Combined Immune Checkpoint Blockade and Helixor^® Therapy in Oncology: Real-World Tolerability and Subgroup Survival (ESMO GROW)

Point-by-point Response to Reviewer 1 Comments

Dear Reviewer, thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corrections highlighted in track changes in the re-submitted files. In addition, please note, that we expanded the flowchart of the study (figure 1). Furthermore, the introduction was improved (page 2, line 69-92). Please see all other point-by-point responses to your comments below.

With kind regards,

The authors

Comments 1: This article investigates very interesting topic. In order to improve the manuscript I suggest: The abstract should be shorter. According to journal guidelines it should be maximum 200 words long. This one is app 500 words.

Response 1: Thank you for this comment. We have shortened the abstract according to your suggestions, and it contains now 192 words, please see page 1 lines 17-55.

Comments 2: In the section 2.1 study design the author should better describe used abbreviations for the groups (CTRL? and COMB?).

Response 2: Thank you for this point. We have improved section 2.1, study design, and also the description of the abbreviations CRTL and COMB in the study design, see pages 3-4, lines 119-161.

Comments 3: In the section statistic, it should only be stated which statistical tests were used. Other information should be transferred to study design section.

Response 3: Agree. We have therefore moved other information than statistical tests to the study design section, as suggested. Please see pages 4-5, lines 189-209.

Comments 4: In the section results table 2 should be reorganized and the applied therapies should be listed by therapy line in which they were applied. Further the authors should add the information in which treatment line immunotherapy with or without HVA was applied for whole group. Further the authors should add the table with data about stage of diseases for NSCLC patients and therapy line in which immunotherapy with or without HVA was applied. Same data are needed for female patients with NSCLC. But it is unclear why the authors presented demographic data about whole group of patients and the survival analysis was done only for NSCLC patients. It is stated that the aim of the study was to evaluate the safety and efficacy of combining ICB with HVA therapy in a cohort of 405 oncological patients?! According to the presented results the title should also be modified.

Response 4: Thank you for this point. We have reorganized Table 2 as suggested (see page 8, line 287). Additionally, as recommended, we have included two new sections: one on NSCLC patients (Chapter 3.3) and another specifically addressing female NSCLC patients (Chapter 3.4), both with information on their anti-neoplastic treatment as well as on the disease stage, see page 8, lines 292-310 and page 9, lines 312-332. We have also added two tables detailing therapy lines for immunotherapies with or without HVA—see Table 3 (page 8) and Table 4 (page 9).. Furthermore, we have revised the title as suggested. The terms "tolerability and effectiveness" have been removed, as effectiveness applies only to NSCLC patients. The revised title now reads: Combined Immune Checkpoint Blockade and Helixor® Viscum album Therapy in Oncological Patients: Real-World Evidence in Line with ESMO GROW Guidelines.”

Comments 5: The section discussion should be improved

Response 5: Agree. We have, accordingly, modified the section discussion. Please see pages 13-14, lines 431-443 and lines 507-513.

Comments 6: Limitations should also be improved

Response 6: Thank you for this suggestion. We have, accordingly, improved the limitations, see page 15, lines 535-548.

Point-by-point Response to Reviewer 2 Comments

Dear Reviewer, thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corrections highlighted in track changes in the re-submitted files. In addition, please note, that we expanded the flowchart of the study (figure 1). Furthermore, the introduction was improved (page 2, line 69-92). Additionally, as recommended by reviewer 1, we have included two new sections: one on NSCLC patients (Chapter 3.3) and another specifically addressing female NSCLC patients (Chapter 3.4), both with information on their anti-neoplastic treatment as well as on their disease stages, see page 8, lines 292-305 and page 9, lines 312-328. We have also added two tables detailing therapy lines for immunotherapies with or without HVA—see Table 3 (page 8) and Table 4 (page 9). Please see all other point-by-point responses to your comments below.

With kind regards,

The authors

Comments 1: General Comments: This manuscript is an interesting study examining the use of Real-world data (RDW) in assessing multimodal therapies in oncology. The study is well done with convincing data and suggests a potential therapeutic benefit of the complementary therapy viscum album extract alongside immune checkpoint blockades. There are a few areas in which clarification would be helpful as well as some minor editorial changes that should be made to enhance the manuscript’s quality. Please see below for detailed comments:

Point-by-point Response to Reviewer 1 Comments

Dear Reviewer, thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corrections highlighted in track changes in the re-submitted files. Please see all other point-by-point responses to your comments below.

With kind regards,

The authors

Comment 1: I still do not understand why you named the group who did not received HVA CTRL?

Response 1: We designated the group receiving immune checkpoint inhibitors as the control (CTRL) group serving as the appropriate clinical benchmark against which we evaluated the potential additive or synergistic effects of Helixor Viscum album (HVA) therapy. The aim of our study was to assess whether the addition of HVA to an established therapeutic backbone (ICIs) could confer additional clinical benefits. Labeling the ICI-only group as “CTRL” therefore reflects its role as the reference treatment arm, consistent with conventions in clinical and translational research where the control group receives the current standard therapy.

Comment 2: In the section results the captions of the tables 3 and 4 are not correct.

Response 2: Thank you for pointing this out. We have reviewed and corrected the captions for Tables 3 and 4 to accurately reflect their contents. The updated captions are now consistent with the data presented and align with the text in the Results section.

Comment 4: Line 211- A higher proportion of patients in the CTRL group (71.6%) received 17.7% more first-line ICB compared to COMB (52.9%), and the difference was significant.

What 17.7% means?

Response 4: Thank you for your comment. The 17.7% refers to the absolute difference in the proportion of patients receiving first-line immune checkpoint blockade (ICB) therapy between the CTRL and COMB groups. We have revised the sentence for clarity as follows: “A significantly higher proportion of patients in the CTRL group (71.6%) received first-line immune checkpoint blockade (ICB) therapy compared to the COMB group (52.9%), representing a 17.7% absolute difference between the groups, p=0.03", see line 211-223.

Comment 5: it is unclear why the authors presented demographic data about whole group of patients and the survival analysis was done only for NSCLC patients. It is stated that the aim of the study was to evaluate the safety and efficacy of combining ICB with HVA therapy in a cohort of 405 oncological patients?! According to the presented results the title and the aim of the study still need to be modified.

Response 5: We appreciate the reviewer’s comments and the opportunity to clarify this point. The primary objective of our study was to assess the tolerability/safety of the combined ICB and HVA therapy, which was evaluated in the entire cohort of 405 oncological patients, as stated in the manuscript. Accordingly, demographic data were presented for the whole group to support the tolerability analysis, which applies to all included patients regardless of tumor type. The secondary objective—to explore efficacy—was performed in a subgroup of patients with NSCLC, as this subgroup had adequate follow-up and sample size to allow for meaningful survival analysis. We believe this approach is methodologically sound and appropriately reflects the hierarchy of study objectives. However, in order that the title accurately reflect its design and focus, we changed it to the following: “Combined Immune Checkpoint Blockade and Helixor® Viscum album Therapy in Oncology: Real-World Tolerability and Subgroup Survival (ESMO GROW)”.

Comment 6: In the discussion you should not repeat your results but you should compare them with the results of other studies.

Response 6: We thank the reviewer for this observation. However, we respectfully disagree with the comment. In the Discussion section, we have made a conscious effort to avoid merely repeating our results. Where results are mentioned, they are presented in the context of comparison with existing literature, see in detail below, in order to highlight similarities and differences, and to place our findings into a broader clinical and scientific framework. Specifically, we have compared our tolerability data and survival outcomes with those reported in prior studies involving ICB and HVA therapy, and have discussed how our findings align with or diverge from these, see below:

Lines 345-347: “The safety profile observed in this study, with fewer discontinuations of ICB therapy in the COMB group, aligns with literature highlighting the tolerability of add-on VA therapy when used alongside conventional cancer treatments (50, 51).”
Lines 347-353: “Reduced discontinuation rates for the PD-1 inhibitor pembrolizumab, in particular, suggest that HVA therapy might help manage adverse events, potentially improving adherence to immunotherapy. This aligns with findings from clinical and real-world data studies suggesting that VA therapy can reduce chemotherapy- and radiotherapy-induced toxicities (29, 30), reduce antibody-related toxicities (31) and does not increase ICB-associated toxicities (6,7,8,9). In addition, add-on VA helps to maintain adherence to standard oncological therapy (15).”
Lines 354-362: “Tumor representation differed between the two groups, with only 33% of tumor entities documented in both. This is because our data reflect tumor entities from the registry that received real-life ICB or combinational ICB/Helixor VA therapies. Additionally, our intention was to highlight gaps in the real-life utilization of ICB and combinational ICB/Helixor VA therapy, which could be addressed in the future. Furthermore, younger patients were more frequently represented in the COMB group, suggesting a trend toward younger patients opting for complementary therapies. This aligns with previous findings that younger patients tend to integrate these therapies while adhering to guideline-based standard oncological treatments (56).”
Lines 367-377: “The findings of this RWD study suggest a positive association of the combinational ICB plus Helixor^® VA therapy with improved overall survival in females with NSCLC. The hazard rate and the restricted mean survival time based measures were in agreement regarding the statistical significance of the effect. The adjusted survival regression analysis balancing confounding effects revealed a significant effect of the combinational therapy on the adjusted hazard of death in female lung cancer patients. This effect was independent of age, stage, treatment line, or non-ICB neoplastic treatment. Interestingly, the 5-year survival rate in these patients increased more than fourfold when Helixor^® VA was added to ICB. Recent RWD studies of our group have already demonstrated that combining immune checkpoint inhibitors with VA therapy was linked to enhanced overall survival outcomes in NSCLC (4,5).”
Lines 377-388: “The significant improvement of the adjusted hazard of death observed in the female lung cancer subgroup adds an interesting dimension to our analysis so far. Several studies have suggested that gender plays a role in the response to immune checkpoint inhibitors (32, 33, 34). For example, a study published in 2020 found gender differences in response to immune checkpoint inhibitors also in NSCLC patients (32). A systematic review and meta-analysis on the other hand did not find a significant difference between male or female NSCLC patients when treated with PD-1/PD-L1 checkpoint inhibitors (33). Another study published in 2022 in the journal Frontiers in Immunology observed different expression patterns of PD-1 between male and female gender in NSCLC (34). Thus, scientific results are inconsistent so far and the reason behind the potential gender disparity in immune checkpoint blockade response is not yet fully understood.”

Comment: Lines 373-381- you just mentioned some molecular pathways, but you did not perform that analysis. Thus you should move this part to the introduction, but with more details.

Comment: Lines 382-394- also should be moved to the introduction.

Response: We respectfully disagree. The molecular mechanisms mentioned were not intended to represent novel findings from our own analyses but were included to contextualize and support our interpretation of potential gender-specific effects observed in our results. These mechanisms are discussed to underscore the plausibility of gender differences based on existing literature and biological pathways. Therefore, we believe it is appropriate to retain this discussion in the Discussion section, where we interpret our findings and integrate them with known mechanisms. In addition, it was made clear for every statement that these molecular pathways were discovered by other groups. Please see here: Line 389-396: “Several genes and molecular pathways have been implicated in gender-related differences in immune responses and PD-L1 expression such as estrogen-related, sex hormone-related, immune-related, oncogene and tumor suppression related molecular pathways (35, 36). Genetic alterations of these genes may influence the expression of PD-L1 and immune checkpoint blockade (35, 37). While gender-specific responses to both immunotherapy and complementary treatments have been explored in the literature (52, 53), more evidence is needed to elucidate whether female patients derive greater benefit from combinational approaches involving VA therapy.”

Comment: Same comment for the next paragraph.

Response: Thank you for your suggestion. We have moved the following text, as suggested, to the introduction, see line 56-62, as it rather describes the overall potential of VA therapy, which may not be directly connected to our findings: “Findings suggest the potential of VA therapy in improving patient prognosis across various settings (11, 29, 38, 39, 29). However, outcomes may be different between tumor types, i.e. immune-modulators such as PD-1 inhibitors or VA may be more effective in cancers like NSCLC because these tumors often have a higher mutational burden, producing more neo-antigens that can trigger an immune response (42). In contrast, pancreatic cancer tends to have a lower mutational burden and a dense, immunosuppressive TME, which may limit effectiveness of PD-1 inhibitors (43) and VA (44).”

Comment: In the discussion you should compare your results with the results of other studies!

Response: Thank you for your comment, which is the same as your comment 6. Please see our response 6.

Comment 7: The conclusion in the abstract is not in accordance with the results.

Response 7: Thank you for your comment. We have adapted the conclusion in the abstract and also in the manuscript text at the end. Please see abstract, lines 32-33 and in the manuscript text, lines 450-460.

Comment You cannot state suggest a potential gender-specific survival benefit since you did not perform analysis for male patients

Response: Thank you for your feedback. We respectfully disagree with this suggestion. In our analysis, we did not observe these changes in male patients. To support our statements, we have now included supplementary data. Additionally, we have updated the results section and the discussion section to include the following: 'This effect was not observed in male NSCLC patients, as shown in Supplementary Figure 2 and Supplementary Table 3.”, see lines 300-301 and line 378.

Point-by-point Response to Reviewer 1 Comments

Dear Reviewer,

thank you very much for taking the time to review this manuscript. Please find our response below and the corrections highlighted in track changes in the re-submitted file (word file).

With kind regards,

The authors

Comment 1: Lines 82-85- It is stated that the aim of the study was to evaluate the safety and efficacy of combining ICB with HVA therapy in a cohort of 405 oncological patients?! Please clarify that the safety was investigated in the whole group and the efficacy in 312 NSCLC patients. 

Response 1: Thank you for your helpful comment. We have revised the sentence accordingly and it reads now: “Our study utilized real-world registry data to evaluate the safety of combining ICB with HVA therapy in a cohort of 405 oncological patients and the efficacy of this combination in a subgroup of 312 patients with non-small cell lung cancer (NSCLC).” Please refer to lines 80-81 in the marked-up text (word file).