGURE 1. IDH1 Mutation in a Low-Grade Astrocytoma A B (A) Hematoxylin and eosin stained section of a World Health Organization grade II gemistocytic astrocytoma. (B) Immunohistochemistry using a mutation-specific antibody that recognizes IDH1 R132H mutant protein (brown staining), which is frequently mutated in low-grade astrocytomas, oligodendrogliomas, and secondary glioblastomas. clinically (Fig. 2B and D), but they can also be identified by sequencing technologies when they occur in atypical presentations. Although both high- and low-grade histologic phenotypes are thought to carry a dismal prognosis based on current clinicopathologic data and are correspondingly classified as WHO grade IV, further assessment of the prognostic and predictive value of such histologic features in cohorts of molecularly characterized cases is underway and may reveal stratification of outcomes based on histologic phenotype. Medulloblastoma The 2007 WHO classification defined multiple histopathologic variants of medulloblastoma with different clinical outcomes. Systematic transcriptomic, microrna, cytogenetic, and epigenomic studies sought to understand the differences between subtypes and had established by 2012 that multiple biologically distinct genotypic clusters could be defined with excellent cross-modality concordance, including those with activation of the Sonic hedgehog signaling pathway (SHH) or Wnt pathways Clinicopathologic studies subsequently showed that distinct biologic clusters had different clinical outcomes. For instance, there was an excellent prognosis for Wnt pathway-driven tumors, intermediate prognosis for SHH pathway-driven tumors and poor prognoses for non-shh/wnt tumors: these genotypic clusters frequently, but not always, correlated with specific histologic categories. 31 The revised 2016 WHO classification for the first time incorporates these advances into integrated diagnoses. Tumors are substratified into four histologic categories (classic, desmoplastic/nodular, extensive nodularity, and large cell/anaplastic) and four principle molecular categories (WNT-activated, SHH-activated, group 3, and group 4), allowing for treatment and prognostic stratification by pathway activity and histology. 32 As with the grading of diffuse gliomas, histologic descriptors remain useful for the diagnosis of medulloblastoma. Nearly all medulloblastomas with desmoplastic/nodular or extensive nodularity histology fall into the SHH-activated molecular group; these tumors have lower risk profiles and may be eligible for trials of hedgehog pathway inhibitors. WNT-activated tumors, a particularly low-risk group, nearly always exhibit classic histology. Anaplastic/large cell tumors are typically SHH-activated or group 3 tumors, and these tumors are associated with poor prognosis Other Tumors In addition to the examples listed above, the revised 2016 WHO classification includes several other molecularly defined entities, including RELA fusion-positive ependymoma and embryonal tumor with multilayered Rosettes, C19MC-altered. These entities follow a similar paradigm as described above, and extended discussion may be found elsewhere. 8 In addition, a not otherwise specified classification was added for lesions with atypical genotypes or cases lacking molecular data ASCO EDUCATIONAL BOOK asco.org/edbook

4 EVOLVING ROLE OF GENOMICS IN NEURO-ONCOLOGY EVOLVING PARADIGMS AND FUTURE DIRECTIONS IN BRAIN TUMOR HISTOLOGIC CLASSIFICATIONS The integrated diagnoses in the 2016 WHO classification incorporate a large number of canonical molecular alterations; however, the theoretical limit of biologic findings that may have clinical relevance is vast. Integrated molecular diagnoses may see a rapid expansion as novel assays expand the scope of genetic, molecular, and phenotypic profiling. Recent years have seen an explosion of genome-level sequencing data whose clinicopathologic contextualization is still in its infancy, and rapid advances in epigenomic and proteomic profiling and acute sensitivity testing approaches promise to enable far greater granularity of tumor classification. Glioblastomas, for instance, may prove to be effectively substratified for prognostication or clinical treatment by the presence of specific pathway alterations or combinations of alterations (e.g., EGFR amplification), epigenetic profiles such as the glioma CpG island methylator phenotype (G-CIMP) or O-6-methylguanine methyltransferase (MGMT) promoter methylation (a prognostic and possibly predictive factor), and numerous other as-yet undiscovered alterations. To address this complexity, future classification systems may alternatively seek to develop diagnoses with succinct condensations of the most clinically relevant features, or exploit computational methods to develop increasingly high resolution precision classification and treatment plans for each unique tumor. As in the taxonomy of all tumor types, the challenge for brain tumors will also be in establishing the merits of when to lump or split. 33 In the context of evolving classification systems, the establishment of clinical practice guidelines by the Society for Neuro- Oncology and the College of American Pathologists will foster the appropriate level of genetic testing for patients with brain tumors. Given the superiority of molecular analyses in discriminating a variety of lesions, an important question is whether histologic evaluation of tissue will remain necessary. We believe that histologic review will still be required for the foreseeable future. First, histologic evaluation of specimens remains the most efficient and accurate methodology for the initial diagnosis of neoplasia and is essential for determining the tissue regions most amenable to molecular profiling. Although molecular studies may become more precise at identifying specific cellular classifications of tumors, including possible epigenetic identification of tumor cell lineage, 34 histologic evaluation of tissue also reveals spatial information that is not available by molecular studies, such as the FIGURE 2. H3K27M-Mutant Diffuse Midline Glioma A B C D (A and B) Hematoxylin and eosin stained section (A) of an H3K27M-mutant diffuse midline glioma in the thalamus displaying high-grade morphology supported by immunohistochemistry (B) using an H3K27M mutation specific antibody (brown staining). (C and D) Hematoxylin and eosin stained section (C) of an H3K27M-mutant diffuse midline glioma displaying low-grade morphology in the cingulate gyrus supported by immunohistochemistry (C) using an H3K27M mutation specific antibody (brown staining). asco.org/edbook 2018 ASCO EDUCATIONAL BOOK 151

5 GALANIS ET AL spatial relationships between tumor cells and subgroups of immune cells may become increasingly important in the age of high-dimensional pathology. In addition, as illustrated by the present grading of diffuse gliomas, histologic features may retain superior diagnostic utility in specific situations, although continued analysis of genomic or epigenetic alterations may reveal more effective paradigms. TISSUE ACQUISITION BEFORE, DURING, AND AFTER TREATMENT: INCORPORATING GENOMICS INTO NEURO-ONCOLOGY PRACTICE The 2016 WHO classification of CNS tumors incorporates and mandates genomic analysis in routine neuropathology and neuro-oncology practice. There are two associated challenges, however. First, this improved classification paradigm applies primarily to initial diagnosis: the impact of treatment interventions on these molecular markers remains poorly characterized. Second, additional molecular alterations of prognostic or predictive significance (MGMT methylation in glioblastoma, V600E mutation in gliomas, TSC mutations in subependymal giant cell astrocytoma, etc.) were not included. Furthermore, this classification was only in part able to capitalize and include existing information on genetic, transcriptional, and epigenetic changes, which occur during glioma evolution. Therefore, serial tissue acquisition to guide clinicians in tailoring treatment to the dynamic changes seen in the tumor through the continuum of care is increasingly becoming a clinical mandate. Available technology, including next-generation sequencing, which is increasingly available for clinical use, 35 can ensure that the clinician s knowledge of the biology of the tumor is at least as up to date as the dynamic changes occurring in these malignancies and can create the foundation for future therapeutic advances. We anticipate that this approach could hold the greatest immediate impact in the treatment of patients with glioma. A substantial body of existing evidence supports that recurrent gliomas undergo transformation and clonal evolution. For example, Riehmer et al 36 used genome-wide array comparative genomic hybridization analysis and showed that 75% of non-idh mutant recurrent glioblastomas in their series acquired new genomic aberrations while either maintaining or losing their primary tumor aberrations, referred to as sequential and discrepant pairs, respectively. Similarly, recent work by Johnson et al 37 comparing 23 matched primary to recurrent lower-grade gliomas by genome sequence analysis found an average of 33 somatic mutations in each primary tumor, of which only an average of 54% were also detected in the matched recurrent tumor. Of these reported somatic mutations, only the prognostically favorable IDH mutation was shared in every matched recurrent case, suggesting that IDH mutation remains intact through serial tissue acquisition. Because IDH mutation is believed to be an early initiation event in gliomagenesis and is maintained through serial tissue acquisition, downstream molecular alterations tightly coexpressed with IDH mutation, such as 1p/19q codeletion, ATRX, and TP53 mutations, are also shared in most but not all cases, despite treatment and tumor recurrence. 37,38 In contrast, genomic events that occur later in glioma evolution, such as aberrations in receptor tyrosine kinases platelet-derived growth factor receptor A and EGFR, as well as phosphatase and tensin homolog (PTEN) mutation, have been reported to switch with tumor progression due to divergent clonal selection and their predominantly mutually exclusive presence in glioblastoma cells Of practical importance, in the series by Johnson et al, 37 exposure of these patients with low-grade glioma to the commonly used chemotherapeutic agent temozolomide resulted in the acquisition of mutations in the DNA mismatch-repair pathway and the development of a hypermutated phenotype. In addition to the information gathered from bulk tumor analysis, important information can be derived from rapidly evolving state-of-the-art technology that now allows for genomic profiling of single cells derived from a bulk tumor. Results from the single-cell-omics approach in gliomas support that primary tumors represent a heterogeneous group of subclones that, upon treatment, undergo selective pressure resulting in divergent clonal evolution at recurrence. This theory highlights the importance of developing clonalspecific treatments, as opposed to a bulk one-stop-fitsall paradigm, and can better explain primary and secondary resistance to chemotherapy. For example, Meyer et al 43 demonstrated that treatment-naïve patients with glioblastoma harbor temozolomide-resistant clones regardless of MGMT promoter status. This may explain the variability of responses to temozolomide even within this predominantly sensitive group. 43 Taken together, these studies suggest that genomic evolution of recurrent gliomas could impact treatment decisions, including eligibility for targeted therapies or combinations, and support the rationale for tissue acquisition at recurrence. In conjunction with tissue-deriving information, ongoing research on liquid biopsies from blood or cerebrospinal fluid, which characterize genetic material in exosomes or circulating DNA, and advanced imaging, including functional or metabolic imaging, can create the foundation for the development of reproducible noninvasive measures for monitoring molecular evolution in gliomas (Fig. 3) INTEGRATING TUMOR GENOMICS INCLUDING WHO GUIDELINES INTO NEURO- ONCOLOGY CLINICAL TRIAL DESIGN Diffuse Astrocytic and Oligodendroglial Tumors Glioblastoma. Both the 2016 WHO classification of CNS tumors and our expanding knowledge of CNS tumor genomics are impacting neuro-oncology clinical trial design for many primary CNS tumors. The 2016 WHO classification of CNS tumors resulted in a major restructuring of diffuse gliomas with incorporation of genetically defined entities. As such, ongoing and future glioma trials must comply with this classification. For glioblastoma trials, ASCO EDUCATIONAL BOOK asco.org/edbook

6 EVOLVING ROLE OF GENOMICS IN NEURO-ONCOLOGY FIGURE 3. Workflow Demonstrating an Optimal Future Diagnostic and Treatment Selection Paradigm for Patients With Glioma in the New Molecular Era the impact overall is expected to be minor: the new classification introduces tumor characterization for the IDH status; nevertheless, less than 5% of glioblastomas harbor IDH mutations and as such, it is unlikely that the addition of this diagnostic criterion, even during the conduct of ongoing randomized trials, will impact results or imbalance groups. Despite the fact that it was not included in the 2016 WHO classification, MGMT methylation status at diagnosis represents a prognostic and possibly predictive factor for patients with glioblastoma. Methylation of the promoter of MGMT, a DNA repair enzyme, leads to decreased enzyme activity and impaired ability to repair temozolomide-induced DNA methylation. This is important because temozolomide in combination with radiation therapy is currently the standard-of-care treatment regimen for newly diagnosed glioblastoma based on the results of the EORTC/NCIC CE3 randomized phase III trial. 50 Retrospective analysis in a subgroup of 206/572 study patients for whom baseline tumor samples were available demonstrated that patients with MGMT promoter methylation were more likely to benefit from the addition of temozolomide. 51 Long-term results of this trial 52 showed that MGMT promoter methylation status was the strongest predictive factor for survival. At 3 and 5 years, 27.6% and 15.8%, respectively, of patients with MGMT methylation who received combination treatment were alive compared with 11.1% and 8.3% of patients without MGMT methylation who received combination treatment. For patients treated with radiation alone, MGMT promoter methylation was associated with a 7.8% and 5.2% survival at 3 and 5 years, respectively, whereas no patient without MGMT methylation survived beyond 3 years. Analysis of progression-free survival showed an advantage only for patients whose tumor had a methylated MGMT promoter and who were treated with temozolomide and radiotherapy (RT). 53 These data support the prognostic value of MGMT methylation for patients with newly diagnosed glioblastoma and a possible predictive value because it impacted progression-free survival after temozolomide therapy, butthe impact on overall survival did not reach statistical significance. 52 A number of subsequent phase III trials, including RTOG 0525, 54 RTOG 0825, 55 and AVAGLIO, 56 confirmed the prognostic value of MGMT methylation: patients without MGMT methylation have a median survival ranging from 14 to 16.2 months, whereas patients with MGMT methylation have a median survival ranging from 22 to 26 months. As such, stratification based on MGMT status is viewed as mandatory in newly diagnosed glioblastoma trials because imbalance of this prognostic factor can confound the results. In addition, MGMT promoter methylation can lead to greater sensitivity of glioma cells to other agents such as PARP inhibitors that also block DNA repair. 57 Based on preclinical data confirming that the PARP inhibitor veliparib statistically significantly enhanced the efficacy of temozolomide in patient-derived glioblastoma xenografts with MGMT promoter methylation, 57 MGMT promoter methylation is used as an eligibility criterion for A (NCT ), a biomarker enrichment design based phase II/III Alliance clinical trial evaluating temozolomide/veliparib versus placebo in the adjuvant treatment of patients with newly diagnosed glioblastoma. This trial uses a marker by treatment interaction seamless phase II/III design and has currently progressed to the randomized phase III stage. 58 Despite the minimal clinical progress with only three new therapies having been approved by the U.S. Food and Drug Administration for the treatment of glioblastoma in the last 15 years (temozolomide, bevacizumab, and Optune), substantial molecular knowledge has been gained as a result of large-scale genome sequencing projects such as The Cancer Genome Atlas. 59 In parallel, many new therapies have been developed for clinical testing. These advances lead to optimism that molecularly based precision medicine may improve outcomes for patients with glioblastoma, but they also highlight the limitations of current clinical trial designs that do not test multiple therapies and biomarker combinations simultaneously. At least two clinical trials in glioblastoma are attempting to address this gap: INSIGhT (Individualized Screening Trial of Innovative Glioblastoma Therapy) is ongoing 60 and AGILE (Adaptive Global Innovative Learning Environment for Glioblastoma) is in the late planning stages. 61 In both trials, comprehensive multiplex genomic analysis will be conducted for each patient to identify biomarker signatures prior to treatment assignments. For example, for the INSIGhT trial, initial biomarker classifiers are based on four specific pathway markers: EGFR amplification mutation; PI3K asco.org/edbook 2018 ASCO EDUCATIONAL BOOK 153

7 GALANIS ET AL activation (PTEN loss through analogous deletion or mutation plus deletion), PIK3CA mutation, or PIK3R1 mutation; p53 status (MDM2-4 amplification or p53 wildtype); and CDK (CDK4/6 amplification or CDKN2A disomy). Both trials follow Bayesian adaptive designs, which allow continuous evaluation of a priori biomarker hypothesis and associations with drug efficacy, with subsequent adaptation of randomization should an association be found. In the AGILE trial, which targets both patients with newly diagnosed and recurrent glioblastomas, effective therapies identified in this first learning stage will also transition in an inferentially seamless manner to a second confirmatory stage that uses fixed randomization to confirm findings and support regulatory registration. 61 These trial designs could potentially have substantial applicability in the development of personalized treatments for patients with glioblastoma and could expedite new drug development. 62,63 Oligodendroglioma. The diagnosis of oligodendroglioma and anaplastic oligodendroglioma according to the 2016 WHO classification requires demonstration of both an IDH gene family mutation and combined whole arm losses of 1p and 19q (1p/19q codeletion). The RTOG and EORTC trials convincingly demonstrated that 1p/19q codeletion is both a prognostic factor as well as a predictive factor of the efficacy of procarbazine, CCNU, and vincristine (PCV) chemotherapy, administered either prior to or following radiation for newly diagnosed patients. For example, the addition of PCV led to doubling of overall survival for these patients (from 7.3 years in the RT-only group to 14.7 years in the RT/PCV group) and increased progression-free survival (from 2.9 years in the RT group to 8.4 years in the RT/PCV group) in RTOG 9402, establishing a new standard of clinical care for these patients. 64 IDH mutation was also predictive of response. 65,66 Importantly, survival and progression-free survival curves in these two anaplastic oligodendroglioma studies were identical to RTOG 9802, 67 a trial for patients with high-risk, low-grade glioma with similar treatment arms as in RTOG 9402, supporting that genotyping in this context is more important than grade in determining prognosis and therapy efficacy. These data, which have clearly associated 1p/19q and IDH mutations with both a better prognosis as well as chemotherapy efficacy, have played a critical role in shaping the design of the CODEL trial/ Alliance N0577 (NCT ), an international intergroup trial conducted by Alliance for Clinical Trials in Oncology, NRG Oncology, the Eastern Cooperative Oncology Group, and the Cooperative Trials Group for Neuro-Oncology. This trial started as a three-arm study comparing RT/PCV versus RT versus single-agent temozolomide among patients with 1p/19q codeleted anaplastic glioma. Based on evolving molecular and clinical knowledge gained from the RTOG 9402, EORTC 2695, and RTOG 9802 trials and the 2016 WHO classification, the N0577 trial has now modified eligibility rules that allow enrollment of all patients with oligodendroglioma, 1p/19q codeleted and IDH mutant, independent of grade. In its current design, the N0577 trial randomly assigns patients to RT followed by PCV versus RT with temozolomide followed by temozolomide and represents a prime example of how our evolving knowledge in tumor genomics can inform and alter clinical trial design in neuro-oncology. Medulloblastoma The 2016 WHO classification of CNS tumors has led to major restructuring of medulloblastomas with the incorporation of genetically defined entities that describe subgroups with distinct molecular characteristics, demographics, and prognosis. Use of this classification in clinical trials is expected to both increase the likelihood of informative results, because by definition clinical trial populations in medulloblastoma trials will be more homogenously defined, and also allow investigators to better tailor development of new treatments. Aggressive treatments with high toxicity should be reserved for subgroups with poor prognosis (e.g., group 3 or 4) in which the outcomes remain dismal, although a potential decrease of treatment intensity could be acceptable in groups such as WNT that have excellent prognosis in order to mitigate unnecessary exposure to short- and long-term treatment-related side effects. In addition, this new classification encourages incorporation of targeted treatments for groups where such treatments exist. For example, an ongoing St. Jude trial (A Clinical and Molecularly Risk-Directed Therapy for Newly Diagnosed Glioblastoma, NCT ) uses both a clinical risk and a molecular subtype based classification to customize treatment of patients with medulloblastoma, including use of a targeted agent, the SHH pathway inhibitor vismodegib for patients in the SHH subgroup. Other Tumor Types Despite the fact that they are not included in the 2016 WHO classification, a number of driver mutations have been identified in other primary brain tumors, creating hope for effective targeted treatments. One such example is meningiomas, which represent the most common primary brain tumors among adults, with an incidence of 140,000 cases in the United States. 68 Although surgery represents the mainstay of treatment of these tumors, there is a high risk of recurrence ranging from 20% for grade I up to 80% for grade III tumors, even following gross total resection. 69 For tumors that are refractory to RT and surgery, no medical treatment has been shown to be of proven benefit. NF2 inactivation represents the most common alteration observed in approximately 50% of meningiomas, whereas other less common alterations include AKT mutations (8% 13% of meningiomas) with 15% of meningiomas exhibiting PI3K/AKT1/mTOR pathway activation. 70,71 In addition, approximately 5% of meningiomas, especially skull base, olfactory groove meningiomas, can harbor SMO mutations. 70,71 Mutations in TRAF7, a proapoptotic ubiquitin ligase, were also identified in approximately one-quarter of these tumors in one study, 71 but the role of the TRAF7 mutations in the pathogenesis of meningioma is less clear. Based on this information, the Alliance for Clinical Trials in Oncology launched an umbrella trial (Alliance A071401, NCT ) in which tumors of patients with recurrent ASCO EDUCATIONAL BOOK asco.org/edbook

8 EVOLVING ROLE OF GENOMICS IN NEURO-ONCOLOGY meningioma are sequenced and, depending on the predominant genetic alteration, patients are assigned to one of three treatment arms: (a) patients with NF2 mutations are treated with a FAK inhibitor, based on data supporting a synthetic lethal relationship of FAK inhibition in the context of Merlin (NF2 gene product) deficiency, 72 (b) patients with AKT mutations are treated with an AKT inhibitor, whereas (c) patients with SMO or PTCH mutations are treated with a SMO inhibitor. The fact that these driver genetic alterations are mutually exclusive increases the enthusiasm for the therapeutic potential of this approach. Craniopharyngiomas are another primary CNS tumor with molecular subtypes for which targeted treatments are being developed. These are rare suprasellar tumors that occur among children and adults and can cause substantial impairment through compression of critical structures and morbidity of treatments. 73 Subtypes are recognized based on single driver mutations and include the 600E BRAF mutation in approximately 95% of papillary craniopharyngiomas (seen predominantly in adults), whereas catenin beta-1 (CTNNB1) mutations were identified in 96% of adenomatous craniopharyngiomas (more common among children). 74 The combination of the BRAF inhibitor dabrafenib with the MEK inhibitor trametinib resulted in a rapid objective response and symptom resolution for a young adult with a papillary craniopharyngioma. 75 This led to a development of a phase II trial of BRAF/MEK inhibitors (Alliance A071601, NCT ) for this patient population. The study includes two separate cohorts and the BRAF/MEK inhibitor combination (vemurafenib/ cobimetinib) is administered either in the neoadjuvant setting (cohort A), with the goal being tumor regression to decrease the morbidity of standard-of-care treatment and improve long-term control, or in the recurrent disease (cohort B) setting for which no good treatment options exist. CONCLUSION Dramatic advances in our understanding of the molecular biology of tumors have driven a rapid evolution in the taxonomy of CNS tumors, with concomitant improvements in therapeutic decision-making and clinical outcomes. The revised 2016 WHO classification of CNS tumors clearly illustrates the modern paradigm of integrated molecular and histologic diagnoses, provides an effective framework for continued incorporation of molecular findings into pathologic diagnoses, and highlights the importance of surgical sampling not only at initial presentation but also at recurrence to more accurately capture the molecular evolution of these tumors and customize treatment. Future analysis of the increasingly vast amounts of information obtained from genomic sequencing, epigenetic profiling, and proteomic analyses is likely to greatly increase the precision and complexity of tumor diagnoses. Incorporation of this accumulating information in ongoing and future clinical trials is expected to result in better outcomes for patients with primary CNS tumors and lead to personalized treatment options. References 1. Akbani R, Ng PK, Werner HM, et al. A pan-cancer proteomic perspective on The Cancer Genome Atlas. Nat Commun. 2014;5: Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic landscape of glioblastoma. Cell. 2013;155: Weinstein JN, Collisson EA, Mills GB, et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. 2013;45: Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144: Wheeler DA, Wang L. From human genome to cancer genome: the first decade. Genome Res. 2013;23: Zack TI, Schumacher SE, Carter SL, et al. Pan-cancer patterns of somatic copy number alteration. Nat Genet. 2013;45: Louis DN. The next step in brain tumor classification: let us now praise famous men or molecules? Acta Neuropathol. 2012;124: Louis DN, Ohgaki K, Wiestler OD, et al (eds). World Health Organization Histological Classification of Tumours of the Central Nervous System, 4th ed. Lyon, France: International Agency for Research on Cancer; Kernohan JW, Sayre GP. Tumors of the Central Nervous System. Washington, DC: Armed Forces Institute of Pathology; International Union Against Cancer. Illustrated Tumor Nomenclature. Berlin, Germany: Springer-Verlag; Zulch KJ. Histological Typing of Tumours of the Central Nervous System. Geneva, Switzerland: World Health Organization; Brierley JD, Gospodarowicz MK, Wittekind C. TNM Classification of Malignant Tumours. Chichester, UK: Wiley-Blackwell; Kleihues P, Burger PC, Scheithauer BW. Histological typing of tumours of the central nervous system. In Sobin PH (series ed). World Health Organization International Histological Classification of Tumours, 2nd ed. Heidelberg, Germany: Springer-Verlag; 1993; Kleihues P, Cavenee WK (eds). Pathology and Genetics of Tumours of the Nervous System, WHO Classification of Tumours, 3rd ed. Lyon, France: International Agency for Research on Cancer; Louis DN, Ohgaki H, Wiestler OD, et al. WHO Classification of Tumours of the Central Nervous System, 4th ed. Lyon, France: International Agency for Research on Cancer; Bailey P, Cushing H. A Classification of Tumours of the Glioma Group on a Histogenetic Basis With a Correlated Study of Prognosis. Philadelphia, PA: JB Lippincott; Reifenberger J, Reifenberger G, Liu L, et al. Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. Am J Pathol. 1994;145: Cairncross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Inst. 1998;90: Parsons DW, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321: Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360: asco.org/edbook 2018 ASCO EDUCATIONAL BOOK 155

9 GALANIS ET AL 21. Liu XY, Gerges N, Korshunov A, et al. Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 mutations. Acta Neuropathol. 2012;124: Ozawa T, Riester M, Cheng YK, et al. Most human non-gcimp glioblastoma subtypes evolve from a common proneural-like precursor glioma. Cancer Cell. 2014;26: Ceccarelli M, Barthel FP, Malta TM, et al. Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell. 2016;164: Brat DJ, Verhaak RG, Aldape KD, et al. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N Engl J Med. 2015;372: Ohgaki H, Kleihues P. The definition of primary and secondary glioblastoma. Clin Cancer Res. 2013;19: Chen L, Voronovich Z, Clark K, et al. Predicting the likelihood of an isocitrate dehydrogenase 1 or 2 mutation in diagnoses of infiltrative glioma. Neuro Oncol. 2014;16: Louis DN, Perry A, Reifenberger G, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016;131: Northcott PA, Jones DT, Kool M, et al. Medulloblastomics: the end of the beginning. Nat Rev Cancer. 2012;12: Northcott PA, Korshunov A, Witt H, et al. Medulloblastoma comprises four distinct molecular variants. J Clin Oncol. 2011;29: Pomeroy SL, Tamayo P, Gaasenbeek M, et al. Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature. 2002;415: Ellison DW, Dalton J, Kocak M, et al. Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-shh/wnt molecular subgroups. Acta Neuropathol. 2011;121: Rudin CM, Hann CL, Laterra J, et al. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC N Engl J Med. 2009;361: Santagata S, Thakkar A, Ergonul A, et al. Taxonomy of breast cancer based on normal cell phenotype predicts outcome. J Clin Invest. 2014;124: Kundaje A, Meuleman W, Ernst J, et al; Roadmap Epigenomics Consortium. Integrative analysis of 111 reference human epigenomes. Nature. 2015;518: Byron SA, Tran NL, Halperin RF, et al. Prospective feasibility trial for genomics-informed treatment in recurrent and progressive glioblastoma. Clin Cancer Res. 2018;24: Riehmer V, Gietzelt J, Beyer U, et al; German Glioma Network. Genomic profiling reveals distinctive molecular relapse patterns in IDH1/2 wildtype glioblastoma. Genes Chromosomes Cancer. 2014;53: Johnson BE, Mazor T, Hong C, et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science. 2014;343: Watanabe T, Nobusawa S, Kleihues P, et al. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am J Pathol. 2009;174: Little SE, Popov S, Jury A, et al. Receptor tyrosine kinase genes amplified in glioblastoma exhibit a mutual exclusivity in variable proportions reflective of individual tumor heterogeneity. Cancer Res. 2012;72: Snuderl M, Fazlollahi L, Le LP, et al. Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma. Cancer Cell. 2011;20: Sottoriva A, Spiteri I, Piccirillo SG, et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad Sci USA. 2013;110: Szerlip NJ, Pedraza A, Chakravarty D, et al. Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response. Proc Natl Acad Sci USA. 2012;109: Meyer M, Reimand J, Lan X, et al. Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity. Proc Natl Acad Sci USA. 2015;112: Giusti I, Di Francesco M, Dolo V. Extracellular vesicles in glioblastoma: role in biological processes and in therapeutic applications. Curr Cancer Drug Targets. 2017;17: Pentsova EI, Shah RH, Tang J, et al. Evaluating cancer of the central nervous system through next-generation sequencing of cerebrospinal fluid. J Clin Oncol. 2016;34: Wang J, Bettegowda C. Applications of DNA-based liquid biopsy for central nervous system neoplasms. J Mol Diagn. 2017;19: Branzoli F, Di Stefano AL, Capelle L, et al. Highly specific determination of IDH status using edited in vivo magnetic resonance spectroscopy. Neuro Oncol. Epub 2017 Nov Huang KT, Ludy S, Calligaris D, et al. Rapid mass spectrometry imaging to assess the biochemical profile of pituitary tissue for potential intraoperative usage. Adv Cancer Res. 2017;134: Neill E, Luks T, Dayal M, et al. Quantitative multi-modal MR imaging as a non-invasive prognostic tool for patients with recurrent low-grade glioma. J Neurooncol. 2017;132: Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352: Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352: Stupp R, Hegi ME, Mason WP, et al; National Cancer Institute of Canada Clinical Trials Group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10: Fallon KB, Palmer CA, Roth KA, et al. Prognostic value of 1p, 19q, 9p, 10q, and EGFR-FISH analyses in recurrent oligodendrogliomas. J Neuropathol Exp Neurol. 2004;63: Gilbert MR, Wang M, Aldape KD, et al. Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial. J Clin Oncol. 2013;31: Gilbert MR, Dignam JJ, Armstrong TS, et al. A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med. 2014;370: Chinot OL, Wick W, Mason W, et al. Bevacizumab plus radiotherapytemozolomide for newly diagnosed glioblastoma. N Engl J Med. 2014;370: Gupta SK, Kizilbash SH, Carlson BL, et al. Delineation of MGMT hypermethylation as a biomarker for veliparib-mediated temozolomidesensitizing therapy of glioblastoma. J Natl Cancer Inst. 2015;108: djv ASCO EDUCATIONAL BOOK asco.org/edbook

10 EVOLVING ROLE OF GENOMICS IN NEURO-ONCOLOGY 58. Sargent DJ, Conley BA, Allegra C, et al. Clinical trial designs for predictive marker validation in cancer treatment trials. J Clin Oncol. 2005;23: Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455: Alexander BM, Galanis E, Yung WK, et al. Brain Malignancy Steering Committee clinical trials planning workshop: report from the Targeted Therapies Working Group. Neuro Oncol. 2015;17: Alexander BM, Ba S, Berger MS, et al. Adaptive global innovative learning environment for glioblastoma: GBM AGILE. Clin Cancer Res. 2018;24: Berry DA. Bayesian clinical trials. Nat Rev Drug Discov. 2006;5: Galanis E, Wu W, Sarkaria J, et al. Incorporation of biomarker assessment in novel clinical trial designs: personalizing brain tumor treatments. Curr Oncol Rep. 2011;13: Cairncross G, Wang M, Shaw E, et al. Phase III trial of chemoradiotherapy for anaplastic oligodendroglioma: long-term results of RTOG J Clin Oncol. 2013;31: van den Bent MJ, Brandes AA, Taphoorn MJ, et al. Adjuvant procarbazine, lomustine, and vincristine chemotherapy in newly diagnosed anaplastic oligodendroglioma: long-term follow-up of EORTC brain tumor group study J Clin Oncol. 2013;31: Cairncross JG, Wang M, Jenkins RB, et al. Benefit from procarbazine, lomustine, and vincristine in oligodendroglial tumors is associated with mutation of IDH. J Clin Oncol. 2014;32: Buckner JC, Shaw EG, Pugh SL, et al. Radiation plus procarbazine, CCNU, and vincristine in low-grade glioma. N Engl J Med. 2016;374: Ostrom QT, Gittleman H, Liao P, et al. CBTRUS Statistical Report: primary brain and other central nervous system tumors diagnosed in the United States in Neuro Oncol. 2017;19(suppl 5): v1-v Pasquier D, Bijmolt S, Veninga T, et al. Atypical and malignant meningioma: outcome and prognostic factors in 119 irradiated patients. A multicenter, retrospective study of the Rare Cancer Network. Int J Radiat Oncol Biol Phys. 2008;71: Brastianos PK, Horowitz PM, Santagata S, et al. Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations. Nat Genet. 2013;45: Clark VE, Erson-Omay EZ, Serin A, et al. Genomic analysis of non-nf2 meningiomas reveals mutations in TRAF7, KLF4, AKT1, and SMO. Science. 2013;339: Shapiro IM, Kolev VN, Vidal CM, et al. Merlin deficiency predicts FAK inhibitor sensitivity: a synthetic lethal relationship. Sci Transl Med. 2014;6:237ra Lober RM, Harsh GR IV. A perspective on craniopharyngioma. World Neurosurg. 2013;79: Brastianos PK, Taylor-Weiner A, Manley PE, et al. Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas. Nat Genet. 2014;46: Brastianos PK, Shankar GM, Gill CM, et al. Dramatic response of BRAF V600E mutant papillary craniopharyngioma to targeted therapy. J Natl Cancer Inst. 2015;108:djv310. asco.org/edbook 2018 ASCO EDUCATIONAL BOOK 157