Combination Chemoradiotherapy with Temozolomide, Vincristine, and Interferon-β Might Improve Outcomes Regardless of O6-Methyl-Guanine-DNA-Methyltransferase (MGMT) Promoter Methylation Status in Newly Glioblastoma

doi:10.21203/rs.3.rs-484091/v1

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Research article

Combination Chemoradiotherapy with Temozolomide, Vincristine, and Interferon-β Might Improve Outcomes Regardless of O6-Methyl-Guanine-DNA-Methyltransferase (MGMT) Promoter Methylation Status in Newly Glioblastoma

https://doi.org/10.21203/rs.3.rs-484091/v1

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published 28 Jul, 2021

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Background: This investigator-initiated, open-label, single-arm, single-institute study was conducted to investigate the effectiveness of induction combination chemoradiotherapy for newly glioblastoma and also the effectiveness of long-term maintenance therapy with temozolomide (TMZ) plus interferon (IFN)-b.

Methods: The initial induction combination chemoradiotherapy comprised radiotherapy plus TMZ plus vincristine plus IFN-b. Maintenance chemotherapy comprised monthly TMZ, continued for 24–50 cycles, plus weekly IFN-b continued for as long as possible. The primary endpoint was 2-year overall survival (2y-OS). A 2y-OS exceeding 38%, with upper limit of the 95% confidence interval (CI) exceeding 31.7%, and lower limit of the 95%CI exceeding 21.2% as compared to historical controls, was considered as the criterion for treatment effectiveness. Secondary endpoints were median progression-free survival (mPFS), median OS (mOS), 5-year OS rate (5y-OS), and mPFS and mOS classified according to MGMT promoter methylation status.

Results: Forty-seven patients were analyzed. The 2y-OS was 40.7% (95%CI, 27.5–55.4%), suggesting that this protocol was effective. The mPFS and mOS were 11.0 months and 18.0 months, respectively, and 5y-OS was 20.3% (95%CI, 11.0–34.6%). The mPFS in groups with and without MGMT promoter methylation in the tumor was 10.0 months and 11.0 months (p=0.59), respectively, and mOS was 24.0 months and 18.0 months (p=0.88), respectively. The frequency of grade 3/4 neutropenia was 19.1%.

Conclusions: These results suggest the efficacy of induction multicombination chemoradiotherapy, as well as of long-term maintenance therapy comprising TMZ plus IFN-b. This protocol would have the possibility to deny the MGMT promoter methylation status as prognostic factors.

Trial registration: The University Hospital Medical Information Network (Number UMIN000040599)

Cancer Biology

Oncology

newly glioblastoma

combination therapy

temozolomide

interferon-β

MGMT

Glioblastoma (GBM) remains an incurable disease. The reported median overall survival (mOS) and 2-year OS rate (2y-OS) in patients receiving treatment according to the Stupp protocol (temozolomide [TMZ] plus radiotherapy [RT]), which is regarded as the international standard of care, were 14.6 months and 26.5%[1].That protocol represented a great paradigm shift in chemotherapy as compared to the treatment with alkylating agents used prior to the introduction of TMZ. The introduction of TMZ has significantly improved treatment outcomes. However, continued follow-up after 2 years has shown that the 5-year OS rate (5y-OS) in patients receiving RT plus TMZ was only 9.8%, not significantly different from results obtained under previous conventional regimens[2]. Also, according to a systematic review by Tykocki et al.[3], the 10-year OS rate is a dismal 0.71%. Complete cure of GBM thus still seems to be a far-fetched goal. In addition, the incidence of GBM has not decreased. According to the Brain Tumor Registry of Japan (2005–2008)[4], about 1000 patients are newly diagnosed with GBM in Japan each year. Improving the treatment outcomes of GBM is thus an urgent issue worldwide.

The primary treatment strategy for newly diagnosed patients with GBM is resection; that is, removal of as much of the tumor as possible[5, 6], followed by combined RT plus TMZ therapy. Alternatively, the latest treatment method can be used; namely, a combination of the Novo Tumor Treating Fields system plus TMZ maintenance treatment[7]. However, tumor O6-methyl-guanine-DNA-methyltransferase (MGMT) promoter methylation status is a prognostic factor, and an obstacle that cannot be overcome by the initial induction therapy and 6 subsequent cycles (C) of maintenance treatment with TMZ[8, 9]. In addition, interferon (IFN)-β is known to downregulate MGMT via p53[10]. We have therefore devised a new treatment strategy involving the use of a multidrug combination, including IFN-β in the initial induction therapy administered with RT, followed by maintenance therapy with TMZ and IFN-β administered for as long as possible, aimed at depleting tumor MGMT. This post-hoc analysis investigated the efficacy and safety of our proposed treatment, which we expected to yield better treatment outcomes than the conventional regimen.

Patients

Inclusion criteria for this study were as follows: 1) pathologically confirmed newly diagnosed GBM (including giant cell glioblastoma or gliosarcoma) as defined by the World Health Organization classification of tumors 2007 (IARC 4th edition); 2) age, 16–80 years; 3) tumor located without involvement of the optic nerve, hypothalamus, or ventricles (containing most of the tumor mass, but not involving the brainstem), and without cerebrospinal fluid dissemination at initial diagnosis; 4) no history of malignant tumors, and no previous or current history of chemotherapy; 5) no previous history of RT; 6) no multiple primary cancers; 7) Eastern Cooperative Oncology Group performance scale (ECOG-PS), 0–2, or 3 in the presence of neurological symptoms; 8) adequate organ functions; 9) no serious infectious diseases; 10) patient suitable to receive treatment within 28 days of undergoing surgery; 11) regardless of the extent of surgical resection (biopsy is also acceptable); 12) provision of informed consent for participation in the study by the patient or their legal representative.

Treatments

The initial induction therapy was RT and concomitant chemotherapy with TMZ + VCR + IFN-β. RT (at 2.0 Gy/fr/day, 30 fr) and TMZ (75 mg/m²/day) were started simultaneously on Day 1. Oral TMZ was administered daily before breakfast and continued for 42 days. Up to 45 days of TMZ treatment was accepted, allowing for holidays and rest days for maintenance of the RT equipment. On days 2 and 3, vincristine (VCR) was administered by intravenous injection at 0.6 mg/m². On day 5, a thrice-weekly regimen of IFN-β was started; the drug was administered by intravenous drip infusion at 3 MU/body over 1 h. After completion of the initial induction therapy, study patients were followed-up for 28 days until the start of maintenance therapy. RT comprised fractionated focal irradiation administered at a fractional dose of 2 Gy given once a day, 5 days a week (Monday through Friday) for a period of 6 weeks, to a total dose of 60 Gy. The radiation treatment plan was the same as applied in the Stupp protocol and JCOG0911[1, 11].

Maintenance therapy was started with oral TMZ (150 mg/m²/day, for 5 days) administered before breakfast when the patient met all blood test criteria. However, for patients who developed grade 3/4 neutropenia or thrombocytopenia during the initial induction therapy and those who did not meet the blood test criteria within 28 days, TMZ (at 100 mg/m²/day, for 5 days) was started within 56 days of the initial induction therapy. The permissible treatment interruption period for TMZ was set at 23 days. Dose-increases of TMZ after the second cycle, from 100 to 150 mg/m²/day (for 5 days), then from 150 to 200 mg/m²/day (for 5 days), were allowed in patients who met the blood test criteria, but no further dose increase was allowed from the third cycle onward. TMZ treatment was continued for at least 24 cycles. Even after that, whether the patient was willing to continue treatment was assessed at 30 and 40 cycles, and continuation of TMZ treatment was allowed up to 50 cycles. IFN-β was administered at 3 MU/body once weekly. Dose adjustment of IFN-β within 1–3 MU/ body was allowed, taking into account body weight and myelosuppression. IFN-β treatment was continued for at least 2 years, and for as long as possible thereafter. Treatment after recurrence was not specified in the protocol, and patients were given the liberty to choose any course of treatment.

Study Design

This study was an investigator-initiated, open-label, single-arm, single-institute trial. The main research hypothesis of this study was that combining RT with chemotherapy using TMZ + VCR + IFN-β might yield better survival outcomes than the results reported by Stupp et al.[1], as historical controls.

Based on the study results reported by Stupp et al.[1] and other study results[12–14], the 2y-OS of RT + TMZ was set as 30% of the 2-year survival threshold. The 2y-OS in patients receiving chemoradiotherapy with the conventional combination of ACNU + VCR at our hospital is 33% (unpublished data). We used IFN-β as add-on therapy in our proposed treatment, as the so-called toxic new regimen. Also, taking into account patient burden and medical expenses, survival rate in patients receiving the combined chemoradiotherapy (RT with TMZ + VCR + IFN-β) should exceed that in the historical control by ≥5%. Therefore, for the 2y-OS threshold set at 30% for this therapy, the expected 2y-OS was set at 38%, with a registration period covering 3 years and a follow-up of 5 years. With the one-tailed significance level of tests in the main analysis set at 10%, the number of eligible patients necessary to obtain a statistical power of 80% was calculated as 44 according to the formula of Simon et al[15]. With allowance for ineligible patients, the number of patients to be enrolled in this study was set at 50.

This study was conducted in compliance with the Declaration of Helsinki and guidelines on Good Clinical Practice, and was conducted with the approval of The Committee of Medical Ethics of University Graduate School of Medicine, Hirosaki, Japan (Approval No. 2007 − 142). After the confirmation of histopathological diagnosis, patients who provided informed consent were enrolled in the Neurosurgical Clinical Study Registry of Hirosaki University Hospital, and trial treatment was started within 28 days after surgery.

Statistical analysis

The primary endpoint was the 2y-OS. Secondary endpoints were progression-free survival (PFS), 5y-OS, overall survival (OS), and PFS and OS classified according to tumor MGMT promoter methylation status, and the frequency/nature of adverse events. PFS was defined as the period from the date of surgery to the date of MRI confirmation of recurrence. OS was defined as the period from the date of surgery to the date of death or last date of confirmed survival. Adverse events were coded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. MGMT promoter methylation status was determined by methylation-specific polymerase chain reaction (MS-PCR) using surgical specimens, as described later[8, 16]. The extent of surgical resection was determined from findings on MRI performed within 72 h after surgery. In addition, tumor immunostaining was performed after surgery to detect IDH1^R132H mutations (Anti-IDH1^R132H antibody [H09]; DIANOVA GmbH, Hamburg, Germany).

In the main analysis, the efficacy of our proposed treatment was determined based on an expected 2y-OS of ≥ 38% as the primary endpoint. If the upper limit of the 95% confidence interval (CI) for 2y-OS (which was 26.5% according to the Kaplan-Meier method for the protocol used in the clinical study by Stupp et al.[1], which was regarded as the standard treatment) were to exceed 31.7% (with 95%CI including 31.7%), and if the lower limit of the 95%CI were to exceed 21.2%, our proposed treatment would be determined as effective. OS and PFS were examined using Kaplan-Meier methods. Determination of the OS and PFS according to MGMT promoter methylation status was performed by log-rank test. All statistical analyses were performed on a Mac OSX version 10.14.5 operating system, using JMP version 14 statistical software (SAS Institute, Cary, NC).

Ms-pcr For Determining Mgmt Promoter Methylation Status

DNA extraction from formalin-fixed, paraffin-embedded tissue sections and subsequent bisulfite conversion were performed using the Methylamp Whole Cell Bisulfite Modification Kit, in accordance with the instructions from the manufacturer (EPIGENTEK, Farmingdale, NY). PCR was performed using ZymoTaq PreMix (ZYMO RESEARCH, Irvine, CA) and a C1000 Thermal Cycler (Bio-Rad, Hercules, CA). Amplified products were separated on 4% agarose gels, stained with ethidium bromide, and visualized using the Gel Doc EZ Imager (Bio-Rad). For each PCR reaction, a Human Methylated & Non-methylated DNA Set (ZYMO RESEARCH, Irvine, CA) was used as the control. These reaction conditions and the sequences of the PCR primers were as previously described in the literature[8, 16].

Patients

Patients were enrolled in this study between April 2008 and March 2011. Originally, 53 patients were enrolled to the study and a final total of 47 patients thus received the protocol treatment (Fig. 1).

The median age of patients was 62 years (range, 16–80 years), and 10 patients (21.3%) were elderly (≥71 years old). Twenty patients (42.6%) had an ECOG-PS of 2, indicating that many patients were in a serious condition. Total tumor resection was achieved in 27 patients (57.4%). Immunostaining of the resected tumor revealed wild-type IDH1^R132H in 41 patients (87.2%) and a mutant gene in 6 patients (12.8%). MGMT promoter methylation status as determined by MS-PCR was rated as “methylated” in 16 patients (34.0%) and “unmethylated” in 31 patients (66.0%). No dropouts or treatment discontinuations due to emergence of adverse events to the initial induction therapy were encountered. The median number of TMZ maintenance therapy cycles administered was 13 (range, 3–50), and the median number of IFN-β maintenance therapy cycles administered was 51 (range, 10–328) (Table 1).

Clinical Course

Six of the 47 patients remained alive, 39 had died, and 2 were lost to follow-up. Of the 39 patients who died, 2 died of comorbid diseases, including myocardial infarction and acute aortic dissection, and the remaining 37 died of the tumor or tumor-related complications. Under this protocol, 10 patients (21.3%) continued TMZ maintenance therapy for 2 years without recurrence, and 5 patients (10.6%) continued TMZ maintenance therapy for 5 years without recurrence (Table 1). Six patients (12.8%) discontinued after 50 cycles of TMZ, including 5 patients who completed 5 years of relapse-free treatment and 1 patient who relapsed during treatment and continued to use TMZ.

The 2-year PFS rate (2y-PFS) was 18.2% (95%CI, 9.5–32.2%). The primary endpoint, 2y-OS, was 40.7% (95%CI, 27.5–55.4%). The 2y-OS was 7.7% higher than that recorded for the in-house treatment (unpublished data). Our proposed treatment was thus determined to be effective on the basis of fulfilling our efficacy criteria (2y-OS ≥ 38%; lower limit of 95%CI > 21.2%; and upper limit of 95%CI ≥31.7%). The 5-year PFS rate (5y-PFS) was 9.1% (95%CI, 3.5–21.8%), and the 5y-OS was 20.3% (95%CI, 11.0–34.6%). Median PFS (mPFS) was 11.0 months, and mOS was 18.0 months (Fig. 2a, Table 2).

Pfs And Os According To Mgmt Promoter Methylation Status

Examined by MGMT promoter methylation status as determined by MS-PCR, mPFS was 11.0 months (95%CI, 7.0–13.0 months) in the 31 patients of the unmethylated group and 10.0 months (95%CI, 4.0–27.0 months) in the 16 patients of the methylated group. Log-rank test revealed no significant difference (p = 0.59) in PFS between groups. The mOS was 18.0 months (95%CI, 14.0–27.0 months) in the unmethylated group and 24.0 months (95%CI, 11.0–40.0 months) in the methylated group. Log-rank test showed no significant difference (p = 0.88) in OS between groups (Fig. 2b, c).

Post-protocol Treatments

With regard to treatment course after recurrence, which the patients were given the liberty to choose, the majority (26 patients; 61.9%) opted to continue maintenance therapy with TMZ and IFN-β. Five patients underwent additional surgery, 12 underwent additional stereotactic radiosurgery, and 2 received bevacizumab (BEV). Most patients (40 patients; 95.2%) continued combination therapy with TMZ and IFN-β (Table 3).

Adverse Events

Table 4 shows adverse events observed throughout the initial induction therapy and maintenance therapy periods. One patient developed IFN-associated retinopathy (CTCAE G3) in the maintenance phase. Although this patient, who had underlying hypertension and diabetes mellitus, was a high-risk patient, the causal relationship between the event and IFN-β was assessed as “probable,” and the patient therefore stopped IFN-β. Three patients developed CTCAE grade 1 peripheral sensory neuropathy, which was considered to have been caused by VCR, and the causal relationship with the drug was assessed as “probable.” In addition, 2 patients developed CTCAE grade 3 hydrocephalus, with the causal relationship to the treatment assessed as “unlikely”. As common toxicities, CTCAE grade 1–4 lymphopenia occurred in all patients, and grade 3/4 lymphopenia was noted in 87.3% of patients. Grade 3/4 neutropenia occurred in 19.1% of patients, while no patients developed febrile neutropenia. The incidence of thrombocytopenia was also high, at 72.5% overall, with 8.6% developing grade 3/4 thrombocytopenia. Anorexia also occurred at a relatively high incidence of 57.4% overall, with 8.5% developing grade 3/4 anorexia. Incidence of constipation was high, at 80.8%, but was controllable with medications in most cases.

PCV multidrug combination therapy with procarbazine, CCNU and vincristine was widely used for the treatment of high-grade glioma (HGG) in Western countries[17]. In Japan, ACNU which was developed in Japan,[18] had been used as a community standard therapy in synchronous chemotherapy (RT + ACNU + VCR)[12] and IAR therapy (RT + ACNU + IFB-β)[13, 19] for HGG.

Treatment with VCR has been reported to induce a high accumulation of tumor cells during the highly radiosensitive G2-M phase of the cell cycle. In addition, a study of the growth pattern by flow cytometry revealed that about 10% of cells in the mitotic phase were in the G2-M phase, suggesting the promise of combination therapy with VCR[20]. IFN-β is classified as a type I IFN and was discovered as a cytokine with antiviral activity. IFN-β has since been shown to exert various biological activities, such as immunostimulatory activity, angiogenesis-inhibitory activity, antiproliferative activity, and anti-tumor activity mediated by induction of apoptosis[21]. IFN-β was found to be useful in the treatment of not only HGG[13, 19] but also low-grade glioma, with minor adverse drug reactions and a high response rate[22]. Clinical trials were also conducted overseas for recurrent HGG and showed a PFS of 23 weeks and a 23% partial response rate[23].

Utilizing these advantages, Aoki et al.[14] conducted a phase-II study of combined chemoradiotherapy, that is, RT with ACNU + carboplatin + VCR + IFN-β, in patients with newly diagnosed GBM, with the expectation of additive and synergistic effects of the drugs. They reported a PFS of 10 months, and OS of 16 months. We also investigated the clinical usefulness of RT administered with the combination regimen of ACNU + VCR (unpublished data). Meanwhile, Stupp et al. reported the results of treatment with TMZ for newly diagnosed cases of GBM[1]. The study reported by Stupp et al.[1] was definitely an epoch-making event, demonstrating significant prolongation of OS by a single agent in patients with newly diagnosed GBM. However, the results were still far from satisfactory. By that time, basic experiments had revealed TP53-mediated inactivation of MGMT by IFN-β[10]. We therefore developed a new toxic multidrug combination by replacing the conventionally used ACNU with TMZ and adding IFN-β, resulting in TMZ + VCR + IFN-β.

The primary endpoint of 2y-OS in the present trial of a new treatment regimen we developed, that is, RT combined with TMZ + VCR + IFN-β, was 40.7% (95%CI, 27.5–55.4%) and the initial study purpose was achieved. In addition, mPFS and mOS, set as the secondary endpoints, were 11.0 and 18.0 months, respectively, both of which were superior to the results reported by Stupp et al[1]. In addition, 5y-OS was also higher (20.3%; 95%CI 11.0–33.6) than the result of the follow-up study reported by Stupp et al[2]. Nevertheless, the mPFS and mOS of 11.0 and 18.0 months, respectively, which were longer than those (6.9 months and 14.6 months, respectively) reported by Stupp et al.[1], did not represent significant improvements. This could be because the entry criteria for our study population were less stringent, to better represent actual clinical settings. For example, the study population included elderly people up to 80 years old, and no limit was placed on tumor size, so inclusion of even very large tumors with volume ≥100 mL was permitted. The study population thus included patients with poor preoperative PS and patient ineligible for total resection (gross total removal rate, 57.4%). In other words, the study population included patients more likely to die earlier. However, not only were the survival rates/duration (2y-OS to 5y-OS, PFS, OS, etc.) superior to those reported by Stupp et al.[1, 2], but also survival rates after 2 years were better. This appears to be the first paper to conclude that PFS and OS were unassociated with the presence/absence of MGMT promoter methylation, a known prognostic factor. We discuss the potential reasons below.

First, one reason could be our continuation of maintenance therapy with TMZ for as long as possible. There were 6 responders (12.8%), with the inclusion of 1 case with recurrence, and treatment was discontinued after 50 cycles of TMZ. In addition, 40 patients (95.2%) received TMZ plus IFN-β therapy in combination with BEV, surgery or stereotactic radiosurgery, etc., even after the development of recurrence. In actual clinical practice in Japan, 6 treatment cycles of TMZ as specified in the protocol reported by Stupp et al.[1] is not sufficient, and TMZ treatment is actually continued for longer in many cases. In the literature, one report has described administration of TMZ for 101 cycles[24], and another indicated that long-term treatment with TMZ until progression is more cost-effective than a treatment protocol that recommends completion of TMZ treatment after 6 cycles[25]. Many publications have discussed the efficacy of long-term treatment[24, 26–33]. A meta-analysis has indicated that long-term treatment is more beneficial in terms of both OS and PFS[34]. In addition, according to one study, MGMT-mediated TMZ resistance may be attenuated by continuous therapy[35]. Certainly, long-term administration of TMZ would enhance the accumulation of TMZ in the body, and may have the same significance as the use of dose-dense TMZ, which depletes MGMT. On the other hand, some studies reporting on the benefits of long-term treatment with TMZ have also indicated that prognosis varies depending on tumor MGMT promoter methylation status[24, 26–28]. We cannot definitively conclude that long-term treatment with TMZ in our study resulted in good prognosis regardless of tumor MGMT promoter methylation status. Of course, TMZ could be considered ineffective for patients with unmethylated MGMT. Hegi et al, also reported that combined TMZ plus RT as compared to RT alone in the unmethylated group yielded a significantly prolonged PFS statistically, and combined TMZ plus RT as compared to RT alone in the unmethylated group prolonged OS, although the differences were not significant[8]. Furthermore, MGMT expression is heterogeneous and varies depending on the assay, and the site and timing of the assay[36, 37]. Suggesting that long-term treatment with TMZ contributes to improvement of PFS and OS may thus be acceptable.

No data have since demonstrating the efficacy of VCR alone in patients with brain tumors[38]. However, VCR is always used together with other antitumor agents, to deliver synchronous chemotherapy[12] or to obtain additive or synergistic effects[14, 39–41]. A meta-analysis revealed that VCR can become an antagonist for ACNU, BCNU, and cytosine arabinoside (Ara-C), and can synergize with CCNU, procarbazine, and cyclophosphamide. Only limited reports have described the use of VCR in combination with TMZ, but it is not an antagonist[38]. We still need to accumulate more cases in the future, but for now, we can say that combined therapy with TMZ may contribute to the prolongation of PFS and OS.

Why then did the results of this study suggest that OS was unrelated to MGMT promoter methylation status? As mentioned above, continued treatment with IFN-β could be one reason. The INTEGRA study[42] was conducted using combined TMZ plus IFN-β therapy, based on the results of basic experiments reported by Natsume et al.[10], aimed at depleting MGMT, followed by the phase-II JOCG0911 study[11]. In this study of JCOG0911, the add-on efficacy of IFN-β was denied, because no superiority of TMZ plus IFN-β could be established. However, a definitive difference from our study was seen in the dose of IFN-β. While the dose used for induction therapy was the same, that used for maintenance therapy was 4-fold higher in our study, leading to differences in continuing treatment. This may be the reason for the significantly superior results in our study.

In addition, continued treatment with TMZ causes lymphopenia in most cases. Likewise in this study, lymphopenia was observed in all patients, reaching grade 3/4 in 87.3%. Lymphopenia caused by TMZ is said to be characterized by depletion of a particularly high proportion of CD4-positive helper T cells[43]. If helper T cells disappear, the response of CD8-positive killer T cells also disappears, probably resulting in decreased antitumor effects. However, IFN-β can compensate for this, exerting cytocidal activity and providing immunotherapeutic advantages[21]. In other words, during the first 2 years or so of treatment, the main effect is produced by anticancer agents, such as TMZ, which has cytocidal effects. Thereafter, IFN-β may exert immune antitumor actions. However, this remains speculative.

Verification of the present findings requires a well-designed prospective study based on patient condition under certain statuses in a collaborative study with other institutions, and we have to identify the populations in which IFN-β is most effective by performing sub-analyses for each MGMT methylation status and other biomodulators.

With regard to adverse drug reactions, interferon-associated retinopathy have been reported. Since the development of irreversible visual dysfunction has been reported, special attention to patients with comorbid hypertension and diabetes mellitus should be necessary[44]. No serious adverse events such as interstitial pneumonia were encountered, and all observed adverse events including hematologic adverse events were considered tolerable.

Overall, the adverse events observed in this study were tolerable, and the 2y-OS, as the primary endpoint, was 40.7% (95%CI, 27.5–55.4%). These findings suggest our proposed treatment is an effective treatment regimen. This appears to represent the first report of a treatment yielding improvements in PFS and OS regardless of tumor MGMT promoter methylation status, suggesting that our proposed treatment could hold promise even in patients with unmethylated tumors. However, a well-designed and large scale randomized controlled rial with bio-molecular studies is required in the future.

CTCAE; Common Terminology Criteria for Adverse Events, CI; Confidence interval, C; Cycles, ECOG-PS; Eastern Cooperative Oncology Group performance scale, GBM; Glioblastoma, HGG; High-grade glioma, IFN-β; Interferon beta, IDH; Isocitrate dehydrogenase, MGMT; O6-methyl-guanine-DNA-methyltransferase, mOS; Median Overall survival, mPFS; Median Progression-free survival, MS-PCR; Methylation-specific polymerase chain reaction, OS; Overall survival, PFS; Progression-free survival, RT; Radiotherapy, TMZ; Temozolomide, VCR; Vincristine,

Acknowledgements

We thank all staff at Hirosaki University Graduate School of Medicine. And also, we express our deep gratitude to Dr. Masashi Matsuzaka, Department of Cancer epidemiology and Community health, Hirosaki University Graduate School of Medicine, for his advice on statistical analyses.

Authors' contributions

K Asano and H Ohkuma contributed to concept and design of the study.

K Asano and H Ohkuma contributed to tumor removal operations.

K Asano, K Katayama, and H Ohkuma contributed to management of patients after operation.

K Asano, Katayama, S Hasegawa, N Suzuki, and K Akasaka contributed to follow-up after treatment.

A Kurose and A Kamataki contributed to review pathological diagnosis.

T Fumoto and A Kamataki contributed to molecular analysis.

K Asano, K Katayama, and H Ohkuma contributed to acquisition and analysis of the data.

K Asano, T Fumoto, A Kurose, and H Ohkuma contributed to drafting the text and preparing the figure.

Funding

No funding was received for this study. No authors have any financial relationships to disclose.

Availability of data and material

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Ethics approval and consent to participate

Consent for publication:

The authors and the Committee of Medical Ethics of Hirosaki University Graduate School of Medicine affirm that human research participants provided informed consent for the publication of this project.

Conflicts of interest

All authors have no conflicts to report.

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Table 1 Patient baseline characteristics and treatment details

Characteristic	The number of patients (N=47)
Age	-
Median (IQR), Range	62 (54, 69), 16-80
Age ≤ 70	37 (78.7%)
Age < 71	10 (21.3%)
Gender	-
M : F	21:26
ECOG performance status	-
0	13 (27.7)
1	14 (29.8)
2	20 (42.6)
3	0 (0.0)
Operation removal rate	-
Biopsy	6 (12.8)
Partial resection	14 (29.8)
Gross total removal	27 (57.4)
Pathological diagnosis	-
Glioblastoma	43 (91.5)
Giant cell glioblastoma	3 (6.4)
Gliosarcoma	1 (2.1)
IDH status (IDH1^R132H)	-
Wild	41 (87.2)
Mutant	6 (12.8)
MGMT status	-
Methylated	16 (34.0)
Unmethylated	31 (66.0)
Concomitant induction phase	-
Complete protocol	47 (100)
Incomplete protocol	0 (0.0)
Maintenance phase	-
Complete protocol of TMZ 24C + IFN-b 96C	10 (21.3)
Complete protocol of TMZ 50C + IFN-b 200C	5 (10.6)
Maintenance cycles of TMZ (Median (IQR) Range)	13 (8, 23), 3-50
Maintenance cycles of IFN-b (Median (IQR) Range)	51 (32, 86), 10-328

Data are number (%).

Table 2 Progression free survival and overall survival

Variable	PFS (M / %)	95 %CI
Median PFS (M)	11.0	7.0-13.0
At 1 year (%)	40.4	27.5-54.9
At 2 year (%)	18.2	9.5-32.2
At 3 year (%)	16.0	7.9-29.6
At 4 year (%)	11.4	4.9-24.5
At 5 year (%)	9.1	3.5-21.8
Median OS (M)	18.0	15.0-26.0
At 1 year (%)	80.9	67.1-89.7
At 2 year (%)	40.7	27.5-55.4
At 3 year (%)	24.9	14.4-39.5
At 4 year (%)	20.3	11.0-34.6
At 5 year (%)	20.3	11.0-34.6

Table 3 Second line treatments after the first recurrence

Treatment Regimen	The Number of Patients N=42 (%)
Same as protocol of TMZ+IFN-b	26 (61.9)
TMZ+IFN-b+SRS	8 (19.0)
TMZ+IFN-b+SRS+BEV	1 (2.4)
TMZ+IFN-b+SRS+Surgery	3 (7.1)
TMZ+IFN-b+Surgery+BEV	1 (2.4)
TMZ+INF-b+Surgery	1 (2.4)
Subtotal (TMZ+IFN-b)	40 (95.2)
Surgery alone	1 (2.4)
Best supportive care	1 (2.4)

SRS: Stereotactic radiosurgery

BEV: Bevacizumab

Table 4 Adverse events

	Grade 1 (%)	Grade 2 (%)	Grade 3 (%)	Grade 4 (%)	Grade 3+4 %
Hematological
Anemia	14 (29.8)	19 (40.4)	0	0	0.0
Neutropenia	6 (12.8)	18 (38.3)	8 (17.0)	1 (2.1)	19.1
Lymphopenia	3 (6.4)	3 (6.4)	28 (59.6)	13 (27.7)	87.3
Thrombopenia	21 (44.7)	9 (19.2)	2 (4.3)	2 (4.3)	8.6
Non-hematological
Nausea	7 (14.9)	5 (10.6)	2 (4.3)	0	4.3
Vomiting	4 (8.5)	4 (8.5)	1 (2.1)	0	2.1
Anorexia	16 (34.0)	7 (14.9)	4 (8.5)	0	8.5
Constipation	12 (25.5)	26 (55.3)	0	0	0.0
ALT elevation	16 (34.0)	10 (21.3)	2 (4.3)	0	4.3
Hyponatremia	26 (55.3)	3 (6.4)	2 (4.3)	0	4.3
Hyperpotassemia	11 (23.4)	1 (2.1)	1 (2.1)	0	2.1
Skin rush	3 (6.4)	4 (8.5)	0	0	0.0
Fever	15 (31.9)	0	0	0	0.0
Febrile neutropenia	-	-	0	0	0.0
Others	3 (6.4)	0	3 (6.4)	0	6.4

Others: Interferon-associated retinopathy (G3) 1 case, hydrocephalus(G3) 2 cases, and Peripheral sensory neuropathy(G1) 3 cases

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Combination Chemoradiotherapy with Temozolomide, Vincristine, and Interferon-β Might Improve Outcomes Regardless of O6-Methyl-Guanine-DNA-Methyltransferase (MGMT) Promoter Methylation Status in Newly Glioblastoma

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Ms-pcr For Determining Mgmt Promoter Methylation Status

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Pfs And Os According To Mgmt Promoter Methylation Status

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