1140

Neuro-Oncology
24(7), 1140–1149, 2022 | https://doi.org/10.1093/neuonc/noab282 | Advance Access date 8 December 2021

Clinical utility of targeted next-generation sequencing

assay in IDH-wildtype glioblastoma for therapy

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decision-making
  

Mary Jane Lim-Fat, Gilbert C. Youssef, Mehdi Touat, J. Bryan Iorgulescu, Sydney Whorral,

Marie Allen, Rifaquat Rahman , Ugonma Chukwueke, J. Ricardo McFaline-Figueroa,
Lakshmi Nayak, Eudocia Q. Lee, Tracy T. Batchelor, Omar Arnaout , Pier Paolo Peruzzi,
E. Antonio Chiocca, David A. Reardon, David Meredith, Sandro Santagata , Rameen Beroukhim,
Wenya Linda Bi, Keith L. Ligon, and Patrick Y. Wen

Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, Toronto,
Ontario, Canada (M.J.L-F.); Center for Neuro-Oncology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital
and Harvard Medical School, Boston, Massachusetts, USA (G.C.Y., J.B.I., S.W., M.A., R.R., U.C., J.R.M-F., L.N., E.Q.L.,
T.T.B., D.A.R., D.M., S.S., R.B., K.L.L., P.Y.W.); Sorbonne Université, Inserm, CNRS, UMR S 1127, Institut du Cerveau et
de la Moelle épinière, ICM, AP-HP, Hôpitaux Universitaires La Pitié Salpêtrière - Charles Foix, Service de Neurologie
2-Mazarin, Paris, France (M.T.); Department of Pathology, Brigham and Women’s Hospital and Harvard Medical
School, Boston, Massachusetts, USA (J.B.I., D.M., S.S., K.L.L.); Department of Neurosurgery, Brigham and Women’s
Hospital, Harvard Medical School, Boston, Massachusetts, USA (O.A, P.P.P., E.A.C, W.L.B); Department of Oncologic
Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA (K.L.L.)

Corresponding Author: Mary Jane Lim-Fat, MD, MSc, Division of Neurology, Sunnybrook Health Sciences Centre, University of
Toronto, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada (maryjane.limfat@sunnybrook.ca).

Abstract
Background. Targeted gene NGS testing is available through many academic institutions and commercial entities
and is increasingly incorporated in practice guidelines for glioblastoma (GBM). This single-center retrospective
study aimed to evaluate the clinical utility of incorporating NGS results in the management of GBM patients at a
clinical trials-focused academic center. 
Methods.  We identified 1011 consecutive adult patients with pathologically confirmed GBM (IDHwt or IDHmut) who had
somatic tumor sequencing (Oncopanel, ~500 cancer gene panel) at DFCI from 2013–2019. Clinical records of all IDHwt
GBM patients were reviewed to capture clinical trial enrollment and off-label targeted therapy use based on NGS results. 
Results.  Of the 557 IDHwt GBM patients with sequencing, 182 entered clinical trials at diagnosis (32.7%) and 213 (38.2%)
entered after recurrence. Sequencing results for 130 patients (23.3%) were utilized for clinical trial enrollment for either
targeted therapy indications (6.9 % upfront and 27.7% at recurrent clinical trials and 3.1% for off-label targeted therapy)
or exploratory studies (55.4% upfront and 6.9% recurrent clinical trials). Median overall survival was 20.1 months with no
survival difference seen between patients enrolled in clinical trials compared to those who were not, in a posthoc analysis. 
Conclusions. While NGS testing has become essential for improved molecular diagnostics, our study illustrates
that targeted gene panels remain underutilized for selecting therapy in GBM-IDHwt. Targeted therapy and clinical
trial design remain to be improved to help leverage the potential of NGS in clinical care.

Key Points
1. Beyond diagnostic confirmation, use of NGS to select therapies in GBM remains
constrained by lack of effective targeted therapy options.
2. Development of more effective targeted therapies could help leverage the potential of
NGS in GBM.

© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved.
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 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM
Importance of the Study
With the increased use of targeted gene NGS panels
in clinical care for the diagnosis of glioblastoma, the
therapeutic and clinical trial utility of these panels for
patient management has not been well studied. This
single-center retrospective study evaluates the practice
In 2016, a revision to the WHO classification incorporated
molecular diagnostics to complement histological diagnosis
and grading.1 Molecular testing is now integral to prac-
tice guidelines,2 endorsed by the National Comprehensive
Cancer Network (NCCN) and expounded upon by the
Consortium to Inform Molecular and Practical Approaches
to CNS Tumor Taxonomy (cIMPACT-NOW). The 2021 WHO
classification (5th Edition) reflects updates from cIMPACT-
NOW3,4—seven of which have now been published—and
relies on testing for molecular alterations for the diagnosis
of gliomas.5–11 Furthermore, in glioma, molecular signatures
acquired longitudinally can help broaden our understanding
of each tumor’s natural history, vulnerabilities, response to
treatment, and progression pattern, potentially paving the
way to improved precision medicine.12,13
The concept of targeted therapy based on individual-
ized tumor genotyping has long been attractive in glioma.
However, only certain targeted therapies are currently FDA
approved for use in gliomas, such as mammalian target of
rapamycin (mTOR) inhibitors in tuberous sclerosis associ-
ated subependymal giant cell astrocytoma, neurotrophic ty-
rosine receptor kinase (NTRK)-inhibitors for NTRK fusions,
and programmed cell death protein 1 (PD-1) inhibitors in
mismatch repair deficient-/high tumor mutational burden/
high microsatellite instability tumors. Many other targets
are attractive due to their identification as key oncogenic
drivers in gliomas such as IDH mutations in astrocytoma
and oligodendroglioma. In IDH-wildtype (IDHwt) gliomas,
specifically, glioblastoma (GBM), a number of different
targets have been identified including epidermal growth
factor receptor (EGFR) amplification and mutations, cyclin
dependent kinase inhibitor 2A and 2B (CDKN2A/B), and
phosphoinositide 3-kinase (PI3K) pathways, the v-Raf mu-
rine sarcoma viral oncogene homolog V1 (BRAF) V600E
mutations, and fusion events such as in fibroblast growth
factor receptor (FGFR) and neurotrophic tyrosine receptor
kinase (NTRK).12 However, efforts to find effective therapies
have so far fallen short when it comes to life-prolonging
treatments in GBM. There are a number of possible explan-
ations for this including the redundant nature of several on-
cogenic pathways, the lack of CNS penetrance from tested
therapies due to the blood-brain barrier, and substantial
intratumoral and intertumoral heterogeneity.14 Outside of
these challenges, it is also important to consider that the
targetable mutations can be rare, for example, only about
2% of adult GBM harbor a BRAF V600E mutation and
better characterization of such subtypes may be helpful
in screening patients for such mutations.15 In the broader
world of oncology, and across tumor sites, precision on-
cology has seen some success stories, but widespread suc-
cess has been elusive, particularly in brain tumors.16
1141
Oncology
Neuro-
patterns and utility of incorporating targeted gene
sequencing results into therapeutic decision-making
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for GBM patients at a clinical trials-focused academic
center.
In recent years, molecular diagnostics in the form of
next-generation sequencing (NGS)-based targeted gene
assays have become commercially available and has been
incorporated into routine clinical care for a number of
cancer types at many institutions. However, given the spe-
cialized laboratory infrastructure associated with NGS, as
well as its limited availability and accessibility, the current
impact of widespread targeted NGS assays for IDHwt GBM
patients in treatment remains unclear. In this study, we de-
scribe our single-center experience of incorporating tar-
geted gene NGS testing into therapeutic decision-making
for patients with IDHwt GBM.
Materials and Methods
This study was conducted with approval from the Dana-
Farber/Harvard Cancer Center Institutional Review Board.
A waiver was granted for informed consent due to the ret-
rospective nature of the study.
Cohort Selection
This retrospective study was carried out with methodology
consistent with the STROBE (Strengthening the Reporting
of Observational studies in Epidemiology) guidelines.17
Adult patients (≥18-years-old) with a pathological di-
agnosis of glioblastoma WHO grade 4, treated at Dana-
Farber Cancer Institute/Brigham and Women’s Hospital
from January 1, 2013 to July 31, 2019, and had molecular
information through the institutional NGS targeted gene
panel (ie, OncoPanel, ~500 cancer-causing genes) were
screened for this study. Exclusion criteria were: i) pro-
gression of previous low-grade glioma (secondary glio-
blastoma), ii) IDHmut high-grade glioma, and iii) lack of
consistent (ie, >2) clinical visits with documented clinical
notes after the first consultation (Figure 1).
Data Collection
A review of the medical records and clinical charts of
each patient was performed (MA, ML, SW, GY), and clin-
ical data including sex, age, histomolecular diagnosis, all
tumor-directed therapy, progression dates based on clin-
ical notes, progression-free survival, and overall survival
were collected retrospectively. Specifically, all therapeutic
clinical trial enrollment information was captured, and re-
quired biomarkers for enrollment purposes including O6-
methylguanine-DNA methyltransferase (MGMT) promoter
1142 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM
  
All GBM patients with NGS from
2013–2019 at DFCI/BWH
(n = 1011)
Secondary GBM
(N = 83)
IDH wildtype on IHC and NGS
IDH mutant
(n = 50)
Reliable follow-up data (>2
documented visits)
No consistent follow-
up
(n = 321)
Selected cohort
(n = 557)
Fig. 1.  Patient selection: 1011 patients with glioblastoma at
Dana-Farber Cancer Institute who had in-house targeted panel
sequencing between 2013 and 2019 were retrospectively identi-
fied. All secondary GBMs, IDH-mutant tumors and patients without
consistent follow-up were excluded. This algorithm resulted in 557
patients which were included in the final cohort.
methylation status, or specific mutations were annotated.
Clinical trials not investigating tumor-directed treatments
were not included.
Pathology and Molecular Data
Histomolecular diagnosis in accordance with the 2016
WHO classification1 was collected. Isocitrate dehydro-
genase (IDH) mutations by either immunohistochemistry
for IDH1 R132H or NGS were assessed and all IDH1 or
IDH2-mutant tumors were excluded. MGMT promoter
methylation status was recorded where available. All pa-
tients included in this retrospective cohort had their tumor
sequenced using the previously validated OncoPanel18,19
at the Center for Cancer Genome Discovery (Dana-Farber
Cancer Institute) and the Center for Advanced Molecular
Diagnosis (Brigham and Women’s Hospital). This institu-
tional targeted gene NGS assay surveys cancer-associated
genes for single nucleotide variants, insertions, and de-
letions, and copy number variants; as well as intronic re-
gions for rearrangement detection and is based on the
Illumina HiSeq 2500 platform (targeted genes are listed in
Supplementary Appendix 1).
Statistical Analysis
Statistical analyses were performed using Stata/SE Version
16. Kaplan-Meier estimator and log-rank test were per-
formed to assess for difference in survival data in a posthoc
fashion due to the retrospective nature of the study. Time-
to-event analyses for overall survival (OS) were calculated
using the Kaplan-Meier method from time of glioblastoma
diagnosis to date of death.
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Results
Patient Characteristics
From 1011 patients with newly diagnosed GBM and avail-
able NGS OncoPanel data, 878 were newly diagnosed
IDHwt patients (Figure 1). 321 patients were excluded due
to lack of reliable follow-up at our institution; 557 patients
were included for the final cohort. Baseline and relevant
characteristics of the whole cohort are summarized in Table
1. 508 patients (91.2%) had documented MGMT promoter
methylation data, including 198 (39.0%) with methylated
tumors, 30 (5.9%) with partially methylated tumors, and
280 (55.1%) with unmethylated tumors.
Reported Molecular Alterations of Clinical
Significance
Our analysis included the clinically significant Tier 1 and
Tier 2 single nucleotide variants (SNV) and estimated copy
number variants (CNV) based on the tiering system of our
institutional panel.20 Relevant genes required for eligibility
in GBM clinical trials at our institution (EGFR, PTEN, MET,
FGFR1, FGFR3, H3F3A, PDGFRA, CDKN2A/B, RB1, TP53)
and the frequency of patients with each molecular alter-
ation are shown in Table 2. The most common alterations
included mutations in TERT promoter (n  =  180, 63.6%),
CDKN2A (n = 254, 45.6%), CDKN2B (n = 229, 41.1%), PTEN
(n = 196, 35.2%) and EGFR amplification (n = 186, 33.4%).
Results directly relevant for FDA approved therapies in-
cluded high tumor mutational burden (TMB high defined
as >17 mut/Mb)21 (n = 11, 2%), BRAF V600E (n = 12, 2.2%)
and FGFR3-TACC fusions. (n = 4; ~1%).
Standard of Care vs Clinical Trial Enrollment
The vast majority of our patients received concurrent
chemoradiation with temozolomide as upfront treatment
(n = 530, 95.2%; Table 1). While 417 (74.8%) of patients re-
ceived adjuvant therapy with temozolomide, 182 (32.7%)
were enrolled in clinical trial immediately after first diag-
nosis (“upfront setting”), of which 112 (61.5%) received
temozolomide as part of the trial (with or without another
agent). Of these 182 patients, 75 (41.2%) also enrolled in
subsequent clinical trials at recurrence. In total, 213 (38.2%)
of patients were enrolled in a clinical trial at first or subse-
quent recurrence. Altogether, 314 (56.4%) patients enrolled
in 77 trials upfront or at recurrence (Appendix 2).
Figure 2 shows the different investigational drug agents
used in the context of a clinical trial in this cohort. The dem-
ographics of patients enrolled in therapeutic clinical trials
in the upfront setting or at any point during their diagnosis
were also assessed and compared (Table 1). Patients in clin-
ical trials (both in the upfront setting and at any timepoint)
 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM 1143

Oncology
Neuro-
  
Table 1  Patient Demographic for Cohort of 557 Glioblastoma Patients

Total Upfront Clinical Trial Any Clinical Trial

(N = 557)
Yes (N = 182) No Yes No

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(N = 375) (N = 314) (N = 243)
Median age at diagnosis (95% CI) 60.6 58.5 61.5 58.7 62.9
(59.1–61.7) (57.4–60.6) (60.2–62.9) (57.3–60.3) (61.2–64.6)
P = .01 P < .01
Sex (%) 227 (40.8) 108 222 193 137
Male
(59.3) (59.2) (61.4) (56.3)
P = .98 P = .23
KPS at baseline (%) N = 462 150 (82.4) 241 (64.2) 229 (72.9) 162 (66.7)
≥ 80 396 (85.7) P < .01 P = .11
Extent of resection (%) N = 549 83 (46.4) 146 (39.2) 153 (49.5) 76 (31.4)
GTR 229 (41.6) P = .11 P < .01
MGMT methylation status (%) N = 508 63 (38.7) 135 (39.1) 115 (40.8) 83 (36.7)
Methylated 198 (39.0)
Partially methylated 30 (5.9)
Unmethylated 280 (55.1) 11 (6.7) 19 (5.5) 18 (6.4) 12 (5.3)
89 (54.6) 191(55.4) 149 (52.8) 131 (58.0)
P = .86 P = .50
Concurrent chemoradiation with N = 553 169 (92.9) 361 (97.3) 299 (95.2) 231 (96.7)
TMZ
530 (95.2) P = .01 P = .40
Adjuvant treatment 417 (74.8) 112 (61.9) 305 (83.6) 233 (74.9) 184 (78.2)
TMZ P < .01 P = .36
Median number of TMZ cycles 6 (6–6) 6 (6–6) 6 (5–6) 6 (6–6) 5 (5–6)
(95% CI)
P = .41 P < .01
  
were younger and exhibited better Karnofsky Performance NCT01480479, NCT02573324, NCT03107780) and a phase
Scale (KPS) scores at baseline (P < .05). Patients enrolled 0/1 study of AMG 232, an MDM2 inhibitor for patients with
in clinical trials also were more likely to have had a gross p53 wild type glioblastoma (NCT03107780). The remaining
total resection (P < .01) and were more likely to receive 65 patients (85.5%) required sequencing information for
more cycles of temozolomide compared to those who did exploratory/biomarker analyses and this information did
not participate in a clinical trial (P < .01). not guide therapy selection. Thirty-four (52.3%) of these
patients were enrolled in the Individualized Screening
Trial of Innovative Glioblastoma Therapy (INSIGhT) trial22
Utilization of Molecular Data for Tumor-Directed (NCT02977780), a Bayesian adaptive platform trial for
Therapy or Trial Enrollment newly diagnosed glioblastoma patients with unmethylated
MGMT promoter. In this design, randomization at diag-
Among the 182 patients who were enrolled in clinical nosis occurs to multiple experimental arms or one control
trials in the upfront setting, 100 (54.9%) required MGMT arm, and patient subtypes including biomarkers are iden-
methylation status information and 81 (44.5%) required tified, after which adaptive randomization occurs based on
sequencing information for enrollment (Figure 3). Twelve accumulating trial results.
of the 77 trials required molecular testing for eligibility In the recurrent setting, only 45 patients out of the
based on 10 genes: EGFR, PTEN, MET, FGFR1, FGFR3, 213 (21.1%) who were enrolled in clinical trials required
H3F3A, PDGFRA, CDKN2A/B, RB1, TP53. Of these 81 pa- sequencing data for enrollment eligibility. Of these, 36
tients requiring sequencing information, only 9 patients (80%) required specific mutations as inclusion criteria for
(11.1%) required a specific mutation to meet inclusion biologically driven therapies (Figure 2). All trials and their
criteria. Of the 4 molecularly selective upfront trials that requirements with regards to MGMT methylation status or
selected patients based on the identification of specific molecular screening are summarized in Appendix 2.
genetic aberrations, several were trials with EGFR mu- Out of the 11 patients identified to have hypermutated
tation/amplification-directed treatments (NCT01800695, tumors, 3 (27.3%) received immune checkpoint inhibition.
1144 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM

   (20.4 months [95% CI: 17.3–23.3] vs 20.2 months [95% CI:

Table 2  Frequency of Molecular Alteration in Select Genes in Cohort 18.9–22.3]), P = .13) (Figure 4).

Gene Total (%)

TERT promoter (N = 283) 180 (63.6)
CDKN2A 254 (45.6) Discussion

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CDKN2B 229 (41.1)
With the implementation of the 2021 WHO Classification
PTEN 196 (35.2) of Central Nervous Tumours 5th edition, molecular testing
EGFR amplification 186 (33.4) is an increasingly crucial component of the standard-of-
TP53 161 (28.9) care diagnostic work-up of an infiltrative glioma. National
Comprehensive Cancer Network guidelines (CNS cancers
MTAP (N = 283) 108 (38.2)
v1.2021) additionally encourages the molecular testing of
CDK4 61 (11.0) glioblastomas to help identify driver mutations that may
NF1 54 (9.7) be targetable in the context of a clinical trial or on a com-
PIK3CA 51 (9.2) passionate use basis. Targeted NGS gene panel assays can
help simultaneously test for the growing number of diag-
PDGFRA 43 (7.7)
nostically important—and potentially targetable—molec-
RB1 41 (7.4)
ular alterations in infiltrative gliomas in a straightforward
MDM2 36 (6.5) and cost-effective manner. However, although access
MDM4 31 (5.6) to targeted gene sequencing panels for gliomas has in-
PIK3C2B 31 (5.6) creased in the past decade, beyond their marked value
in diagnosis, the utility and clinical benefit for guiding
PIK3R1 21 (3.8)
therapy based on these tests remain unclear. Molecular
STAG2 16 (2.9) testing in brain tumors has become increasingly feasible in
ATRX 14 (2.5) the timeline of real-world best clinical practice, with some
BRAF V600E 12 (2.2) groups reporting an integrated workflow of 5  days from
PTPN11 7 (1.3)
tissue biopsy to finalized report of a 130 gene NGS panel.23
Given the increase in the number of trials for newly diag-
NOTCH1 3 (0.5)
nosed glioblastoma, molecular information, including
POLE (N = 283) 2 (0.7) MGMT status, is typically helpful at the first consultation
APC 2 (0.4) with the neuro-oncologist prior to radiation planning; how-
SETD2 1 (0.2) ever, this is not always feasible and can present a logistical
challenge.
   A retrospective cross-sectional study using publicly
available data across cancer types found that about 4.5%
Only four patients received compassionate use targeted
of patients derived benefit from “genome-targeted” drugs
therapy: one with dabrafenib/trametinib (BRAF V600E mu-
(where the drug targeted the specific aberration identified
tant), one with abemaciclib (CDK4/6 amplification), and
by the genomic test) and 6% benefited from “genome-
two with rindopepimut (EGFRvIII amplification).
informed” therapy (all genome-targeted drugs or drugs
given after genomic test, regardless of whether drug tar-
Survival Times and Follow-Up geted aberration or acted via an alternate mechanism of
action).24 In our study of IDHwt GBM, we found that the
Given that patients without consistent follow-up were ex- targeted gene sequencing results were utilized for clinical
cluded for this analysis, the median follow-up time could decision-making in 23% of patients—mostly as a prereq-
be estimated by the survival time until death or last cen- uisite for clinical trial enrollment. In a subset of these pa-
sored date. Median overall survival for this cohort was tients (8% overall), specific mutations or molecular results
20.1 months (95% confidence interval [CI]: 18.9–22.0); spe- were required for enrollment due to mechanism of action
cifically, 17.1 months for the MGMT unmethylated patients of the drugs. Most of these trials were studies of recurrent
(n = 280; 95% CI:15.5–18.0); 20.4 months for the MGMT par- gliomas and the rate was much lower in the upfront clinical
tially methylated patients (n  =  30, 95% CI: 10.3–22.3) and trial setting.
28.3 months for the MGMT methylated patients (n = 198, In our analysis, there was no survival difference be-
95% CI: 25.7–34.2). Median progression-free survival was tween patients who enrolled in clinical trials versus those
9.1  months (95%CI: 8.5–9.7) for the cohort. There was no that did not. This finding is worth highlighting as previous
difference in overall survival in patients who were enrolled studies, with smaller patient numbers have indicated that
in clinical trials in the upfront setting compared to those patients benefit from enrollment in clinical trials compared
in the recurrent setting (19.0 months [95% CI: 17.1–22.2] vs to standard therapy.25,26 It is possible that this survival dif-
20.2  months [95% CI: 19.0–22.3]), P  =  .60) in our posthoc ference may be less pronounced in centers where routine
survival analysis. In addition, there was no difference in care of patients receiving standard-of-care therapy does
overall survival for patients who were enrolled in a clinical not differ significantly from those on clinical trials. In addi-
trial (either in the upfront or recurrent settings) compared tion, a more relevant survival outcome would be a compar-
to those who were not part of any therapeutic clinical trial ison of survival for patients that used molecular selection
 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM
Experimental agents in upfront
trials (enrolled 182 patients)
• Abemaciclib
• ABT-414*
• BGB290 Clinical trial enrollment for 557 patients
• BKM120
• CC-115
• Durvalumab
• GDC0084 Upfront and
recurrent, 81,
• ICT-107
• INO-5401 15%
• Neovax
• Neratinib
• Nivolumab
• Pembrolizumab
• Veliparib
None, 243,
44%
* Trial for which NGS was used for
patient selection based on molecular
alteration
Fig. 2  Clinical trial enrollment for the 557 patients included in our study and the experimental agents used in the clinical trial setting.
for enrollment, although this was not feasible in our study
due to the small sample sizes within these groups. This
analysis would be a helpful addition to the understanding
of the utility of NGS in clinical care and could be performed
through multi-institutional review of such trials.
Most of the trials involved sequencing of patients’ tu-
mors for exploratory biomarker purposes, and while such
efforts may eventually further our understanding of poten-
tial targets and inform future patient selection, the enrolled
individual generally does not undergo mutation-derived
targeted therapy in those cases. A unique example of the
use of molecular biomarkers in an adaptive setting is the
Individualized Screening Trial of Innovative Glioblastoma
Therapy (INSIGhT) trial22 (ClinicalTrials.gov Identifier:
NCT02977780), which is a Bayesian adaptive platform trial
for newly diagnosed GBM patients with a two-phase ap-
proach. In this trial, biomarkers are identified, after which
adaptive randomization occurs based on accumulating
trial results. In this trial, and in the larger similarly designed
GBM-AGILE study, while the biomarker-specific probability
of treatment impact on progression-free survival is used
to drop arms that would have a low probability of treat-
ment impact on overall survival, molecular biomarkers are
not used at the time of accrual for specific arm assignment
until specific criteria have been met. Therefore, in this par-
ticular trial, “targeted therapy” is not the basis of the first
phase of the trial for each individual patient.27
Only four patients in our cohort had their molecular in-
formation used for off-label targeted therapies (which are
FDA-approved for other cancer types but not for GBM as
a specific indication) during their follow-up time at DFCI.
The accessibility of off-label therapies to individual pa-
tients is often dependent on third-party coverage and
FDA approval. This landscape has been evolving in recent
1145
Oncology
Neuro-
Experimental agents in
recurrent trials (enrolled 213
patients)
• Abemaciclib*
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• ABT-414*
• AMG232*
• Anti LAG3
• AZD1390
• Bevacizumab
Upfront,
101, 18% • BGB290
• BGJ398*
• BKM120*
• Dabrafenib/Trametinib*
• Lapatinib*
• Neratinib*
Recurrent, • ONC201*
132, 24% • Pembrolizumab
• Pembrolizumab + Re-RT
• Rindopepimut*
• rQ-Nestin
• Selinexor
• TAK-228*
• TAS-120*
• VBI 1901
years and there are now several tumor type-agnostic ther-
apies approved by the FDA, such as NTRK inhibitors (eg,
larotrectinib and entrectinib) for NTRK fusions and immune
checkpoint inhibitors (eg, pembrolizumab) for mismatch
repair deficient/microsatellite instability-high solid tumors.
Initiating off-label treatments outside of clinical trials in the
upfront setting remains controversial if it is done in lieu of
or in addition to standard-of-care therapy. Therefore, anec-
dotally, off-label targeted therapies are often initiated by
the provider if there is available sequencing data to guide
this decision at the time when salvage treatment is justi-
fied. This window may be very short for patients with an
aggressive tumor type such as GBM, therefore obtaining
targeted gene sequencing results “for future use” at the
time of initial surgery has been an emerging phenomenon
in clinical practice, catalyzed in part by growing access to
NGS testing. This approach may be feasible for molecular
targets such as EGFR amplification and CDKN2A/B dele-
tion, both of which are usually unchanged at the time of
recurrence in GBM but may be less effective in tumors
where targets such as EGFR mutations may be lost in re-
current tumors.28,29 The overall clinical utility of this “for fu-
ture use” sequencing strategy—given GBM’s increasingly
recognized proclivity for continued tumor evolution and
acquiring mutations due to standard-of-care therapy21,28—
as well as the strategy’s cost-effectiveness, remain to be
determined.
Utilization of MGMT methylation status is now well
established both as a predictive marker of response to
alkylating therapy and a prognostic biomarker. MGMT
methylation status can be determined by quantitative or
semi-quantitative PCR methods and is mandated by the
NCCN guidelines, although uniform and routine imple-
mentation can remain challenging.30,31 While methylation
1146 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM

  

IDH wt GBM patients at DFCI/BWH

(n = 557)

Downloaded from https://academic.oup.com/neuro-oncology/article/24/7/1140/6456497 by Universidade Federal de Minas Gerais user on 15 July 2022
Clinical Trials (n = 314) Off label targeted therapy (n = 4)
Upfront trial (n = 182) Dabrafenib/trametinib (n = 1) No clinical trial participation or
Recurrent trial (n = 213) Abemaciclib (n = 1) targeted therapy (n = 239)
Both (n = 81) Rindopepimut (n = 2)

Upfront trial requirements (n = 182) Recurrent trial requirements (n = 213)

MGMT methylation status: 100 MGMT methylation status: 0
Required NGS: 81 Required NGS: 45
- 9 for specific mutation - 36 for specific mutation
- 72 for exploratory analysis - 9 for exploratory analysis

Fig. 3  Flow chart showing utilization of NGS and MGMT methylation data for patient enrollment in clinical trial or for off-label therapy.
  

  
A OS for upfront clinical trial enrollment B OS for any clinical trial enrollment
100
100
80
80
Survival (%)
Survival (%)

60
60
40
40
20
20

0 0
0 6 12 18 24 30 36 0 6 12 18 24 30 36
Overall survival (mos) Overall survival (mos)
Number at risk
Number at risk
Any clinical trial = 0 243 225 181 116 75 53 35
Adjuvant clinical trial = 0 375 344 272 183 121 83 55
Any clinical trial = 1 314 290 230 151 103 64 49
Adjuvant clinical trial = 1 182 171 139 84 57 34 29

95% CI 95% CI 95% CI 95% CI

Adjuvant clinical trial = 0 Adjuvant clinical trial = 1 Adjuvant clinical trial = 0 Any clinical trial = 1

Fig. 4  Survival curves of patients enrolled in upfront clinical trials vs not (A) and patients enrolled in any clinical trial vs not (B). There was no dif-
ference in overall survival in patients who were enrolled in clinical trials upfront compared than those who did not (19.0 months [95% CI: 17.1–22.2]
vs 20.2 months [95% CI: 19.0–22.3]), P = .60). In addition, there was no difference in overall survival in patients who were enrolled in a clinical trial
(at any point—upfront or at the recurrent stage) compared to those who were not part of any clinical trial (20.4 months [95% CI: 17.3–23.3] vs
20.2 months [95% CI: 18.9–22.3]), P = .13).
  

status has anecdotally been more helpful in treatment de- MGMT methylation status, due to an appreciation of the
cisions for elderly or frail patients with glioblastoma,32,33 unique risks and benefits posed by standard therapy vs un-
there is an impetus to rethink the role of standard-of- proven therapies to different MGMT methylation statuses.
care concurrent and adjuvant chemoradiation in MGMT While we did not specifically evaluate the utility of MGMT
unmethylated patients with glioblastoma, especially in methylation status in guiding treatment decisions in this
the post-COVID19 era.34 Most upfront GBM trials with in- study, most upfront clinical trials at our institution utilized
vestigational drug therapies stratify enrollment based on this information for initial enrollment, with MGMT status
 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM 1147

representing perhaps the most commonly utilized bio-
marker at that juncture.
Many institutional sequencing panels and commercial
panels are now available, each of which can examine sev-
eral hundreds of selected GBM relevant genes.35 While our
results show that targeted NGS may be currently under-
Oncology
Neuro-
availability of targeted gene NGS testing in a clinical set-
ting, access to clinical trials, and use of off-label therapies.
However, the Center for Neuro-Oncology at Dana-Farber
Cancer Institute is a comprehensive brain tumor pro-
gram that sees large patient volumes, and at which next-
generation sequencing is performed on most glioblastoma

Downloaded from https://academic.oup.com/neuro-oncology/article/24/7/1140/6456497 by Universidade Federal de Minas Gerais user on 15 July 2022
utilized for selecting targeted therapies, this study does specimens, multiple clinical trials are available both in the
not argue against the benefits of sequencing in the clinical upfront and recurrent setting, and off-label therapies are
care of patients. The diagnostic and prognostic value to also often discussed with patients. While we estimate that
patients are clear and targeted sequencing panels can be other large comprehensive brain tumor programs may
a cost-effective solution compared to individual selected have similar opportunities for NGS testing in IDH-wt GBM,
gene panels addressing specific alterations that define our results may in fact be an overestimate the use of NGS
gliomas within the WHO 2021 classification3 (eg, IDH1, testing for access to therapies outside of specialized cen-
IDH2, ATRX, T53, CDKN2A/B, 1p/19q, TERT promoter, CIC, ters. Evaluation of potential survival benefits will also re-
FUBP1, NOTCH1, chromosome 7 gain or 10 loss, EGFR, quire assessment in the context of each individual trial as
MYB, MYBL1, BRAF, FGFR, H3K27, H3G34). Additionally, in more results from such studies emerge over time.
the face of a growing number of molecular alterations/sig-
natures that are potentially targetable (eg, mismatch repair
deficiency/microsatellite instability-high, NTRK, PIK3CA,
CDK4/6, PDGFRA, etc.) in glioblastoma, NGS gene panel Conclusions
assays can provide a more scalable solution for neuropa-
With the rise of more cost-effective and efficient sequencing
thology laboratories than the continuous validation and
technology, comprehensive repositories of molecular data
implementation of new single-gene or single-purpose
combined with clinical data could provide further insight
molecular assays. The answer in better leveraging the po-
into the therapeutic impact of molecular typing in glioblas-
tential of multi-gene NGS assay data may ultimately lie in
toma patients. However, until effective targeted therapies
creating computational platforms that automatically match
against the more common molecular targets such as EGFR,
a patient’s identified molecular alterations with relevant
CDKN2A/B, and PI3kinase can be developed, the thera-
clinical trials and – at a fundamental level –in improving
peutic utility of targeted gene NGS testing in GBM patients
the drug development pipeline for targeted agents in GBM.
will not be leveraged to its full potential. Patients need to be
Certain subgroups within IDHwt GBM may in particular
included in a discussion with regards to the expectations
benefit from multi-gene NGS testing, for example, younger
and to emphasize that these tests are highly valuable for
patients may be more likely to have targetable mutations
diagnostic purposes and for consideration of clinical trials.
such as those in BRAF or FGFR, and the clinical yield of
sequencing patients with pediatric type IDHwt gliomas
would be more pronounced.
Designing precision medicine-focused clinical trials
Supplementary Material
for GBM to find more effective agents remains an area of
pressing need, and while accrual to molecular selective Supplementary material is available at Neuro-Oncology
trials is often a challenge due to the low frequency of cer- online.
tain driver mutations in high-grade gliomas (eg, NTRK and
BRAF), multi-institutional trials with novel trial designs in-
cluding basket studies are approaches that can be effective.
Novel adjunctive technologies to overcome the blood- Keywords
brain-barrier that are currently under investigation such as
focused ultrasound (FUS)36 may also represent effective GBM | next-generation sequencing | targeted therapy
combinatorial strategies to be used with targeted agents.
Beyond clinical trials, molecularly annotated and clini-
cally annotated “real-life” cohorts offer a unique chance to
study precision medicine in GBM patients, guided by NGS Funding
data, whether or not patients receive targeted therapy or
standard-of-care therapy. Lastly, this type of data may be The authors received no financial support for the research, au-
particularly valuable to study patterns of response, identify thorship and/or publication of this article.
and understand biomarkers of long-term response and re-
sistance and sequencing of serum or CSF37,38 may also rep-
resent an emerging application that will help in these efforts.

Limitations
This single-institution experience at an NCI-Designated
Cancer Center does not necessarily reflect the current prac-
tice patterns at other institutions, specifically in terms of
Acknowledgments
J.B.I.  gratefully acknowledges support from the NIH/NCI
(K12CA090354) and the Conquer Cancer Foundation/Sontag
Foundation.
1148 Lim-Fat et al. NGS for therapy decision-making in IDHwt GBM
Conflict of interest statement. M.T.  reports consulting or advi-
sory role from Agios Pharmaceutical, Integragen, and Taiho
Oncology, outside the submitted work; research funding from
Sanofi, outside of submitted work.
Authorship statement. All authors contributed to the experi-
mental design, data interpretation, draft writing, and revision,
and have approved the final version.
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