Bevacizumab rechallenge for recurrent glioblastoma - characterization of patients presenting multiple responses

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This study aimed to characterize such repeat responders and identify potential predictive biomarkers. Methods A total of 254 glioblastoma IDHwt patients treated with bevacizumab-based combination therapy were evaluated for eligibility. Five patients who demonstrated repeated responses to bevacizumab and had available tumor tissue for molecular analysis were included. Histopathological re-assessments were performed to confirm glioblastoma diagnoses. Angiotensinogen (AGT) promoter methylation status was analyzed in all primary tumor samples, while bulk RNA sequencing and TSO500 assays were conducted on all available samples. Results In our cohort, 40% of patients who were rechallenged with bevacizumab following a treatment pause exhibited a response both during the initial course and upon rechallenge. Bulk RNA sequencing revealed downregulation of HILPDA, HHLA2, and IGF2 as potential predictive biomarkers for repeated bevacizumab response. Additionally, AGT promoter methylation analysis identified high methylation levels as another potential predictive biomarker. Conclusion A subgroup of glioblastoma patients respond to bevacizumab at least up to five times in the recurrent setting, and these repeated responders exhibit prolonged survival. No definitive prognostic variables or histopathological features were found in this study. Further investigations into the downregulation of HILPDA, HHLA2 and IGF2 along with high AGT promoter methylation levels and other potential predictive biomarkers, are warranted to better understand the mechanisms underlying repeated bevacizumab response. glioblastoma bevacizumab rechallenge reinduction response Figures Figure 1 Figure 2 Figure 3 Introduction Newly diagnosed glioblastoma (GBM) patients in good performance status are treated with maximal safe surgery followed by radiation therapy and temozolomide [ 1 ]. However, no standard treatment exists at disease recurrence. Multiple treatments have been tested in the recurrent setting, including re-surgery, re-irradiation, chemotherapeutic and targeted agents, but most regimens have shown limited efficacy [ 2 ]. Bevacizumab, an antibody targeting The Vascular Endothelial Growth Factor A (VEGF), has shown clinical activity with response rates of approximate 30%, improved progression-free survival (PFS) when given in combination with irinotecan or lomustine, but no improvement in overall survival (OS) when considering the total population of recurrent GBM patients [ 3 – 5 ]. These data demonstrate that not all patients benefit from bevacizumab combination therapy with irinotecan or lomustine. However, a subset of recurrent GBM patients who respond to bevacizumab combination therapy experience improved survival and enhanced clinical status [ 6 – 8 ]. This highlights the importance of characterizing patients benefitting from treatment with the overall objective to identify predictive biomarkers for efficacy. Recently, we identified low expression of the angiotensinogen (AGT) gene, regulated by methylation of its promoter region, as being associated with a higher likelihood of response to bevacizumab [ 9 – 10 ]. In patients who initially respond to bevacizumab, rechallenge with the therapy has been reported to induce a new response in around 60% of cases [ 11 – 13 ], and even a third response in selected cases 9 . These studies reported longer survival for rechallenged patients [ 11 – 12 ], indicating that re-exposure to bevacizumab could be beneficial for prolonging survival in responders whose treatment was discontinued prior to tumour progression. Such findings contrast with studies testing the continuation of bevacizumab in patients who had previously progressed on bevacizumab therapy. In these studies, bevacizumab failed to produce a second treatment response [ 14 – 16 ] and showed no impact on survival [ 16 – 17 ]. Here, we report clinical history, pathological, and genomic features for patients with progressive GBM treated with up to five individual bevacizumab courses resulting in clinical benefit. Patients and Methods Patients From our clinical glioblastoma database [ 18 ], patients treated with bevacizumab with either lomustine or irinotecan combination therapy between year 2007-18 at Rigshospitalet for progressive GBM IDH wild type were screened for eligibility. Patients were included in the study if they achieved treatment response during both the initial bevacizumab course and the rechallenge, and if GBM tissue was available for analysis. The control group used for comparison with the repeated bevacizumab responders in Kaplan Meier plots were 38 GBM patients, who had formalin-fixed paraffin-embedded (FFPE) tissue from either primary or relapse tumour available in the biobank. Treatment First-line treatment included maximal safe surgical resection followed by radiation therapy plus concomitant and adjuvant temozolomide according to the Stupps regimen [ 1 , 18 ]. At time of progression, patients were evaluated by a multidisciplinary team for relapse surgery or second-line line therapy. Patients in good ECOG performance status (ECOG PS 0–1) and with measurable tumour on MRI was offered bevacizumab in combination with irinotecan or lomustine. For the irinotecan combination, 10 mg/kg bevacizumab was given every 2 weeks together with 125 mg/m 2 irinotecan [ 19 ]. For the lomustine combination 10 mg/kg bevacizumab was given every 2 weeks while lomustine treatment was delivered every 6 weeks at a concentration of 90 mg/m 2 [ 8 ]. A treatment cycle was defined as 4 weeks treatment in patients receiving bevacizumab and irinotecan and 6 weeks in patients receiving bevacizumab and lomustine. A new treatment course was defined when treatment was initiated after disease progression following a treatment pause of at least 3 months. Treatment stop was registered as the last day in the treatment course where patients received treatment. Clinical follow-up Patients were clinically evaluated every 2 weeks when receiving therapy, and MRI evaluation was performed after 2 cycles, being every 8 weeks in patients receiving irinotecan and every 12 weeks in patients receiving lomustine [ 8 , 19 ]. Patient and tumour data was retrospectively reviewed via medical journals, pathology reports and imaging descriptions. Response evaluation was performed according to the RANO criteria [ 20 ]. OS was calculated from the day of initial diagnostic surgery to the day of death and to the date the patient started the first bevacizumab treatment. PFS was calculated from the day of diagnosis to the day of 1st relapse confirmed by MRI. Pathology In addition to routine pathological evaluations, DNA purified from macro-dissected FFPE tissue – enriched for tumour content - was bisulfite-treated and analysed using Infinium MethylationEPIC v1.0 BeadChip (Illumina, Ca, USA) according to the manufacturer’s instructions. Methylation profiling report containing methylation classification, copy number variations and MGMT promoter methylation status were generated using app.epignostix.com (v12.8), Heidelberg based on previously described method [ 21 ]. AGT promoter methylation status was assessed using the generated iDAT files to analyse AGT promoter CpG site cg12469306. Immunohistochemistry for mutation in IDH using anti-IDH1 R132H (clone H09, Dianova, 1:700 dilution), and in ATRX using anti-ATRX (HPA001906, Sigma, 1:150 dilution). One patient was tested for H3F3A mutation by dideoxysequencing covering codon 28–35. RNA sequencing and TSO500 Archived FFPE tissue from primary tumour of case 1, case 2, case 4 and case 5 were accessible for bulk RNA sequencing and TSO500. For case 3 only FFPE tissue from 1st relapse was accessible and for case 5 FFPE tissue from 1st relapse was also accessible for bulk RNA sequencing and TSO500. For best comparison of samples and to avoid the least amount of batch effect only FFPE tissue from the primary tumours (4 samples) were evaluated and analysed in downstream differential expression analysis on the bulk RNA sequencing data. A control group consisting of 11 other FFPE primary GBM tumours that were available in the biobank were used for comparison. RNA was extracted from FFPE GBM tumour tissues from archived patient samples which were sequenced on Illumina NovaSeq. FASTQ files were processed using Nextflow [ 22 ] nf-core [ 23 ] pipeline nf-core/rnaseq (v3.8.1). STAR and Salmon were used for alignment and quantification with Singularity (v3.6.2) as the profile option and GRCh38 as the reference genome. Downstream bulk RNA-seq analysis was performed using R (v4.2.1). Batch correction and differential expression analysis was performed using R package limma [ 24 ](v3.52.4). For TSO500 all cases were evaluated with regards to most common GBM mutations [ 25 ], except for case 5 relapse tumour sample because of bad quality. Results Clinical history of patients with repeated bevacizumab response A total of 254 GBM IDHwt patients treated with bevacizumab combination therapy for progression after Stupp’s regimen were screened for eligibility. As shown in the REMARK diagram (Supplementary Figure S1), 15 patients were rechallenged with bevacizumab therapy after a treatment pause and six (40%) of these achieved response to both first course bevacizumab and bevacizumab rechallenge. A total of five patients with repeated bevacizumab response and with available tumour tissue for molecular analysis were included in the study (Table 1). Treatment covered in total 172 bevacizumab cycles divided over 20 courses, with 18 courses resulting in clinical benefit and 15 courses being rechallenges. Pauses between individual bevacizumab treatment courses were due to either clinical benefit with MRI confirmed reduction in tumour size or other non-progression-related reasons. Case 1 A 60-year-old woman with GBM, methylated MGMT (RTK2 subtype), survived 77 months post-diagnosis and 45 months after initiating bevacizumab. Initial treatment (Figure 1A) included partial resection followed by standard radio-chemotherapy, achieving a partial response (PR). After 16 months of stable disease (SD), progression near the right lateral ventricle occurred. Temozolomide reinduction (3 cycles) failed due further tumor progression, and bevacizumab/irinotecan was initiated. The patient underwent 24 bevacizumab cycles over four courses. Prior to bevacizumab courses, ECOG PS declined from 0 (course 1) to 3 (course 4) (Figure 1B). Neurocognitive deficits fluctuated throughout but improved prior to course 3. No corticosteroids were used. The first course achieved PR after two cycles, and microspots remained after eight cycles, prompting a treatment pause. After five months, local progression led to a second course (six cycles), again with PR. After a 12-month pause a third course (bevacizumab plus lomustine) achieved a durable complete response for six cycles but was paused due to clinical decline. Four months later, recurrence prompted a fourth course (bevacizumab/irinotecan), which failed due to refractory disease. The patient died shortly after. Case 2 A 34-year-old woman with GBM, methylated MGMT, survived 130 months from diagnosis and 112 months after starting bevacizumab. The patient achieved SD upon standard therapy. Over the years, the patient received 55 bevacizumab/irinotecan cycles in five courses, with 9–12 month treatment pauses. ECOG PS was 0–1 at each course start; corticosteroids were required before course 2. No neurocognitive deficit was observed. Multifocal disease appeared before courses 4 and 5. The first course (10 cycles) began six months after adjuvant therapy due to MRI-confirmed progression, achieving PR. The second course (20 cycles) achieved SD by cycle 2 and PR by cycle 6. A third course (10 cycles) achieved SD with tumour shrinkage. Course 4 was started after MRI revealed a new isolated lesion, achieving SD after two cycles and PR after 11 cycles. Progression with new tumour lesions led to a fifth course, achieving SD with lesion reduction until cycle 4, when infection and facial nerve palsy interrupted treatment. After 12 months with SD, the disease progressed with new lesions and leptomeningeal spread. The patient developed epilepsy and ventriculitis and died five months later. Case 3 A 58-year-old man with GBM, MGMT methylated (mesenchymal subtype), survived 72 months, including 61 months post-bevacizumab. Initial standard treatment led to PR with disappearance of contrast-enhancing lesions. At completion of adjuvant temozolomide MRI showed progression, which advanced to measurable lesions within two months, prompting initiation of bevacizumab/irinotecan. The patient received 30 cycles across four courses. ECOG PS was 0 and no neurocognitive deficits were noted prior to each course. Corticosteroid use and multifocal disease were observed at later courses. The first bevacizumab course resulted in PR after two cycles and further tumor reduction after eight cycles, followed by a treatment pause. Eleven months later, new progressive lesions led to a second course (5 cycles), achieving SD. An infection interrupted treatment, after which tumor progressed. A third course (7 cycles) again achieved SD with tumor regression. A 17-month treatment-free interval followed. The fourth course produced durable PR, but after ten cycles progression with a new lesion prompted surgery. The patient experienced postoperative complications and died shortly thereafter. Case 4 A 58-year-old woman with non-resectable, multifocal GBM (MGMT status unknown) involving the left temporal lobe and corpus callosum survived 48 months, with 43 months post-bevacizumab. Initial treatment included standard therapy. Three months post-radiation, MRI revealed progression with a new lesion in the contralateral hemisphere, prompting bevacizumab-based therapy. The patient received 27 bevacizumab cycles over three courses. Prior to course 1, ECOG PS was 1 and the patient used corticosteroids. Before subsequent courses, PS had improved to 0 and corticosteroids were no longer needed. The first course led to clinical improvement and PR after two cycles, with further tumor reduction seen after nine cycles. After 11 cycles, the treatment was paused. After a six-month break, a new measurable lesion appeared. A second course (bevacizumab with lomustine) achieved durable PR for eight cycles but treatment was interrupted due to grade 3 hepatic enzyme elevation attributed to lomustine. Following a five-month pause, MRI showed progression, and a third course (bevacizumab/irinotecan) was initiated, achieving SD over eight cycles. The patient clinically deteriorated two months after the final pause and died shortly thereafter. Case 5 A 60-year-old woman with GBM, MGMT methylated (mesenchymal subtype) located in the left occipital and right parietal lobe survived 57 months, with 52 months post-bevacizumab. Standard therapy was followed by early progression (focal seizures and two new lesions), leading to bevacizumab-based therapy. The patient received 36 cycles over four treatment courses. Multifocal disease was present from the outset. ECOG PS was 0–1 at courses 1 and 2, declining to 2 at course 4. Corticosteroids were used prior to course 3 and 4. The first course with bevacizumab/lomustine achieved durable PR for six cycles until paused due to grade 3 thrombocytopenia. After five months, progression prompted reinduction with lomustine (discontinued due to thrombocytopenia) and a switch to irinotecan. A durable PR was achieved over nine total cycles, including bevacizumab monotherapy. Following a three-month pause, progression led to 15 additional bevacizumab monotherapy cycles, with SD as the best response. A final rechallenge after another five-month break resulted in progression after six cycles. Surgery confirmed recurrent GBM (RTK2 subtype). The patient died three months later. Pathological examination for confirmation of the GBM diagnosis Initial GBM diagnosis was for all five cases based on histological features as having pleomorphic cells, necrosis, vascular proliferation and a prominent mitotic index . Selected molecular analyses conducted at the time of diagnosis supported the GBM diagnosis, however, as the molecular pathology were limited at this time (2008-2013), we re-reviewed the material with up-to-date pathological methods for diagnosis confirmation in the cases where it was relevant (Table 3). AGT promoter methylation status was analysed in the four cases with primary diagnostic tumour tissues. In these cases, AGT promoter was found methylated with a median of 26% (range: 16% - 49%). Bulk RNA sequencing and TSO500 Differential expression analysis on bulk RNA sequencing data showed significant downregulation (Figure 2A) of 3 genes: HILPDA (logFC -5.58 and adjusted p-value of 0.012), HHLA2 (logFC -8.09 and adjusted p-value of 0.040) and IGF2 (logFC -18.80 and adjusted p-value of 0.048) in the 4 repeated bevacizumab therapy responders compared to the 11 control samples. The genes are marked with blue colour in the volcano plot. TSO500 data showed no specific mutation pattern in the most common GBM-mutated genes e.g. TERT, PTEN, EGFR and TP53 (Figure 2B). Discussion We present five recurrent GBM patients showing clinical benefit from bevacizumab rechallenge up to five times after treatment stop for other reasons than progression. This is beyond the one to two rounds of successful bevacizumab rechallenges previously reported in GBM [ 11 – 12 ] and show a long treatment perspective of bevacizumab in sensitive patients. Combined with previous reports of exceptional high response rates under bevacizumab rechallenge in previously responding patients following non-progression related treatment pause [ 11 – 12 ], our data support the existence of GBM tumours with particularly high bevacizumab sensitivity. All five patients experienced OS (from diagnosis) far beyond the median OS of 15 months (Fig. 3 A) and OS from start of bevacizumab relapse treatment far beyond the median OS of 8–9 months (Fig. 3 B) normally observed for GBM patients [ 4 – 5 , 19 ]. Negative result for survival in later years clinical trials testing bevacizumab for GBM [ 4 , 26 ] has fired under speculations that MRI observed bevacizumab responses are not related to direct anti-tumour effects. Rather, it may be pseudo-responses caused by bevacizumab induced decrease in vessel permeability and thereby reduced contrast enhancement [ 27 ]. Furthermore, theories have been that bevacizumab initiates rebound progression with a more fast-growing phenotype [ 28 ]. Multiple mechanisms for tumour acquirement of bevacizumab resistance have been presented [ 29 ], including a bevacizumab induced phenotypic shift from a vascular growth pattern to an aggressively growing invasive phenotype which could be a cause for more rapid progression [ 30 – 31 ]. Our and previous [ 11 – 12 ] findings of combined bevacizumab responsiveness and long survival argues that above theories do not cover all patients, but that some GBM patients could obtain a real anti-tumour response from bevacizumab combination therapy. The DNA methylation-based classification system for CNS tumours has together with other molecular methods been assessed useful supplements to regular histopathological examination for diagnosing gliomas [ 32 ]. Consequently, re-evaluation of cases presenting rare clinical history diagnosed before implementation of these methods is relevant. For our cases, re-assessment by integrative molecular and histological examination maintained the GBM diagnosis, ruling out the long patient survival resulting from a misdiagnosis of GBM. Neither did these analyses or TSO500 reveal any consisting traits differing these patients from other GBMs. Among the consistent traits found in this small patient cohort, primary tumour tissues exhibited high methylation levels of the AGT promoter ranging from 16–49%, supporting a prior hypothesis from our institution suggesting that AGT gene silencing by promoter methylation serve as a predictive biomarker for bevacizumab response [ 9 ]. This hypothesis is currently being tested in an ongoing randomized phase III trial (EudraCT No. 2020-003545-11). Bulk RNA sequencing analysis revealed three genes: HILPDA, HHLA2 and IGF2 were significantly downregulated compared to control samples. HILPDA has been generally connected to a hypoxic phenotype in gliomas [ 33 – 34 ] and it could make sense that a less hypoxic environment is more likely to undergo bevacizumab induced vascular normalization compared to more irreversible hypoxic and necrotic tumours [ 35 – 36 ]. Whether the three genes HILPDA, HHLA2 and IGF2 could be predictive biomarkers of bevacizumab response would have to be tested in a larger prospective cohort. Regarding, previously reported independent prognostic variables for bevacizumab treated GBM (PS, tumour multifocality, steroid intake and neurocognitive deficit) [ 19 , 37 – 38 ], our cases overall showed good prognostic status before each effective bevacizumab treatment. At least four out of the five patients had MGMT promoter methylated GBM tumours as well. Still, compared to other non-responding patients they were not extraordinary good and consequently, the positive clinical outcome can’t alone be explained by the belonging to a good prognostic group. This is further underlined by PFS following standard therapy was not significantly different from the control group in this cohort (Supplementary Figure S2). We could also not establish a connection between responsiveness to first-line radiation/temozolomide therapy and bevacizumab therapy in a larger population in a previous study from our institution [ 19 ] again indicating that a separate response mechanism for bevacizumab therapy exist for some patients. Conclusion GBM patients can respond to bevacizumab with clinical benefit at least five times in the recurrent setting. These repeated responders stand out by having long survival. In this study, no secure prognostic variables and histopathological features differ the responders from other GBM patients. However, HILPDA, HHLA2, IGF2 were downregulated compared with the controls. Due to the small sample size, further studies must be carried out to determine whether downregulated gene expression of HILPDA, HHLA2, IGF2 and high level of AGT promoter methylation could be predictive biomarkers of bevacizumab response. Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests The authors declare no conflicts of interest. Author contributions MJSL, SRM, CSDE and TU evaluated the cases and drafted the paper. HSP, UL, JW, MJSL, SRM and TU concepted the study. MJSL and AL performed bioinformatic analysis. DS and LCM contributed to pathological re-evaluation. All authors edited and approved the final manuscript. Data availability The data generated during and analysed during the current study are available from the corresponding author on reasonable request. 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Urup et al. , “Transcriptional changes induced by bevacizumab combination therapy in responding and non-responding recurrent glioblastoma patients.,” BMC Cancer , vol. 17, no. 1, p. 278, Apr. 2017, doi: 10.1186/s12885-017-3251-3. S. Jain, Y. Li, S. Patil, and A. Kumar, “HNF-1alpha plays an important role in IL-6-induced expression of the human angiotensinogen gene.,” Am J Physiol Cell Physiol , vol. 293, no. 1, pp. C401-10, Jul. 2007, doi: 10.1152/ajpcell.00433.2006. E. Tabouret et al. , “Limited impact of prognostic factors in patients with recurrent glioblastoma multiforme treated with a bevacizumab-based regimen.,” J Neurooncol , vol. 114, no. 2, pp. 191–8, Sep. 2013, doi: 10.1007/s11060-013-1170-y. J. Duerinck et al. , “Patient outcome in the Belgian medical need program on bevacizumab for recurrent glioblastoma.,” J Neurol , vol. 262, no. 3, pp. 742–51, Mar. 2015, doi: 10.1007/s00415-014-7633-z. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 16 Jul, 2025 Read the published version in Journal of Neuro-Oncology → Version 1 posted Editorial decision: Revision requested 14 Jun, 2025 Reviews received at journal 12 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviewers agreed at journal 29 May, 2025 Reviewers agreed at journal 28 May, 2025 Reviewers agreed at journal 28 May, 2025 Reviews received at journal 28 May, 2025 Reviewers agreed at journal 27 May, 2025 Reviewers invited by journal 27 May, 2025 Editor assigned by journal 27 May, 2025 Submission checks completed at journal 27 May, 2025 First submitted to journal 27 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6757864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":463327449,"identity":"72bf49ce-1f71-4bdc-b68a-5fe99edd6f02","order_by":0,"name":"Maya Jeje Schuang Lü","email":"","orcid":"","institution":"The Finsen Laboratory, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Maya","middleName":"Jeje Schuang","lastName":"Lü","suffix":""},{"id":463327450,"identity":"3ae94302-d58c-4fd5-838e-d087b2d22495","order_by":1,"name":"Signe Regner Michaelsen","email":"","orcid":"","institution":"Department of Pathology, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Signe","middleName":"Regner","lastName":"Michaelsen","suffix":""},{"id":463327451,"identity":"e226d623-0c57-4639-9679-01d41b470bf7","order_by":2,"name":"Alessio Locallo","email":"","orcid":"","institution":"The Finsen Laboratory, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Alessio","middleName":"","lastName":"Locallo","suffix":""},{"id":463327452,"identity":"398c3276-8bdc-40d0-b749-d865b20bd94e","order_by":3,"name":"Christina Schjellerup Eickhart-Dalbøge","email":"","orcid":"","institution":"The Regional Department of Clinical Microbiology, University Hospital of Region Zealand","correspondingAuthor":false,"prefix":"","firstName":"Christina","middleName":"Schjellerup","lastName":"Eickhart-Dalbøge","suffix":""},{"id":463327453,"identity":"0ba2ce0b-b239-43aa-ba2c-ef4ded3fdfe6","order_by":4,"name":"David Scheie","email":"","orcid":"","institution":"Department of Pathology, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Scheie","suffix":""},{"id":463327454,"identity":"49016bea-b12e-4b21-8f0f-082c1af4e2b8","order_by":5,"name":"Linea Cecilie Melchior","email":"","orcid":"","institution":"Department of Pathology, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Linea","middleName":"Cecilie","lastName":"Melchior","suffix":""},{"id":463327455,"identity":"856360e4-71f5-481c-96e1-80bb1fe54294","order_by":6,"name":"Joachim Weischenfeldt","email":"","orcid":"","institution":"The Finsen Laboratory, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Joachim","middleName":"","lastName":"Weischenfeldt","suffix":""},{"id":463327456,"identity":"74e8e6fe-9c51-495c-91df-bc1f6935682f","order_by":7,"name":"Ulrik Lassen","email":"","orcid":"","institution":"Department of Oncology, Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Ulrik","middleName":"","lastName":"Lassen","suffix":""},{"id":463327457,"identity":"8a8d00b4-1d9b-4d3d-a68b-9f4de768c658","order_by":8,"name":"Hans Skovgaard Poulsen","email":"","orcid":"","institution":"The Danish Comprehensive Cancer Center, Brain Tumor Center (DCCC-BTC)","correspondingAuthor":false,"prefix":"","firstName":"Hans","middleName":"Skovgaard","lastName":"Poulsen","suffix":""},{"id":463327458,"identity":"8def5443-73e1-4c37-b737-b73e628131e7","order_by":9,"name":"Thomas Urup","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACCQST8QGQ4IGgA7h18CBpYTYgWQsblE1Ai71089ENP/4wJG6fkXusuqKiVoZfuvcAw5szeGyROZZ2s7eNIXHOjby0m2fOHOeRnHMugXHODXwOyzG7wdvAkDgDyLjZ2HaMx+BGjgEzzwf8Wm7++QPRUtj4j0gtt3nYIFoYGxtqoFrwOexGWtpt2TYJ4xk875IlG44d4JGckWNwcA4e77PPSD52880fG9kZ7LkHPzbU1NnzS+QYPnhzDLcWKADFCA+IcRjMPUBQA9SNIKKOSMWjYBSMglEwkgAASfpSYGjpkb8AAAAASUVORK5CYII=","orcid":"","institution":"The Danish Comprehensive Cancer Center, Brain Tumor Center (DCCC-BTC)","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Urup","suffix":""}],"badges":[],"createdAt":"2025-05-27 09:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6757864/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6757864/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11060-025-05162-2","type":"published","date":"2025-07-16T16:05:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83623808,"identity":"b3e82c13-3c26-4220-a420-b954d29ca13f","added_by":"auto","created_at":"2025-05-29 16:01:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Timelines of treatment divided into cycles and courses for repeated bevacizumab responders. \u003cstrong\u003eB\u003c/strong\u003e Tables of repeated bevacizumab responders including number of bevacizumab courses (Bev course) and clinical status prior to bevacizumab courses: ECOG Performance status (PS), use of cortisteroids (Steroids), multifocal disease (Multifocal) and neurocognitive deficit (Neu.defi.).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6757864/v1/52cab4b8eb333edfb1f1d87c.jpg"},{"id":83623803,"identity":"0701588d-18ec-4b99-871b-a3c67f81a83b","added_by":"auto","created_at":"2025-05-29 16:01:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eVolcano plot showing 3 significantly downregulated genes (dodgerblue) in primary patient samples from 4 repeated bevacizumab responders compared to 11 control samples. X-axis: Average log2 Fold Change. Y-axis: -log10 transformed adjusted p-values. \u003cstrong\u003eB\u003c/strong\u003e Table of common GBM mutations from TSO500 panel of all 5 repeated bevacizumab responders.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6757864/v1/83bc0ba0ae67488163ecdcbe.jpg"},{"id":83623811,"identity":"2d6d611a-7f50-4805-aea6-e7ef4c5b9ef4","added_by":"auto","created_at":"2025-05-29 16:01:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eKaplan-Meier curves of overall survival between date of diagnosis and death in repeated responders (orange) and control samples (light blue). \u003cstrong\u003eB\u003c/strong\u003e Kaplan-Meier curves of overall survival between relapse and death in repeated responders (orange) and control samples (light blue).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6757864/v1/d6953815349086a060f7ea8d.jpg"},{"id":88506124,"identity":"c911d4d7-0ac4-44d2-80ac-51b892447935","added_by":"auto","created_at":"2025-08-07 07:31:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":806059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6757864/v1/14bae5ab-ca53-4f09-b82f-de466a55d191.pdf"},{"id":83623809,"identity":"dbac8e41-8de7-4f82-9ce5-6f1770ee2e1d","added_by":"auto","created_at":"2025-05-29 16:01:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":357715,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6757864/v1/0d2e5236b6b95cb6e978d7aa.docx"},{"id":83623813,"identity":"e8994ffc-684b-41a2-9178-4d25c3a7dc74","added_by":"auto","created_at":"2025-05-29 16:01:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":461064,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6757864/v1/24023e8d8d0c8953d063db3b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bevacizumab rechallenge for recurrent glioblastoma - characterization of patients presenting multiple responses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNewly diagnosed glioblastoma (GBM) patients in good performance status are treated with maximal safe surgery followed by radiation therapy and temozolomide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, no standard treatment exists at disease recurrence. Multiple treatments have been tested in the recurrent setting, including re-surgery, re-irradiation, chemotherapeutic and targeted agents, but most regimens have shown limited efficacy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Bevacizumab, an antibody targeting The Vascular Endothelial Growth Factor A (VEGF), has shown clinical activity with response rates of approximate 30%, improved progression-free survival (PFS) when given in combination with irinotecan or lomustine, but no improvement in overall survival (OS) when considering the total population of recurrent GBM patients [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These data demonstrate that not all patients benefit from bevacizumab combination therapy with irinotecan or lomustine. However, a subset of recurrent GBM patients who respond to bevacizumab combination therapy experience improved survival and enhanced clinical status [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This highlights the importance of characterizing patients benefitting from treatment with the overall objective to identify predictive biomarkers for efficacy. Recently, we identified low expression of the angiotensinogen (AGT) gene, regulated by methylation of its promoter region, as being associated with a higher likelihood of response to bevacizumab [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn patients who initially respond to bevacizumab, rechallenge with the therapy has been reported to induce a new response in around 60% of cases [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and even a third response in selected cases\u003csup\u003e9\u003c/sup\u003e. These studies reported longer survival for rechallenged patients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], indicating that re-exposure to bevacizumab could be beneficial for prolonging survival in responders whose treatment was discontinued prior to tumour progression. Such findings contrast with studies testing the continuation of bevacizumab in patients who had previously progressed on bevacizumab therapy. In these studies, bevacizumab failed to produce a second treatment response [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and showed no impact on survival [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we report clinical history, pathological, and genomic features for patients with progressive GBM treated with up to five individual bevacizumab courses resulting in clinical benefit.\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eFrom our clinical glioblastoma database [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], patients treated with bevacizumab with either lomustine or irinotecan combination therapy between year 2007-18 at Rigshospitalet for progressive GBM IDH wild type were screened for eligibility. Patients were included in the study if they achieved treatment response during both the initial bevacizumab course and the rechallenge, and if GBM tissue was available for analysis. The control group used for comparison with the repeated bevacizumab responders in Kaplan Meier plots were 38 GBM patients, who had formalin-fixed paraffin-embedded (FFPE) tissue from either primary or relapse tumour available in the biobank.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreatment\u003c/h3\u003e\n\u003cp\u003eFirst-line treatment included maximal safe surgical resection followed by radiation therapy plus concomitant and adjuvant temozolomide according to the Stupps regimen [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. At time of progression, patients were evaluated by a multidisciplinary team for relapse surgery or second-line line therapy. Patients in good ECOG performance status (ECOG PS 0\u0026ndash;1) and with measurable tumour on MRI was offered bevacizumab in combination with irinotecan or lomustine. For the irinotecan combination, 10 mg/kg bevacizumab was given every 2 weeks together with 125 mg/m\u003csup\u003e2\u003c/sup\u003e irinotecan [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For the lomustine combination 10 mg/kg bevacizumab was given every 2 weeks while lomustine treatment was delivered every 6 weeks at a concentration of 90 mg/m\u003csup\u003e2\u003c/sup\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA treatment cycle was defined as 4 weeks treatment in patients receiving bevacizumab and irinotecan and 6 weeks in patients receiving bevacizumab and lomustine. A new treatment course was defined when treatment was initiated after disease progression following a treatment pause of at least 3 months. Treatment stop was registered as the last day in the treatment course where patients received treatment.\u003c/p\u003e\n\u003ch3\u003eClinical follow-up\u003c/h3\u003e\n\u003cp\u003ePatients were clinically evaluated every 2 weeks when receiving therapy, and MRI evaluation was performed after 2 cycles, being every 8 weeks in patients receiving irinotecan and every 12 weeks in patients receiving lomustine [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Patient and tumour data was retrospectively reviewed via medical journals, pathology reports and imaging descriptions. Response evaluation was performed according to the RANO criteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. OS was calculated from the day of initial diagnostic surgery to the day of death and to the date the patient started the first bevacizumab treatment. PFS was calculated from the day of diagnosis to the day of 1st relapse confirmed by MRI.\u003c/p\u003e\n\u003ch3\u003ePathology\u003c/h3\u003e\n\u003cp\u003eIn addition to routine pathological evaluations, DNA purified from macro-dissected FFPE tissue \u0026ndash; enriched for tumour content - was bisulfite-treated and analysed using Infinium MethylationEPIC v1.0 BeadChip (Illumina, Ca, USA) according to the manufacturer\u0026rsquo;s instructions. Methylation profiling report containing methylation classification, copy number variations and MGMT promoter methylation status were generated using app.epignostix.com (v12.8), Heidelberg based on previously described method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. AGT promoter methylation status was assessed using the generated iDAT files to analyse AGT promoter CpG site cg12469306.\u003c/p\u003e \u003cp\u003eImmunohistochemistry for mutation in IDH using anti-IDH1 R132H (clone H09, Dianova, 1:700 dilution), and in ATRX using anti-ATRX (HPA001906, Sigma, 1:150 dilution). One patient was tested for H3F3A mutation by dideoxysequencing covering codon 28\u0026ndash;35.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing and TSO500\u003c/h3\u003e\n\u003cp\u003eArchived FFPE tissue from primary tumour of case 1, case 2, case 4 and case 5 were accessible for bulk RNA sequencing and TSO500. For case 3 only FFPE tissue from 1st relapse was accessible and for case 5 FFPE tissue from 1st relapse was also accessible for bulk RNA sequencing and TSO500. For best comparison of samples and to avoid the least amount of batch effect only FFPE tissue from the primary tumours (4 samples) were evaluated and analysed in downstream differential expression analysis on the bulk RNA sequencing data. A control group consisting of 11 other FFPE primary GBM tumours that were available in the biobank were used for comparison. RNA was extracted from FFPE GBM tumour tissues from archived patient samples which were sequenced on Illumina NovaSeq.\u0026nbsp;FASTQ files were processed using Nextflow [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] nf-core [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] pipeline nf-core/rnaseq (v3.8.1). STAR and Salmon were used for alignment and quantification with Singularity (v3.6.2) as the profile option and GRCh38 as the reference genome. Downstream bulk RNA-seq analysis was performed using R (v4.2.1). Batch correction and differential expression analysis was performed using R package limma [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e](v3.52.4). For TSO500 all cases were evaluated with regards to most common GBM mutations [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], except for case 5 relapse tumour sample because of bad quality.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical history of patients with repeated bevacizumab response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 254 GBM IDHwt patients treated with bevacizumab combination therapy for progression after Stupp’s regimen were screened for eligibility. As shown in the REMARK diagram (Supplementary Figure S1), 15 patients were rechallenged with bevacizumab therapy after a treatment pause and six (40%) of these achieved response to both first course bevacizumab and bevacizumab rechallenge. A total of five patients with repeated bevacizumab response and with available tumour tissue for molecular analysis were included in the study (Table 1). Treatment covered in total 172 bevacizumab cycles divided over 20 courses, with 18 courses resulting in clinical benefit and 15 courses being rechallenges. Pauses between individual bevacizumab treatment courses were due to either clinical benefit with MRI confirmed reduction in tumour size or other non-progression-related reasons.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCase 1\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eA 60-year-old woman with GBM, methylated MGMT (RTK2 subtype), survived 77 months post-diagnosis and 45 months after initiating bevacizumab. Initial treatment (Figure 1A) included partial resection followed by standard radio-chemotherapy, achieving a partial response (PR). After 16 months of stable disease (SD), progression near the right lateral ventricle occurred. Temozolomide reinduction (3 cycles) failed due further tumor progression, and bevacizumab/irinotecan was initiated.\u003c/p\u003e\n\u003cp\u003eThe patient underwent 24 bevacizumab cycles over four courses. Prior to bevacizumab courses, ECOG PS declined from 0 (course 1) to 3 (course 4) (Figure 1B). Neurocognitive deficits fluctuated throughout but improved prior to course 3. No corticosteroids were used.\u003c/p\u003e\n\u003cp\u003eThe first course achieved PR after two cycles, and microspots remained after eight cycles, prompting a treatment pause. After five months, local progression led to a second course (six cycles), again with PR. After a 12-month pause a third course (bevacizumab plus lomustine) achieved a durable complete response for six cycles but was paused due to clinical decline. Four months later, recurrence prompted a fourth course (bevacizumab/irinotecan), which failed due to refractory disease. The patient died shortly after.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCase 2\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eA 34-year-old woman with GBM, methylated MGMT, survived 130 months from diagnosis and 112 months after starting bevacizumab. The patient achieved SD upon standard therapy. Over the years, the patient received 55 bevacizumab/irinotecan cycles in five courses, with 9–12 month treatment pauses.\u003c/p\u003e\n\u003cp\u003eECOG PS was 0–1 at each course start; corticosteroids were required before course 2. No neurocognitive deficit was observed. Multifocal disease appeared before courses 4 and 5.\u003c/p\u003e\n\u003cp\u003eThe first course (10 cycles) began six months after adjuvant therapy due to MRI-confirmed progression, achieving PR. The second course (20 cycles) achieved SD by cycle 2 and PR by cycle 6. A third course (10 cycles) achieved SD with tumour shrinkage. Course 4 was started after MRI revealed a new isolated lesion, achieving SD after two cycles and PR after 11 cycles. Progression with new tumour lesions led to a fifth course, achieving SD with lesion reduction until cycle 4, when infection and facial nerve palsy interrupted treatment. After 12 months with SD, the disease progressed with new lesions and leptomeningeal spread. The patient developed epilepsy and ventriculitis and died five months later.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCase 3\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eA 58-year-old man with GBM, MGMT methylated (mesenchymal subtype), survived 72 months, including 61 months post-bevacizumab. Initial standard treatment led to PR with disappearance of contrast-enhancing lesions. At completion of adjuvant temozolomide MRI showed progression, which advanced to measurable lesions within two months, prompting initiation of bevacizumab/irinotecan.\u003c/p\u003e\n\u003cp\u003eThe patient received 30 cycles across four courses. ECOG PS was 0 and no neurocognitive deficits were noted prior to each course. Corticosteroid use and multifocal disease were observed at later courses. The first bevacizumab course resulted in PR after two cycles and further tumor reduction after eight cycles, followed by a treatment pause. Eleven months later, new progressive lesions led to a second course (5 cycles), achieving SD. An infection interrupted treatment, after which tumor progressed. A third course (7 cycles) again achieved SD with tumor regression. A 17-month treatment-free interval followed. The fourth course produced durable PR, but after ten cycles progression with a new lesion prompted surgery. The patient experienced postoperative complications and died shortly thereafter.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCase 4\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eA 58-year-old woman with non-resectable, multifocal GBM (MGMT status unknown) involving the left temporal lobe and corpus callosum survived 48 months, with 43 months post-bevacizumab. Initial treatment included standard therapy. Three months post-radiation, MRI revealed progression with a new lesion in the contralateral hemisphere, prompting bevacizumab-based therapy.\u003c/p\u003e\n\u003cp\u003eThe patient received 27 bevacizumab cycles over three courses. Prior to course 1, ECOG PS was 1 \u0026nbsp;and the patient used corticosteroids. Before subsequent courses, PS had improved to 0 and corticosteroids were no longer needed. The first course led to clinical improvement and PR after two cycles, with further tumor reduction seen after nine cycles. After 11 cycles, the treatment was paused. After a six-month break, a new measurable lesion appeared. A second course (bevacizumab with lomustine) achieved durable PR for eight cycles but treatment was interrupted due to grade 3 hepatic enzyme elevation attributed to lomustine. Following a five-month pause, MRI showed progression, and a third course (bevacizumab/irinotecan) was initiated, achieving SD over eight cycles. The patient clinically deteriorated two months after the final pause and died shortly thereafter.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCase 5\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eA 60-year-old woman with GBM, MGMT methylated (mesenchymal subtype) located in the left occipital and right parietal lobe survived 57 months, with 52 months post-bevacizumab. Standard therapy was followed by early progression (focal seizures and two new lesions), leading to bevacizumab-based therapy.\u003c/p\u003e\n\u003cp\u003eThe patient received 36 cycles over four treatment courses. Multifocal disease was present from the outset. ECOG PS was 0–1 at courses 1 and 2, declining to 2 at course 4. Corticosteroids were used prior to course 3 and 4. The first course with bevacizumab/lomustine achieved durable PR for six cycles until paused due to grade 3 thrombocytopenia. After five months, progression prompted reinduction with lomustine (discontinued due to thrombocytopenia) and a switch to irinotecan. A durable PR was achieved over nine total cycles, including bevacizumab monotherapy. Following a three-month pause, progression led to 15 additional bevacizumab monotherapy cycles, with SD as the best response. \u0026nbsp;A final rechallenge after another five-month break resulted in progression after six cycles. Surgery confirmed recurrent GBM (RTK2 subtype). The patient died three months later.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathological examination for confirmation of the GBM diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitial GBM diagnosis was for all five cases based on histological features as having pleomorphic cells, necrosis, vascular proliferation and a prominent mitotic index\u003cem\u003e.\u0026nbsp;\u003c/em\u003eSelected molecular analyses conducted at the time of diagnosis supported the GBM diagnosis, however, as the molecular pathology were limited at this time (2008-2013), we re-reviewed the material with up-to-date pathological methods for diagnosis confirmation in the cases where it was relevant (Table 3).\u003c/p\u003e\n\u003cp\u003eAGT promoter methylation status was analysed in the four cases with primary diagnostic tumour tissues. In these cases, AGT promoter was found methylated with a median of 26% (range: 16% - 49%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulk RNA sequencing and TSO500\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential expression analysis on bulk RNA sequencing data showed significant downregulation (Figure 2A) of 3 genes: HILPDA (logFC -5.58 and adjusted p-value of 0.012), HHLA2 (logFC -8.09 and adjusted p-value of 0.040) and IGF2 (logFC -18.80 and adjusted p-value of 0.048) in the 4 repeated bevacizumab therapy responders compared to the 11 control samples. The genes are marked with blue colour in the volcano plot. TSO500 data showed no specific mutation pattern in the most common GBM-mutated genes e.g. TERT, PTEN, EGFR and TP53 (Figure 2B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe present five recurrent GBM patients showing clinical benefit from bevacizumab rechallenge up to five times after treatment stop for other reasons than progression. This is beyond the one to two rounds of successful bevacizumab rechallenges previously reported in GBM [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and show a long treatment perspective of bevacizumab in sensitive patients. Combined with previous reports of exceptional high response rates under bevacizumab rechallenge in previously responding patients following non-progression related treatment pause [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], our data support the existence of GBM tumours with particularly high bevacizumab sensitivity.\u003c/p\u003e \u003cp\u003eAll five patients experienced OS (from diagnosis) far beyond the median OS of 15 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and OS from start of bevacizumab relapse treatment far beyond the median OS of 8\u0026ndash;9 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) normally observed for GBM patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Negative result for survival in later years clinical trials testing bevacizumab for GBM [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] has fired under speculations that MRI observed bevacizumab responses are not related to direct anti-tumour effects. Rather, it may be pseudo-responses caused by bevacizumab induced decrease in vessel permeability and thereby reduced contrast enhancement [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, theories have been that bevacizumab initiates rebound progression with a more fast-growing phenotype [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Multiple mechanisms for tumour acquirement of bevacizumab resistance have been presented [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], including a bevacizumab induced phenotypic shift from a vascular growth pattern to an aggressively growing invasive phenotype which could be a cause for more rapid progression [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our and previous [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] findings of combined bevacizumab responsiveness and long survival argues that above theories do not cover all patients, but that some GBM patients could obtain a real anti-tumour response from bevacizumab combination therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe DNA methylation-based classification system for CNS tumours has together with other molecular methods been assessed useful supplements to regular histopathological examination for diagnosing gliomas [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consequently, re-evaluation of cases presenting rare clinical history diagnosed before implementation of these methods is relevant. For our cases, re-assessment by integrative molecular and histological examination maintained the GBM diagnosis, ruling out the long patient survival resulting from a misdiagnosis of GBM. Neither did these analyses or TSO500 reveal any consisting traits differing these patients from other GBMs.\u003c/p\u003e \u003cp\u003eAmong the consistent traits found in this small patient cohort, primary tumour tissues exhibited high methylation levels of the AGT promoter ranging from 16\u0026ndash;49%, supporting a prior hypothesis from our institution suggesting that AGT gene silencing by promoter methylation serve as a predictive biomarker for bevacizumab response [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This hypothesis is currently being tested in an ongoing randomized phase III trial (EudraCT No. 2020-003545-11). Bulk RNA sequencing analysis revealed three genes: HILPDA, HHLA2 and IGF2 were significantly downregulated compared to control samples. HILPDA has been generally connected to a hypoxic phenotype in gliomas [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and it could make sense that a less hypoxic environment is more likely to undergo bevacizumab induced vascular normalization compared to more irreversible hypoxic and necrotic tumours [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Whether the three genes HILPDA, HHLA2 and IGF2 could be predictive biomarkers of bevacizumab response would have to be tested in a larger prospective cohort. Regarding, previously reported independent prognostic variables for bevacizumab treated GBM (PS, tumour multifocality, steroid intake and neurocognitive deficit) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], our cases overall showed good prognostic status before each effective bevacizumab treatment. At least four out of the five patients had MGMT promoter methylated GBM tumours as well. Still, compared to other non-responding patients they were not extraordinary good and consequently, the positive clinical outcome can\u0026rsquo;t alone be explained by the belonging to a good prognostic group. This is further underlined by PFS following standard therapy was not significantly different from the control group in this cohort (Supplementary Figure S2). We could also not establish a connection between responsiveness to first-line radiation/temozolomide therapy and bevacizumab therapy in a larger population in a previous study from our institution [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] again indicating that a separate response mechanism for bevacizumab therapy exist for some patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGBM patients can respond to bevacizumab with clinical benefit at least five times in the recurrent setting. These repeated responders stand out by having long survival. In this study, no secure prognostic variables and histopathological features differ the responders from other GBM patients. However, HILPDA, HHLA2, IGF2 were downregulated compared with the controls. Due to the small sample size, further studies must be carried out to determine whether downregulated gene expression of HILPDA, HHLA2, IGF2 and high level of AGT promoter methylation could be predictive biomarkers of bevacizumab response.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMJSL, SRM, CSDE and TU evaluated the cases and drafted the paper. HSP, UL, JW, MJSL, SRM and TU concepted the study. MJSL and AL performed bioinformatic analysis. DS and LCM contributed to pathological re-evaluation. All authors edited and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated during and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Danish National Committee on Health Research Ethics and granted an exemption from the requirement for informed consent (2000021). The study was done in accordance with the Helsinki Declaration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. 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Li, S. Patil, and A. Kumar, \u0026ldquo;HNF-1alpha plays an important role in IL-6-induced expression of the human angiotensinogen gene.,\u0026rdquo; \u003cem\u003eAm J Physiol Cell Physiol\u003c/em\u003e, vol. 293, no. 1, pp. C401-10, Jul. 2007, doi: 10.1152/ajpcell.00433.2006.\u003c/li\u003e\n\u003cli\u003eE. Tabouret \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Limited impact of prognostic factors in patients with recurrent glioblastoma multiforme treated with a bevacizumab-based regimen.,\u0026rdquo; \u003cem\u003eJ Neurooncol\u003c/em\u003e, vol. 114, no. 2, pp. 191\u0026ndash;8, Sep. 2013, doi: 10.1007/s11060-013-1170-y.\u003c/li\u003e\n\u003cli\u003eJ. Duerinck \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Patient outcome in the Belgian medical need program on bevacizumab for recurrent glioblastoma.,\u0026rdquo; \u003cem\u003eJ Neurol\u003c/em\u003e, vol. 262, no. 3, pp. 742\u0026ndash;51, Mar. 2015, doi: 10.1007/s00415-014-7633-z.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"glioblastoma, bevacizumab, rechallenge, reinduction, response","lastPublishedDoi":"10.21203/rs.3.rs-6757864/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6757864/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eBevacizumab rechallenge in glioblastoma patients who initially responded at recurrence has shown renewed responses in up to 60% of cases, with associated survival benefits. This study aimed to characterize such repeat responders and identify potential predictive biomarkers.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 254 glioblastoma IDHwt patients treated with bevacizumab-based combination therapy were evaluated for eligibility. Five patients who demonstrated repeated responses to bevacizumab and had available tumor tissue for molecular analysis were included. Histopathological re-assessments were performed to confirm glioblastoma diagnoses. Angiotensinogen (AGT) promoter methylation status was analyzed in all primary tumor samples, while bulk RNA sequencing and TSO500 assays were conducted on all available samples.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn our cohort, 40% of patients who were rechallenged with bevacizumab following a treatment pause exhibited a response both during the initial course and upon rechallenge. Bulk RNA sequencing revealed downregulation of HILPDA, HHLA2, and IGF2 as potential predictive biomarkers for repeated bevacizumab response. Additionally, AGT promoter methylation analysis identified high methylation levels as another potential predictive biomarker.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eA subgroup of glioblastoma patients respond to bevacizumab at least up to five times in the recurrent setting, and these repeated responders exhibit prolonged survival. No definitive prognostic variables or histopathological features were found in this study. Further investigations into the downregulation of HILPDA, HHLA2 and IGF2 along with high AGT promoter methylation levels and other potential predictive biomarkers, are warranted to better understand the mechanisms underlying repeated bevacizumab response.\u003c/p\u003e","manuscriptTitle":"Bevacizumab rechallenge for recurrent glioblastoma - characterization of patients presenting multiple responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 16:01:00","doi":"10.21203/rs.3.rs-6757864/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-14T12:21:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T17:54:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42064212371583524120252271216782808444","date":"2025-06-03T04:49:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261296353848233096543975372113575390655","date":"2025-05-29T11:51:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255370405895830711701001025996360846250","date":"2025-05-29T02:27:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188285990382248954317862133515356914140","date":"2025-05-29T00:39:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-28T18:26:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319485234630509695343439444899104923530","date":"2025-05-27T14:10:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-27T11:20:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-27T10:38:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-27T10:37:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuro-Oncology","date":"2025-05-27T09:26:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1efa5828-5e4e-4432-88d4-f4936d6eced7","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:13:43+00:00","versionOfRecord":{"articleIdentity":"rs-6757864","link":"https://doi.org/10.1007/s11060-025-05162-2","journal":{"identity":"journal-of-neuro-oncology","isVorOnly":false,"title":"Journal of Neuro-Oncology"},"publishedOn":"2025-07-16 16:05:10","publishedOnDateReadable":"July 16th, 2025"},"versionCreatedAt":"2025-05-29 16:01:00","video":"","vorDoi":"10.1007/s11060-025-05162-2","vorDoiUrl":"https://doi.org/10.1007/s11060-025-05162-2","workflowStages":[]},"version":"v1","identity":"rs-6757864","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6757864","identity":"rs-6757864","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}