Tertiary Lymphoid Structures as Mediators of Immunotherapy Response
Round 1
Reviewer 1 Report
The topic of TLS is one of the most important issues in cancer immunotherapy, and it is also attracting attention as a biomarker in the latest clinical trials. It has a charm that attracts the reader's interest. This reviewer's critisisim can be summarized in the following three points.
1) TLS heterogeneity: TLS has various morphological characteristics due to its maturity, such as lymphocyte aggregation, early-TLS, primary/seconadry folicle like TLS, in the process of its formation, but it is not recognized well by clinicians and basic researchers; thereby it is indispensable information to clarify this non-uniformity of TLS when discussing TLSs.
2) As immunosensitive tumors, the topic of urogenital cancer (e.g., kidney cancer and urothelial cancer) is indispensable. A new heading should be created to deepen the discussion on these gentiourinary cancers associated with TLSs.
3) It is useful information for the reader to clarify the relationship between TLS and the prognosis in pan-cancer as much as possible at this stage (if possible, it is more attractive to describe it in meta-analysis etc.).
Author Response
Dear Editors and Reviewers;
Your time, consideration, and feedback are overwhelmingly appreciated. Attached is a revised draft that includes the editorial changes requested (Abstract, Simple Summary, Reference Formats). Also please find below the responses to the specific comments made by the reviewers.
Sincerely,
Joseph J. Skitzki MD, FACS
Associate Professor of Oncology
Department of Surgical Oncology
Department of Immunology
Roswell Park Cancer Institute
Reviewer 1
Comment 1: TLS heterogeneity: TLS has various morphological characteristics due to its maturity, such as lymphocyte aggregation, early-TLS, primary/secondary follicle like TLS, in the process of its formation, but it is not recognized well by clinicians and basic researchers; thereby it is indispensable information to clarify this non-uniformity of TLS when discussing TLSs.
Authors response: The following lines were added to the manuscript-
“Despite these characteristic features, the metrics used to identify TLS within tissue samples is variable. This can lead to the obfuscation of clinical applicability while also making mechanistic investigations difficult to compare. Much like SLO organogenesis, TLS develop on a spectrum and can be identified at various stages of maturation (i.e. lymphoid aggregates or aggregates with germinal centers). Beyond this structural heterogeneity there are also factors such as immune cell composition, the presence of HEVs, and numerous gene signatures that are variably used to identify the presence of TLS. The forthcoming summary of TLS within specific malignancies highlights the importance of recognizing the heterogeneity of TLS as it has been applied clinically thus far.”
Comment 2: As immunosensitive tumors, the topic of urogenital cancer (e.g., kidney cancer and urothelial cancer) is indispensable. A new heading should be created to deepen the discussion on these gentiourinary cancers associated with TLSs.
Authors response: The following lines were added to the manuscript-
“TLS and urothelial cancer:
Urothelial bladder cancer has had a long-standing connection with immunology beginning with the treatment of malignancy with Bacillus Calmette-Guerin (BCG). Additionally, there has been a focus on the immune infiltration patterns of bladder cancers, especially given the recognition that low grade variants and high-grade variants may be distinct clinical entities with significant impact on patient prognosis. In an early study examining immune cell infiltration in a cohort of both high grade (muscle invasive bladder cancer, MIBC) and low-grade bladder cancer (non-muscle invasive bladder cancer, NMIBC), TLS was only present in 25% of NMIBC cases while it was present in 75% of MIBC cases [32]. And although specific patient outcomes was not examined in this study, the relationship of TLS and prognosis may be more nuanced. In a study of patients with locoregionally advanced urothelial cancer who underwent treatment with ipilimumab and nivolumab, pretreatment samples revealed that clinical non-responders tended to enrich immature TLS more than responders [33]. Alternatively, another trial looking at pretreatment samples of urothelial carcinoma patients who received neoadjuvant durvalumab (anti PD-L1) and tremelimumab (anti CTLA-4) found that responders tended to have higher TLS [34]. In an exploration of these dialectic findings, data indicates that the specific cellular composition of TLS (defined as distinct clusters) differed among immunotherapy responders versus non-responders (FoxP3 T-cell low TLS) and that additionally, post treatment samples demonstrated cluster changes based on exposure to immunoactive agents (more TLS that were macrophage low in treated versus untreated) [35]. The immense diversity in TLS characteristics described in bladder cancer highlights the importance of recognizing TLS heterogeneity as it relates to patient prognosis.”
Comment 3: It is useful information for the reader to clarify the relationship between TLS and the prognosis in pan-cancer as much as possible at this stage (if possible, it is more attractive to describe it in meta-analysis etc.).
Authors response: One of the purposes of reviewing the different cancer types and the role of
TLS was to expound on the extraordinarily nuanced role that TLS play. The location, maturity, and composition of TLS as well as the specific malignancy appear to drive prognosis in different ways. In aggregate, the is no simple way to clarify the relationship between TLS and pan-cancer and other publications which have sought to do so have reverted to analyzing TLS by disease site due to the aforementioned complexity.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors provide a concise review of the role TLS in cancer. The paper is well written and balanced.
Following the title, which refers specifically to immunotherapy, the authors should discuss more in depth the studies evaluating TLS as predictive/prognostic factors in cancer patients undergoing immunotherapy with ICIs. A table summarizing the results of the (few) published clinical studies on this specific issue should be useful.
Author Response
Dear Editors and Reviewers;
Your time, consideration, and feedback are overwhelmingly appreciated. Attached is a revised draft that includes the editorial changes requested (Abstract, Simple Summary, Reference Formats). Also please find below the responses to the specific comments made by the reviewers.
Sincerely,
Joseph J. Skitzki MD, FACS
Associate Professor of Oncology
Department of Surgical Oncology
Department of Immunology
Roswell Park Cancer Institute
Reviewer 2
Comment 1: Following the title, which refers specifically to immunotherapy, the authors should discuss more in depth the studies evaluating TLS as predictive/prognostic factors in cancer patients undergoing immunotherapy with ICIs. A table summarizing the results of the (few) published clinical studies on this specific issue should be useful.
Authors response: We attempted to organize the current review of the existing literature by disease site with discussion of immunotherapy with ICI within each section. As the literature is extremely heterogeneous in terms of ICI, disease sites treated, and detection and evaluation of TLS a summary table would be difficult to generate. This is an excellent comment though and speaks to the need for a more systematic approach in detecting and reporting TLS in future immunotherapy studies.
Author Response File: Author Response.docx
Reviewer 3 Report
TLS are potential biomarkers of patient prognosis and response to IO treatments. In addition to utilizing TLS as a therapeutic intervention to potentiate the anti-tumoral immunity, this review discussed the functional and prognostic relevance of TLS in some solid tumor types.
Minor revisions:
Figure 1: It would be relevant to add explanation in the text regarding mature versus immature TLS phenotypes.
line 178: this is not a correct statement, TLS could be found within normal alveolar epithelial areas (PMIDs: 20864612, 28002730).
line 188-190: please add reference about the survival benefit of TLS/B-cell level regardless of the treatment modalities.
Figure 2: Neither ref 50 nor 51 explained about TLS induction by implanting TLS into local tumor microenvironment. The authors should elaborate in detail about this technology – implantable TLS – and cite original works.
Author Response
Reviewer 3
Comment 1: According the literature (10.1172/JCI71611 and 10.1016/j.autrev.2021.102980) the vessels in TLS divide to lymphatic and blood vessels. It seems that the reviewer excluded the role of lymphatic vessels in structure and development of TLS and its role in different cancer types? It would be necessary to write a short introduction and address the role of lymphatic vessels in each cancer types.
Authors response: We fully agree that a discussion of lymphatic vessels would be relevant in the setting of TLS in cancer; but, to date, there is almost no published data examining true lymphatic vessels in cancer TLS. There are however several investigations of lymphatic vessels associated with TLS in non-malignant inflammatory conditions (as described in the second citation 10.1016/j.autrev.2021.102980). The first citation (10.1172/JCI71611) primarily focuses on lymphatic vessels in inflammatory conditions and makes a single reference to lymphatics in a malignant setting of breast cancer (J Immunol. 2013;191(4):2001–2008.); however, the referenced article only discusses HEV-like vessels and not lymphatic vessels. A more detailed review of the literature does not reveal any additional studies that examined lymphatic vessels in association with TLS of cancer.
Comment 2: Figure number 1, the structure of TLS is not illustrated completely and properly. Highly recommend that the authors revise it.
Authors response: Please find embedded in the text a revised set of figures.
Comment 3: Figure number 2 is mainly based on ref. nr. 52. It was written in this reference: “by complete TLS manufacture ex vivo prior to adoptive transfer/retransfer” and was referred to figure:1. But not only in Fig.1 but also none of the mentioned references could be tracked how the complete TLS platform will be implanted. The author should suggest a high quality and solid reference or revise the induction method of TLS.
Authors response: Please find embedded in the text a revised set of figures.
Author Response File: Author Response.docx
Reviewer 4 Report
The authors presented a comprehensive minireview about the role of Tertiary Lymphoid Structure (TLS) in different cancer types. They emphasized TLS as a prognostic and survival factor in cancer patients. However, there are several issues that require to be addressed.
1- According the literature (10.1172/JCI71611 and 10.1016/j.autrev.2021.102980) the vessels in TLS divide to lymphatic and blood vessels. It seems that the reviewer excluded the role of lymphatic vessels in structure and development of TLS and its role in different cancer types? It would be necessary to write a short introduction and address the role of lymphatic vessels in each cancer types.
2- Figure number 1, the structure of TLS is not illustrated completely and properly. Highly recommend that the authors revise it.
3- Figure number 2 is mainly based on ref. nr. 52. It was written in this reference: “by complete TLS manufacture ex vivo prior to adoptive transfer/retransfer” and was referred to figure:1. But not only in Fig.1 but also none of the mentioned references could be tracked how the complete TLS platform will be implanted. The author should suggest a high quality and solid reference or revise the induction method of TLS.
Author Response
Reviewer 4
Comment 1: Figure 1: It would be relevant to add explanation in the text regarding mature versus immature TLS phenotypes.
Authors response: Please find embedded in the text a revised set of figures. Also, new lines were added highlighting the range of TLS reported in the literature including maturity.
“…Much like SLO organogenesis, TLS develop on a spectrum and can be identified at various stages of maturation (i.e., lymphoid aggregates or aggregates with germinal centers)…”
Comment 2: Line 178: this is not a correct statement, TLS could be found within normal alveolar epithelial areas (PMIDs: 20864612, 28002730).
Authors response: The included references are much appreciated. The distinction of TLS in lung cancer is particularly nuanced as the lungs are a natural site of inflammation and immune response and thus TLS has been reported in both non-cancer and cancerous settings within “normal” lung parenchyma. The manuscript text has been revised to remove the implication of the non-existence of TLS in normal parenchyma.
Comment 3: line 188-190: please add reference about the survival benefit of TLS/B-cell level regardless of the treatment modalities.
Authors response: The reference has been updated.
Comment 4: Figure 2: Neither ref 50 nor 51 explained about TLS induction by implanting TLS into local tumor microenvironment. The authors should elaborate in detail about this technology – implantable TLS – and cite original works.
Authors response: Please find embedded in the text a revised set of figures.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
None.
Reviewer 2 Report
Following the reviewers' suggestions, the authors have improved the quality of the manuscript. I still think that a Table summarizing the data on TLS in studies with ICIs on cancer patients should have been useful to the readers, but I agree that it is not mandatory.
