Transcriptional comparison of testicular adrenal rest tumors with fetal and adult tissues

  1. Mariska A M Schröder
  2. Fred C G J Sweep
  3. Antonius E van Herwaarden
  4. Rod T Mitchell
  5. Jitske Eliveld
  6. Ans M M van Pelt
  7. Alan E Rowan
  8. Darren Korbie
  9. Nike M M L Stikkelbroeck
  10. Hedi L Claahsen-van der Grinten
  11. Paul N Span

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Abstract

Background

Testicular adrenal rest tumors (TART) are a common complication of unknown cellular origin in patients with congenital adrenal hyperplasia (CAH). These benign tumors have both adrenal and testicular characteristics and are hypothesized to either derive from cells of adrenal origin from the fetal adrenogonadal primordium or by atypical differentiation of adult Leydig-progenitor cells.

Objective

This study aims to unravel the identity and etiology of TART.

Methods

Co-expression of adrenal-specific CYP11B1 and Leydig cell-specific HSD17B3 in TART was studied using immunohistochemistry. We studied the possibility of TART being derived from atypical differentiation of adult Leydig-progenitor cells by the quantification of adrenal-specific enzyme expression upon adrenocorticotrophic hormone (ACTH)-like stimulation of ex vivo cultured platelet-derived growth factor receptor alpha-positive cells. By comparing the transcriptome of TART (n = 16) with the transcriptome of fetal adrenal (n = 13), fetal testis (n = 5), adult adrenal (n = 11), and adult testis (n = 10) tissues, we explored the identity of TART.

Results

We demonstrate co-expression of adrenal-specific CYP11B1 and testis-specific HSD17B3 in TART cells, indicating the existence of a distinct TART cell exhibiting both adrenal and testicular characteristics. Ex vivo cultured adult Leydig-progenitor cells did not express the ACTH-receptor MC2R but did express CYP11B1 upon stimulation. Unsupervised clustering of transcriptome data showed that TART was most similar to adult adrenal tissue, followed by adult testis tissue, and least similar to either fetal tissue.

Conclusion

Our data suggest that TART is induced — most likely via activation of a cAMP/protein kinase A-dependent receptor — from a progenitor cell into a unique mature adrenal-like cell type, sometimes exhibiting both adrenal and testicular features.

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  1. Version published to 10.1530/eje-22-0143
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    __Rebuttal to reviewers ReviewCommons manuscript # RC-2020-00281 __

    We would like to thank the reviewers and editors of Review Commons for evaluating our manuscript entitled “Transcriptional comparison of Testicular Adrenal Rest Tumors with fetal and adult tissues” and providing their valuable comments. We have listed the reviewers’ comments along with our response and amendments below.

    __Board Advice on initial submission: __

    *This seems to be a study mainly relevant to the field of Testicular Adrenal Rest Tumors (TART). It presents the first RNAseq profiling of these tumors in multiple human samples at different stages. This has the potential to advance knowledge in this particular field. It would be less interesting to researchers interested in tissue spatial transcriptomics in general, since the experimental and computational tools are quite standard, but the findings may be important to the TART field. *

    Response: Indeed, this is the first study using transcriptomics to characterize Testicular Adrenal Rest Tumors, a frequent occurrence in patients with Congenital Adrenal Hyperplasia. It is also the first to find that the reported adrenal and testicular features of these tumors can be found in a single cell. We therefore believe this study is not only of interest to those working in the TART field, but also in development, endocrinology and andrology in general.

    Comments Reviewer #1:

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

    __**Summary:** __

    *The manuscript by Schroder M., et al describes the whole transcriptome of testicular adrenal rest tumors (TART) and shows that TART tissue is characteristically similar to adult adrenal and testicular rather than fetal adrenal and testicular tissues. The authors propose that their previous claim that TART is derived from an undifferentiated pluripotent progenitor is likely untrue and claim that TART likely originates from a mature cell type with both adrenal and testicular characteristics. The authors describe a unique cell type most similar to the adult adrenal, but with variable testis-specific gene expression patterns. The finding of overexpressed genes associated with ECM remodeling is interesting and may provide insight into the natural history of these tumors. A strength of the study is the number of tissue samples since surgery for these rare tumors is usually not performed. *

    __**Major Comments:** __

    *The key conclusions are mostly based on RNA studies, thus their claims are preliminary. *

    Response: We agree that a major part of our conclusions is based on RNA studies. Although indeed primarily based on transcriptomics, this claim is, in our opinion, not preliminary as the identity of TART cells can definitively be deduced from their expression profile. Our second key conclusion, i.e. that TART cells comprise both adrenal and testicular features within the same, unique, TART specific cell, is based on immunohistochemistry of adrenal and testis-specific enzymes.

    In Figure 1/Result p. 3: Authors claim that there were no exclusive HSD17B3 staining cells without CYP11B1, however Figure 1 looks like there are exclusively green (HSD17B) areas (especially TART3). The authors need to address this. It appears as if there are mature Leydig cells. This is important because the presence of Leydig cells would affect the interpretation of the findings.

    Response: We do understand the concern of the reviewer. Aspecific background staining for HSD17B3 in TART samples complicated the differentiation between specific and background staining. This can be seen when comparing the staining in HSD17B3-positive (Leydig) cells with the background staining in non-Leydig cells in testis tissue and in a portion of TART cells. In TART, we found that cells with high intensity, specific HSD17B3 staining all also showed CYP11B1 staining, but not vice-versa. However, we do acknowledge that due to this -most likely background- staining, the occurrence of mature Leydig cells in TART cannot be completely excluded based on our results.

    Therefore, we have tried to be more careful in our claims in the results section (page 3; TART cells express adrenal- and Leydig cell-specific steroidogenic enzymes paragraph) and we have addressed this in the discussion section (page 5/6):

    High background staining for HSD17B3 complicated the differentiation between specific and background staining. For some cells this exclusive HSD17B3 staining might have been specific and therefore, despite that most HSD17B3-positive cells were positive for CYP11B1, the absence of mature Leydig cells in TART could not be guaranteed by these results.

    *Discussion: authors state that based on their previous observations that fetal Leydig cells have both adrenal and testis developmental potential. It was speculated that TART might have been derived from a totipotent progenitor cell type, but the current study shows that these tumors lack similarities with fetal tissues. Thus, the authors claim that these tumors are not derived from the transdifferentiation of pluripotent cells. However what is the origin of this mature distinct cell type? Is it not possible that this distinctive cell type is derived from a common progenitor since the testis and adrenal gland are derived from the same adrenogonadal primordium? Lack of similarities with fetal tissues at this late stage of development does not necessarily rule out a common progenitor origin. *

    Response: In this study, we compared the TART transcriptome with fetal tissues, as we hypothesized these might be similar considering the likely progenitor origin of TART cells. However, this was not the case, and we showed that the transcriptomic profile of TART resembles the transcriptomic profile of mature cell types, rather than their fetal counterparts. Therefore, we conclude that the hypothesis that TART arises from progenitor cells is not supported by our data. The reviewer is correct that we did not prove that it is not derived from pluripotent cells. We have therefore added the following text to the discussion:

    Although we here find that the transcriptome of TART tissues are clearly distinct from fetal tissues, we did not prove that TART does not originate from fetal Leydig cells. TART being derived from a multipotent progenitor cell is still possible as we initially hypothesized, given the fact that TART is likely already present in utero and its resemblance to both testis and adrenal tissues which derive from a common primordium. Therefore, we were surprised to find TART to be more like adult adrenal and testis tissue, raising the possibility of TART being derived from a ‘mature’ progenitor cell type, i.e. adult stem Leydig cells or adrenal progenitor cells, that under influence of high ACTH levels and/or the localization in the testicular region might differentiate into a distinct cell type that expresses both adrenal- and testis-specific markers. However, this remains to be established.

    __**Minor Comment:** __

    *In Methods: Was RNA isolated from FFPE sections or frozen tissue? *

    We agree that this was not clearly mentioned enough in our original manuscript, as both frozen (RNA isolation) and FFPE (IHC) material was used. *We have now clarified in the methods section that the RNA was retrieved from frozen tissue samples (page 8; RNA isolation, library preparation, and sequencing paragraph). *

    __Reviewer #1 (Significance (Required)): __

    *This first study of transcriptome analysis of TART provides useful insight into the characteristics of these rare tumors that commonly develop in males with classic CAH. This study provides a foundation for further investigation of the biological pathways contributing to the development of TART, the most common cause of male infertility in CAH. This study is of interest to endocrinologists. Reviewed by a pediatric endocrinologist and molecular biologist - we are not completely aware of the sequencing analysis but are familiar with clustering and enrichment analysis. *

    Comments Reviewer #3:

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    *The manuscript by Schröder et al describes the transcriptome sequencing of TARTs in CAH/CS in order to sort out the origin of TARTs. This is an interesting subject and the manuscript is well-written but I have a few comments that could be addressed. *

    Some parts of the Results should be in the Methods and some in the Discussion. In the Results only the results should be given.

    Response: We agree that we have incorporated some methodological sentences and some concluding remarks in the results sections to, in our opinion, improve the flow of the manuscript. As the manuscript guidelines differ between journals, we have for now decided not to change this. We will do so if this is wanted by the concerning journal.

    *Normally TARTs are not removed or biopsied, if not by mistake... Thus, most centers would not have tissue samples of TARTs at all. How come you have so many samples available? *

    Response: We thank the reviewer for highlighting this. As indeed TARTs are not routinely removed, the number of TART tissues included in our dataset is unique. Most of the TART samples were already obtained in 2004 because of reported pain and discomfort and in an attempt to improve semen quality in these patients. Removal of those particular TART samples have led to new insights that removal of longstanding TART did not improve semen parameters, nor parameters of pituitary-gonadal function (Claahsen-van der Grinten et al., 2007). Therefore, to date, the only indication for surgery for the removal of longstanding TART is the relief of pain or discomfort.

    *Ref 2 and 3 are rather old and similar. Could newer review references be used instead? *

    Response: We have changed those two references for a more recent review by Dr. Witchel on Congenital Adrenal Hyperplasia, who addresses both statements in a more recent review (Witchel, 2017).

    __Reviewer #3 (Significance (Required)): __

    *New and significant study. Very interesting for people dealing with CAH patients. *

    References

    Claahsen-van der Grinten, H. L., Otten, B. J., Takahashi, S., Meuleman, E. J. H., Hulsbergen-van de Kaa, C., Sweep, F. C. G. J., & Hermus, A. R. M. M. (2007). Testicular adrenal rest tumors in adult males with congenital adrenal hyperplasia: Evaluation of pituitary-gonadal function before and after successful testis-sparing surgery in eight patients. Journal of Clinical Endocrinology & Metabolism, 92(2), 612-615. doi:10.1210/jc.2006-1311

    Witchel, S. F. (2017). Congenital Adrenal Hyperplasia. J Pediatr Adolesc Gynecol, 30(5), 520-534. doi:10.1016/j.jpag.2017.04.001

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Schröder et al describes the transcriptome sequencing of TARTs in CAH/CS in order to sort out the origin of TARTs. This is an interesting subject and the manuscript is well-written but I have a few comments that could be addressed.

    1. Some parts of the Results should be in the Methods and some in the Discussion. In the Results only the results should be given.
    2. Normally TARTs are not removed or biopsied, if not by mistake... Thus, most centers would not have tissue samples of TARTs at all. How come you have so many samples available?
    3. Ref 2 and 3 are rather old and similar. Could newer review references be used instead?

    Significance

    New and significant study. Very interesting for people dealing with CAH patients.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Schroder M., et al describes the whole transcriptome of testicular adrenal rest tumors (TART) and shows that TART tissue is characteristically similar to adult adrenal and testicular rather than fetal adrenal and testicular tissues. The authors propose that their previous claim that TART is derived from an undifferentiated pluripotent progenitor is likely untrue and claim that TART likely originates from a mature cell type with both adrenal and testicular characteristics. The authors describe a unique cell type most similar to the adult adrenal, but with variable testis-specific gene expression patterns. The finding of overexpressed genes associated with ECM remodeling is interesting and may provide insight into the natural history of these tumors. A strength of the study is the number of tissue samples since surgery for these rare tumors is usually not performed.

    Major Comments:

    1. The key conclusions are mostly based on RNA studies, thus their claims are preliminary.

    2. In Figure 1/Result p. 3: Authors claim that there were no exclusive HSD17B3 staining cells without CYP11B1, however Figure 1 looks like there are exclusively green (HSD17B) areas (especially TART3). The authors need to address this. It appears as if there are mature Leydig cells. This is important because the presence of Leydig cells would affect the interpretation of the finidings

    3. Discussion: authors state that based on their previous observations that fetal Leydig cells have both adrenal and testis developmental potential. It was speculated that TART might have been derived from a totipotent progenitor cell type, but the current study shows that these tumors lack similarities with fetal tissues. Thus, the authors claim that these tumors are not derived from the transdifferentiation of pluripotent cells. However what is the origin of this mature distinct cell type? Is it not possible that this distinctive cell type is derived from a common progenitor since the testis and adrenal gland are derived from the same adrenogonadal primordium? Lack of similarities with fetal tissues at this late stage of development does not necessarily rule out a common progenitor origin.

    Minor Comment:

    In Methods: Was RNA isolated from FFPE sections or frozen tissue?

    Significance

    This first study of transcriptome analysis of TART provides useful insight into the characteristics of these rare tumors that commonly develop in males with classic CAH. This study provides a foundation for further investigation of the biological pathways contributing to the development of TART, the most common cause of male infertility in CAH. This study is of interest to endocrinologists. Reviewed by a pediatric endocrinologist and molecular biologist - we are not completely aware of the sequencing analysis but are familiar with clustering and enrichment analysis.

  5. Version published to 10.1101/2020.05.07.082313v1 on bioRxiv