Integrative Use of Cannabidiol, Melatonin, and Oxygen–Ozone Therapy in Triple-Negative Breast Cancer with Lung and Mediastinal Metastases. A Case Report

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors report an interesting case of a 27 year old patient with TNBC who after initial treatment experienced disease relapse with lung and mediastinal lymph node metastases. This patient in addition to standard of care, received melatonin, cannabidiol, and oxygen–ozone therapy. This combinational approach led to the complete disappearance of the lung nodules. Subsequently, stereotactic radiotherapy was performed and, in association with the ongoing integrative treatment led to significant reduction of mediastinal adenopathy. Finally, treatment with PARP inhibition in addition to integrative treatment was associated with the patient achieving a complete remission. This case highlights the utility of melatonin, cannabidiol, and oxygen–ozone therapy as part of an integrative/supplemental approach with standard of care treatment.

This case is novel and the mechanisms at play are of interest making it highly relevant. My overall impressions are positive; however, I do think that some areas of the manuscript/some statements require revision.

Major Revisions:

-Please make figure markings/indications more prominent as it was hard to see

- Line 76-77: Please cite clinical trials you are referring to. Also regarding the statement “potentiated the efficacy of chemotherapeutic agent” when going through the reference provided (reference 6) and looking up the trials directly, I did not see sufficient evidence to support this statement. I would suggest that it be removed. The rest of the statement is fine.

- Line 142: Olaparib is a PARP inhibitor, not referred to as immunotherapy. Please make manuscript modifications to reflect this.

-Line 207: For this concluding statement, please look at including some variation of the below statement as it is important to accurately delineate the patient’s treatment course and role MLT/CDB and O2/O2 played in it in accordance with published literature:

It is important to make it clear that the patient received carboplatin and gemcitabine administered every 21 days in addition to stereotactic radiotherapy in addition with Olaparib. This regiment was taken together with the melatonin, cannabidiol, and oxygen–ozone therapy and the contribution of melatonin, cannabidiol and oxygen-ozone therapy towards tumor efficacy cannot accurately be delineated in this case. Additional comprehensive, well-powered clinical trials are required to elicit efficacy and effect of this treatment. The combination of melatonin, cannabidiol and oxygen-ozone therapy however was safe for use in this patient, and it has evidence supporting its utility as an augmentative agent in patients with TNBC.               

Minor Revisions:

Line 23: What do you mean by advanced? Can you clarify or rephrase this line.

Line 27: Space before “:”

Line 27-29: Rephrase for reader clarity

Line 51: Italicize BRCA1 and BRCA2 for proper genomic nomenclature (other instances throughout manuscript to fix as well)

Line 52: My interpretation that family history and the BRCA1/2 mutations are the main risk factors from this line. I think this is a bit bold of a statement and would rephrase while also including risk factors of prior chemotherapy/radiation and other genes such as CDH1, TP53, PTEN, etc.

Line 57: Would recommend using Luminal A/B/Basal/HER-2 descriptions of breast cancer sub-types

Line 61: Grammer issues, please rephrase

Line 63: Per other literature it seems to be more around 10-15%

Line 66-68: Ensure that it is clear that the PARP Inhibitor effects were in addition with other treatments not in isolation. Provide more information regarding “pretreatment” and rather state the prior treatment requirements for inclusion in these trials. I would also reference and refer to the actual clinical trials you are referring to (ex. DOI: 10.1056/NEJMoa2105215)

Line 68: Double spacing issue

Line 91: “standard chemo-, radio-, and immunotherapy” it would be helpful to list her treatment course briefly prior to delving into the case.

Line 104: BRCA1 italicize

Line 107: Any other molecular/genetic workup and results to share? If so please include.

Line 107-108: Were there any biopsies and pathology work up of the metastatic lesions? If so please include.

Line 116-117: Include dosage of chemotherapeutic agents

Line 142: Olaparib is a PARP inhibitor, not referred to as immunotherapy  

Line 197: italicize in vitro and in vivo throughout manuscript

Comments for author File: Comments.pdf

Author Response

Major Revisions:

-Please make figure markings/indications more prominent as it was hard to see

1. High resolution (a minimum of 1000 pixels in width/height, and a resolution of 600 dpi were deposited as zip. file .

- Line 76-77: Please cite clinical trials you are referring to. Also regarding the statement “potentiated the efficacy of chemotherapeutic agent” when going through the reference provided (reference 6) and looking up the trials directly, I did not see sufficient evidence to support this statement. I would suggest that it be removed. The rest of the statement is fine.

1. Thank you for the comment, which allows us to provide additional information regarding the clinical case. We have added references to the clinical studies and removed the requested sentence.

- Line 142: Olaparib is a PARP inhibitor, not referred to as immunotherapy. Please make manuscript modifications to reflect this.

1. R. Done.

-Line 207: For this concluding statement, please look at including some variation of the below statement as it is important to accurately delineate the patient’s treatment course and role MLT/CDB and O2/O2 played in it in accordance with published literature:

1. Done

It is important to make it clear that the patient received carboplatin and gemcitabine administered every 21 days in addition to stereotactic radiotherapy in addition with Olaparib. This regiment was taken together with the melatonin, cannabidiol, and oxygen–ozone therapy and the contribution of melatonin, cannabidiol and oxygen-ozone therapy towards tumor efficacy cannot accurately be delineated in this case. Additional comprehensive, well-powered clinical trials are required to elicit efficacy and effect of this treatment. The combination of melatonin, cannabidiol and oxygen-ozone therapy however was safe for use in this patient, and it has evidence supporting its utility as an augmentative agent in patients with TNBC.

1. We have revised the text to better clarify the potential role of the integrated therapy described in this case report.

Minor Revisions:

Line 23: What do you mean by advanced? Can you clarify or rephrase this line.

1. We rephrase the sentence.

Line 27: Space before “:”

R: Done

Line 27-29: Rephrase for reader clarity

1. Done

Line 51: Italicize BRCA1 and BRCA2 for proper genomic nomenclature (other instances throughout manuscript to fix as well)

1. Done

Line 52: My interpretation that family history and the BRCA1/2 mutations are the main risk factors from this line. I think this is a bit bold of a statement and would rephrase while also including risk factors of prior chemotherapy/radiation and other genes such as CDH1, TP53, PTEN, etc.

1. Comments have been added

Line 57: Would recommend using Luminal A/B/Basal/HER-2 descriptions of breast cancer sub-types

1. Done

Line 61: Grammer issues, please rephrase

1. Done

Line 63: Per other literature it seems to be more around 10-15%

1. Done

Line 66-68: Ensure that it is clear that the PARP Inhibitor effects were in addition with other treatments not in isolation. Provide more information regarding “pretreatment” and rather state the prior treatment requirements for inclusion in these trials. I would also reference and refer to the actual clinical trials you are referring to (ex. DOI: 10.1056/NEJMoa2105215)

R: We added more information in the text

Line 68: Double spacing issue

1. Done

Line 91: “standard chemo-, radio-, and immunotherapy” it would be helpful to list her treatment course briefly prior to delving into the case.

1. At the appearance of metastases, the patient underwent chemotherapy according to the carboplatin and gemcitabine regimen; however, in the submitted medical records the drug dosages for the two regimens are not described (including any dose reductions due to toxicity, as had been reported for olaparib). In any case, the standard dosages described in the protocols are as follows:
   EC (epirubicin 90 mg/m² and cyclophosphamide 600 mg/m²) on day 1 every 21 days for 4 cycles, followed by paclitaxel 80 mg/m² administered weekly.
   Gemcitabine 1000 mg/m² IV on days 1 and 8 every 21 days plus carboplatin AUC 4 (for unfit, elderly, or previously treated patients) or [references: 1.A phase II trial of gemcitabine/carboplatin with or without trastuzumab in the first-line treatment of patients with metastatic breast cancer; 2.Carboplatin and gemcitabine combination in metastatic triple-negative anthracycline- and taxane-pretreated breast cancer patients: a phase II study].

We preferred not to describe the dosages in detail due to the lack of complete documentation on this matter.

Line 104: BRCA1 italicize

1. Done

Line 107: Any other molecular/genetic workup and results to share? If so please include.

1. No more information provided

Line 107-108: Were there any biopsies and pathology work up of the metastatic lesions? If so please include.

1. No metastatic lesions analysis has been provided by the patient.

Line 116-117: Include dosage of chemotherapeutic agents

Line 142: Olaparib is a PARP inhibitor, not referred to as immunotherapy

1. Modified 

Line 197: italicize in vitro and in vivo throughout manuscript

1. Done

Reviewer 2 Report

Comments and Suggestions for Authors

I think it is a nice work, it can be accepted. only typesetting and fomration of the main text should be addressed.

Author Response

Thanks for the comments. The text has been revised for grammar and formatting.

Reviewer 3 Report

Comments and Suggestions for Authors

Strengths and Scientific Merit

• Rigour in longitudinal follow-up: The manuscript stands out for its 60-month observation period, which significantly exceeds the median overall survival reported for metastatic TNBC, providing valuable prognostic information.
• Precision of the therapeutic protocol: The authors rigorously detail the dosages and routes of administration of the integrative regimen, allowing for a clear assessment of pharmacological exposure (specifically the 2 g/day of melatonin and the ozone concentrations).
• Robustness of the diagnostic evidence: The clinical response is documented in a chronological sequence of high-resolution images (PET-CT), allowing clear visual correlation between the interventions and tumour regression.
• Relevance in precision oncology: The study addresses a critical niche in current oncology: the management of patients with homologous recombination deficiency (BRCA mutation) and the potential synergy of non-conventional agents with PARP inhibitors.

Limitations and Critical Considerations

• Presence of significant confounding factors: The main limitation lies in the difficulty of isolating the therapeutic effect of the integrative protocol. The patient received olaparib and stereotactic radiotherapy, interventions that constitute the standard of care and have demonstrated high efficacy in patients with BRCA1 mutations. This is a critical confounding factor in establishing a direct causal relationship between CBD and melatonin.
• Generalizability limitations: As is inherent in case reports (n=1), the findings have limited external validity. The exceptional response could be linked to the patient's specific tumour biology rather than to the protocol's reproducibility in the general TNBC population.
• Uncertainty regarding safety and tolerability at high doses: The use of 2 g of melatonin daily represents an extreme deviation from conventional clinical doses. The text requires further discussion on the neuropsychiatric tolerability and long-term metabolic safety of such concentrations.
• Gaps in interaction analysis: The potential impact of phytocannabinoids and melatonin on cytochrome P450 enzymes is not described, which is vital to rule out alterations in metabolism and to assess the potential for potentiation of olaparib toxicity.

Clarification requirements:

The authors are requested to address the following points to strengthen the quality of the manuscript:

1. Pharmacodynamic justification: What was the clinical criterion or specific preclinical evidence that supported the melatonin escalation to 2 g daily?
2. Differentiation of response: Given that the reduction in mediastinal lymphadenopathy coincided with radiotherapy, are there diagnostic elements to differentiate a local effect of radiation from a possible abscopal effect mediated by integrative therapy?
3. Toxicity and adherence monitoring: Given the reported haematological toxicity, how was adherence to the integrative protocol assessed, and were there any dose adjustments due to minor side effects (nausea, drowsiness)?
4. Quality of life methodology: Were standardised tools (such as the EORTC QLQ-C30 questionnaire) used to monitor the patient's well-being during these five years?
5. Biomarker analysis: In addition to imaging, were tumour markers or liquid biopsy (circulating tumour DNA) performed to confirm the absence of minimal residual disease?

Author Response

1. Pharmacodynamic justification: What was the clinical criterion or specific preclinical evidence that supported the melatonin escalation to 2 g daily?

1. Thanks for the question. The selected melatonin doses were determined based on the patient’s response, on preclinical data from oncological studies (references provided in the text), and on published pharmacodynamic data showing low toxicity up to certain daily dose levels.

1. Differentiation of response: Given that the reduction in mediastinal lymphadenopathy coincided with radiotherapy, are there diagnostic elements to differentiate a local effect of radiation from a possible abscopal effect mediated by integrative therapy?

10. Thanks for the question. Based on current clinical knowledge regarding the use of ozone therapy, medical cannabis, or melatonin, there are no diagnostic elements that allow one to distinguish a local effect of radiation from a possible abscopal effect mediated by integrative therapy. However, have been published scientific studies that highlight a possible role of melatonin. For example, it has been described that melatonin shows potential in cancer therapy, as it can protect healthy tissue from radiation damage (radioprotection) and also sensitize cancer cells to treatment, potentially enhancing the effects of radiation (including abscopal effects) by boosting anti-tumor immunity and inducing cancer cell death through pathways such as reactive oxygen species (ROS) production (as also observed with O₂/O₃ therapy) (Masoud Moslehi, Reza Moazamiyanfar, Mohammad Sedigh Dakkali, et al. Modulation of the immune system by melatonin; implications for cancer therapy, International Immunopharmacology, 2022, Volume 108, https://doi.org/10.1016/j.intimp.2022.108890).

1. Toxicity and adherence monitoring: Given the reported haematological toxicity, how was adherence to the integrative protocol assessed, and were there any dose adjustments due to minor side effects (nausea, drowsiness)?

1. R. Since the patient did not show any adverse effects, the treatments were maintained over the years. The patient fully adhered to the integrated protocol without interruptions throughout the entire period.

1. Quality of life methodology: Were standardised tools (such as the EORTC QLQ-C30 questionnaire) used to monitor the patient's well-being during these five years?

1. R. Thanks for the suggestion. Standardized tools were not applied. The patient was monitored over the years using the diagnostic assessments described in the text.

1. Biomarker analysis: In addition to imaging, were tumour markers or liquid biopsy (circulating tumour DNA) performed to confirm the absence of minimal residual disease?

1. R. No, other biomarkers analysis were not performed.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Line 52: Rephrase this part “they represent only a fraction of total cases” as it diminishes the proportion of breast cancer attributable to these mutations. Change it instead to something like “however, other risk factors such as exist such as certain chemotherapy regiments…”

Line 54- Reference consistency (4)

Line 222-223: The sentence “While the patient underwent a robust conventional protocol—including carboplatin/gemcitabine, stereotactic radiotherapy, and Olaparib—the integration of MLT, CBD, and O₂/O₃ therapy played a key role in her treatment course.” How could these agents have played a key role if we cannot delineate their effects.

Please change to “may have contributed to her treatment efficacy” or something similar.

Line 231-231: “particularly in aggressive and hard-to-treat cancers such as triple-negative metastatic breast cancer”

Why particularly in aggressive/hard-to treat cancers? Those patients may be more sensitive/have comorbidities making these approaches unsuitable based on current evidence. Therefore, I would suggest that this quoted part of the sentence is removed to not overstate. The prior part of the sentence is fine.

Author Response

Dear Reviewer,

We thank you for the time you devoted to evaluating our manuscript and for the constructive comments provided.
For clarity, the changes made to the text are highlighted in the revised version.
We remain at your disposal for any further clarification and thank you again for your valuable contribution.

Kind regards,

Margherita Luongo

Author Response File: Author Response.docx