Human Germ Cell Tumors are Developmental Cancers: Impact of Epigenetics on Pathobiology and Clinic

Round  1

Reviewer 1 Report

The review article titled “The pathobiological and clinical impact of epigenetics on human germ cell tumors as a developmental cancer”, presents a unique perspective on the underlying cause of human germ cell tumors (GCTs), with emphasis on testicular GCT (TGCT). Specifically, authors argue that metastatic GCTs arise from an interplay between epigenetic (e.g., imprinting), developmental, and environmental factors, and not attributed to somatic abnormalities. They have meticulously elaborated on genetic factors, like, deregulated parental genetic imprinting, defects in germline programming, and developmental abnormalities in gonadal locale/maturation, as critical mediators in tumor causation. In addition, the review also details the possible utilization of two epigenetic factors – methylation patterns and miRNAs as possible biomarkers. However, there are a few issues with the manuscript as mentioned below.

Major Comments

1.      The ‘Genvironmental’ Model (section 1.2) should be clearly described under genetic and environmental factors separately first (under distinct sub-heading), and then as an interplay between the two. In addition, the environmental factors need to categorized clearly into external environment (postnatal factors like exposure to diet, physical exercise, marijuana etc.), vs. internal developmental/medical factors, like, cryptorchidism. This section is presented in a very confusing manner.

2.      Section (3.2) detailing the utilization of methylation patterns and miRNAs as possible biomarkers should additionally be represented in a tabular format, for convenience.

Minor Comments

1.      The title of the manuscript is confusing. It needs to be clear. Similarly, “a developmental cancer” in the title for section 2 is confusing.

2.      All the abbreviations used in the main manuscript should be described in a tabular format separately.

3.      Individual rows in Table 1 should be separated with a line in between.

4.      The abbreviation GCNIS first appears in Line# 117. However, its is described later in Line# 146. It should be described earlier.

5.      The epidemiology statistics (Section 1.1) should additionally be presented in a tabular format.

6.      The text in Figure 3 is not legible enough due to the miRNA watermark.

7.      Line# 227 – “In practice, this unifying model for GCTs…..”. Replace "means they can be" by "enables them to be".

8.      Line# 254 – replace the word “progress” with a suitable term or rephrase.

9.      Line# 539 – provide appropriate reference9s) for the histological evaluation of TEs.

Author Response

Reviewers' comments:

Reviewer #1:

The review article titled “The pathobiological and clinical impact of epigenetics on human germ cell tumors as a developmental cancer”, presents a unique perspective on the underlying cause of human germ cell tumors (GCTs), with emphasis on testicular GCT (TGCT). Specifically, authors argue that metastatic GCTs arise from an interplay between epigenetic (e.g., imprinting), developmental, and environmental factors, and not attributed to somatic abnormalities. They have meticulously elaborated on genetic factors, like, deregulated parental genetic imprinting, defects in germline programming, and developmental abnormalities in gonadal locale/maturation, as critical mediators in tumor causation. In addition, the review also details the possible utilization of two epigenetic factors – methylation patterns and miRNAs as possible biomarkers.

However, there are a few issues with the manuscript as mentioned below.

Major Comments

1.      The ‘Genvironmental’ Model (section 1.2) should be clearly described under genetic and environmental factors separately first (under distinct sub-heading), and then as an interplay between the two. In addition, the environmental factors need to be categorized clearly into external environment (postnatal factors like exposure to diet, physical exercise, marijuana etc.), vs. internal developmental/medical factors, like, cryptorchidism. This section is presented in a very confusing manner.

Reply:  Based on the well appreciated suggestion of this reviewer, we subdivided this section into three subsections, first dealing with genetic factors, second with environmental factors (including both external and internal), followed by an integrated section combining all. We strongly agree that this improved the readability of this part of the review.

2.      Section (3.2) detailing the utilization of methylation patterns and miRNAs as possible biomarkers should additionally be represented in a tabular format, for convenience.

Reply: We thank the Reviewer for his/her suggestion, which was also mentioned by Reviewer #2. We have added Table 4 summarizing these aspects discussed in the text (see below in the reply to Reviewer #2).

Minor Comments

1.      The title of the manuscript is confusing. It needs to be clear. Similarly, “a developmental cancer” in the title for section 2 is confusing.

Reply: The original title “The pathobiological and clinical impact of epigenetics on human germ cell tumors as a developmental cancer” has been adjusted to “Human germ cell tumors are developmental cancers: impact of epigenetics on pathobiology and clinic.”

Also, the title of section 2 was adjusted to “Pathobiology of Germ Cell Tumors and their developmental potential”.

2.      All the abbreviations used in the main manuscript should be described in a tabular format separately.

Reply: We thank the Reviewer for the suggestion. It has been added at the end of the Manuscript.

3.      Individual rows in Table 1 should be separated with a line in between.

Reply: We thank the Reviewer for his/her suggestion. It has been changed.

4.      The abbreviation GCNIS first appears in Line# 117. However, its is described later in Line# 146. It should be described earlier.

Reply: It has been corrected, accordingly.

5.      The epidemiology statistics (Section 1.1) should additionally be presented in a tabular format.

Reply: We thank the Reviewer for his/her suggestion, which will make the paper easier to read. We have added Table 1, accordingly. We decided to summarize on the Table only the figures concerning incidence, prevalence and mortality, so not to repeat every figure stated in the text right beneath it.

Table 1. Epidemiology of germ cell tumors: incidence, prevalence and mortality data

6.      The text in Figure 3 is not legible enough due to the miRNA watermark.

Reply: We thank the Reviewer for calling our attention to this matter. It has been corrected by turning the miR watermark more transparent.

7.      Line# 227 – “In practice, this unifying model for GCTs…..”. Replace "means they can be" by "enables them to be".

Reply: It has been corrected in accordance.

8.      Line# 254 – replace the word “progress” with a suitable term or rephrase.

Reply: We have rephrased accordingly: “will become invasive tumors”.

9.      Line# 539 – provide appropriate references for the histological evaluation of TEs.

Reply: We thank the Reviewer for this suggestion. Proper references were added.

Reviewer 2 Report

This is an interesting article dealing with the contribution of epigenetic modifications to human germ cell tumors. There are however several sections of the manuscript that could be improved. The conclusions are really short and merely speculative: the authors must provide a more integrated overview of the described points.

Major points:

1) Section 1.2 The genvironmental model: authors should better explain the contribution of genetic and environmental factors related to TGCTs. They mention diet, physical exercise or environmental exposure as potential risk factors, but should clarify what kind of diet, physical activity or exposure. Similarly, in line 117 it is unclear what "GCNIS" is for. The genetics of TGCTs is briefly summarized. I would encourage the authors to include a Table and/or figure showing all the genetic and environmental risk factors linked to TGCT and provide the relative/risk or odds ratio for each of them.

2) Authors should include a paragraph explaining epigenetic mechanisms in details.

3) Section 3.1 The use of high-throughput... The authors describe epigenome-wide studies in this section. It is therefore unclear why these studies are not described in section 3.2 The role of epigenetics.

4) One or two Tables are required to summarize both genome-wide and candidate-gene methylation investigations, including the used techniques and the main findings of each study.

5) Also for section 3.2.2 a Table summarizing the studies is required.

6) Conclusions: the manuscript lacks an integrated overview of the use ob genetic, cytogenetic and epigenetic biomarkers for TGCT subtyping. Authors must be able to integrate the various articles described in the study and provide a discussion (as well as a diagram) of the major genetic, cytogenetic and epigenetic marks that could be used to stratify human germ cell tumors into different subtypes and clinical entities.

Author Response

Reviewer #2:

This is an interesting article dealing with the contribution of epigenetic modifications to human germ cell tumors.

Reply: We thank the Reviewer for his/her positive opinion on our work.

There are however several sections of the manuscript that could be improved. The conclusions are really short and merely speculative: the authors must provide a more integrated overview of the described points.

Major points:

1) Section 1.2 The genvironmental model: authors should better explain the contribution of genetic and environmental factors related to TGCTs. They mention diet, physical exercise or environmental exposure as potential risk factors, but should clarify what kind of diet, physical activity or exposure. Similarly, in line 117 it is unclear what "GCNIS" is for. The genetics of TGCTs is briefly summarized. I would encourage the authors to include a Table and/or figure showing all the genetic and environmental risk factors linked to TGCT and provide the relative/risk or odds ratio for each of them.

Reply: We thank the Reviewer for his/her suggestion, which was also pointed out by Reviewer #1. In order to comply with both Reviewers we have divided the section in the manuscript in subsections, described the influences of environmental factors in more detail and have added a Table 2 to summarize this section.

Table 2. Genetic and environmental risk factors for germ cell tumors

Abbreviations: KITLG – KIT-ligand; OR – odds ratio; TGCT – testicular germ cell tumor

2) Authors should include a paragraph explaining epigenetic mechanisms in detail.

Reply: We thank the Reviewer for pointing this out. We have added such a paragraph:    Epigenetics encompasses an array of processes that change gene expression without altering the DNA sequence, leading to a change in phenotype without changing the genotype. It comprises covalent modifications of DNA (such as DNA methylation), histone variants, histone post-translational modifications and non-coding RNAs (ncRNAs). DNA methylation, one of the most studied mechanisms, occurs by addition of methyl groups to the fifth carbon of cytosines, occurring preferentially at CpG sites, which are unevenly distributed in the genome – being concentrated in the so-called CpG islands. Differential methylation of gene promoters ultimately affect gene expression. Similarly, a number of ncRNAs are involved in the dynamic and environmentally sensitive regulation of gene expression. These molecules are known to interact (directly or indirectly) with the other established epigenetic mechanisms and can also directly interfere with messenger RNA (mRNA); this way, they can be seen as an extension of the complex epigenetic network, establishing important bridges between related modifications and truly influencing gene expression [85-87]. In this review, we will be focusing on methylation and a subtype of ncRNAs – the microRNAs (miRs).

3) Section 3.1 The use of high-throughput... The authors describe epigenome-wide studies in this section. It is therefore unclear why these studies are not described in section 3.2 The role of epigenetics.

Reply: We thank the Reviewer for his/her suggestion. Accordingly, the studies on the section “Use of high-throughput methodologies” were discussed in “The role of epigenetics” section.

4) One or two Tables are required to summarize both genome-wide and candidate-gene methylation investigations, including the used techniques and the main findings of each study.

AND

5) Also for section 3.2.2 a Table summarizing the studies is required.

Reply: We thank the reviewer for his/her suggestion, which was also pointed out by Reviewer #1. In accordance, we have added a Table (Table 4) summarizing these studies. Only one Table, joining both methylation and miRNAs was added as the article contains already many “Figure and Table” elements.

Table 4. Summary of studies on testicular germ cell tumor biomarkers regarding methylation and microRNAs

Abbreviations: AR – androgen receptor; AUC – area under the curve; COBRA – combined bisulfite restriction analysis; DMR – differentially methylated region; EC – embryonal carcinoma; GCTs – germ cell tumors; MeDIP - methylated DNA immunoprecipitation; miR – microRNA; NST – non-seminomatous tumor; qMS-PCR – quantitative methylation-specific polymerase chain reaction; RT-qPCR – real-time quantitative polymerase chain reaction; SE – seminoma; ST – spermatocytic tumor; TE – teratoma; TGCTs – testicular germ cell tumors; TVB – testicular vein blood; YST – yolk sac tumor

6) Conclusions: the manuscript lacks an integrated overview of the use of genetic, cytogenetic and epigenetic biomarkers for TGCT subtyping. Authors must be able to integrate the various articles described in the study and provide a discussion (as well as a diagram) of the major genetic, cytogenetic and epigenetic marks that could be used to stratify human germ cell tumors into different subtypes and clinical entities.

Reply: We thank the Reviewer for his/her suggestion. We have added such a paragraph and an integrative diagram:

“An integrated model (Figure 4) for defining TGCTs as distinct subtypes, concerning both genetic, cytogenetic and epigenetic biomarkers, is warranted. We have showed that both DNA methylation profiles and miRs expression differ greatly among histological TGCT subtypes, and their detection in liquid biopsies has proved its use, such as miR-371a-3p. Somatic mutations are scarce in TGCTs, and are present mainly in SE components, especially those concerning KIT (which define a specific subset of SEs). Extensive aneuploidy (and frequent presence of i(12p)) is a hallmark of TGCTs, regardless the histologic type. Only by integrating all these factors can we reveal novel unappreciated diversity within TGCTs as clinical entities”. 
                                

Round  2

Reviewer 2 Report

The authors have nicely addressed my criticisms