Limited Clinical Efficacy with Potential Adverse Events in a Pilot Study of Autologous Adoptive Cell Therapy in Canine Oral Malignant Melanoma

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Thank you for the revised manuscript and response to my questions. The manuscript is improved and many parts now have a much better explanation and the results are completely understandable. Thank also for a very clear M&M-section. I have no further questions.

Author Response

Thank you so much for your warm encouragement and all your precious suggestions!

Reviewer 2 Report (Previous Reviewer 1)

The authors adequately responded to my comments.

Author Response

Many thanks for your invaluable opinions and advice that helped us improve our manuscript!

Reviewer 3 Report (New Reviewer)

The authors of this article YuanYuan Xia et al. present a pilot clinical trial in dogs with malignant oral melanoma.

It seems to me that the study is well structured, however, as the authors mention, it has many limitations, such as the sample size and the heterogeneity of characteristics among the ten dogs that represent the sample under study.

It is understandable that this is a pilot trial, which involves a smaller sample, however, the heterogeneity of the sample makes it difficult to evaluate and analyze the results. There are animals with lesions at different stages, therefore associated with different prognoses, which will certainly respond differently to therapy.

In conclusion, this article needs to be improved, essentially with regard to the sample under study, so that the results can be improved.

Author Response

Thank you for your suggestion. We totally understand your concern that the study scale was too small to draw a conclusion about treatment response and outcome. As you mentioned, this was only a pilot study, and we would like to assess the treatment safety as our first endpoint, while the outcome was the second. Honestly, it was not easy for us to recruit many patients, and after seeing our results, we think we have to modify our treatment protocol first to prevent the treatment-related anaphylaxis as possible as we can, and this is also a point that we would like to report in our manuscript. Actually, we do have future plans to recruit a group of oral melanoma dogs with more homogeneous characteristics, for example, dogs with stage I/II oral melanoma and receive the cell therapy as an adjuvant treatment post-surgery, or even combine the cell therapy with other treatments. Therefore, we are not planning to recruit more dogs into the current project but want to report the pilot result first, and conduct further specified trials with a better design. We do consider your advice important and appreciate your help in improving our paper quality, so we have modified our manuscript by incorporating your concern and our future plan into the discussion section and adjusting the abstract mildly. Please refer to page 14, lines 536-542.

Round 2

Reviewer 3 Report (New Reviewer)

Thank you for responding to my comments.

I think that the fact that the sample is small, since it is a pilot trial, is not the main problem. The heterogeneity of the sample seems much more worrying to me as it makes it difficult to analyze the results.

Author Response

Thank you for discussing this issue with us. We agree that the population in our study was heterogeneous that it is impossible to have specific outcome or prognostic information in one specific group. However, as the study was initially designed to evaluate treatment toxicity and the outcome was not our major aim, considering that the patient recruitment process was not very easy, the dogs were not limited to a specific tumor status. Thus, for outcome analysis, we just hope to provide as much information as we can.

But we totally understand your concern, we tried to only analyze patients in a later clinical stage (stage III/IV), or patients with macroscopic disease before treatment, but this will exclude two out of three patients who had suspected treatment-related anaphylaxis, which we would like to report. Therefore, to incorporate your advice, we modified our manuscript to make the outcome analysis to be more like supplemental descriptive information rather than an informative conclusion. We adjusted the prognosis part in our main text by removing the univariable analysis table to supplementary data and simplifying the result and discussion, and just some points in the discussion that were necessary to describe or were advised by other reviewers were kept. We invite you to refer to lines 362-374 and 487-509, for the revised paragraphs.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This article suggested the limitations and improvements of the adoptive cell therapy for canine oral melanoma, which had not been objectively indicated for a long time, although novelty was relatively low. Therefore, I considered that this article was worth publishing as a pilot study. Please revise some minor points.

1. Please add the references regarding anti-PD-1 antibody against canine cancers. Such as Camerino M, et al. Vet Comp Oncol. 2022.

2. Why was bone invasion added in Table 1? Please indicate the rationale with references, e.g. Noguchi S et al. Vet Sci 2022. 

3. What was the difference between debulking and partial debulking in Table 2?

In line 437 and 439, 'however' was repeated in consecutive two sentences.

Author Response

Thank you for taking the time to review our article.

Please see the attachment for the point-by-point response.

Author Response File: Author Response.docx

Reviewer 2 Report

Although interesting, this research is premature and has several technical drawbacks that make it difficult to publish immediately. In particular deeper phenotypic and functional in vitro characteristics of expanded cells has to be provided.

Major concerns:

1. PBMC expansion has to be better characterized and quantified. How uniform it was between different dogs? 

2. Deeper characteristics of the immunophenotype (i.e. activation markers, CD16 expression, degranulation marker CD107a) of expanded cells has to be determined and shown in a separate figure

4. Puromycin has to be withdrawn from the conditioned medium because it may affect expansions of PBMCs

5. In vitro cytotoxic capacity of expanded cells to kill canine melanoma cells has to be provided

Reviewer 3 Report

The authors present a clinical trial in 10 dogs with malignant melanoma of different stages and anatomical locations. Some cases were pre-treated with other immunotherapies and some received cyto reductive surgery before adoptive cell therapy (ACT), containing autologous cell transfusion of predominantly non-B non-T cells via in vitro stimulations of IL-15, IL-2 and 30 IL-21. Three dogs experienced anaphylaxis during later stages of the treatment protocol (one 30 min infusion weekly q4w). The response rate in the group varied widely, as expected by the wide range of stages included. The authors concluded that there is a need for protocol refinement and that the toxicity currently was not acceptable compared to the (minimal) overall response reported in this cohort of dogs.

While the study is interesting there are some comments I would like the authors to address before potential re-submission.

General:

This report describe the same type of results as Dr Steven Rosenberg did before he developed/detected the potential with TILs in human melanoma patients S.A. Rosenberg et al., N Engl J Med. 1988 Dec 22;319(25):1676-80. Even if the authors briefly mention TILs in the introduction, the authors should expand this more (intro and/or discussion) and acknowledge the likely same path forward as was described in humans with this type of ACT, before switching to TILs.

Specific:

To be even more contemporary regarding the discussion of PD-1 inhibitors in dogs the current reference should be added and possibly discussed: S. Yshimoto et al., MABS. 2023, VOL. 15, NO. 1, 2287250.

The finding in Table 3 that dogs experience PD had a significantly longer OS should be better mentioned in the text (also in the result as well as expended in the discussion). Otherwise, the fast reader may think that this is an error in the table.

It is recommended to more specific sort on CD5. This is described in a recent publication by E.M.V. Forsberg et al., Cancers (Basel). 2023 Jan 20;15(3):648. Either the authors can perform this experimentally, or likely in the current situation, comment this possibility in the discussion.

P 11 L 425: Please change CAT-T to CAR-T, or explain what CAT-T is.

P 11 LL 435: The significant finding of PD having longer OS should be further explained and the group with progressive dissse better described. Possibly the (failed?) pre-treatment immunotherapies could be explained in further detailed and the potential for the underlying immune activation of these could have potentiated the ACT described in the current manuscript.

I am not native English, but detected some minor edits needed, particularly regarding past tense. Before re-submission the text is recommended to be proof read.