Olive Leaf Extract (OLE) Anti-Tumor Activities Against Hematologic Tumors: Potential Therapeutic Implications for Pediatric Patients with B-Acute Lymphoblastic Leukemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an in vitro study of the antileukemic properties of olive leaf extract (OLE), with a focus on pediatric acute lymphoblastic leukemia (B-ALL). The study combines leukemia cell lines, primary patient blast cells, and comprehensive molecular profiling.

While the in vitro findings are robust, the manuscript would benefit from a clearer statement that any therapeutic implications remain preliminary and require further validation before extrapolation to the clinical setting.

Translational Context- A brief discussion linking effective in vitro OLE concentrations to available pharmacokinetic or bioavailability data would facilitate the reader's assessment of the potential clinical relevance of the findings.

Methodological Limitations- Lack of Intestinal Digestion and Absorption Steps.
Although the study is robust as an in vitro model, the lack of simulated intestinal digestion or absorption is a significant limitation that should be considered. Because OLE is an orally administered and chemically heterogeneous plant extract, digestive transformation and intestinal absorption significantly influence which components become bioavailable. Authors should clearly justify the interpretation of their results in the absence of these processes and explain how this limitation impacts translational conclusions.

Extract heterogeneity- Adding a brief note recognizing the inherent variability of plant extracts and the importance of extract standardization would contextualize the results and increase reproducibility.

B-ALL vs. AML- Because this study demonstrates significant differences in reactivity between these malignancies, a brief extension of the discussion of potential mechanisms underlying resistance in AML would further enrich the interpretation.

The above suggestions are intended to clarify and strengthen the translational dimension of the manuscript.

Comments on the Quality of English Language

I recommend light proofreading, focusing on grammar, punctuation, and shortening complex or long sentences.

Author Response

Comments and Suggestions for Authors

This manuscript presents an in vitro study of the antileukemic properties of olive leaf extract (OLE), with a focus on pediatric acute lymphoblastic leukemia (B-ALL). The study combines leukemia cell lines, primary patient blast cells, and comprehensive molecular profiling.

 

COMMENT 1. While the in vitro findings are robust, the manuscript would benefit from a clearer statement that any therapeutic implications remain preliminary and require further validation before extrapolation to the clinical setting.

RESPONSE 1. We thank the Reviewer for this comment. We agree with this criticism and we have modified this statement in the Conclusions (page 11 lines 487-488 and 496-499).

 

COMMENT 2. Translational Context- A brief discussion linking effective in vitro OLE concentrations to available pharmacokinetic or bioavailability data would facilitate the reader's assessment of the potential clinical relevance of the findings.

RESPONSE 2. We agree with this comment. We have now added this information in the discussion (page 11 Lines 475-481) and we have cited two papers focused on pharmaokinetic and bioavaliability of oleuropein (references 98-99).

 

COMMENT 3. Methodological Limitations- Lack of Intestinal Digestion and Absorption Steps.

Although the study is robust as an in vitro model, the lack of simulated intestinal digestion or absorption is a significant limitation that should be considered. Because OLE is an orally administered and chemically heterogeneous plant extract, digestive transformation and intestinal absorption significantly influence which components become bioavailable. Authors should clearly justify the interpretation of their results in the absence of these processes and explain how this limitation impacts translational conclusions.

RESPONSE 3. We thank the Reviewer for having highlighted this crucial point. Indeed, we have now added in the discussion a new section regarding the bioavailability of oleuropein and other compounds after the administration of OLE, by citing two different studies that address this issue (references 98-99). Accordingly, we modified our conclusions regarding therapeutic implications (page 11. Lines 475-481).

 

COMMENT 4. Extract heterogeneity- Adding a brief note recognizing the inherent variability of plant extracts and the importance of extract standardization would contextualize the results and increase reproducibility.

RESPONSE 4. We thank the Reviewer for this comment. We agree that the concentration of biologically active compounds in OLE may be variable. However, we reported the mean concentration of Oleuropein, Hydrohytyrosol, Tyrosol, Elenolic Acid and Rutin in different batches, and the manufacturers’ disclosed a low variability among different batches analyzed. We added a comment in the Materials and Methods section (page 3 lines 99-100) and in the Discussion at page 8 lines 339-342.

 

COMMENT 5. B-ALL vs. AML- Because this study demonstrates significant differences in reactivity between these malignancies, a brief extension of the discussion of potential mechanisms underlying resistance in AML would further enrich the interpretation.

RESPONSE 5. We agree with this comment. Some of these differences have been highlighted in the Discussion (page 8-11 lines 355-356, 406-407, 426-429, 458-461). However, we updated the Discussion by adding a new sentence regarding this issue (page 11 Lines 462-468).

 

 COMMENT 6. The above suggestions are intended to clarify and strengthen the translational dimension of the manuscript.

RESPONSE 6. We really appreciated the Reviewer’s effort in helping us to improve the manuscript.

 

 

Comments on the Quality of English Language

COMMENT 7. I recommend light proofreading, focusing on grammar, punctuation, and shortening complex or long sentences.

RESPONSE 7. We thank the Reviewer for this comment and we have modified many sentences, as requested.

Reviewer 2 Report

Comments and Suggestions for Authors

The study presents interesting observations, but the work remains largely descriptive and lacks the mechanistic integration and analytical rigor required for publication. The central claims are not sufficiently supported by validated signaling data or coherent connections across experiments. Substantial conceptual and experimental strengthening is needed

Author Response

Comments and Suggestions for Authors

COMMENT 1. The study presents interesting observations, but the work remains largely descriptive and lacks the mechanistic integration and analytical rigor required for publication. The central claims are not sufficiently supported by validated signaling data or coherent connections across experiments. Substantial conceptual and experimental strengthening is needed

RESPONSE 1. We thank the Reviewer for His/Her comments and the recognition that the study presents interesting observations. Regarding the central claim related to the coherent connection, we believe that the experiments here reported followed a logical sequence, since we first explored whether OLE could exert an anti-tumor activity against lymphoproliferative disorders in terms of inhibition of cell proliferation and induction of apoptosis, moving forward to the underlying mechanisms of action, by analyzing the modulation of 126 molecules and finally asking whether OLE could be used as nutraceutical compound in combination with the standard of care chemotherapy used in leukemia patients. Accordingly, we have reported the results with the same logical view.  Although we would be happy to improve our manuscript, it is difficult to do so without any specific comments regarding our experimental setting and/or conceptualization of the study.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

What control was used in this study for OLE, and why did the authors not choose an available drug to compare the efficacy of OLE with a marketed therapeutic? The manuscript does not explain how the OLE was obtained, and the experimental section is poorly described. Details on flow cytometry and cell separation procedures are missing.

Additionally, what are the other potential bioactive compounds present in OLE? Using crude extracts is not generally recommended for clinical applications; it would strengthen the study if the authors identified specific active compounds and compared their effects to the extract.

Author Response

Comments and Suggestions for Authors

COMMENT 1. What control was used in this study for OLE, and why did the authors not choose an available drug to compare the efficacy of OLE with a marketed therapeutic?

RESPONSE 1. We thank the Reviewer for this comment. The aim of our study was to analyze the anti-tumor effects of OLE in the context of lymphoproliferative disorders, similarly to what we reported some years ago in Neuroblastoma (Morandi et al. 2021 Nutrients). In addition, we were interested in finding possible addictive/synergistic effects of OLE with common chemotherapeutic drugs rather than comparing OLE with other drugs.

COMMENT 2. The manuscript does not explain how the OLE was obtained, and the experimental section is poorly described. Details on flow cytometry and cell separation procedures are missing.

RESPONSE 2. We agree with the comment regarding how OLE was obtained, and we added this information in materials and methods in the new paragraph 2.1 (page 3 lines 93-100). We also added some details regarding flow cytometric analysis (page 3 lines 125-128 and page 4 lines 140-143). As described in materials and methods (page 3, lines 102-114) we used commercial lymphoma and leukemia cell lines and primary leukemic blasts obtained from bone marrow aspirates following mononuclear cell separation.

 

COMMENT 3. Additionally, what are the other potential bioactive compounds present in OLE? Using crude extracts is not generally recommended for clinical applications; it would strengthen the study if the authors identified specific active compounds and compared their effects to the extract.

RESPONSE 3. We thank the Reviewer for this suggestion and we agree that bioactive compounds should be identified. However, it has been already demonstrated that oleuropein is the most effective anti-tumor compound in OLE (Boss et al, Nutrients 2016) and we based our experiments on the concentration of this molecule. However, as described at page 3 Lines 97-99, other biologically active compounds, such as Hydrohytyrosol, Tyrosol, Elenolic Acid and Rutin are present at high concentrations. We would like to stress out that testing each compound separately is beyond the scope of this study, which was focused on anti-tumor activities of OLE as nutraceutical food supplement. Furthermore, the stability of these compounds in the leaf extract and their interaction should be taken into account and cannot be comparable to single compounds tested separately.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a timely and relevant topic by exploring the anti-tumor potential of olive leaf extract (OLE) in pediatric B-acute lymphoblastic leukemia (B-ALL), including analyses in cell lines and patient-derived primary blasts, which strengthens its translational relevance.

• The comparative approach between B-ALL, AML, and lymphoma models is a clear strength and highlights a disease-selective response to OLE; however, the mechanistic conclusions appear to go beyond the level of direct experimental support currently provided.

• The conclusion that OLE predominantly activates the extrinsic apoptotic pathway is mainly based on antibody array correlations; direct functional validation (e.g., caspase-8/-3 activation, TRAIL or death receptor blocking experiments) is lacking and should be addressed or explicitly acknowledged as a limitation.

• The concentrations of OLE (oleuropein-equivalent) required to induce biological effects in vitro are relatively high, and the physiological or clinical relevance of these doses is insufficiently discussed in the context of achievable plasma levels following oral administration.

• While the synergistic effect between OLE and cytarabine is an important and interesting finding, the absence of synergy with cyclophosphamide is not mechanistically explained; additional discussion on cell-cycle dependence, DNA damage response, or apoptosis priming would strengthen result interpretation.

• The antibody array analyses cover a large number of targets, but key findings (e.g., TRAIL R1, FADD, p53 phosphorylation changes) are not independently validated by orthogonal methods such as Western blotting, limiting confidence in the mechanistic claims.

• The manuscript would benefit from clearer distinction between proof-of-concept in vitro observations and clinically translatable conclusions, particularly in the Discussion and Conclusions sections.Overall, the study presents promising and potentially impactful data, but significant clarification and mechanistic strengthening are required before the conclusions can be fully supported.

Overall, the study presents promising and potentially impactful data, but significant clarification and mechanistic strengthening are required before the conclusions can be fully supported.

Author Response

The manuscript addresses a timely and relevant topic by exploring the anti-tumor potential of olive leaf extract (OLE) in pediatric B-acute lymphoblastic leukemia (B-ALL), including analyses in cell lines and patient-derived primary blasts, which strengthens its translational relevance. The comparative approach between B-ALL, AML, and lymphoma models is a clear strength and highlights a disease-selective response to OLE; however, the mechanistic conclusions appear to go beyond the level of direct experimental support currently provided.

COMMENT 1. The conclusion that OLE predominantly activates the extrinsic apoptotic pathway is mainly based on antibody array correlations; direct functional validation (e.g., caspase-8/-3 activation, TRAIL or death receptor blocking experiments) is lacking and should be addressed or explicitly acknowledged as a limitation.

RESPONSE 1. We thank the Reviewer for this comment. We completely agree that our conclusion regarding the activation of extrinsic apoptotic pathway is based only on data obtained with antibody array and lack of functional experiments. We would be happy to perform these experiments on caspases and using blocking antibodies against different receptors, and we plan to perform these studies in the future. However, due to the need of many months to perform these additional studies and the limited time available for the revision (5 days) we choose to explicit such limitation, as suggested. Thus, we removed a sentence regarding this issue in the Results (paragraph 3.4 at page 7) and we modified the Discussion at page 9 Line 403 and page 10, lines 457-460.

COMMENT 2. The concentrations of OLE (oleuropein-equivalent) required to induce biological effects in vitro are relatively high, and the physiological or clinical relevance of these doses is insufficiently discussed in the context of achievable plasma levels following oral administration.

RESPONSE 2. We thank the Reviewer for this criticism and we totally agree. Indeed, the same criticism has been previously raised by other Reviewers in the first revision. Thus, we added a sentence in the conclusions (page 11, lines 490-494) and we cited two papers addressing this issue.

COMMENT 3. While the synergistic effect between OLE and cytarabine is an important and interesting finding, the absence of synergy with cyclophosphamide is not mechanistically explained; additional discussion on cell-cycle dependence, DNA damage response, or apoptosis priming would strengthen result interpretation.

RESPONSE 3. We thank the Reviewer for this comment and we totally agree. Thus, we now added a new paragraph addressing possible mechanisms underlying the synergistic/asdditive effects observed with cytarabine but not with cyclophosphamide (page 11, lines 479-489).

COMMENT 4. The antibody array analyses cover a large number of targets, but key findings (e.g., TRAIL R1, FADD, p53 phosphorylation changes) are not independently validated by orthogonal methods such as Western blotting, limiting confidence in the mechanistic claims.

RESPONSE 4. We thank the Reviewer for this comment. We agree that data obtained with antibody arrays could be additionally validated through Western blotting. However, we would like to stress the fact that the validation of a large number of proteins which we found to be modulated by OLE (at least 40) would require a very high cost and additional very long time. Moreover, Western blot does not invole a different scientific principle or tecnique compared to antibody array, since both methods involed the assessment of proteins on a nitrocellulose membrane using whole protein extracts from cell lines, primary antibodies and biotin-conjugated secondary antibodies, followed by the use of HRP-conjugated streptavidin. Furthermore, we believe that antibody arrays imply a standardized and validated method based on the use of multple and quantified positive and negative controls.

COMMENT 5. The manuscript would benefit from clearer distinction between proof-of-concept in vitro observations and clinically translatable conclusions, particularly in the Discussion and Conclusions sections.Overall, the study presents promising and potentially impactful data, but significant clarification and mechanistic strengthening are required before the conclusions can be fully supported. Overall, the study presents promising and potentially impactful data, but significant clarification and mechanistic strengthening are required before the conclusions can be fully supported.

RESPONSE 5. We totally agree with the Reviewer. This issue has been raised by another Reviewer in the first revision. Thus, we have already coped with this criticism and we have mitigated our conclusions (page 11). However, we now added a new sentence at page 11 lines 500-503.

Reviewer 3 Report

Comments and Suggestions for Authors

In my view, the editor may make the final decision, as research involving crude extracts has been subject to considerable criticism in recent years.

Author Response

COMMENT 1. In my view, the editor may make the final decision, as research involving crude extracts has been subject to considerable criticism in recent years.

RESPONSE 1. We thank the Reviewer for this comment and we agree that the use of crude extracts may generate criticism especially when used in clinical settings. However, we proposed OLE as food supplement and possibly adjuvant for patients with B-ALL only in combination with standard-of-care treatments. We totally agree with the concept that OLE and other crude extracts must be used exclusively as support and not as alternative to chemotherapy. Nonetheless, different studies (more than 400) have addressed beneficial effects of OLE in vitro, and some of them support its anti-cancer effects alone or in combination with conventional chemotherapeutic drugs (Boss et al, Nutrients 2016; De Cicco et al, Phytother Res. 2022; Tezcan et al, Biomed Pharmacother. 2017; Ruzzolini et al, Nutrients. 2018; Campo et al, Life (Basel). 2024).