1 Introduction

Radiotherapy (RT) plays an integral role in the multimodality management of adult-type diffuse gliomas. Radiation is indicated in adult-type low-grade gliomas with high-risk features or high-grade gliomas following maximal safe resection [1-3]. Higher doses of RT can lead to symptomatic radio-necrosis (RN) in approximately 5%–15% of patients, typically within the first 2 years of RT completion [4-7]. The development of RN can lead to significant morbidity, with new-onset or worsening of pre-existing neurodeficits having substantial implications for quality of life (QOL), and in extreme situations, can lead to mortality. The pathogenesis of RN is multifactorial, with a complex interplay of vascular-mediated damage and injury to glial cells primarily postulated through the activation of several inflammatory markers like tumor necrosis factor, interleukins, and vascular endothelial growth factor [6]. Corticosteroids, preferably dexamethasone, form the first line of management of RN, with variable response rates ranging from 25%–60%, impacted by several factors like the dose of RT and response evaluation methods (neurological/radiographic) [8, 9]. The response rate in our practice concurs with the reported literature, with combined clinical and radiological responses seen in approximately 50% of patients from institutional experience and audit. It is important to note that the long-standing use of corticosteroids comes at the cost of complications like hyperglycemia, myopathy, and increased risk of infections precluding prolonged use. Also, a proportion of patients remain refractory to steroids or turn out to be dependent on steroids, where bevacizumab (anti-angiogenic agent) can be used as second-line therapy in appropriately selected patients [10, 11]. However, the major disadvantages of bevacizumab remain intravenous administration, requiring regular hospital visits, treatment costs, and concerns for related toxicities like hypertension and intracranial or extracranial hemorrhage. Other agents like hyperbaric oxygen therapy, pentoxifylline, and tocopherol have been suggested in refractory radionecrosis, with questionable benefits [6].

Sodium-copper-chlorophyllin is a phytopharmaceutical drug obtained from the green plant pigment chlorophyll. It is a semi-synthetic mixture of sodium copper salts derived from chlorophyll. Chlorophyllin scavenges RT-induced free radicals and reactive oxygen species. It has been used as a food colorant and over the counter in the USA, Japan, Australia, and China for many years for various health benefits, including the prevention of body odor in geriatric patients, enhanced wound healing, antibacterial action, etc. [12]. Studies have shown that CHL has immunostimulatory, anti-inflammatory, and antiviral effects in addition to antioxidant and radioprotective properties [13]. It increases the expression of a transcription factor (protein) Nrf2, improving lymphocyte survival and enabling efficient detoxification after RT exposure [14, 15].

The current study is a phase 2 study to investigate the role of CHL as an anti-inflammatory agent in treating brain radionecrosis, a long-term toxicity of cranial radiotherapy.

2 Study Methodology

2.1 A. Study Design/Population

This is a prospective phase 2 open-label study conducted in a single institute. Patients with a histopathological diagnosis of adult-type diffuse glioma treated with conventionally fractionated high-dose radiotherapy developing radionecrosis will be eligible for the current study. Radionecrosis will be diagnosed using standard institutional protocols based on magnetic resonance imaging (MRI) and/or positron emission tomography (PET) as decided clinically. MRI will include standard brain protocol per institutional practice, including T1w pre- and post-contrast, T2w propeller, T2 FLAIR, GRE/susceptibility sequences, ADC, DWI, spectroscopy, and perfusion-weighted imaging. All the images will be reviewed independently by a neuroradiologist and nuclear medicine physician (as appropriate) and discussed in the multidisciplinary tumor board (in cases of indeterminate findings) to reach a consensus of RN and exclude the possibility of disease progression. For evaluating disease progression, the updated Response Assessment in Neuro-Oncology criteria (RANO 2.0) will be used [16]. A patient with typical radiological findings of radionecrosis is presented in Figure 1. Patients will be assessed for the eligibility criteria below prior to inclusion in the study.

2.5 Response Evaluation

The study endpoints have been summarized in Table 1. Response to CHL will be considered as the absence of all three criteria as follows:

1. Imaging progression of radionecrosis: Defined as an increase in T1-contrast or T2w hyperintensity of the index lesion on the MRI brain, as assessed by a neuroradiologist. The PET scan at 1 month will be assessed by the nuclear medicine physician, and findings will be interpreted based on the avidity of the index lesion.
2. Clinical deterioration will be considered as a ≥ 2-point drop in the NPS at assessment points (1 and 3 months) from baseline NPS.
3. Dexamethasone requirement: Any increase in the dexamethasone requirement during the predetermined tapering schedule will be considered refractory RN for stratum A. Any requirement of dexamethasone after CHL will be considered a failure of CHL in stratum B. Similarly, after the 1st month (when no dexamethasone use is planned in stratum A), the use of dexamethasone will be considered a failure of CHL for stratum A during the analysis of response at the 3rd month.

TABLE 1. Objectives and endpoints of CHROME study.

Study objective	To assess the efficacy of oral CHL in the treatment of radionecrosis in patients with diffuse glioma
Study endpoints
Primary endpoint	Response rates (clinical-radiological) at 1 month with the use of CHL
Secondary endpoint	Clinical-radiological response rates at 3 months Biological response rates using functional imaging (PET and MRI) Survival analysis (Progression-free survival and overall survival) EORTC QOL C-30 and BN-20 questionnaire at baseline, 1-month, and at 3-month follow-ups NANO scale score QTWIST score
Exploratory endpoint	Temporal changes of inflammatory and tumor-related biomarkers obtained from the serum

Therefore, for study analysis, combined evaluation (radiological, clinical, steroid requirement) will serve as a method of testing the efficacy of CHL, with progression in any single domain of the above three considered a failure, as mentioned above. Patients with unequivocal disease progression on imaging at 1 or 3 months will be excluded from RN response assessment (since the possibility of recurrence component is likely to contribute to neurological functioning and outcomes). Any discrepancies or equivocal findings will be discussed in a joint neuro-oncology meeting (JNOM) for a consensus decision. No additional follow-up visits will be required as per study protocol since patients with RN are called at 1 month for clinical evaluation and at 3 months for radiological and clinical evaluation. However, additional imaging will be undertaken with MRI and PET scan at 1 month after starting CHL. Imaging with MRI brain tumor protocol will be done at 3 months as the standard of care following institutional practice. The study workflow has been presented in Figure 2. A subgroup analysis of response to chlorophyllin will be done for patients with IDH-mutant and IDH-wild gliomas for both strata. As a part of the study (exploratory endpoint), 12–15 mL blood samples (EDTA and plain tubes) will be collected at baseline, 1 month, and 3 months. The blood will be analyzed for inflammatory indices from routine hemogram serum biochemistry, including albumin, CRP, and ferritin, along with additional exploratory markers like cytokines, chemokines (GM-CSF, IFN-gamma, TNF-beta, interleukins, etc.), oxidative stress markers, proteomics, and metabolomics.

3 Discussion

Brain radionecrosis is encountered in patients receiving high-dose cranial radiotherapy, either with fractionated radiotherapy or ablative high-dose radiosurgical treatments. Symptomatically, RN can present with an entire spectrum from radiologically evident clinically silent asymptomatic to life-threatening situations refractory to treatment [22]. There are limited pharmacological options for effective treatment of RN other than corticosteroids and bevacizumab, which have their limitations regarding side effects. The current study will address the gap in investigating the role of chlorophyllin, a phytopharmaceutical with potential anti-inflammatory effects in treating brain radionecrosis.

Radionecrosis is mostly seen within the first 2 years, with the most common time being 6–18 months post-radiation. Factors influencing the incidence and severity of RN include radiation dose (with higher rates > 60 Gy EQD2), radiotherapy technique, radiation volume, fractionation, and other biological factors, including intrinsic radiosensitivity [22, 23]. The diagnosis of radionecrosis can often be challenging due to the similar radiological appearance with disease recurrence, which should be ruled out as prognosis and treatment implications are completely different. The use of multiparametric imaging, including advanced functional sequences like perfusion-weighted imaging and MR spectroscopy, can help differentiate radionecrosis from recurrence [24]. Typically, the changes appearing within the initial few months from radiation in the high-dose region, the presence of Swiss cheese appearance on the T1-contrast sequence, and hypointense to hypointense signal of T2-weighted imaging should lead to suspicion for radionecrosis. Compared to recurrence, radionecrosis is typically hypoperfused on perfusion imaging and has a lower Choline: NAA peak on MR spectroscopy. Dynamic susceptibility contrast MRI has proven to be useful in differentiating radionecrosis from tumor recurrence, and standardized protocols have been recommended by consensus [25, 26]. Novel imaging techniques like treatment response assessment maps (TRAMs) which utilize multiple T1-contrast sequences to explore differential washout of contrast from viable tumor and necrotic regions, or T1 mapping techniques can provide further clarity in differentiating radiation-induced changes from disease recurrence [27, 28]. Amino acid PET with carbon-11 methionine, DOPA, and FET can add to the MRI findings in improving the diagnostic performance in differentiating between radionecrosis and disease progression, with sensitivity and specificity of 80%–95%, as shown in different studies [29-34]. In the current study, the patients will undergo multiparametric MRI, including spectroscopy, perfusion, and diffusion-weighted imaging, to diagnose radionecrosis. In equivocal cases, additional imaging with FET or F-DOPA will be considered for confirmation of the diagnosis. All the patients will be discussed in the multidisciplinary joint neuro-oncology meeting before accrual in the study. The interval imaging after starting intervention (starting CHL) will be considered at 1 month with both MRI and PET for assessment of both morphological and functional response for primary endpoint assessment. Further imaging will be considered at 3 months for evaluation of long-term response to CHL. Since there is always a possibility of inclusion of patients with progression, these patients will be excluded from analysis regarding the efficacy of CHL if recurrence is documented within 3 months from study accrual.

The most commonly used medication for RN is corticosteroids, which can relieve symptoms and lead to reversal or stability of radiological findings in a proportion of patients. There are no guidelines regarding the dose and duration of use of corticosteroids and also the type of steroids (with dexamethasone preferred by the majority). Typically, in symptomatic patients, at least a few weeks of steroid treatment are required for optimal clinical benefit. There are concerns about certain adverse effects both with short-and long-term use of steroids, including insomnia, acute psychosis, hyperglycemia, osteoporosis, risk of infections, skin changes, myopathy, and adrenocortical insufficiency, which can itself lead to new complications affecting quality of life. Also, some patients often turn into a state of steroid dependence with relapse of symptoms upon withdrawal of steroids. In refractory cases, the anti-angiogenic agent bevacizumab can provide significant benefit; however, the major challenges remain the need for frequent hospital visits for intravenous administration (typically delivered every 2–3 weeks), associated costs, and side effects including hypertension or life-threatening hemorrhages. Some agents with questionable benefits include edaravone, pentoxifylline, and hyperbaric oxygen therapy [8, 35]. The current study will use CHL to treat radionecrosis, which has demonstrated anti-inflammatory and antioxidant properties with a free radical scavenging effect in preclinical studies, with the pathophysiology of radionecrosis being considered a pro-inflammatory environment [15, 36]. In the study, we are considering patients with symptomatic and asymptomatic radionecrosis in two different strata since the burden of necrosis, tempo of disease, and clinical outcomes are expected to be different. In the symptomatic stratum, patients will be treated with CHL in addition to oral dexamethasone, which is otherwise used as the standard first-line therapy for symptomatic RN in our institution, while in the other stratum (asymptomatic), patients will receive CHL alone to explore the role of CHL as a steroid-sparing agent.

The primary endpoint used in the study includes combined clinical and radiological criteria at 1 month for assessment of response. Other endpoints include 3-month response and additional patient-reported outcomes, survival outcomes, toxicity, and exploratory analysis for blood markers, which can predict the response to treatment. If proven useful and the primary endpoint is met, further phase 3 randomized studies will be planned to determine the efficacy of CHL in the treatment of radionecrosis. If proven useful, this will open a new avenue for the treatment of radionecrosis as a well-tolerated oral therapy, avoiding or reducing the side effects of current standard treatment with corticosteroids.

4 Conclusion

Chlorophyllin is an orally administered phytopharmaceutical that has anti-inflammatory effects. In the current phase 2 study, the efficacy of CHL will be investigated in both symptomatic and asymptomatic radionecrosis, with clinico-radiological response at 1 month as the primary endpoint.

Author Contributions

Archya Dasgupta: conceptualization (lead), data curation (lead), formal analysis (lead), funding acquisition (lead), investigation (lead), methodology (lead), project administration (lead), resources (lead), writing – original draft (lead). Saranga Sawant: data curation (equal), investigation (equal), methodology (equal), writing – original draft (equal). Abhishek Chatterjee: conceptualization (equal), data curation (equal), formal analysis (equal), investigation (equal), methodology (equal), project administration (equal), writing – review and editing (equal). Vikram Gota: data curation (equal), funding acquisition (lead), investigation (equal), methodology (equal), writing – review and editing (equal). Arpita Sahu: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Amitkumar Choudhari: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Kajari Bhattacharya: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Ameya Puranik: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Indraja Dev: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Aliasgar Moiyadi: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Prakash Shetty: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Vikas Singh: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Nandini Menon: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Sridhar Epari: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Ayushi Sahay: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Aekta Shah: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Nazia Bano: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Farnaz Shaikh: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Aabha Jirage: data curation (equal), investigation (equal), methodology (equal), writing – review and editing (equal). Tejpal Gupta: conceptualization (equal), data curation (equal), formal analysis (equal), investigation (equal), methodology (equal), project administration (equal), writing – review and editing (equal).

Ethics Statement

The study is being conducted in accordance with ICMR (2017) “National Ethical Guidelines for Biomedical and Health Research Involving Human Participants, International Conference on Harmonization Good Clinical Practice (ICH-GCP)” guidelines, Good Clinical Practice, and the principles of the Declaration of Helsinki. The study, including all the study-related documents, has obtained approval from the Ethics Committee prior to the enrollment of participants. The study is registered with the Clinical Trial Registry, India (CTRI) and ClinicalTrial.gov.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.