Successful application of dietary ketogenic metabolic therapy in patients with glioblastoma: a clinical study

Abstract

Key Aspects

• Introduction and Rationale: This study investigates the use of ketogenic metabolic therapy (KMT) as a treatment for glioblastoma multiforme (GBM), a highly aggressive brain cancer. The rationale is based on the altered metabolism of cancer cells, which often rely heavily on glucose for energy, unlike normal brain cells that can utilize ketones. The study explores whether a ketogenic diet, which is high in fat and low in carbohydrates, can exploit this metabolic difference to slow tumor growth and improve survival.
• Methods and Study Design: The study design was a prospective clinical trial involving 18 GBM patients. The patients were followed from January 2016 to January 2024. The key intervention was the implementation of a ketogenic diet, and the primary outcome measure was survival, with a benchmark of 3 years considered a success.
• Results and Survival Rates: The results showed a statistically significant difference in survival rates between patients who adhered to the ketogenic diet for more than 6 months and those who did not. Among the adherent group, 66.7% achieved the 3-year survival benchmark, compared to only 8.3% in the non-adherent group. This suggests a strong positive association between ketogenic diet adherence and improved survival in GBM patients.
• Discussion and Implications: The discussion section highlights the promising potential of the ketogenic diet as an adjunctive therapy for GBM. The authors acknowledge the limitations of the small sample size but emphasize the statistically significant survival difference. The study contributes to the growing body of evidence supporting the exploration of metabolic therapies for cancer treatment.

Strengths

• Clear Research Question

  The abstract clearly states the research question, focusing on the impact of ketogenic metabolic therapy on glioblastoma progression.

  "A total of 18 patients with GBM, 8 women and 10 men, aged between 34 and 75 years participated in a prospective study, examining the impact of ketogenic diet on tumor progression." (Page 1)
• Concise Summary of Key Findings

  The abstract concisely summarizes the key findings, highlighting the statistically significant difference in survival rates between the adherent and non-adherent groups.

  "Comparing the survival rates of the two groups, we see that the difference is 58.3% (66.7% versus 8.3%) and is statistically significant with p < 0.05 (0.0114) and X2 = 6.409." (Page 1)
• Brief Methodological Overview

  The abstract provides a brief overview of the methods, including the study population, intervention, and primary outcome measures.

  "A total of 18 patients with GBM, 8 women and 10 men, aged between 34 and 75 years participated in a prospective study, examining the impact of ketogenic diet on tumor progression...We considered the therapeutic combination successful if the survival reached at least 3 years." (Page 1)

Suggestions for Improvement

• Highlight Novelty

  High impact. Enhancing the abstract with a statement about the novelty of the work would improve the impact and context for readers. Currently, the abstract does not explicitly state what is new about this study, making it harder for readers to quickly grasp its contribution to the field. This belongs in the abstract as it sets the stage for the entire paper.

  "Recent research has pivoted toward exploring alternative therapeutic methods, such as the ketogenic diet, for GBM." (Page 1)

  Implementation: Include a sentence in the introduction or discussion section of the abstract that highlights the unique aspects of this study. For example, "This clinical study provides novel evidence for the successful application of dietary ketogenic metabolic therapy in patients with glioblastoma, demonstrating a significant improvement in survival rates compared to standard treatment."

• Specify Ketogenic Diet Details

  Medium impact. Adding specific details about the ketogenic diet protocol would improve clarity. The abstract mentions "ketogenic diet" but doesn't specify the type or macronutrient ratios. This belongs in the abstract as it is critical information for understanding the intervention.

  "Methods: A total of 18 patients with GBM, 8 women and 10 men, aged between 34 and 75 years participated in a prospective study, examining the impact of ketogenic diet on tumor progression." (Page 1)

  Implementation: Include a brief phrase indicating the type of ketogenic diet (e.g., classical, modified Atkins) and the target ketogenic ratio or range. For example: "...examining the impact of a classical ketogenic diet (target ratio of 3:1 to 4:1) on tumor progression."

• Contextualize Survival Benchmark

  Medium impact. Providing context for the 3-year survival benchmark would improve reader understanding. While the abstract mentions a 3-year survival target, it doesn't explain why this timeframe is significant. This belongs in the abstract to provide essential context for the primary outcome.

  "We considered the therapeutic combination successful if the survival reached at least 3 years." (Page 1)

  Implementation: Add a brief phrase explaining the relevance of the 3-year survival mark. For example: "We considered the therapeutic combination successful if the survival reached at least 3 years, a clinically meaningful milestone in GBM treatment."

Introduction

Key Aspects

• Glioblastoma and Treatment Challenges: Glioblastoma (GBM) is presented as the most common and aggressive primary malignant brain tumor, with a poor prognosis despite standard treatment involving surgery, radiotherapy, and chemotherapy. This establishes the clinical significance and the urgent need for new therapeutic approaches.
• Introduction of Ketogenic Metabolic Therapy (KMT): The introduction of Ketogenic Metabolic Therapy (KMT) is presented as a novel therapeutic strategy. KMT is defined as a precision nutrition approach that uses biomarker-driven ketogenic diets, fasting, and other lifestyle interventions to target the altered metabolism of cancer cells.
• Warburg's Theory of Carcinogenesis: Warburg's theory of carcinogenesis, which posits that cancer cells have altered metabolism characterized by increased glycolysis and impaired mitochondrial function, is described. This theory provides the scientific foundation for exploring metabolic therapies like KMT.
• Metabolic Differences Between Normal and Cancer Cells: The metabolic differences between normal brain cells and GBM cells are explained. Normal brain cells can utilize ketones for energy, while GBM cells rely heavily on glucose due to mitochondrial dysfunction. This difference forms the basis for KMT's potential to selectively target cancer cells.
• Growing Interest in Metabolism-Based Treatments: The introduction highlights the growing interest in metabolism-based cancer treatments and the limitations of current chemoradiotherapy in improving long-term outcomes for GBM patients. This sets the stage for exploring KMT as a potential adjunctive therapy.
• Cytoplasmic Lipid Droplets and Metabolic Inefficiency: The introduction mentions the presence of cytoplasmic lipid droplets in many malignant cancers as evidence of oxidative phosphorylation inefficiency. This connects to Warburg's theory and supports the idea that KMT could exploit metabolic vulnerabilities in cancer cells.
• Rationale for Offering KMT to GBM Patients: The rationale for offering ketogenic diet therapy to a cohort of 18 GBM patients is presented, based on Warburg's theory and existing experimental and clinical data. The choice of GBM is justified by its metabolic dependency on glycolysis and poor prognosis.

Strengths

• Clear Context and Problem Statement

  The introduction clearly establishes the context by defining glioblastoma (GBM) and its aggressive nature, highlighting the limitations of current treatments and the need for new approaches.

  "Glioblastoma (GBM) is the most common primary malignant tumor of the brain and central nervous system...Despite surgical excision (total or subtotal), followed by adjuvant radiotherapy and chemotherapy with temozolomide, patients with newly diagnosed GBM have a median overall survival of 12-18 months, with <10% surviving beyond 5 years (2, 3)." (Page 2)
• Logical Introduction of KMT

  The introduction logically introduces the concept of ketogenic metabolic therapy (KMT) as a potential alternative treatment strategy, providing a concise definition and linking it to Warburg's theory of carcinogenesis.

  "Thus, in recent years, the research community has turned to the discovery of alternative therapeutic strategies for GBM, such as dietary ketogenic metabolic therapy (KMT). Dietary KMT is defined as a synergistic precision nutrition approach, incorporating biomarker-driven ketogenic diets, fasting and fasting-mimicking diets, as well as other lifestyle interventions (4, 5)." (Page 2)
• Explanation of Metabolic Rationale

  The introduction effectively explains the metabolic rationale behind KMT, highlighting the differences between normal brain cells and GBM cells in utilizing glucose and ketones, thus justifying the potential of KMT to selectively target cancer cells.

  "Unlike normal brain cells, that have evolved to metabolize ketone bodies for energy when glucose availability is low, GBM cells depend on glycolysis for growth and are unable to efficiently metabolize ketones due to impaired mitochondrial function (11-13)." (Page 2)

Suggestions for Improvement

• Explicitly State Study Novelty

  Medium impact. The introduction could be improved by more explicitly stating the novelty of this specific study. While it introduces KMT and its rationale, it doesn't clearly differentiate this study's contribution from existing research. This belongs in the introduction to set the stage for the paper's unique contribution.

  "Based on the above observations, interest is growing in designing metabolism-based treatments for cancer in general and primary brain tumors in particular." (Page 2)

  Implementation: Add a sentence or short paragraph near the end of the introduction that explicitly states what is new or unique about this study. For example: "While previous studies have explored KMT in GBM, this study focuses on [specific aspect, e.g., a specific patient population, a particular dietary protocol, a longer follow-up period, or a combination of factors]."

• State Study Objective or Hypothesis

  Low impact. The introduction could benefit from a brief mention of the study's primary objective or hypothesis. While the rationale for KMT is presented, the specific aim of this study isn't directly stated. This belongs in the introduction to guide the reader's expectations.

  "Based on Warburg's theory and expanding upon the abovementioned experimental and clinical data, we offered ketogenic diet therapy to a cohort of 18 GBM patients." (Page 2)

  Implementation: Add a concise statement of the study's objective or hypothesis at the end of the introduction. For example: "This study aims to investigate the impact of [specific ketogenic diet protocol] on survival and disease progression in patients with newly diagnosed GBM." or "We hypothesize that adherence to a ketogenic diet will be associated with improved survival outcomes in GBM patients."

• Mention Limitations of Previous Research

  Low impact. The introduction could be strengthened by briefly mentioning the limitations of previous research on KMT for GBM. This would further justify the need for the current study. The introduction is the appropriate place for this, as it provides context for the current work.

  "While there is adequate experimental evidence supporting the beneficial effects of KMT on brain tumors, clinical testing is still ongoing (13, 18, 19)." (Page 2)

  Implementation: Add a sentence or two summarizing the limitations of previous KMT studies in GBM. For example, "Previous studies of KMT in GBM have been limited by small sample sizes, heterogeneous patient populations, and variations in dietary protocols." This could be placed before the statement of the study's objective.

Non-Text Elements

FIGURE 1 (A) Simplified scheme of glucose and ketone metabolism in a normal...

Full Caption

FIGURE 1 (A) Simplified scheme of glucose and ketone metabolism in a normal brain cell.

Figure/Table Image (Page 3)

First Reference in Text

Glucose and ketone metabolism in normal and cancer cells is illustrated in Figures 1A, B, 2A, B.

Description

• Overview of glucose and ketone metabolism: Figure 1A illustrates the metabolic processes within a normal brain cell, focusing on glucose and ketone metabolism. Glucose enters the cell via a glucose transporter (Glut-1) and is converted to pyruvate through glycolysis in the cytoplasm. Pyruvate then enters the mitochondria, where it's converted to Acetyl-CoA, initiating the Krebs cycle (also known as the citric acid cycle). This cycle produces NADH and ATP, which are essential for cellular energy. When glucose is limited, ketone bodies enter the cell via monocarboxylate transporters (MCTs) and are converted to Acetyl-CoA, feeding into the Krebs cycle to produce energy.
• Integration of metabolic pathways: The diagram depicts the interconnectedness of glucose and ketone metabolism in a normal brain cell. The figure shows that both glucose and ketone bodies are metabolized to Acetyl-CoA, which enters the Krebs cycle. The Krebs cycle is a series of chemical reactions that extract energy from Acetyl-CoA and generate molecules like NADH and ATP, which the cell uses for fuel.
• Role of transporters: The figure highlights the role of specific transporters, Glut-1 for glucose and MCTs for ketone bodies, in facilitating the entry of these substrates into the cell. These transporters are crucial for cellular metabolism because they regulate the availability of energy substrates inside the cell.

Scientific Validity

• Accuracy of metabolic representation: The diagram accurately represents the core biochemical pathways of glucose and ketone metabolism in a normal brain cell. However, it simplifies the complexity of these processes by omitting regulatory enzymes and intermediate steps, which could be misleading for experts seeking detailed mechanistic insights.
• Completeness of metabolic pathways: The schematic representation does not include all possible metabolic fates of pyruvate or Acetyl-CoA, such as lipid synthesis or amino acid metabolism, which are also relevant in brain cells. This omission could oversimplify the metabolic capabilities of normal brain cells.
• Inclusion of regulatory mechanisms: The figure does not explicitly address the regulation of glucose and ketone metabolism, such as hormonal control or allosteric regulation of key enzymes. Including these regulatory aspects would provide a more complete picture of metabolic control in normal brain cells.

Communication

• Overall clarity and visual representation: The diagram effectively uses visual cues to represent metabolic pathways, making it easier to understand the complex processes involved. The use of arrows and labels is clear, but could benefit from more specific annotations to highlight key regulatory steps or enzyme involvement.
• Caption completeness: The caption provides a basic description of the figure's content, but could be expanded to include the specific context within the study (i.e., how this normal cell metabolism contrasts with that of a cancer cell) to improve its immediate relevance to the reader.
• Accessibility for non-experts: For readers unfamiliar with metabolic pathways, a brief glossary or more detailed introduction to the key molecules and processes (e.g., glycolysis, Krebs cycle) would enhance understanding.

FIGURE 1 (B) Ketogenic diet impact in normal brain cells.

Figure/Table Image (Page 3)

First Reference in Text

Glucose and ketone metabolism in normal and cancer cells is illustrated in Figures 1A, B, 2A, B.

Description

• Overview of ketogenic diet impact: Figure 1B illustrates how a ketogenic diet affects metabolism in a normal brain cell. A ketogenic diet is a diet very low in carbohydrates, moderate in protein, and high in fats. When carbohydrates are restricted, the body produces ketone bodies from fat, which serve as an alternative fuel source. In this diagram, the ketogenic diet is shown to inhibit glucose uptake by the brain cell, represented by a crossed-out glucose transporter (Glut-1).
• Shift in substrate utilization: The figure shows that ketone bodies enter the cell via monocarboxylate transporters (MCTs). Once inside, they're converted to Acetyl-CoA, which then enters the Krebs cycle to produce energy. The ketogenic diet forces the brain cell to rely more heavily on ketone bodies for fuel instead of glucose.
• Metabolic consequences: The diagram highlights the reduced reliance on glucose and increased utilization of ketone bodies in normal brain cells during a ketogenic diet. This metabolic shift is thought to have therapeutic benefits in certain neurological conditions.

Scientific Validity

• Accuracy of metabolic representation: The diagram accurately represents the core concept of reduced glucose utilization and increased ketone body utilization in normal brain cells under a ketogenic diet. However, it simplifies the complex regulatory mechanisms that govern substrate selection and metabolic flux.
• Completeness of adaptive mechanisms: The figure does not address the potential adaptive mechanisms that brain cells might employ to compensate for reduced glucose availability, such as increased expression of ketone body transporters or altered enzyme activity. This omission could limit the figure's scientific rigor.
• Inclusion of energy status: The figure does not explicitly show the effects on ATP production or energy status within the cell, which are key outcomes of the metabolic shift. Including this information would strengthen the figure's scientific validity.

Communication

• Visual representation of metabolic shift: The diagram effectively illustrates the shift in substrate utilization in normal brain cells under a ketogenic diet. The visual representation of reduced glucose entry and increased ketone body entry is clear. However, the figure could benefit from a more explicit depiction of the relative concentrations or flux rates of these substrates to better convey the quantitative impact of the diet.
• Contextual information in caption: The caption provides context for the figure, but it could be enhanced by mentioning the intended benefit or consequence of this metabolic shift in normal brain cells, such as neuroprotection or energy efficiency.
• Accessibility for non-experts: While the diagram is simplified, it assumes a certain level of familiarity with metabolic pathways. Providing a brief explanation of the rationale behind the ketogenic diet's impact on glucose and ketone body utilization would improve accessibility for readers without a strong background in metabolism.

FIGURE 2 (A) Simplified schema of glucose and fat metabolism in a cancer cells.

Figure/Table Image (Page 4)

First Reference in Text

Glucose and ketone metabolism in normal and cancer cells is illustrated in Figures 1A, B, 2A, B.

Description

• Overview of cancer cell metabolism: Figure 2A depicts the metabolic processes within a cancer cell, emphasizing the Warburg effect, which is the preference for glycolysis (the breakdown of glucose) over oxidative phosphorylation (the use of oxygen to generate energy in the mitochondria) even when oxygen is available. Glucose enters the cell and is converted to pyruvate, but instead of primarily entering the mitochondria, it's converted to lactate via anaerobic glycolysis.
• Mitochondrial dysfunction: The figure indicates that the mitochondria in cancer cells are dysfunctional. This is represented by dashed lines around the Krebs cycle and reduced ATP production, implying that oxidative phosphorylation is impaired. The cancer cell relies heavily on glycolysis for its energy needs.
• Lactate production and tumor microenvironment: The diagram shows the production of lactate, which is exported from the cell. Lactate production contributes to an acidic environment around the tumor, which can promote cancer progression and metastasis. Acidosis, lactate and MCTs are highlighted.

Scientific Validity

• Accuracy of metabolic representation: The diagram accurately represents the core features of cancer cell metabolism, including increased glycolysis and mitochondrial dysfunction. However, it is a simplified representation and does not capture the full complexity of metabolic heterogeneity within tumors.
• Completeness of metabolic pathways: The figure does not include other important metabolic pathways that are often dysregulated in cancer cells, such as glutamine metabolism or fatty acid synthesis. Including these pathways would provide a more complete picture of cancer cell metabolism.
• Inclusion of regulatory mechanisms: The figure does not explicitly address the genetic and epigenetic factors that contribute to the metabolic rewiring of cancer cells. Mentioning key oncogenes or tumor suppressor genes that regulate metabolism would enhance the figure's scientific validity.

Communication

• Clarity of visual representation: The diagram clearly illustrates the metabolic differences in cancer cells compared to normal cells, specifically highlighting the reliance on glycolysis and reduced mitochondrial function. The use of simplified schematics effectively conveys the overall metabolic phenotype.
• Caption completeness: The caption provides a basic description, but could be enhanced by explicitly stating the Warburg effect and how it is represented in the figure. Mentioning the therapeutic implications (i.e., targeting glycolysis) would also improve the caption's impact.
• Accessibility for non-experts: For readers unfamiliar with cancer metabolism, a brief explanation of why cancer cells favor glycolysis even in the presence of oxygen (Warburg effect) would improve understanding.

FIGURE 2 (B) Ketogenic diet impact in cancer cells.

Figure/Table Image (Page 4)

First Reference in Text

Glucose and ketone metabolism in normal and cancer cells is illustrated in Figures 1A, B, 2A, B.

Description

• Overview of ketogenic diet impact: Figure 2B illustrates the effects of a ketogenic diet on cancer cell metabolism. The diagram shows a crossed-out glucose transporter (Glut-1), indicating that the ketogenic diet reduces glucose uptake into the cancer cell. This is because the ketogenic diet aims to lower blood glucose levels, thus depriving cancer cells of their preferred fuel source.
• Impaired ketone body oxidation: The diagram indicates that even though ketone bodies are available, the cancer cell's ability to oxidize them efficiently is impaired due to mitochondrial abnormalities. This is represented by the dashed lines around the Krebs cycle, implying that the cancer cell cannot effectively use ketone bodies for energy production.
• Therapeutic consequences: The figure highlights that the combination of reduced glucose uptake and impaired ketone body oxidation leads to reduced proliferation rates in cancer cells. This is the intended therapeutic effect of the ketogenic diet.

Scientific Validity

• Accuracy of metabolic representation: The diagram accurately represents the intended metabolic effects of a ketogenic diet on cancer cells, including reduced glucose availability and impaired ketone body oxidation. However, it is a simplified representation and does not capture the full complexity of metabolic adaptations that cancer cells can undergo.
• Completeness of metabolic pathways: The figure does not address the potential for cancer cells to upregulate other metabolic pathways, such as glutamine metabolism or fatty acid oxidation, to compensate for the reduced glucose availability. Including these alternative pathways would provide a more complete picture of cancer cell metabolism.
• Inclusion of microenvironmental factors: The figure does not explicitly address the role of the tumor microenvironment or systemic factors that can influence cancer cell metabolism. Including these factors would enhance the figure's scientific validity.

Communication

• Clarity of visual representation: The diagram effectively illustrates the intended impact of a ketogenic diet on cancer cells, specifically highlighting the reduction in glucose availability and the potential for reduced proliferation rates. The use of crossed-out arrows effectively conveys the inhibition of glucose uptake.
• Caption completeness: The caption could be enhanced by explicitly mentioning the therapeutic rationale, which is to exploit the metabolic inflexibility of cancer cells and their dependence on glucose. Also, stating the potential limitations (i.e., cancer cells may still use ketone bodies) would improve the caption's completeness.
• Accessibility for non-experts: For readers unfamiliar with cancer metabolism, a brief explanation of why limiting glucose might be detrimental to cancer cell growth would improve understanding. It could also benefit from acknowledging the potential for metabolic adaptation.

Materials and methods

Key Aspects

• Study Design and Patient Population: The study design is a prospective clinical trial involving 18 patients with newly diagnosed glioblastoma multiforme (GBM). The study aimed to evaluate the effects of ketogenic diet therapy on tumor progression, with patients originating from the authors' hospital and a collaborating oncologist between January 2016 and July 2021, and followed until January 2024.
• Inclusion and Exclusion Criteria: Specific inclusion criteria were adult patients (18-75 years old) with newly diagnosed GBM. Exclusion criteria included patients with cachexia (body weight <40 kg), severe cardiovascular or renal disease, and inherited metabolic disorders where a ketogenic diet is contraindicated.
• Ketogenic Diet Administration Protocol: The ketogenic diet administration involved a gradual increase in the fat-to-(protein + carbohydrate) ratio, starting at 1:1 and aiming for 3:1, with target ketone values >3.5 mM/L and glucose values <80 mg/dL. Energy requirements were calculated using the Mifflin-St Jeor equation, and the diet was adapted to Mediterranean diet patterns.
• Clinical Assessment using ECOG Scale: Patients underwent clinical assessments using the ECOG (Eastern Cooperative Oncology Group) scale before starting the diet and every 3 months thereafter. This scale assesses disease progression and its impact on daily living abilities.
• Assessment Criteria and Self-Monitoring: The assessment criterion for successful adherence to the ketogenic diet was set at beyond 6 months. The therapeutic combination was considered successful if survival from diagnosis reached at least 3 years. Patients self-measured blood glucose and ketone levels.
• Laboratory Evaluation: A detailed laboratory evaluation was performed, including complete blood count, biochemical tests (electrolytes, blood glucose, transaminases, cholesterol, triglycerides, thyroid hormones), electrocardiogram, and electroencephalogram (EEG).
• Ethical Considerations: The study was conducted in accordance with the 1975 Declaration of Helsinki and received prior approval from the ethics committee of the authors' university (PN 1232/16). A double-blind design was planned but was not feasible due to the rapid progression of the disease.

Strengths

• Clear Study Design

  The section clearly outlines the study design, stating it was a prospective study evaluating the effects of ketogenic diet therapy on tumor progression in 18 GBM patients.

  "A total of 18 patients with GBM, 8 women and 10 men, between 34 and 75 years of age (median age 57.5 years) participated in our prospective study evaluating the effects of ketogenic diet therapy on tumor progression." (Page 2)
• Specific Inclusion/Exclusion Criteria

  The section provides specific inclusion and exclusion criteria, enhancing the reproducibility of the study and defining the target population.

  "Adult patients, 18–75 years old with newly diagnosed glioblastoma multiforme (GBM) were included. Patients with cachexia (weight body<40 kg), with severe cardiovascular or renal disease, and inherited metabolic disorders for which ketogenic diet is contraindicated, were excluded from the study." (Page 3)
• Detailed Diet Administration Protocol

  The section describes the ketogenic diet administration protocol, including the initial ratio, target ketone and glucose values, and the method for calculating energy requirements. It also mentions adapting to Mediterranean diet patterns.

  "We started with a 1:1 diet (fat: protein+ carbohydrates), and gradually, while monitoring ketonemia and glycemia, increased the ratio with the goal of reaching a 3:1 ratio, aiming for ketone values >3.5 mM/L and glucose values <80 mg/dL. Energy requirements were calculated based on the patients’ body weight at the time we took them on, using the Mifflin-St Jeor equation (which uses the current body weight)." (Page 4)
• Defined Assessment Criterion

  The section specifies the assessment criterion, defining adherence to the ketogenic diet as lasting beyond 6 months and considering a survival of at least 3 years as a successful therapeutic combination.

  "As an assessment criterion, we set an optimistic target for adherence to the ketogenic diet beyond 6 months...We considered the therapeutic combination successful if the survival from diagnosis reached at least 3 years." (Page 5)

Suggestions for Improvement

• Provide More Detail on Diet Composition

  Medium impact. The methods section could be improved by providing more detail about the specific Mediterranean ketogenic diet composition. While it mentions olive oil, mono/polyunsaturated fatty acids, and fish, it lacks precise macronutrient percentages or ranges, food lists, or sample menus beyond a single example. This level of detail is crucial for reproducibility and for understanding the intervention's precise nature. The Methods section is the appropriate place for this information, as it directly pertains to the intervention being studied.

  "The diet was adapted to Mediterranean diet patterns due to the patient’s aggravated status from the disease itself and standard treatments, with the intention of avoiding additional side effects." (Page 4)

  Implementation: Include a more detailed description of the Mediterranean ketogenic diet, specifying the typical macronutrient ranges (e.g., % of calories from fat, protein, and carbohydrates). Consider adding a supplementary table with a more comprehensive list of allowed foods and their typical serving sizes, or providing a more detailed example of a typical daily meal plan. Expand on Table 2 to give more complete information.

• Clarify Objective Adherence Assessment

  Medium impact. The methods section should clarify how adherence to the ketogenic diet was objectively assessed beyond self-reported measurements. While it mentions self-measured blood glucose and ketone levels, it doesn't describe how this data was verified or used to categorize patients as adherent or non-adherent. This is crucial for the validity of the study's conclusions. The Methods section is the correct location for this, as it directly relates to how the primary outcome was measured and interpreted.

  "The patients self-measured their blood glucose and ketone levels with blood glucose and β-ketone test strips, in the morning and afternoon preprandially." (Page 5)

  Implementation: Describe in detail the process for assessing adherence. Specify the frequency of required self-measurements, the criteria for considering a patient adherent (e.g., average ketone levels above a certain threshold, percentage of readings within the target range), and how missing data was handled. Mention if any other methods were used to corroborate self-reported adherence, such as dietician follow-up, food diaries, or urinary ketone measurements.

• Expand Statistical Methods Description

  Low impact. The methods section lacks information on the statistical methods used beyond a very basic statement. It mentions the software used and a p-value threshold but doesn't specify the tests used for comparing survival rates or other outcomes. This information is essential for evaluating the rigor of the analysis. This belongs in the Methods section, specifically in the subsection on statistical analysis.

  "Statistical analysis was done using the Statistical Analysis Systems statistical software package, version 20 (SAS Institute). Results were regarded as significant when p < 0.05. The difference between the two groups was assessed by the t-test for paired comparisons." (Page 5)

  Implementation: Expand the "Assessment criterion: statistics" subsection to include more detail. Specify the statistical tests used to compare survival rates between groups (e.g., Kaplan-Meier analysis, log-rank test). Describe how other variables were analyzed (e.g., t-tests, chi-square tests). Indicate whether any adjustments were made for potential confounders.

• Specify Frequency and Protocols for Assessments

  Low Impact. While the section mentions neurological and MRI evaluations, it doesn't specify the frequency or standardized protocols used for these assessments. This information is crucial for understanding the monitoring process and ensuring consistency across patients. This detail should be included in the Methods section as it pertains to data collection procedures.

  "All subjects were referred to our hospital’s outpatient clinic for a general neurological assessment, including neurological status and MRI evaluation." (Page 2)

  Implementation: Specify the frequency of neurological assessments and MRI evaluations (e.g., every 3 months, every 6 months). If any standardized scales or protocols were used for neurological assessments (beyond the ECOG scale, which is mentioned), name them explicitly. If specific MRI sequences or imaging parameters were used, provide those details.

Non-Text Elements

TABLE 1 Characteristics of all patients who participated in the study.

Figure/Table Image (Page 5)

First Reference in Text

Grades 2 and 3 are intermediary forms (32) (Table 1).

Description

• Overview of patient characteristics: Table 1 presents the characteristics of all 18 patients who participated in the study. The table includes the following information for each patient: patient identifier (P), gender (G), age (A), date of diagnosis (DD), date of surgery and type of operation (DS-TOO), chemotherapy and radiation therapy administered before the ketogenic diet (Chemo + Rad prior KD administration), and molecular biology (MB).
• Patient demographics and surgical information: The table shows a mix of male (M) and female (F) patients, with ages ranging from 34 to 75 years. The 'DS-TOO' column indicates whether the patient underwent total resection or subtotal resection/stereotactic biopsy. Total resection refers to complete removal of the tumor, while subtotal resection means that part of the tumor was left.
• Treatment and molecular biology information: The 'Chemo + Rad prior KD administration' column lists the specific chemotherapeutic agents (Temozolamide) and the use of radiation therapy, specifically mentioning 30 cycles of radiation. The 'MB' column indicates the IDH1 status of the tumor, with results showing either negative (-), positive (+), or IDH 1-2.

Scientific Validity

• Relevance of included variables: The table provides relevant descriptive information about the patient cohort, which is essential for assessing the study's generalizability. The inclusion of molecular biology data (IDH1 status) is important for characterizing the tumor subtypes.
• Completeness of clinical data: The table is missing key clinical data that could influence the outcomes, such as Karnofsky Performance Status (KPS) scores at baseline, ECOG scores, extent of resection, and details of concurrent treatments. Adding these variables would strengthen the table's scientific value.
• Transparency of data collection: The table does not indicate the source of the patient data or the methods used to collect it. Clearly stating the data collection procedures would improve the table's transparency and scientific rigor.

Communication

• Overall organization and presentation: The table is generally well-organized, providing essential information about the study participants. However, the table could benefit from including summary statistics (e.g., mean ± SD for age) to allow for easier comparison between groups.
• Clarity of abbreviations: The use of abbreviations is generally defined, but a glossary of abbreviations within the table or in the caption would ensure clarity for all readers.
• Relevance to study objectives: The table effectively presents the data to support the study's methods. However, consider adding a column indicating whether each patient adhered to the ketogenic diet and for how long, as this is a critical variable in the study.

Results

Key Aspects

• Adherence to the Ketogenic Diet: The primary finding is that 6 out of 18 patients with GBM adhered to the ketogenic diet for more than 6 months. This establishes the feasibility of maintaining the diet for a substantial period in a subset of patients.
• Survival Rates in the Adherent Group: Of the 6 patients who adhered to the diet, 4 achieved the 3-year survival benchmark, representing a 66.7% survival rate in this group. This is the key outcome measure and suggests a potential survival benefit associated with diet adherence.
• Survival Rates in the Non-Adherent Group: In contrast, only 1 out of the 12 patients who did not adhere to the diet for more than 6 months reached 36 months of survival, resulting in an 8.3% 3-year survival rate in this group. This highlights the difference in outcomes between the two groups.
• Statistical Significance of Survival Difference: The difference in survival rates between the adherent and non-adherent groups was found to be statistically significant (p < 0.05), suggesting that the observed difference is unlikely due to chance.
• Individual Case Presentations: Detailed case presentations are provided for each of the 6 patients who adhered to the diet, including their medical history, treatment regimen, ketogenic diet implementation, and clinical outcomes. These case studies offer individual-level insights.
• Patient 1: Long-Term Survival: Patient 1 achieved an 84-month follow-up with no evidence of tumor recurrence, maintaining a ketogenic diet and working as a teacher. This case exemplifies the potential for long-term survival with KMT.
• Patient 2: Sustained Ketosis and No Recurrence: Patient 2 maintained nutritional ketosis for 43 months with no evidence of GBM recurrence, continuing to work as a primary school teacher. This case further supports the potential benefits of sustained ketosis.
• Patient 3: Recurrence After Diet Discontinuation: Patient 3 experienced GBM recurrence after discontinuing the ketogenic diet, but reinitiated the diet after relapse with improved adherence. This case highlights the potential importance of continuous adherence.
• Patient 4: Relapse and Continued Diet: Patient 4 experienced a GBM relapse 32 months after diagnosis but continued the ketogenic diet and underwent surgical resection and second-line chemotherapy. The patient ultimately passed away 43 months post diagnosis.
• Patient 5: Late Recurrence: Patient 5 had no evidence of disease progression for almost 3 years while on the ketogenic diet but experienced a GBM recurrence 36 months post-diagnosis. The patient is undergoing further treatment.
• Patient 6: Recurrence and Death: Patient 6 maintained satisfactory ketosis on the ketogenic diet but experienced a recurrence 22 months after diagnosis and ultimately passed away 36 months after diagnosis.
• Summary Table for Adherent Patients: Table 4 summarizes the key data for the six patients who followed the diet beyond 6 months, including diagnosis date, KD initiation, KD maintenance, disease progression, GKI, survival, and most recent ECOG Grade.
• Survival Data for Non-Adherent Patients: Table 5 provides survival data for the patients who did not follow the diet, including gender, age, and overall survival.

Strengths

• Clear Presentation of Main Result

  The section clearly presents the main result: 6 out of 18 patients adhered to the ketogenic diet for over 6 months, and of these, 4 out of 6 (66.7%) achieved the 3-year survival benchmark, compared to 1 out of 12 (8.3%) in the non-adherent group. This finding is directly related to the study's primary outcome.

  "Out of the 18 patients, 6 followed the diet for more than 6 months (Figure 10). The effect of diet on the disease evolution of these patients is shown in Table 4...Therefore, the 3-year survival rate is4/6 = 66.7%...Of the 12 patients who did not adhere to the diet, only one reached 36 months of survival, while the rest have died in an average time of 15.7 ± 6.7 months, with a 3-year survival rate of 8.3%." (Page 9)
• Inclusion of Individual Case Summaries

  The section provides individual patient case summaries, detailing their treatment history, adherence to the ketogenic diet, and clinical outcomes. This adds depth and context to the overall findings.

  "3 Cases presentation 3.1 Patient 1: 84-months follow-up A 41-year-old man was diagnosed with GBM of the left temporal lobe on December 2016 following a brain MRI (Figure 3)." (Page 6)
• Reported Statistical Significance

  The section presents the statistical significance of the difference in survival rates between the two groups (p < 0.05).

  "Comparing the survival rates of the two groups, we see that the difference is 58.3% (66.7 versus 8.3%) and is statistically significant with p < 0.05 (0.0114) and X2 = 6.409." (Page 10)

Suggestions for Improvement

• Fully Characterize Outcomes of Non-Adherent Group

  High impact. The Results section should present all relevant results, including those that might not support the main hypothesis. While the section focuses on the 6 adherent patients, it doesn't fully characterize the outcomes of the 12 non-adherent patients beyond overall survival. This omission could introduce bias and limits the reader's ability to fully evaluate the intervention's effects. The Results section is the appropriate place for this, as it's where all findings, both positive and negative, should be presented.

  "The characteristics of the patients who did not comply with the diet beyond 6 months are shown in Table 5." (Page 9)

  Implementation: Include a more detailed summary of the outcomes for the non-adherent group. This could involve reporting the range of survival times, ECOG scores over time, or other relevant clinical observations. Consider creating a table (similar to Table 4) summarizing the key characteristics and outcomes of the non-adherent patients. Report any adverse events or reasons for non-adherence in this group.

• Acknowledge Limitations of the Results

  Medium impact. The Results section should explicitly state the limitations of the presented results. While the individual case summaries are informative, they don't replace a rigorous statistical analysis of potential confounding factors. The Results section is the appropriate location to acknowledge these limitations before they are discussed in more detail in the Discussion section.

  "Out of the 18 patients, 6 followed the diet for more than 6 months (Figure 10)." (Page 9)

  Implementation: Add a paragraph at the end of the Results section acknowledging the limitations of the findings. Specifically mention the small sample size, the non-randomized nature of the study (which makes it difficult to control for confounding factors), and the potential for selection bias (patients who were able to adhere to the diet might have had other characteristics that contributed to their better outcomes). Briefly mention the lack of blinding.

• Report Secondary Outcomes

  Medium impact. The Results section should present data on secondary outcomes if they were measured. The Methods section mentions ECOG scores and MRI evaluations, but the Results section primarily focuses on survival. Reporting these secondary outcomes would provide a more comprehensive picture of the intervention's effects. The Results section is the proper place to present these findings.

  "His most recent ECOG grade is 0." (Page 6)

  Implementation: Include a summary of the ECOG scores and MRI findings for both the adherent and non-adherent groups. This could be presented in tables or figures. For example, you could report the average ECOG score at baseline and at various follow-up time points for each group. For MRI findings, you could describe the frequency of tumor progression or recurrence in each group.

• Present Results of All Statistical Tests

  Low impact. While the section mentions statistical significance (p<0.05), it does not present the results of the t-test mentioned in the methods. This creates inconsistency between the methods and results. The Results section is the appropriate place to present the actual results of all statistical tests performed.

  "The difference between the two groups was assessed by the t-test for paired comparisons." (Page 6)

  Implementation: Include the results of the t-test for paired comparisons that was mentioned in the methods section. Report the t-statistic, degrees of freedom, and p-value. Clarify which groups or variables were compared using this test. If the t-test was not the appropriate test, explain why and describe the correct test that was used (and report its results).

Non-Text Elements

TABLE 2 Example of ketogenic diet 2,150 Kcal 2:1 ratio.

Figure/Table Image (Page 6)

First Reference in Text

An example of the diet applied is shown in Table 2.

Description

• Overview of the ketogenic diet example: Table 2 provides an example of a ketogenic diet with a total daily calorie intake of 2,150 Kcal and a 2:1 ketogenic ratio. The ketogenic ratio refers to the ratio of fat to combined protein and carbohydrates in the diet. The table lists specific food items and their quantities for morning, snack, lunch, snack, and dinner meals.
• Specific food items for main meals: The morning meal consists of 17g tuna in oil and 8g olive oil. The lunch meal includes 93g sardines and 41g olive oil, while dinner consists of 117g raw green salad, 93g salmon, and 41g olive oil.
• Specific food items for snacks: The snack options include items like 20g feta cheese, 60g keto-focaccia, 1 egg fortified with w-3 fatty acids, and 15g avocado. The table provides a 'Daily menu plan' indicating the distribution of food items across the day.

Scientific Validity

• Lack of design criteria: The table provides an example of a ketogenic diet, but it lacks information on the specific criteria used to design the diet (e.g., target blood ketone levels, individual patient needs). Including this information would improve the table's scientific rigor.
• Missing source of food composition data: The table does not specify the source of the food composition data used to calculate the macronutrient content of the diet. Providing this information would enhance the table's transparency and scientific validity.
• Nutritional completeness: The table presents a single example, but it doesn't indicate whether this diet is nutritionally complete or if it was supplemented with vitamins or minerals. Including this information would improve the table's scientific validity.

Communication

• Completeness of macronutrient information: The table provides a useful example of a ketogenic diet, but it lacks information on the macronutrient breakdown (grams of fat, protein, carbohydrates) for each meal. Including this information would allow readers to assess the diet's composition and adherence to the 2:1 ratio more effectively.
• Inclusion of meal timing: The table presents a daily menu plan, but it does not specify the timing of the meals or snacks. Including this information would provide a more complete picture of the dietary regimen.
• Representativeness of the example: The table provides a specific example, but it doesn't indicate whether this was a standard diet or if it was individualized based on patient preferences or tolerances. Clarifying this aspect would enhance the table's value.