Congress of neurological surgeons systematic review and evidence-based guidelines update on the role of neuropathology in the management of progressive glioblastoma in adults

These recommendations apply to adult patients with progressive or recurrent glioblastoma (GBM). For adult patients with progressive glioblastoma does testing for Isocitrate Dehydrogenase (IDH) 1 or 2 mutations provide new additional management or prognostic information beyond that derived from the tumor at initial presentation? Level III: Repeat IDH mutation testing is not necessary if the tumor is histologically similar to the primary tumor and the patient’s clinical course is as expected. For adult patients with progressive glioblastoma does repeat testing for MGMT promoter methylation provide new or additional management or prognostic information beyond that derived from the tumor at initial presentation and what methods of detection are optimal? Level III: Repeat MGMT promoter methylation is not recommended. For adult patients with progressive glioblastoma does EGFR amplification or mutation testing provide management or prognostic information beyond that provided by histologic analysis and if performed on previous tissue samples, does it need to be repeated? Level III: In cases that are difficult to classify as glioblastoma on histologic features EGFR amplification testing may help in classification. If a previous EGFR amplification was detected, repeat testing is not necessary. Repeat EGFR amplification or mutational testing may be recommended in patients in which target therapy is being considered. For adult patients with progressive glioblastoma does large panel or whole genome sequencing provide management or prognostic information beyond that derived from histologic analysis? Level III: Primary or repeat large panel or whole genome sequencing may be considered in patients who are eligible or interested in molecularly guided therapy or clinical trials. For adult patients with progressive glioblastoma should immune checkpoint biomarker testing be performed to provide management and prognostic information beyond that obtained from histologic analysis? Level III: The current evidence does not support making PD-L1 or mismatch repair (MMR) enzyme activity a component of standard testing. For adult patients with progressive glioblastoma are there meaningful biomarkers for bevacizumab responsiveness and does their assessment provide additional information for tumor management and prognosis beyond that learned by standard histologic analysis? Level III: No established Bevacizumab biomarkers are currently available based upon the inclusion criteria of this guideline.


Abstract Target population
These recommendations apply to adult patients with progressive or recurrent glioblastoma (GBM).

Question
For adult patients with progressive glioblastoma does testing for Isocitrate Dehydrogenase (IDH) 1 or 2 mutations provide new additional management or prognostic information beyond that derived from the tumor at initial presentation?

Recommendation
Level III: Repeat IDH mutation testing is not necessary if the tumor is histologically similar to the primary tumor and the patient's clinical course is as expected.
Question For adult patients with progressive glioblastoma does repeat testing for MGMT promoter methylation provide new or additional management or prognostic information beyond that derived from the tumor at initial presentation and what methods of detection are optimal?
Recommendation Level III: Repeat MGMT promoter methylation is not recommended.
Question For adult patients with progressive glioblastoma does EGFR ampli cation or mutation testing provide management or prognostic information beyond that provided by histologic analysis and if performed on previous tissue samples, does it need to be repeated?

Recommendation
Level III: In cases that are di cult to classify as glioblastoma on histologic features EGFR ampli cation testing may help in classi cation. If a previous EGFR ampli cation was detected, repeat testing is not necessary. Repeat EGFR ampli cation or mutational testing may be recommended in patients in which target therapy is being considered.

Introduction
Rationale Glioblastoma is the most common primary brain tumor in adults, it is also one the most malignant and fatal brain cancers with a median survival time of only 15 months. [1; 2] Because of the dismal prognosis, intensive standard therapy including surgical resection, radiotherapy, and chemotherapy is employed early in the course of the disease. [3] Despite this, nearly all glioblastomas will eventually recur and no effective standard treatment strategy against recurrent glioblastoma has been established. [4] While highly variable among institution and clinical setting approximately 25-40% of patients with recurrent glioblastoma will undergo repeat surgery. [5][6][7] Unfortunately the radiation and adjuvant temozolomide therapy that is part of the current standard treatment for primary glioblastoma may produce tissue injury and necrosis that can be di cult to distinguish from recurrence radiologically.[8-10] These same therapy related effects that can cause radiographic uncertainty can also cause challenges for classi cation and grading of progressive glioblastoma histologically as well. Knowledge of the patient's clinical history and treatment status, neurosurgical impression, and the neuroradiologic ndings are crucial. [8; 11] Additionally, it frequently means the patient has progressed after primary standard therapy and there may be increased interest in determining if the patient would be eligible for or may bene t from alternative treatment options.
We evaluated the current literature addressing the diagnosis of progressive GBM, including histologic alterations present in response to therapy. Ancillary studies including immunohistochemistry and molecular diagnostic techniques in this setting will also be evaluated. With the success of immunotherapy and targeted treatment options in other tumor types there is a greater interest in more comprehensive molecular-genetic evaluation so literature pertaining to these subjects will be reviewed.
Since the publication of the previous guideline the impact of select molecular features on the prognosis and progression of primary malignant brain tumors have been better recognized, some of which have been integrated into the revised 4th edition of the World Health Organization (WHO) Classi cation of Tumors of the Central Nervous System (CNS). [12] Objectives While the previously published guidelines[8] thoroughly delineated the histologic and immunohistochemical features of progressive glioblastoma limited studies were available at that time to assess what additional molecular diagnostics should be considered. Since that publication numerous studies looking at the role of ancillary and molecular studies for the diagnosis, prognosis, and treatment of glioblastoma have been published. This review addresses these advancements and reviews the recent literature to evaluate what ancillary testing is most appropriate in progressive glioblastoma to help guide treatment and prognosis and when ancillary testing is most appropriate.

Writing Group and Question Establishment
The evidence-based clinical practice guideline taskforce members and the Joint Tumor Section of the American Association of Neurological Surgeons (AANS) and the Congress of Neurological Surgeons (CNS) have prioritized an update of the guidelines for management of progressive glioblastoma. A series of writers were identi ed and screened for con ict of interest. This group in turn agreed on a set of questions addressing the role of neuropathology in the diagnosis of progressive GBM and conducted a systematic review of the literature relevant to the histopathologic diagnosis of progressive GBM in addition to immunohistochemical and molecular testing that can be used for either for diagnostic, prognostic, or treatment markers.

Literature Search
A search of PubMed, EMBASE, and Cochrane Library searches of the National Library of Medicine database of scienti c literature published between July 1, 2012 and March 31, 2019. A broad search strategy using the following search terms was employed: "Progressive glioblastoma" OR "recurrent glioblastoma" OR "relapsing glioblastoma" OR "treated glioblastoma" OR "recurrent glioma" OR "relapsing glioma" OR "treated glioma" AND diagnosis OR pathology OR cytopathology OR "frozen section" OR radionecrosis OR "radiation necrosis" OR pseudoprogression OR gliosis OR immunohistochemistry OR proliferation OR genetics OR genomics OR prognosis OR accuracy OR "predictive value" OR sensitivity OR grading OR histology OR molecular OR genetic OR IDH OR biomarker OR ki-67 OR morphology." We limited our searches to human studies published in the English language. Key words were searched in multiple combinations. Links to "related articles" from highly relevant studies were utilized to broaden the search. Articles were also identi ed from the reference lists from articles uncovered in initial searches.

Study Selection and Eligibility Criteria
Original articles providing information to establish histopathologic diagnostic criteria for progressive glioblastomas and addressing immunohistochemical and molecular testing, and biomarkers in in ltrating gliomas and progressive glioblastomas were selected for review. A greater focus was put on studies looking at progressive and recurrent IDH-wildtype glioblastomas.
The citations were screened for the following inclusion and exclusion criteria: Those abstracts that met with the selection criteria mentioned above were retrieved in full text form. The adherence to the selection criteria were con rmed. The information was then used for construction of the evidence tables the text below.

Data Collection Process
The search resulted in 923 articles, which were reviewed yielding 283 potentially eligible articles. Links to ''related articles'' from highly relevant studies were utilized to broaden the search. Articles were also identi ed from the reference lists from references uncovered in initial searches. We also analyzed the references from prior evidence-based reports on glioblastomas and progressive glioblastomas.

Assessment for Risk of Bias
Inherent in research related to pathologic studies in patients with progressive glioblastoma is that they represent patients who were able to undergo a second surgery, likely those who were in a better state of health or younger patients at the time of 2nd surgery. Additionally, these frequently represent patients with a relatively de nable lesion, patients who present with more in ltrative disease are often less optimal patients for repeat surgery. The relatively recent identi cation of IDH mutations and MGMT promoter methylation status and their marked prognostic implications and the reclassi cation of in ltrating gliomas based on IDH and other molecular features has made it di cult to use historical samples and publications in which molecular testing was not performed. Thus there is a relatively limited time frame of data in which publications assessing progressive glioblastomas with molecular features incorporated into the results and makes using older studies that did not differentiate between these entities di cult to apply to the current classi cation system. Additionally, pathologic studies are very frequently retrospective, these biases are noted by the authors and the evidence level strati cation does attempt to highlight these drawbacks to the reader.

Classi cation of Evidence and Recommendation Levels
The concept of linking evidence to recommendations has been further formalized by the American Medical Association (AMA) and many specialty societies, including the American Association of Neurological Surgeons (AANS), the Congress of Neurological Surgeons (CNS), and the American Academy of Neurology (AAN). This formalization involves the designation of speci c relationships between the strength of evidence and the strength of recommendations to avoid ambiguity. We utilized the "Classi cation of Evidence on Diagnosis" to evaluate the literature and a summary of this classi cation can be viewed at https://www.cns.org/guidelines/guideline-development-methodology. Much of the pathology literature addressed below are well designed and studied large cohorts giving meaningful outcome data. However, due to the retrospective nature and lack of prospective validation will qualify them as Class III. Generally, Level I recommendations are based on Class I evidence, Level II recommendations are based on Class II evidence and Level III recommendations are based on Class III evidence.

Summary and Commentary on Previously Published Neuropathology Guideline
As noted above this review is an update to the previously published guidelines for progressive glioblastoma by Brat et al.[8] It is useful to brie y review the questions and results from that paper.
The rst topic addressed the diagnostic considerations in reporting progressive glioblastoma. A level III recommendation that the pathologist consider the patient's previous diagnosis and treatment, as well as the current clinical and neuroimaging features that led to a second biopsy or resection. In the setting of prior radiation and chemotherapy, it was recommended the pathologist adhere to strict histologic criteria for microvascular proliferation and necrosis in order to establish a diagnosis of a glioblastoma.
For patients undergoing biopsy or neurosurgical resection at the time of radiologic or clinical progression, reporting the presence and extent of progressive neoplasm as well as the presence and extent of necrosis within the pathologic material examined was recommended.
[8] This recommendation continues to be supported in this update and with recent publications adding further credence to these as described below.
There is often a combination of radiation necrosis and progressive glioma in biopsy and resection specimens and it can be di cult to histologically assess disease status. Additional manuscripts published since the last version of this guideline re ne the understanding of this issue. This was particularly emphasized by the study conducted by Holdhoff, et al., in which only "marginal agreement" by Fleiss' kappa statistics was observed when 48 pathologists (92% of whom were neuropathologists) reviewed 13 cases of suspected recurrence of glioblastoma. [14] Much of this may be due to differential understanding of the terminology used in this this study. The terminology of "active tumor", "inactive tumor", and "treatment effect" are not well established in the literature [14] yet this is commonly used terminology.  Table II). Thus, it is recommended that the percentage of viable tumor and the percentage of radiation necrosis is documented. If available the proliferative index as assessed by MIB-1 immunohistochemistry may also be informative, but it is not felt there is enough evidence at this time to include this as part of the recommendation (See Table II Classi cation of Tumors of the Central Nervous System [12] which incorporates selected immunohistochemical and genetic features in the classi cation system. Thus, while the prior review focused on using these markers to determine the presence and in some cases quantity of tumor present, several of these studies are now indicated for classi cation and prognostic uses as well.
The integration of ancillary studies is expected to expand with the publication of the next World Health Organization Classi cation of Tumors of the Central Nervous System with some of the expected updates already published in the form of cIMPACT-NOW update recommendations.
[18] For the current review of progressive glioblastoma, criteria of the World Health Organization classi cation with the addition of the published cIMPACT-NOW update 3 recommendations will be used, since they represent a recent and updated international standard for classifying and grading. [12; 18] An additional cIMPACT-NOW update 5 was published after the designated time interval of the search for this guideline and the information in this document are not used to formulate the updated recommendations. [19] It is of value to brie y expand on the evolution of the immunohistochemical and molecular markers alluded to in the second question of the prior guideline as it provides background on the new questions asked in this update as noted below. The 2016 WHO classi cation divides the diffuse gliomas into IDHwildtype astrocytomas, IDH-mutant astrocytomas, IDH-mutant and 1p/19q codelted oligodendrogliomas, and H3K27M-mutant diffuse midline glioma. A 3-tiered grading system is used in grading diffuse astrocytic gliomas, and a 2-tiered grading system of oligodendrogliomas. [12] IDH-wildtype diffuse astrocytomas tend to arise in an older patient population with large majority of them presenting as de novo glioblastomas.
[20] As such IDH-wildtype diffuse astrocytoma, WHO grade II and anaplastic astrocytoma (WHO grade III) are recognized as provisional entities in the 2016 WHO and multiple studies have concluded that a substantial subset of these demonstrate an aggressive clinical course most akin to IDH-wildtype glioblastoma, WHO grade IV. Recently the cIMPACT-NOW update 3 addressed this by recommending that IDH-wildtype in ltrating astrocytomas that would histologically be classi ed as WHO grade II or III which carry and EGFR ampli cation, combined whole chromosome 7 gain and whole chromosome 10 loss, or a TERT promoter mutation be given an integrated diagnosis of "diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV." It is critical to note that other IDH-wildtype glial tumors that may enter the differential have been reported to harbor TERT promoter mutations and thus histologic examination of the specimen remains critical.  Table III).

Synthesis
Due to the early occurrence and conserved nature of IDH mutations repeat testing is not necessary if the tumor is histologically similar to the primary tumor and the patient's clinical course is as expected.
Question: For adult patients with progressive glioblastoma does repeat testing for MGMT promoter methylation provide new or additional management or prognostic information beyond that derived from the tumor at initial presentation and what methods of detection are optimal?
Study selection and Characteristics Thirteen papers were uncovered in the screening process that discussed alterations in MGMT promoter methylation status in progressive/recurrent glioblastoma. Eleven were selected for inclusion as 1 was a review article and 1 paper only had four paired tumors and did not speci cally note if MGMT promoter methylation status was retained in those four cases.
Results of individual studies, discussion of study limitations and risk of bias  Table IV).

Synthesis
Repeat MGMT promoter methylation testing does not need to be repeated upon recurrence. Either pyrosequencing or methylation speci c PCR can be used to assess MGMT promoter methylation, with pyrosequencing being the preferred method. Immunohistochemical testing for MGMT protein expression is not recommended for clinical use.
Question: For adult patients with progressive glioblastoma does EGFR ampli cation or mutation testing provide management or prognostic information beyond that provided by histologic analysis and if performed on previous tissue samples, does it need to be repeated?

Study selection and Characteristics
Twenty articles were uncovered in the screening process of which 9 articles were selected for inclusion in this review and 1 additional article was identi ed from the references of the included articles. Reasons for exclusion from the included publications included animal or in-vitro studies, phase 1 clinical trials that were ended early or in which therapeutic outcomes were not discussed, review articles or single case studies, or did not address pathology (studies with only imaging data).
Results of individual studies, discussion of study limitations and risk of bias  Table V).

Synthesis
Testing for EGFR ampli cation should be performed in cases of IDH-wildtype tumors that are di cult to grade as it may help classify the tumor as a glioblastoma. If a previous EGFR ampli cation was detected, retesting is not necessary. Repeat EGFR testing may be indicated for patients in which targeted therapy is being considered, particularly therapies targeting speci c EGFR mutations.
Question: For adult patients with progressive glioblastoma does whole genome or large panel sequencing provide management or prognostic information beyond that derived from histologic analysis?

Study selection and Characteristics
16 studies were uncovered by the search criteria and ten studies were included in this review. The remaining studies were excluded because they were review articles (4) or were focused on lower grade in ltrating gliomas (2).
Results of individual studies, discussion of study limitations and risk of bias The increase in genetic understanding has also sparked hope that more effective novel or targeted therapies may be available for particular subsets of patients. To this end multiple studies looking at possible targets, alterations in recurrence, and clinical trials involving targeted therapies have been performed or are underway. Most studies were performed using next generation sequencing to asses genomic alterations either using data from whole transcriptome sequencing, whole exome sequencing, or targeted panels. Targeted panels have the advantage of being the most cost effective with the analysis of the data being the most straight forward and having relatively high speci city however the genes included on the targeted panels can vary and requires enrichment of the target regions. Whole exome sequencing requires enrichment of the exons and analysis of the data to ensure optimal processing and sequencing reaction. Whole genome sequencing while giving a comprehensive view of all alterations is the most expensive and requires the most data analysis and interpretation and many of the identi ed alterations may not be therapeutically targetable or relevant to diagnosis.  Table VI).

Synthesis
Primary or repeat whole genome or large panel sequencing should be considered in patients in whose management may be impacted including those who are eligible or interested in targeted therapy based on a particular oncogenic pathway anomaly or pathway member or for assessment of eligibility in clinical trials based on a particular molecular characteristic.
Question: For adult patients with progressive glioblastoma should immune checkpoint biomarker testing be performed to provide management and prognostic information beyond that obtained from histologic analysis?

Study selection and Characteristics
Twenty-seven articles were uncovered during the screening process, however the majority of these represented review articles and in vitro or animal models. Other studies were excluded due to a lack of pathology data (radiology studies), phase 1 studies that did not include pathology or outcomes, and studies focused on pediatric patients. Two additional studies were identi ed from references of review articles identi ed in the screening process. Ultimately eight studies were included in the review.
Results of individual studies, discussion of study limitations and risk of bias: Immune checkpoint inhibitors have shown marked success in the treatment of a variety of solid cancer types by blocking immune checkpoint signaling and allowing a T-cell response against the tumor. Loss of MMR enzyme expression is also associated with a hypermutant genotype, while this is a small subset of patients (~10%), it is hypothesized that the increased mutagenesis may make these tumors more immunogenic and thus more amenable to immunotherapy[74; 84] (See Table VII). However, the e cacy of pembrolizumab or other immune checkpoint inhibitors has yet to be investigated in MMR de cient progressive glioblastomas has yet to be fully investigated.

Synthesis
If immune checkpoint inhibitors are being considered PD-L1 expression or loss of MMR enzyme activity should be determined but due to the limited bene t demonstrated by immune checkpoint agents in glioblastoma (primary or progressive) standard testing is not currently necessary.
Question: For adult patients with glioblastoma are Bevacizumab biomarkers available and should they be performed in the setting of progressive glioblastoma?

Study selection and Characteristics
Numerous studies (greater than 75) looking at Bevacizumab were identi ed however most were clinical trial papers, without examination of pathology, studies looking at radiologic biomarkers, or review articles. Eight articles looking at tissue biomarkers were identi ed and included in this review.

Results of individual studies, discussion of study limitations and risk of bias
Glioblastoma demonstrates marked up-regulation of VEGF-A and displays rapid vascularization. [4; 87] Bevacizumab is a monoclonal antibody against vascular endothelial growth factor (VEGF) that has been approved for treatment of recurrent glioblastoma. However, bevacizumab has not been shown to improve OS and there is concern that glioblastomas treated with bevacizumab are more aggressive and show increased in ltration.

Synthesis
Bevacizumab biomarker testing at this time remains experimental, markers which may be indicative of response include YKL-40, COL4A2, and FMO4 expression although con rmatory studies are warranted.

Discussion
If prior ancillary and molecular testing including IDH mutation status, MGMT promoter methylation status, and other relevant testing including chromosomal alterations and histone mutations were not performed on the primary resection specimen, it is recommended they be performed at the time of progression for de nite classi cation.
All of the recommendations presented within this review are based on class III data, usually due to the retrospective nature of the studies or small cohort size, however, the recommendations are based upon multiple class III studies whose results are coherent. Repeat testing of commonly assessed alterations, including IDH mutation, MGMT promoter methylation, and EGFR ampli cation, usually do not need to be repeated upon progression as they tend to remain stable, or

Conclusions And Key Issues For Future Investigations
Continuing work using whole genome, large-scale molecular studies, and methylation pro ling continues to further elucidate the alterations that occur in glioblastomas over time and following therapy. Further understanding of the genetic landscape of in ltrating gliomas has already allowed us a greater understanding of the heterogeneity within these tumors and allowed us to begin integrating these molecular alterations into a recent clinical trials where molecular characteristics were not part of the eligibility criteria and yet the overall results have been disappointing with only a minority of patients demonstrating a meaningful response. Identi cation of useful biomarkers that predict response or resistance for these targeted therapeutic agents will be critical for further progress.
One of the major hindrances within studies related to progressive glioblastoma is that patients who are candidates for re-resection are often younger and healthier patients with a de nable mass. Tissue from these cases may not re ect alterations present in more aggressive diffuse glioblastomas and further work to identify what alterations are present in these tumors will need to be undertaken.

WHO World Health Organization
Declarations Con ict of Interest (COI) All Guideline Task Force members were required to disclose all potential COIs prior to beginning work on the guideline, using the COI disclosure form of the AANS/CNS Joint Guidelines Review Committee. The CNS Guidelines Committee and Guideline Task Force Chair reviewed the disclosures and either approved or disapproved the nomination and participation on the task force. The CNS Guidelines Committee and Guideline Task Force Chair may approve nominations of task force members with possible con icts and restrict the writing, reviewing, and/or voting privileges of that person to topics that are unrelated to the possible COIs. The authors have no personal, nancial, or institutional interest in any of the drugs, materials, or devices described in this series of articles.

Data transparency
The author has ensured all data and materials as well as software applications or custom code supports their published claims and comply with eld standards.

Author Contributions
The author listed on this publication agrees with the content included and gives explicit consent to the submission of this publication. The author obtained consent from the responsible authorities at the institute/organization where the work has been carried out, before the work was submitted.
The author whose name appear on this submission: 1) made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; 2) drafted the work or revised it critically for important intellectual content; 3) approved the version to be published; and 4) agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Compliance with Ethical Standards
Funding: These guidelines were funded exclusively by the Congress of Neurological Surgery and the Joint Section on Tumors of the Congress of Neurological Surgeons and the American Association of Neurological Surgeons, which received no funding from any outside commercial sources to support the development of this document.

Ethical Approval
This article does not contain any studies with human participants performed by any of the authors.

Data Availability
The data generated during and/or analyzed during the current study are available via www.cns.org/guidelines.

Disclosures
These guidelines were funded exclusively by the Congress of Neurological Surgery and the Joint Section on Tumors of the Congress of Neurological Surgeons and the American Association of Neurological Surgeons, which received no funding from any outside commercial sources to support the development of this document.

Disclaimer of Liability
This clinical systematic review and evidence-based guideline was developed by a multidisciplinary physician volunteer task force and serves as an educational tool designed to provide an accurate review of the subject matter covered. These guidelines are disseminated with the understanding that the recommendations by the authors and consultants who have collaborated in their development are not meant to replace the individualized care and treatment advice from a patient's physician(s). If medical advice or assistance is required, the services of a competent physician should be sought. The proposals contained in these guidelines may not be suitable for use in all circumstances. The choice to implement any particular recommendation contained in these guidelines must be made by a managing physician in light of the situation in each particular patient and on the basis of existing resources.     Author's Conclusions: Formal criteria and terminology need to be developed to help improve consistency and reliability.

Comments and Conclusions:
Classi ed as Class III because study is a prospective study with determination of Kappa statistics, however, the cut-off points and delineations made by the authors limited the utility of the study and they either did not report or did not analyze all the data collected. presence of treatment effect was signi cantly associated with more recent RT therapy (8.9 mo. vs 13.8 mo., p=0.003). Patients treated with BEV prior to reoperation were less likely to have treatment effect (20% vs 65%, P < .001). The presence of treatment effect was not correlated with MGMT promoter methylation status or IDH-1 mutation. Treatment effect was associated with increased survival after re-operation (p=0.014) but not OS from primary resection (p=0.172).
Author's Conclusions: Treatment related changes are present in the majority of patients and is associated with earlier re-resection, improved survival from re-resection and unchanged OS from primary resection.

Comments and Conclusions:
Classi ed as Class III because study is a retrospective study and % tumor/treatment effect was not reported for the majority of pt's nor considered in the statistics, however there is a large number of pts. IDH mutation was retained in all 23 cases. 54% of mutations detected in the primary tumor were present upon recurrence. IDH1, TP53, and ATRX were the most commonly retained mutations. Of the 10 pts treated with TMZ 6 (60%) demonstrated a hypermutated phenotype upon progression and progressed to glioblastoma. 97% of TMZ associated mutations were C>T/G>A. Mutations involving the RB and AKT-mTOR pathways were identi ed only in pts who progressed to glioblastoma and none of the grade II-III recurrences. In a subset of patients these mutations seem to be linked to TMZ induced mutations.

Authors' Conclusions:
A signi cant proportion of mutations in primary gliomas are lost at recurrence. TMZ-treated pts showed TMZ-induced mutagenesis and a proportion hypermutagenasis and appeared to follow an evolutionary path to high-grade glioma distinct from that in untreated patients.

Comments and Conclusions:
Classi ed as Class III as it is a retrospective study with a limited sample size and little reported demographic information. This study attempted to get predominantly astrocytomas but likely included some oligodendrogliomas. driver genes, such as TP53, ATRX, CIC and FUBP1, in which mutations were observed in their lower-grade counterparts. During glioma progression, the receptor tyrosine kinase (RTK)-RAS-phosphoinositide 3-kinase (PI3K) pathway, the NOTCH pathway and FAT receptors were most frequently altered (in 22%, 12% and 10% of the patients, respectively). The most frequently ampli ed genomic region during progression was the MYC locus on chromosome 8q (22% of pts), and ampli cation was signi cantly associated with progression (q = 1.7 × 10−3, likelihood-ratio test) Author's Conclusions: During progression numerous convergent alterations occur, including activation of the MYC and RTK-RAS-PI3K signaling pathways, alterations in cell cycle regulators such as CDKN2A-CDKN2B, upregulation of FOXM1-and E2F2mediated cell cycle transitions, and epigenetic silencing of key developmental transcription factors.

Comments and Conclusions:
Classi ed as Class III as it is a retrospective study with a limited samples size. The cohort included oligodendrogliomas and not all tumors progressed to glioblastoma.  There is a strong association between the methods and methylation levels (p<0.001, p=0.002).
Methylation by qMSP and pyrosequencing and IHC was strongly associated (p=0.009 and P<0.001 respectively), IHC was positive in 43.9% and 48.8% of qMSP and pyrosequencing unmethylated samples, and negative in the majority of methylated 78% and 84.4% respectively.
MGMT promoter methylation was associated with better OS (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse OS (p = 0.009).
Author's Conclusions: Use of both MGMT promoter methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with TMZ-treated glioblastoma.

Comments and Conclusions:
Classi ed as Class III because it is a retrospective study with a relatively large number of specimens, however, factors other than methylation status were not evaluated. Author's Conclusions: MGMT promoter methylation conferred a profound delay in tumor progression compared to unmethylated (8.9 to 4.6 mo., p=0.016) and survival bene t (OS 14 vs 9 mo., p= 0.03). MGMT promoter methylation was also an independent prognostic factor for progression and survival after GKS.

Comments and Conclusions:
Classi ed as Class III because it is a retrospective study with a small number of pts, they only measure methylation at initial resection, and did not have a control arm of untreated pts Does not address re-assessing methylation at recurrence. 49% (55) of pts were MGMT promoter unmethylated (<10%), and 51% (56) were methylated (>10%). mPFS and OS in unmethylated GBM was 7.2 mo. and 13.4, for low level methylation (10-20%) PFS and OS was 10.04 and 17.9, respectively, and high level methylation (>20%) has PFS and OS of 19.83 and 29.93. PFS was signi cantly different in all 3 groups but OS was only signi cantly different between unmethylated and high level methylated. Methylation was also determined by sqMSP and dBisequ and results were discordant as compared to pyrosequencing in 53.1% and 54.5% of cases, however in cases >16% good consistency was seen between the 3 methods.
Author's Conclusions: MGMT promoter methylation between 10-20% represents a transition zone in terms of PFS and OS relative to unmethylated or highly methylated patients. Of patients with low methylation PSQ results could only be validated in 51.5% of cases by another method to be clearly methylated. Recommend a 3-tier system of unmethylated (0-9%), low level methylation (10-20%), and high level methylation (>20%).

Comments and Conclusions:
Classi ed as Class III as it is a retrospective single center study with a large relatively homogenous pt population.
The RR was 70% of cases (7/10), with 5 CRs and 2 PRs. PFS-6 was 70% (7/10 cases). Median PFS and OS were 8.0 mo.  Radiosensitive pts had a superior OS compared with RR pts either in radiotherapy-treated subset or in the patient subset that did not receive RT. Nevertheless, in the multivariate Cox regression analysis to assess for independent predictors of the relation between the gene signature and clinicopathologic features, we found that the gene signature is the strongest predictor (p=0.0093) in the subgroup of patients with radiotherapy, whereas it does not remain signi cant (p=0.202) in the non RT group when taking age and KPS into account.
Author's Conclusions: The radiosensitivity gene signature is mainly predictive in patients treated with radiation therapy. Radioresistant phenotype was enriched for genes of EMT, whereas radiosensitive phenotype correlated strongly with decrease of genes of EMT.

Comments and Conclusions:
Classi ed as Class III as it is a retrospective study, but does have a very large cohort, however, not all pts within the GEO cohort have GBM. Author's Conclusions: Gene set enrichment analysis revealed that chromatin fracture, repair, and remodeling genes were enriched in recurrent glioblastoma.

Comments and Conclusions:
Classi ed as Class III as it is a retrospective study with a very young population and conclusions are drawn that likely re ect reoperation bias more so than natural progression of the disease. All matched tumor pairs demonstrated differences in GA between the primary and recurrence including one resected without any intervening therapy. Four genes that were commonly altered in both primary and recurrent GBM CDKN2A (86%) and CDKN2B (86%) deletions, EGFR activating mutation (52%) or ampli cation (81%), and TERT mutation (95%). PI3K pathway activating mutations were also commonly seen in our cohort (67%). EGFR alterations correlated with shorter pt. survival but statistics were not performed due to low pt. # Author's Conclusions: Genetic alterations in GBM changed over time and with treatment, although some mutations are common to both the primary and recurrence. The loss of CDKN2A inhibits both the p53 and Rb pathways, in cases that did not show CDKN2A deletion other mechanisms of p53 and Rb disruptions was present (CDKN2A mutation, p53 + Rb mutations, and MDM2 + CDK4 ampli cations). TERT promoter mutation was the most common mutation (20/21). Screening both the primary and recurrent GBM may provide information that guides therapy.

Comments and Conclusions:
Classi ed as Class III as it is a retrospective study with a limited sample size.  (15) of recurrent GBMs and were primarily located in perivascular areas and zones of tumor invasion into surrounding parenchyma. PD-1 positive TIL density correlated positively with CD3 (P < .001), CD8 (P < .001), and CD20 TIL density (P < .001), and PTEN expression (P = .035) but not MGMT promoter methylation status or PD-L1 expression. PD-L1 expression did not correlate with MGMT promoter methylation status or PTEN expression. There was decreased epithelioid tumor cells with membranous PD-L1 labeling at recurrence but no differences were identi ed in diffuse/ brillary PD-L1 staining or PD-1 positive TILs. Proneural and G-CIMP glioblastoma subtypes had lower levels of PD-L1 gene expression. The mesenchymal subtype GBMs had high PD-L1 gene expression (P = 5.966e-10). No signi cant differences in TIL density between initial and recurrent GBM specimens were evident. Diffuse/ brillary PD-L1 expression did not differ between newly diagnosed and recurrent glioblastoma specimens (P = .411), however, epithelioid tumor cells with anti-PD-L1 membrane labeling were more common in initial tumors (9/18, 50%) than in recurrent tumors (3/18, 16.7%; P = .034).
Author's Conclusions: TILs and PD-L1 expression are detectable in the majority of glioblastoma samples and provides rationale for investigating immune checkpoint inhibitors in GBM. The results between primary and recurrent GBM needs to be further investigated. PD-L1 expression was enriched in the mesenchymal subtype.
Author's Conclusions: in the adjuvant-only group had a mOS of 228 days (7.5 mo.), whereas those in the neoadjuvant arm had a mOS of 417 days (13.7 mo.). mPFS was 72.5 days (2.4 mo.) in the adjuvant-only group and 99.5 d (3.3 mo.) in the neoadjuvant group (P = 0.03). In pts that received surgery and had histologic evidence of tumor (n = 15 patients per group), the mOS of the neoadjuvant treatment cohort was 400 days (13.2 mo.) from registration date, while that of the adjuvant treatment cohort was 192 d (6.3 mo.) (P = 0.03).
The density of tumor-in ltrating CD8+ T cells was not different between groups but demonstrated signi cant variability in the neoadjuvant cohort.
Author's Conclusions: PD-1 monoclonal antibody blockade was associated with statistically signi cant improvements in OS and PFS when administered in the neoadjuvant setting to pts with recurrent GBM. Neoadjuvant PD-1 monoclonal antibody blockade induces functional activation of tumor-in ltrating lymphocytes, producing an interferon response within the tumor microenvironment.

Comments and Conclusions:
Classi ed as Class III as it is a prospective study but has a limited number of participants and control arm is not standard therapy. PD-L1 expression was not assessed.
kinase. RR and PFS-6 of 70% was achieved in the whole cohort, 100% in the group treated with bevacizumab and erlotinib, and 50% in the group treated with bevacizumab.

Comments and Conclusions:
Classi ed as Class III, while it is a prospective study there is a very limited number of pts.
Takano, S, et al., 2014 Study Description: Retrospective study of 19 pts to examine the expression of VEGF in brain tumors and if it correlates with survival.

III
Results: Strong expression of VEGF is seen in the blood vessels in the tumor and in the edge of GBM. In addition, strong expression is seen in the cytoplasm of tumor cells and around the areas of necrosis. Expression is weaker in the anaplastic astrocytoma and in the low-grade astrocytoma than in the GBM. VEGF is not expressed in the normal brain. VEGF concentration in the tissue of GBMs is signi cantly higher than that in the tissues of other types of tumors, and in the tissue of the normal brain. (*P<0.01). A VEGF concentration of more than 1,000 pg/mg was a prognostic factor. Median OS of the patients with VEGF concentration ≥1,000 pg/mg (number [n] =20) was signi cantly shorter than in those with <1,000 pg/mg (n=17) at 11.8 months and 24.8 months, respectively (P=0.0025).
Authors Conclusions: VEGF is localized in tumor cells and tumor endothelial cells in glioma, especially in GBM, and its concentration predicts malignant glioma survival.

Comments and conclusions:
Classi ed as class II as it is a retrospective study with a few patients and the patient number and demographics are not clear.
Erdem-Eraslan, L., et al., 2016 Study Description: Retrospective study of 114 specimens from the BELOB trial. All pts were treated with temozolomide and radiotherapy and at 1 st recurrence were then treated with BEV, CCNU or BEV+CCNU. The cohorts were interrogated to determine possible biomarkers of response to the different treatment arms.
All studies were performed on the primary tumor specimen