Identification of Age-Associated Transcriptomic Changes Linked to Immunotherapy Response in Primary Melanoma
Round 1
Reviewer 1 Report
Very interesting topics and well design analysis however very very small cohorts used.
The discussion should be improve since actually only resume of the results which is not appropriate for a discussion . You should look at the litterature and mention what was already analyse in such type of analysis on the impact of ageing on transcriptional changes during melanoma and on drug resistance.
Author Response
Response to comments:
Reviewer #1
“Very interesting topics and well design analysis however very small cohorts used. The discussion should be improve since actually only resume of the results which is not appropriate for a discussion. You should look at the litterature and mention what was already analyse in such type of analysis on the impact of ageing on transcriptional changes during melanoma and on drug resistance”.
Thank you for the accurate revision of the manuscript, valuable comments and suggestions regarding the cohort size and the discussion. The small cohort size is indeed limited by the number of patients in the TCGA database that receive immunotherapeutic agents. One factor that have contributed to the sample size is the fact that we aimed to include patients with the most significant difference in their ages, so this limited us to a few old and few young patients (This has been referred to in the conclusion, lines 359-363). We do hope to expand this in future studies and include more patients from different age groups and make wider comparisons.
Regarding the discussion, your comment is indeed pithy and valuable. The discussion has now been improved to include comparative analysis between the current results and the previously published results in the same area. The modifications are highlighted in yellow and included in the following lines
Lines 328-332 “MGST1 plays a critical role in inflammation, overexpressed in cancer and correlates with drug resistance [66]. In our analysis, MGST1 was remarkably upregulated in the young cohort. Prall et. al. reported that MGST1 expression is age-dependent and Zeng et. al. reported overexpression of MGST1 in high-risk melanoma patients”
Lines 333-339 “This is consistent with previous studies that reported significant changes in skin extra-cellular matrix during aging such as collagen loss [69]. Miskolczi et. al. also reported that melanoma cell adhesion and nuclear YAP localization are regulated by the mechanical properties of collagen. Collagen stiffness induced the expression of melanoma differentia-tion genes TRPM1, PMEL, TYR and MLANA as well as well as proliferation and survival genes CDK2 and BCL2A1”
Lines 345-347 “Indeed, Mehta et. al. have reported that inflammation-induced dedifferentiation of melanoma cells contributes to poor survival and resistance to immunotherapy in aged melanoma patients”
Lines 321-357 “These results are consistent with previous results indicating that Wnt5A expression in-creases in old (55-65 years old) melanoma patients compared to young (25-35 years old) patients [73]. Behera at. al. also reported that melanoma resistance to vemurafenib is driven by Wnt5A and PPARγ activation with rosiglitazone upregulated the age-related protein klotho, a Fibroblast Growth Factor-23 (FGF23) receptor, and decreased Wnt5A ex-pression in therapy-resistant old melanoma patients thus reducing tumor burden”
Lines 376-379 “Consistent with our transcriptomic analysis, Wu et. al. reported that aging strongly impacts several biological pathways in cancer including epithelial–mesenchymal transition (EMT), metabolism, KRAS signaling, inflammatory response, glycolysis and Il2-stat5 signaling
Reviewer 2 Report
This is a very interesting and important comparison between the effect of immunotherapy of skin melanoma between young and old patients.This paper may help at understanding the mechanisms of melanoma development, and vulnerability of the tumor to the terapy targeting the PD1/PD1-L and CTLA-4 expression of lymphocytes T undergoing immunological suppression in normally developing tumors. There are several doubts and mistakes, the enlightment of which may improve on the quality of the paper:
1. BRAF and NRAS in the mutated form appear often in some types of melanoma, but they are not necessary to transformation, and their mutations, even the most frequent ones V600K, V600E or V600R do not always and inevitably lead to the transformation. They just are found in the transformed melanocytes, so I would suggest "weakening" of this statement where it has been mentioned. The more important are mutations in genes regulating proliferation, telomerase (as you mentioned) and apoptosis. See e.g. Sini et al, 2018, doi: 10.18632/oncotarget.23989, Davis et al., 2019, Cancer, doi: 10.1002/cncr.31345. In the ocular melanoma completely different scheme of mutations leads to the progression. By the way - was such a comparison carried out? This topic deserves a separate study. See recent Markiewicz et al, Cancers 2022, https://doi.org/10.3390/cancers14112753
2. As activation of pro-inflammatory pathways may indirectly lead to the increase in the activity of IL-1beta, is something known about the increase in the expression or activity of NOS-2? This is a phenomenon associated with pyroptosis,, were there other factors of pyroptosis affected in parallel, eg. caspase-1 expression?
3. Affecting glycosylation via GlcNAc (the actual residue is Glc3Man9GlcNAc2 (G3M9) at N371 of the protein structure) may strongly affect the enzymatic activity of tyrosinase, and its antigenic activity, thus, the invasiveness and pigmentation of the tumor. Was it checked? See Garcia-Borrón & Solano, 2002 doi: 10.1034/j.1600-0749.2002.02012.x.
4. page 9 - in my copy something wrong is with hyphenation.
Author Response
Response to comments:
Reviewer #2
This is a very interesting and important comparison between the effect of immunotherapy of skin melanoma between young and old patients. This paper may help at understanding the mechanisms of melanoma development, and vulnerability of the tumor to the terapy targeting the PD1/PD1-L and CTLA-4 expression of lymphocytes T undergoing immunological suppression in normally developing tumors. There are several doubts and mistakes, the enlightment of which may improve on the quality of the paper:
Thank you very much for the positive and insightful comments about our work.
1. BRAF and NRAS in the mutated form appear often in some types of melanoma, but they are not necessary to transformation, and their mutations, even the most frequent ones V600K, V600E or V600R do not always and inevitably lead to the transformation. They just are found in the transformed melanocytes, so I would suggest "weakening" of this statement where it has been mentioned. The more important are mutations in genes regulating proliferation, telomerase (as you mentioned) and apoptosis. See e.g. Sini et al, 2018, doi: 10.18632/oncotarget.23989, Davis et al., 2019, Cancer, doi: 10.1002/cncr.31345. In the ocular melanoma completely different scheme of mutations leads to the progression. By the way - was such a comparison carried out? This topic deserves a separate study. See recent Markiewicz et al, Cancers 2022, https://doi.org/10.3390/cancers14112753
Thank you for this great point. Indeed, you are right regarding melanoma driver gene mutations. The following sentence has been removed from abstract, lines 12-13 “that develops due to constitutive activation of MAPK signaling pathway driven by BRAF and NRAS mutations”
The following sentence was added in the introduction, lines 34-35 “Melanoma develops due to driver mutations in 48 core melanoma genes”
2. As activation of pro-inflammatory pathways may indirectly lead to the increase in the activity of IL-1beta, is something known about the increase in the expression or activity of NOS-2? This is a phenomenon associated with pyroptosis,, were there other factors of pyroptosis affected in parallel, eg. caspase-1 expression?
Thank you for this interesting point. We have checked RNA-Seq data and found that NOS2 expression is 1.3 fold higher in the young cohort. Please see the figure in the attachment.
3. Affecting glycosylation via GlcNAc (the actual residue is Glc3Man9GlcNAc2 (G3M9) at N371 of the protein structure) may strongly affect the enzymatic activity of tyrosinase, and its antigenic activity, thus, the invasiveness and pigmentation of the tumor. Was it checked? See Garcia-Borrón & Solano, 2002 doi: 10.1034/j.1600-0749.2002.02012.x.
Thank you for this interesting point. Based on RNA-Seq data, there is no difference in TYR expression between young and old groups, however, it is very likely that enzyme activity is different as you suggested. We aim to expand the study in the future and check tyrosinase enzyme activity.
4. page 9 - in my copy something wrong is with hyphenation.
We apologize for this inconvenience.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
the discussion is now complete and well done. I agree for publication
