Predicting recurrent glioblastoma clinical outcome to immune checkpoint inhibition and low-dose bevacizumab with tumor in situ fluid circulating tumor DNA analysis

Objective Most recurrent glioblastoma (rGBM) patients do not benefit from immune checkpoint inhibition, emphasizing the necessity for response biomarkers. This study evaluates whether tumor in situ fluid (TISF) circulating tumor DNA (ctDNA) could serve as a biomarker for response to low-dose bevacizumab (Bev) plus anti-PD-1 therapy in rGBM patients, aiming to enhance systemic responses to immunotherapy. Methods In this phase II trial, 32 GBM patients with first recurrence after standard therapy were enrolled and then received tislelizumab plus low-dose Bev each cycle. TISF samples were analyzed for ctDNA using a 551-gene panel before each treatment. Results The median progression-free survival (mPFS) and overall survival (mOS) were 8.2 months (95% CI, 5.2–11.1) and 14.3 months (95% CI, 6.5–22.1), respectively. The 12-month OS was 43.8%, and the objective response rate was 56.3%. Patients with more than 20% reduction in the mutant allele fraction and tumor mutational burden after treatment were significantly associated with better prognosis compared to baseline TISF-ctDNA. Among detectable gene mutations, patients with MUC16 mutation, EGFR mutation & amplification, SRSF2 amplification, and H3F3B amplification were significantly associated with worse prognosis. Conclusions Low-dose Bev plus anti-PD-1 therapy significantly improves OS in rGBM patients, offering guiding significance for future individualized treatment strategies. TISF-ctDNA can monitor rGBM patients' response to combination therapy and guide treatment. Clinical trial registration This trial is registered with ClinicalTrials.gov, NCT05540275.


Introduction
Glioblastoma (GBM) is the most malignant primary brain tumor and is prone to recurrence.Despite multidisciplinary treatments including surgery, radiotherapy, chemotherapy, targeted therapy, and supportive care, the overall prognosis remains poor [1][2][3][4].Updated guidelines for the management of gliomas still encourage clinical trials for rGBM due to the limited efficacy of available salvage therapies at the time of tumor recurrence, with a median survival of only 6-8 month [5].The search for novel therapeutic options to improve the prognosis of rGBM patients is ongoing, with research focusing on combining antiangiogenic agents with immunotherapy to enhance antitumor immune responses [6,7].
Preliminary results have shown that immune checkpoint inhibitors combined with anti-angiogenesis drugs have a good safety profile in treating rGBM [8,9].Non-clinical studies have demonstrated that bevacizumab, an antiangiogenic targeted agent, can inhibit vascular endothelial growth factor, promote tumor vascular normalization, increase T cell infiltration, and reduce immunosuppressive cell activity, thereby improving immunotherapy efficacy [10].Although bevacizumab combined with immunotherapy has been feasible and safe in treating other solid tumors, it has not improved OS of rGBM patients [11].A complete response to concurrent anti-PD-1 and low-dose anti-VEGF therapy was reported in one patient with rGBM [12].Therefore, larger clinical trials are needed to investigate whether low-dose Bev can promote immunotherapy responses.
Biomarkers are critical to maximizing therapeutic efficacy and minimizing toxicity in rGBM treated with low-dose Bev plus anti-PD-1 therapy [13][14][15][16].Analyzing circulating tumor DNA (ctDNA) as an emerging biomarker in solid tumors faces technical challenges due to the specificity of GBM's location [17,18].The collection of TISF for ctDNA analysis has been reported by our research group multiple times, yet there is limited literature on ctDNA changes in rGBM after immunotherapy combined with low-dose Bev treatment [17][18][19][20].Thus, the feasibility of ctDNA as a biomarker in rGBM patients needs further investigation.with recurrent glioblastoma treated with anti-PD-1 antibody combined with low-dose bevacizumab were significantly higher than those before treatment Combining tislelizumab with low-dose Bev in treating rGBM, we hypothesized that low-dose Bev treatment might improve the immunotherapy response.To assess ctDNA's efficacy for monitoring rGBM patients' response to combination therapy, we collected TISF samples at baseline and each subsequent immunotherapy cycle.

Study design and participants
This open-label phase 2 study (Clinical Trials ID: NCT 05540275) recruited rGBM patients at Zhengzhou University People's Hospital (Zhengzhou University).From March 28, 2022, patients received tislelizumab (200 mg) and bevacizumab (3 mg/kg) intravenously every 3 weeks until disease progression or intolerance.Magnetic resonance imaging (MRI) was performed at baseline and every 4-8 weeks thereafter.Tumor volume measurement and RANO 2.0 assessment were performed using 3D slicer software (National Institutes of Health, Bethesda, USA) [21].
Eligible patients were aged 18-75 years with confirmed rGBM, a Karnofsky Performance Status (KPS) ≥ 70, and had undergone ≥ 1 prior systemic GBM therapy.Exclusion criteria included systemic glucocorticoid or other immunosuppressive therapy within 7 days after enrollment, known or suspected active autoimmune disease, active hepatitis B or C, HIV infection, extracranial metastases, significant leptomeningeal disease, or tumors primarily in the brain stem or spinal cord.
Recurrent GBM: Surgery was recommended.Patients who refused surgery were given bevacizumab (5 mg/kg IV) combined with TMZ (150 mg/m 2 /d orally for 5 days, repeated every 21 days for 6 cycles), followed by bevacizumab (3 mg/kg) and tislelizumab (200 mg IV) every 21 days for six cycles.

Sample collection, DNA extraction, and library preparation
Tumor in situ fluid (TISF) samples were collected as previously described [18][19][20]22].A small amount of TISF (0.5-2 ml) was obtained by syringe from the implanted reservoir sac every 4 to 8 weeks (Fig. 1A).TISF is the fluid present in the local surgical cavity.ctDNA profiles from tumor tissue and TISF samples can be used to assess the dynamic evolution of the tumor in real time, while 5 ml of blood is collected as a germline DNA control.

TMB calculation
To calculate the TMB using the 551-solid cancer-gene targeted next-generation sequencing (NGS) panel, all base substitutions and indels in the coding region of targeted genes were summed, excluding synonymous alterations, alterations with AF < 0.02, and alterations listed as known somatic alterations in COSMIC.

Statistical analysis
The primary outcome was overall survival (OS, defined as time from enrollment to death or last clinical followup).Secondary outcomes included OS rate at 12 months, progression-free survival (PFS, defined as time from treatment initiation to first disease progression, death, or last follow-up imaging), and the objective response rate (ORR, defined as complete response plus partial response).The mutant allele fraction (MAF) was defined as the sum of all mutations detectable in each sample.Exploratory endpoints included drug safety and toxicity (Common Terminology Criteria for Adverse Events, CTCAE 5.0).The functional status of tumor patients was assessed using the Karnofsky Performance Status (KPS) scoring criteria.PFS and OS were analyzed using the Kaplan-Meier method, and the stratified Cox proportional hazards model was employed to calculate the hazard ratio (HR) and 95% confidence interval (CI).Clinical response was assessed using RANO 2.0 criteria, classifying responses as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) [23].The Wilcoxon rank-sum test compared continuous variables between two groups, while Spearman's rank correlation estimated the correlation between two continuous variables.P < 0.05 indicated statistical significance.Statistical analyses were conducted using Prism 9.5 or R, version 4.2.1.

Circulating tumor DNA analysis
Despite challenges posed by the COVID-19 pandemic, at least one TISF or tissue sample from 31 patients was analyzed by ctDNA, with 19 samples containing before-andafter controls.High-throughput sequencing of TISF using a custom panel designed for solid tumors was performed (Fig. 2A).TERT emerged as the most prevalent genetic mutation, consistent with previous studies [24].
Two patients demonstrated H3F3B amplification's ability to track combination therapy response.Patient 21 had a near-complete imaging response at 6.4 months but progressed after being lost to follow-up for 3.1 months due to the COVID-19 pandemic (Fig. 5A).Patient 11 consistently had H3F3B amplification detected during follow-up and progressed 1.7 months after starting therapy (Fig. 5B).These results suggest H3F3B amplification may lead to drug resistance by altering the tumor immune microenvironment, and ctDNA has potential to monitor combination therapy response in rGBM patients.
Finally, 8 patients experienced a second relapse after combination therapy, all from the ctDNA/TMB nonresponse group.KEGG pathway enrichment analysis revealed significant increases in mutated genes associated with cell cycle and transcriptional misregulation pathways in ctDNA at second recurrence and significant decreases in genes associated with microRNAs in cancer pathways (Fig. 5C).More cohort studies are needed to verify these changes in detail, revealing the related mechanisms of lowdose Bev + anti-PD-1 therapy and acquired resistance.
Fig. 3 Analysis of ctDNA in patients treated with low-dose Bev + anti-PD-1.A, Spider plot of ctDNA levels before and after treatment with low-dose Bev + anti-PD-1 treatment.Patients are colored by RANO2.0 response, and the ctDNA allele fraction at each time point was divided by the pre-treatment allele fraction.In the one patient with ctDNA not detected prior to treatment, the pre-treatment limit of detection was used for normalization based on the number of mutations tracked and average sequencing depth as described in the methods.B, Fisher precise test analysis showed that changes in ctDNA levels were significantly correlated with treatment response.C, Correlation between baseline tumor burden measured by 3D slicer and baseline ctDNA mutant allele fraction (MAF).D, Forest plot depicting progression-free survival (PFS) and overall survival (OS) improvements for each variable in patients treated with low-dose Bev + anti-PD-1 therapy.The HRs and statistical significance of the difference were computed using the Cox proportional hazards model and Wald test.E, Kaplan-Meier curves showed that patients in the TISF-ctDNA significant changes group had significantly improved PFS and OS after receiving low-dose Bev plus anti-PD-1 therapy.Spearman's correlation coefficient, 95% confidence interval, and P-value are displayed on the graph ◂ Fig. 4 Oncogenic alterations correlated with fewer benefits from lowdose Bev + anti-PD-1 therapy.A, Kaplan-Meier curves showed that patients in the ctDNA response group had significantly improved PFS and OS after receiving low-dose Bev plus anti-PD-1 therapy.B, Kaplan-Meier curves depict PFS and OS improvements in patients with partial gene wild-type mutations on low-dose Bev + anti-PD-1 therapy.C, Copy number changes in H3F3B amplification in 8 rGBM patients throughout treatment

Discussion
In this study, 32 rGBM patients received tislelizumab plus low-dose Bev, hypothesizing that low-dose bevacizumab would normalize vascular conditions and facilitate immunotherapy, using TISF-ctDNA as a biomarker to track treatment response and gene evolution [8,9].The observed ORR of 56.3% significantly benefited patients, exceeding the 7.8% ORR for GBM with nivolumab in the CheckMate 143 trial [13].This study is the first to perform biomarker analysis in rGBM patients treated with this combination therapy.
Standard-dose bevacizumab combined with anti-PD-1 has been confirmed effective in other solid tumors [28][29][30][31], but efficacy in rGBM is poor [32].Bevacizumab, a humanized monoclonal antibody inhibiting VEGF, enhances tumorspecific immune response by promoting immunosuppressive tumor microenvironment, normalizing vascular structure, increasing T cell infiltration, and activating local immune microenvironment [12,[33][34][35][36].The 2021 ASCO Annual Meeting reported no benefit of low-dose Bev + anti-PD-1 compared with standard Bev for rGBM, and standard Bev can help older rather than younger patients [32].Therefore, it is essential to find suitable biomarkers that guarantee to maximize the therapeutic effect [37].However, TMB and PD-L1 expression has not predicted anti-PD-1 monotherapy efficacy in rGBM [38][39][40][41].Although single time-point MAF and TMB expression did not correlate with response to low-dose Bev plus anti-PD-1, dynamic changes predicted response.Two patients with ctDNA negative (ctDNA -) before or after treatment had better prognosis, and patients with high baseline ctDNA levels (MAF > 5%) had better prognosis with significant post-treatment TISF-ctDNA gene mutation changes.This facilitates screening high-risk recurrence patients and timely treatment regimen adjustments.
This study's limitations include the limited data size and all participants were Chinese.Future genomic data from cohorts with low-dose bevacizumab plus anti-PD-1 therapy are needed to validate identified biomarkers.Validation using a combination therapy dataset could demonstrate intrinsic associations between biomarkers and antitumor immunity, affirming their predictive value for immunostimulatory chemotherapy and anti-PD-1 therapy benefits.Technological advancements are needed to reduce genome sequencing costs and ensure speedy analysis for clinical application.
By performing high-throughput sequencing on samples from 97% of patients, we identified four oncogenic risk alterations as reliable biomarkers for low-dose bevacizumab plus anti-PD-1 therapy outcomes in rGBM patients.These findings provide a basis for individualized treatment and future biological studies of its immuno-oncology characteristics, inspiring biomarker exploration of low-dose bevacizumab + anti-PD-1 in other cancer types.

Conclusions
Anti-PD-1 antibody combined with low-dose bevacizumab can significantly prolong PFS and OS in rGBM patients without significant adverse reactions, improving quality of life and providing a new effective treatment for rGBM.TISF-ctDNA dynamic changes can predict the treatment response, identify drug resistance mechanisms, monitor high-risk recurrence (ctDNA molecular recurrence) populations, and provide a basis for early intervention decision making.TISF-ctDNA characterizes in vivo gene evolution in rGBM patients treated with anti-PD-1 antibody combined with low-dose bevacizumab, providing molecular information for drug resistance mechanism studies in rGBM.
Fig. 5 Monitoring response to low-dose Bev + anti-PD-1 therapy using ctDNA analysis.A-B, Examples of longitudinal radiographic imaging and ctDNA monitoring in A a patient with progressive disease on first surveillance imaging and (B) a patient with sustained disease remission after starting treatment.Circulating tumor DNA allele fraction is shown in teal, and H3F3B copy numbers are shown in red.C, Bubble plots show pathway alterations by KEGG enrichment analysis at baseline and after low-dose Bev plus anti-PD-1 treatment.D, Bubble plots showing pathway alterations by KEGG enrichment analysis at baseline and after low-dose Bev plus anti-PD-1 treatment.There was a significant increase in mutated genes associated with cell cycle and transcription dysregulation pathways and a decrease in mutated genes associated with microRNAs in cancer pathways in TISF-ctDNA at relapse compared with baseline.E, The median PFS and OS of all patients were analyzed ◂

Fig. 1
Fig. 1 Treatment schema and patient characteristics.A, Schematic showing the timing of treatment and tumor in situ fluid (TISF) collection.TISF was collected for ctDNA analysis pre-treatment and prior to each cycle of immunotherapy.B, The KPS scores of 32 patients

Fig. 2
Fig.2Patient treatment events and ctDNA outcomes.A, Oncoplot depicting the genomic alteration of 32 recurrent GBM patients at different time points.Plot of tumor variants identified from 551panel sequencing and tracked using ctDNA analysis for each patient.The top panel shows the total number of single nucleotide variants (SNVs) and copy number alterations (CNAs) tracked, and the left panel shows the number of patients with mutations in each gene.Only the most frequently mutated genes are displayed.B, Event chart showing time points for low-dose Bev + anti-PD-1 treatment, treatment response assessed according to RANO2.0 criteria, and the results of ctDNA testing for each patient with at least one TISF sample or tissue-sample time point analyzed.C, Proportion of patients with ctDNA detected in at least one TISF sample time point.Treatment efficacy (PD progressive disease; PR partial response; SD stable disease)

Table 1
Summary table of patient characteristics

Table 2
Treatment-related adverse events in the safety population