Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

An Innovative Immunotherapy Vaccine with Combination Checkpoint Blockade as a First Line Treatment for Glioblastoma in the Context of Current Treatments.

Glioblastoma is a devastating disease with a dismal prognosis. While recent advancements in cancer immunotherapy have led to improvements in treating other types of cancer, patients with glioblastoma have not benefited from these new therapies and techniques. Fortunately, neurosurgeons and oncologists at Washington University School of Medicine conducting a cutting edge clinical trial are looking to overcome these persistent challenges in treating glioblastoma through combining a personalized vaccine with new immunotherapy drugs.

Rapid Clinical and Radiographic Response With Combined Dabrafenib and Trametinib in Adults With BRAF-Mutated High-Grade Glioma.

BRAF V600E mutations have been successfully treated with targeted therapy in melanoma, non-small cell lung cancer, and thyroid cancer. Interestingly, these mutations have also been identified in a subset of pediatric and adult brain tumors, with several cases reportedly responding to targeted therapy. However, these reports have been limited to single-agent BRAF inhibitor therapy and recurrent disease. Herein, we report dramatic clinical and radiographic responses to combination dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) in 2 adults with high-grade gliomas (HGGs), with 1 patient treated in the first-line setting. These observations, together with prior case reports, advocate for routine screening of BRAF point mutations in adult HGGs, and suggest that treatment with dual-targeted therapy, even in newly diagnosed cases, is safe and effective.

Integrating Multisector Molecular Characterization into Personalized Peptide Vaccine Design for Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Outcomes for patients with glioblastoma (GBM) remain poor despite multimodality treatment with surgery, radiation, and chemotherapy. There are few immunotherapy options due to the lack of tumor immunogenicity. Several clinical trials have reported promising results with cancer vaccines. To date, studies have used data from a single tumor site to identify targetable antigens, but this approach limits the antigen pool and is antithetical to the heterogeneity of GBM. We have implemented multisector sequencing to increase the pool of neoantigens across the GBM genomic landscape that can be incorporated into personalized peptide vaccines called NeoVax. PATIENTS AND METHODS: In this study, we report the findings of four patients enrolled onto the NeoVax clinical trial (NCT0342209). RESULTS: Immune reactivity to NeoVax neoantigens was assessed in peripheral blood mononuclear cells pre- and post-NeoVax for patients 1 to 3 using IFNγ-ELISPOT assay. A statistically significant increase in IFNγ producing T cells at the post-NeoVax time point for several neoantigens was observed. Furthermore, a post-NeoVax tumor biopsy was obtained from patient 3 and, upon evaluation, revealed evidence of infiltrating, clonally expanded T cells. CONCLUSIONS: Collectively, our findings suggest that NeoVax stimulated the expansion of neoantigen-specific effector T cells and provide encouraging results to aid in the development of future neoantigen vaccine-based clinical trials in patients with GBM. Herein, we demonstrate the feasibility of incorporating multisector sampling in cancer vaccine design and provide information on the clinical applicability of clonality, distribution, and immunogenicity of the neoantigen landscape in patients with GBM.

A Phase 1/2 Study of Disulfiram and Copper With Concurrent Radiation Therapy and Temozolomide for Patients With Newly Diagnosed Glioblastoma.

PURPOSE: This phase 1/2 study aimed to evaluate the safety and preliminary efficacy of combining disulfiram and copper (DSF/Cu) with radiation therapy (RT) and temozolomide (TMZ) in patients with newly diagnosed glioblastoma (GBM). METHODS AND MATERIALS: Patients received standard RT and TMZ with DSF (250-375 mg/d) and Cu, followed by adjuvant TMZ plus DSF (500 mg/d) and Cu. Pharmacokinetic analyses determined drug concentrations in plasma and tumors using high-performance liquid chromatography-mass spectrometry. RESULTS: Thirty-three patients, with a median follow-up of 26.0 months, were treated, including 12 IDH-mutant, 9 NF1-mutant, 3 BRAF-mutant, and 9 other IDH-wild-type cases. In the phase 1 arm, 18 patients were treated; dose-limiting toxicity probabilities were 10% (95% CI, 3%-29%) at 250 mg/d and 21% (95% CI, 7%-42%) at 375 mg/d. The phase 2 arm treated 15 additional patients at 250 mg/d. No significant difference in overall survival or progression-free survival was noted between IDH- and NF1-mutant cohorts compared with institutional counterparts treated without DSF/Cu. However, extended remission occurred in 3 BRAF-mutant patients. Diethyl-dithiocarbamate-copper, the proposed active metabolite of DSF/Cu, was detected in plasma but not in tumors. CONCLUSIONS: The maximum tolerated dose of DSF with RT and TMZ is 375 mg/d. DSF/Cu showed limited clinical efficacy for most patients. However, promising efficacy was observed in BRAF-mutant GBM, warranting further investigation.

Glioblastoma-Infiltrating CD8+ T Cells Are Predominantly a Clonally Expanded GZMK+ Effector Population.

Recent clinical trials have highlighted the limited efficacy of T cell-based immunotherapy in patients with glioblastoma (GBM). To better understand the characteristics of tumor-infiltrating lymphocytes (TIL) in GBM, we performed cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing with paired V(D)J sequencing, respectively, on TILs from two cohorts of patients totaling 15 patients with high-grade glioma, including GBM or astrocytoma, IDH-mutant, grade 4 (G4A). Analysis of the CD8+ TIL landscape reveals an enrichment of clonally expanded GZMK+ effector T cells in the tumor compared with matched blood, which was validated at the protein level. Furthermore, integration with other cancer types highlights the lack of a canonically exhausted CD8+ T-cell population in GBM TIL. These data suggest that GZMK+ effector T cells represent an important T-cell subset within the GBM microenvironment and may harbor potential therapeutic implications. SIGNIFICANCE: To understand the limited efficacy of immune-checkpoint blockade in GBM, we applied a multiomics approach to understand the TIL landscape. By highlighting the enrichment of GZMK+ effector T cells and the lack of exhausted T cells, we provide a new potential mechanism of resistance to immunotherapy in GBM. This article is featured in Selected Articles from This Issue, p. 897.

A pilot phase Ib study to evaluate tadalafil to overcome immunosuppression during chemoradiotherapy for IDH-wild-type glioblastoma.

Background: Myeloid-derived suppressor cells (MDSCs) are critical regulators of immunosuppression and radioresistance in glioblastoma (GBM). The primary objective of this pilot phase Ib study was to validate the on-target effect of tadalafil on inhibiting MDSCs in peripheral blood and its safety when combined with chemoradiotherapy in GBM patients. Methods: Patients with newly diagnosed IDH-wild-type GBM received radiation therapy (RT) and temozolomide (TMZ) combined with oral tadalafil for 2 months. A historical cohort of 12 GBM patients treated with RT and TMZ was used as the comparison group. The ratio of MDSCs, T cells, and cytokines at week 6 of RT compared to baseline were analyzed using flow cytometry. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method. Results: Tadalafil was well tolerated with no dose-limiting toxicity among 16 evaluable patients. The tadalafil cohort had a significantly lower ratio of circulating MDSCs than the control: granulocytic-MDSCs (mean 0.78 versus 3.21, respectively, P = 0.01) and monocytic-MDSCs (1.02 versus 1.96, respectively, P = 0.006). Tadalafil increased the CD8 ratio compared to the control (1.99 versus 0.70, respectively, P < 0.001), especially the PD-1-CD8 T cells expressing Ki-67, CD38, HLA-DR, CD28, and granzyme B. Proinflammatory cytokine IL-1ß was also significantly increased after tadalafil compared to the control. The tadalafil cohort did not have significantly different PFS and OS than the historical control. Conclusions: Concurrent tadalafil is well tolerated during chemoradiotherapy for GBM. Tadalafil is associated with a reduction of peripheral MDSCs after chemoradiotherapy and increased CD8 T-cell proliferation and activation.

A pilot study of lymphoscintigraphy with tracer injection into the human brain.

Many groups have reported lymphatic and glymphatic structures in animal and human brains, but tracer injection into the human brain to demonstrate real-time lymphatic drainage and mapping has not been described. We enrolled patients undergoing standard-of-care resection or stereotactic biopsy for suspected intracranial tumors. Patients received peritumoral injections of 99mTc-tilmanocept followed by planar or tomographic imaging. Fourteen patients with suspected brain tumors were enrolled. One was excluded from analysis because of tracer leakage during injection. There was no drainage of 99mTc-tilmanocept to regional lymph nodes in any of the patients. On average, after correcting for radioactive decay, 70.7% (95% CI: 59.9%, 81.6%) of the tracer in the injection site and 78.1% (95% CI: 71.1%, 85.1%) in the whole-head on the day of surgery remained the morning after, with variable radioactivity in the subarachnoid space. The retained fraction was much greater than expected based on the clearance rate from non-brain injection sites. In this pilot study, the lymphatic tracer 99mTc-tilmanocept was injected into the brain parenchyma, and there was no drainage outside the brain to the cervical lymph nodes. Our work demonstrates an inefficiency of drainage from peritumoral brain parenchyma and highlights a therapeutic opportunity to improve immunosurveillance of the brain.

Diffusion basis spectrum imaging as an adjunct to conventional MRI leads to earlier diagnosis of high-grade glioma tumor progression versus treatment effect.

Background: Following chemoradiotherapy for high-grade glioma (HGG), it is often challenging to distinguish treatment changes from true tumor progression using conventional MRI. The diffusion basis spectrum imaging (DBSI) hindered fraction is associated with tissue edema or necrosis, which are common treatment-related changes. We hypothesized that DBSI hindered fraction may augment conventional imaging for earlier diagnosis of progression versus treatment effect. Methods: Adult patients were prospectively recruited if they had a known histologic diagnosis of HGG and completed standard-of-care chemoradiotherapy. DBSI and conventional MRI data were acquired longitudinally beginning 4 weeks post-radiation. Conventional MRI and DBSI metrics were compared with respect to their ability to diagnose progression versus treatment effect. Results: Twelve HGG patients were enrolled between August 2019 and February 2020, and 9 were ultimately analyzed (5 progression, 4 treatment effect). Within new or enlarging contrast-enhancing regions, DBSI hindered fraction was significantly higher in the treatment effect group compared to progression group (P = .0004). Compared to serial conventional MRI alone, inclusion of DBSI would have led to earlier diagnosis of either progression or treatment effect in 6 (66.7%) patients by a median of 7.7 (interquartile range = 0-20.1) weeks. Conclusions: In the first longitudinal prospective study of DBSI in adult HGG patients, we found that in new or enlarging contrast-enhancing regions following therapy, DBSI hindered fraction is elevated in cases of treatment effect compared to those with progression. Hindered fraction map may be a valuable adjunct to conventional MRI to distinguish tumor progression from treatment effect.

TCR-engineered adoptive cell therapy effectively treats intracranial murine glioblastoma.

BACKGROUND: Adoptive cellular therapies with chimeric antigen receptor T cells have revolutionized the treatment of some malignancies but have shown limited efficacy in solid tumors such as glioblastoma and face a scarcity of safe therapeutic targets. As an alternative, T cell receptor (TCR)-engineered cellular therapy against tumor-specific neoantigens has generated significant excitement, but there exist no preclinical systems to rigorously model this approach in glioblastoma. METHODS: We employed single-cell PCR to isolate a TCR specific for the Imp3D81N neoantigen (mImp3) previously identified within the murine glioblastoma model GL261. This TCR was used to generate the Mutant Imp3-Specific TCR TransgenIC (MISTIC) mouse in which all CD8 T cells are specific for mImp3. The therapeutic efficacy of neoantigen-specific T cells was assessed through a model of cellular therapy consisting of the transfer of activated MISTIC T cells and interleukin 2 into lymphodepleted tumor-bearing mice. We employed flow cytometry, single-cell RNA sequencing, and whole-exome and RNA sequencing to examine the factors underlying treatment response. RESULTS: We isolated and characterized the 3×1.1C TCR that displayed a high affinity for mImp3 but no wild-type cross-reactivity. To provide a source of mImp3-specific T cells, we generated the MISTIC mouse. In a model of adoptive cellular therapy, the infusion of activated MISTIC T cells resulted in rapid intratumoral infiltration and profound antitumor effects with long-term cures in a majority of GL261-bearing mice. The subset of mice that did not respond to the adoptive cell therapy showed evidence of retained neoantigen expression but intratumoral MISTIC T cell dysfunction. The efficacy of MISTIC T cell therapy was lost in mice bearing a tumor with heterogeneous mImp3 expression, showcasing the barriers to targeted therapy in polyclonal human tumors. CONCLUSIONS: We generated and characterized the first TCR transgenic against an endogenous neoantigen within a preclinical glioma model and demonstrated the therapeutic potential of adoptively transferred neoantigen-specific T cells. The MISTIC mouse provides a powerful novel platform for basic and translational studies of antitumor T-cell responses in glioblastoma.

The Conventional Dendritic Cell 1 Subset Primes CD8+ T Cells and Traffics Tumor Antigen to Drive Antitumor Immunity in the Brain.

The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

Computational prediction of MHC anchor locations guides neoantigen identification and prioritization.

Neoantigens are tumor-specific peptide sequences resulting from sources such as somatic DNA mutations. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. Because most somatic mutations are single-nucleotide variants, changes between wild-type and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient's specific MHC molecules. Whereas a subset of peptide positions are presented to the T cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T cell responses. We computationally predicted anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6 to 38% of neoantigen candidates are potentially misclassified and can be rescued using allele-specific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline, and improve the identification process for relevant clinical studies.

Regulatory T cell suppressive potency dictates the balance between bacterial proliferation and clearance during persistent Salmonella infection.

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3-4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3(GFP) reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3(+) Treg suppressive potency. In complementary experiments using Foxp3(DTR) mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.

Naturally occurring altered peptide ligands control Salmonella-specific CD4+ T cell proliferation, IFN-gamma production, and protective potency.

T cell activation required for host defense against infection is an intricately regulated and precisely controlled process. Although in vitro studies indicate that three distinct stimulatory signals are required for T cell activation, the precise contribution of each signal in regulating T cell proliferation and differentiation after in vivo infection is unknown. In this study, altered peptide ligands (APLs) derived from the protective Salmonella-specific FliC Ag and CD4+ T cells specific for the immune-dominant FliC(431-439) peptide within this Ag were used to determine how changes in TCR stimulation impact CD4+ T cell proliferation, differentiation, and protective potency. To explore the prevalence and potential use of altered TCR stimulation by bacterial pathogens, naturally occurring APLs containing single amino acid substitutions in putative TCR contact residues within the FliC(431-439) peptide were identified and used for stimulation under both noninfection and infection conditions. On the basis of this analysis, naturally-occurring APLs that prime proliferation of FliC-specific CD4+ T cells either more potently or less potently compared with the wild-type FliC(431-439) peptide were identified. Remarkably, despite these differences in proliferation, all of the APLs primed reduced IFN-gamma production by FliC(431-439)-specific CD4+ T cells after stimulation in vivo. Moreover, after expression of the parental FliC(431-439) peptide or each APL in recombinant Listeria monocytogenes, only CD4+ T cells stimulated with the wild-type FliC(431-439) peptide conferred significant protection against challenge with virulent Salmonella. These results reveal important and unanticipated roles for TCR stimulation in controlling pathogen-specific CD4+ T cell proliferation, differentiation, and protective potency.

Considerations for personalized neoantigen vaccination in Malignant glioma.

Malignant gliomas are the most common primary brain cancer diagnosed and still carry a poor prognosis despite aggressive multimodal management. Despite the continued advances in immunotherapy for other cancer types, however, there remain no FDA approved immunotherapies for cancers such as glioblastoma. OF the many approaches being explored, cancer vaccine programs are undergoing a renaissance due to the technological advances and personalized nature of their contemporary design. Neoantigen vaccines are a form of immunotherapy involving the use of DNA, mRNA, and proteins derived from non-synonymous mutations identified in patient tumor tissue samples to stimulate tumor-specific T-cell reactivity leading to enhance tumor targeting. In the last several years, the study of neoantigens as a therapeutic target has increased, with the routine workflow implementation of comprehensive next generation sequencing and in silico peptide binding prediction algorithms. Several neoantigen vaccine platforms are being evaluated in clinical trials for malignancies including melanoma, pancreatic cancer, breast cancer, lung cancer, and glioblastoma, among others. In this review, we will review the concept of neoantigen discovery using cancer immunogenomics approaches in glioblastoma and explore the disease-specific issues being addressed in the design of effective personalized cancer vaccine strategies.

Impact of CD4 T cells on intratumoral CD8 T-cell exhaustion and responsiveness to PD-1 blockade therapy in mouse brain tumors.

BACKGROUND: Glioblastoma is a fatal disease despite aggressive multimodal therapy. PD-1 blockade, a therapy that reinvigorates hypofunctional exhausted CD8 T cells (Tex) in many malignancies, has not shown efficacy in glioblastoma. Loss of CD4 T cells can lead to an exhausted CD8 T-cell phenotype, and terminally exhausted CD8 T cells (Tex term) do not respond to PD-1 blockade. GL261 and CT2A are complementary orthotopic models of glioblastoma. GL261 has a functional CD4 T-cell compartment and is responsive to PD-1 blockade; notably, CD4 depletion abrogates this survival benefit. CT2A is composed of dysfunctional CD4 T cells and is PD-1 blockade unresponsive. We leverage these models to understand the impact of CD4 T cells on CD8 T-cell exhaustion and PD-1 blockade sensitivity in glioblastoma. METHODS: Single-cell RNA sequencing was performed on flow sorted tumor-infiltrating lymphocytes from female C57/BL6 mice implanted with each model, with and without PD-1 blockade therapy. CD8+ and CD4+ T cells were identified and separately analyzed. Survival analyses were performed comparing PD-1 blockade therapy, CD40 agonist or combinatorial therapy. RESULTS: The CD8 T-cell compartment of the models is composed of heterogenous CD8 Tex subsets, including progenitor exhausted CD8 T cells (Tex prog), intermediate Tex, proliferating Tex, and Tex term. GL261 is enriched with the PD-1 responsive Tex prog subset relative to the CT2A and CD4-depleted GL261 models, which are composed predominantly of the PD-1 blockade refractory Tex term subset. Analysis of the CD4 T-cell compartments revealed that the CT2A microenvironment is enriched with a suppressive Treg subset and an effector CD4 T-cell subset that expresses an inhibitory interferon-stimulated (Isc) signature. Finally, we demonstrate that addition of CD40 agonist to PD-1 blockade therapy improves survival in CT2A tumor-bearing mice. CONCLUSIONS: Here, we describe that dysfunctional CD4 T cells are associated with terminal CD8 T-cell exhaustion, suggesting CD4 T cells impact PD-1 blockade efficacy by controlling the severity of exhaustion. Given that CD4 lymphopenia is frequently observed in patients with glioblastoma, this may represent a basis for resistance to PD-1 blockade. We demonstrate that CD40 agonism may circumvent a dysfunctional CD4 compartment to improve PD-1 blockade responsiveness, supporting a novel synergistic immunotherapeutic approach.

A randomized feasibility study evaluating temozolomide circadian medicine in patients with glioma.

Background: Gliomas are the most common primary brain tumor in adults. Current treatments involve surgery, radiation, and temozolomide (TMZ) chemotherapy; however, prognosis remains poor and new approaches are required. Circadian medicine aims to maximize treatment efficacy and/or minimize toxicity by timed delivery of medications in accordance with the daily rhythms of the patient. We published a retrospective study showing greater anti-tumor efficacy for the morning, relative to the evening, administration of TMZ in patients with glioblastoma. We conducted this prospective randomized trial to determine the feasibility, and potential clinical impact, of TMZ chronotherapy in patients with gliomas (NCT02781792). Methods: Adult patients with gliomas (WHO grade II-IV) were enrolled prior to initiation of monthly TMZ therapy and were randomized to receive TMZ either in the morning (AM) before 10 am or in the evening (PM) after 8 pm. Pill diaries were recorded to measure compliance and FACT-Br quality of life (QoL) surveys were completed throughout treatment. Study compliance, adverse events (AE), and overall survival were compared between the two arms. Results: A total of 35 evaluable patients, including 21 with GBM, were analyzed (18 AM patients and 17 PM patients). Compliance data demonstrated the feasibility of timed TMZ dosing. There were no significant differences in AEs, QoL, or survival between the arms. Conclusions: Chronotherapy with TMZ is feasible. A larger study is needed to validate the effect of chronotherapy on clinical efficacy.

Corrigendum to: A phase II study of laser interstitial thermal therapy combined with doxorubicin in patients with recurrent glioblastoma.

[This corrects the article DOI: 10.1093/noajnl/vdab164.].

Characterization of the Genomic and Immunologic Diversity of Malignant Brain Tumors through Multisector Analysis.

Despite some success in secondary brain metastases, targeted or immune-based therapies have shown limited efficacy against primary brain malignancies such as glioblastoma (GBM). Although the intratumoral heterogeneity of GBM is implicated in treatment resistance, it remains unclear whether this diversity is observed within brain metastases and to what extent cancer cell-intrinsic heterogeneity sculpts the local immune microenvironment. Here, we profiled the immunogenomic state of 93 spatially distinct regions from 30 malignant brain tumors through whole-exome, RNA, and T-cell receptor sequencing. Our analyses identified differences between primary and secondary malignancies, with gliomas displaying more spatial heterogeneity at the genomic and neoantigen levels. In addition, this spatial diversity was recapitulated in the distribution of T-cell clones in which some gliomas harbored highly expanded but spatially restricted clonotypes. This study defines the immunogenomic landscape across a cohort of malignant brain tumors and contains implications for the design of targeted and immune-based therapies against intracranial malignancies. SIGNIFICANCE: This study describes the impact of spatial heterogeneity on genomic and immunologic characteristics of gliomas and brain metastases. The results suggest that gliomas harbor significantly greater intratumoral heterogeneity of genomic alterations, neoantigens, and T-cell clones than brain metastases, indicating the importance of multisector analysis for clinical or translational studies.This article is highlighted in the In This Issue feature, p. 1.