Oncological Properties of Intravenous Leiomyomatosis: Involvement of Mesenchymal Tumor Stem-Like Cells

Round 1

Reviewer 1 Report

Authors did good job to address one of the potential issues related to fibrosis, role of cancer stem cells in the disease progression.

1. In Material and Methods, 2.2 sub section and 2.5 subsections are same authors can remove on of the sub section
2. Authors can provide good contrast IHC images, Figure3-A, B, and C to distinguish clearly positive IHC staining.
3. CD44 is the cancer stem cell marker and most of the tumors express this on the tumor surface, fibrosis is heterogeneous tumors, and it can have immune cell infiltration also, like T lymphocytes. T cells express CD44, Authors did IHC extensively in this study in the human tumor sections, it would be great if authors answer CD44 staining is tumor specific but not T cell staining.

Author Response

Reviewer 1: Comments and Suggestions for Authors

Authors did good job to address one of the potential issues related to fibrosis, role of cancer stem cells in the disease progression.

Q1. In Material and Methods, 2.2 sub section and 2.5 subsections are same authors can remove on of the sub section

A1. Thank you for your comment. We agree with the reviewer’s comments. Accordingly, we have removed “2.5 subsection” and revised “2.2 subsection.”

Q2. Authors can provide good contrast IHC images, Figure3-A, B, and C to distinguish clearly positive IHC staining.

A2. Thank you for your comment. In line with this comment, we used IHC images with good contrast in the revised manuscript.

Q3. CD44 is the cancer stem cell marker and most of the tumors express this on the tumor surface, fibrosis is heterogeneous tumors, and it can have immune cell infiltration also, like T lymphocytes. T cells express CD44, Authors did IHC extensively in this study in the human tumor sections, it would be great if authors answer CD44 staining is tumor specific but not T cell staining.

A3. Thank you for your comment. As suggested, we rewrote the relevant sentences, as shown below:

Although some T-lymphocytes express CD44, it is possible that the CD44-positive cells are not T-lymphocytes. A recent report demonstrated that uterine mesenchymal tumors including uterine leiomyosarcomas were mismatch repair-deficient [1-3]. Uterine leiomyosarcomas, which account for 1.4% of stage I–III malignant tumors and 0.6% of stage IV malignant tumors, were classified as dMMR-positive tumors [1-3] (Figure 1). From these research findings, cases with uterine leiomyosarcoma were found to have a small number of infiltrating CD8-positive T-cells. Therefore, as previously reported, the antitumor effect of therapy by immunocheck point inhibitors (ICIs) on uterine leiomyosarcoma is considered low [1-3].

1. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, Lu S, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science. 2017;357(6349):409-413. doi: 10.1126/science.aan6733.
2. Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, D’Angelo S, et al. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial. The Lancet. Oncology. 2017;18(11):1493-1501. doi: 10.1016/S1470-2045(17)30624-1.
3. Hayashi T, Ichimura T, Sano K, Tonegawa S, Kanai Y, Zharhary D, Aburatani H, et al. Profiling of target molecules for immunotherapy in mesenchymal tumors. Journal of Clinical Medicine Research. 2019; 11(8):609-613. doi: 10.14740/jocmr3886.

Figure 1. Mismatch repair deficiency across tumors

The proportion of tumors deficient in mismatch repair (dMMR) in each malignant tumor subtype, expressed as a percentage. The dMMR tumors were identified in tumor subtypes tested more often in early-stage (defined as stage < IV) disease. The figure was adapted from Le DT, et al. Science. 2017;357(6349):409-413.

Q4. (x) English language and style are fine/minor spell check required

A4. To calibrate English expressions, the manuscript was spellchecked and proofread again by a native English speaker.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript titled "Oncological features of intravenous leiomyomatosis: involvement of mesenchymal tumor stem-like cells", the authors have concluded that 'establishing a treatment method targeting cancer stem cells will lead to  treatment of malignant tumors with low risk of recurrence and metastasis.

Listed below are my comments for the authors:
1. Caution about overstating the objectives and conclusion of the study. The oncologic properties of uterine leiomyosarcoma have been well studied in previous numerous studies. It is overstating that uterine mesenchymal tumor stem-like cells will provide therapeutic and diagnostic insight in treatment of leiomyosarcomas.
2. The authors state that “A unique intracellular factor might be expressed in intravenous leiomyomatosis which explains the peculiar infiltration into blood vessels that uterine leiomyomata does not have”. Please provide references to that sentence.
 3. In the result section, the authors claim that “understanding the oncological properties of intravenous leiomyomatosis contributes to developing new targeted antitumor agents for malignant mesenchymal tumors such as uterine leiomyosarcoma”. This is an overstatement and cannot be explained by the findings of this study.
4.  There are numerous typos throughout the manuscript.

Author Response

Reviewer 2: Listed below are my comments for the authors:
Q1. Caution about overstating the objectives and conclusion of the study. The oncologic properties of uterine leiomyosarcoma have been well studied in previous numerous studies. It is overstating that uterine mesenchymal tumor stem-like cells will provide therapeutic and diagnostic insight in treatment of leiomyosarcomas.

A1. Thank you for your comment. As suggested, we revised relevant parts of the manuscript. Based on the new results obtained, we have rewritten the text to avoid exaggerated conclusions, as shown below:

Results: As intravenous leiomyomatosis is a disease in which tumors grow in veins, presumably, CD44-positive mesenchymal tumor stem-like cells can infiltrate the intravascular space. However, CD44-positive mesenchymal tumor stem-like cells may be sensitive to antitumor agents because of their low positive rates for cyclin E and Ki-67 and resistance to antitumor agents (Figure 5). Understanding the oncological properties of intravenous leiomyomatosis might will contribute to the development of new targeted antitumor agents for malignant mesenchymal tumors such as uterine leiomyosarcoma.

Conclusion: The characteristics of these human uterine mesenchymal tumor stem-like cells might will provide insights into the development of novel therapeutics and diagnostic methods for uterine mesenchymal tumors. Further oncological studies of mesenchymal tumors are required to establish treatments for uterine leiomyosarcoma with a poor prognosis.

Q2. The authors state that “A unique intracellular factor might be expressed in intravenous leiomyomatosis which explains the peculiar infiltration into blood vessels that uterine leiomyomata does not have”. Please provide references to that sentence.

A2. Thank you for your comment. Accordingly, we added the citation and the following references in the revised manuscript.

1. Uterine leiomyoma. Female Genital Tumours WHO Classification of Tumours, 5th ed., Vol.4. WHO Classification of Tumours Editorial Board. World Health Organization. 2020; pp272-276.
2. Intravenous leiomyomatosis. Female Genital Tumours WHO Classification of Tumours, 5th ed., Vol.4. WHO Classification of Tumours Editorial Board. World Health Organization. 2020; pp277-278.

These two reports show the difference between intravenous leiomyoma and uterine leiomyoma based on the results of the genome-wide analysis, gene mutation analysis, and cell surface marker analysis. 

Q3. In the result section, the authors claim that “understanding the oncological properties of intravenous leiomyomatosis contributes to developing new targeted antitumor agents for malignant mesenchymal tumors such as uterine leiomyosarcoma”. This is an overstatement and cannot be explained by the findings of this study.

A3. Thank you for your comment. As per the suggestion, we revised relevant parts of the manuscript. Based on the new results obtained, we have rewritten relevant texts to avoid exaggerated conclusions, as shown below:

Results: As intravenous leiomyomatosis is a disease in which tumors grow in the veins, presumably, CD44-positive mesenchymal tumor stem-like cells can infiltrate the intravascular space. However, CD44-positive mesenchymal tumor stem-like cells may be sensitive to antitumor agents because of their low positive rates for cyclin E and Ki-67 and resistance to antitumor agents (Figure 5). Understanding the oncological properties of intravenous leiomyomatosis might will contribute to the development of new targeted antitumor agents for malignant mesenchymal tumors such as uterine leiomyosarcoma.

Conclusion: The characteristics of these human uterine mesenchymal tumor stem-like cells might will provide insights into the development of novel therapeutics and diagnostic methods for uterine mesenchymal tumors. Further oncological studies of mesenchymal tumors are required to establish treatments for uterine leiomyosarcoma with a poor prognosis.

Q4. There are numerous typos throughout the manuscript.

A4. To calibrate English expressions, the manuscript was spellchecked and proofread again by a native English speaker.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Lines 35-36 - The part about subtypes can be confusing for readers. I would revise the statement. Also, similar mention has been made in the abstract (lines 13-14).

Line 56 - "the blood vessel containing the vein" - I would refine this statement. You can use either blood vessel or vein to be more specific. 

Lines 66-68 - How did you come to conclusion? Is there any evidence to back this statement? 

Line - related to reference 7 - CD133 predominantly was shown to be a reliable marker than CD44. Also authors used this in colorectal cancer cell lines. Is there any reasoning behind being able to extrapolate this to uterine leiomyomas? 

Lines 177-186 - Section 2.5 (Ethical approval and consent to participate) seems to be a repetition of section 2.2 (lines 113-123).

Lines 191-193 - I would change the statement to 'Recently, we treated a patient with intravenous leiomyomatosis (IVL). Even in IVL, we noticed increased tumor growth/burden in the vein.' 

 

Line 194 - Any citation to back this statement of 'distance metastasis leading to antitumor agent resistance' would be helpful. If not, I would remove this. 

Table 1 --> Leiomyoma --> Ordinary has been misspelled as 'ordinally' and potential has been misspelled as 'potentialy'

Lines 407-408 - I would reconsider this statement. One cannot presume the "oncological nature" of IVL. Rather I would use a phrase like "pattern of growth" or "proliferation". 

Lines 410-413. IVL behaves in a quasi-malignant way due to its vascular invasion potential. Because it is not expressing Cyclin E and Ki-67, I do not believe one can presume that IVL carries a poor prognosis. I agree with the statement that they behave different to malignant tumors.

Figure 5: I would provide references for the statement regarding cytotoxic effect of celecoxib. 

Line 428 --> resistance to antitumor agents. Given tumor growth driven by CD44 positivity we can conclude that CD44 has a role to play in the proliferation of IVL. However, I do not think it is safe to extrapolate this finding to antitumor resistance. 

 

 

Author Response

Reviewr 3: Comments and Suggestions for Authors

Q1. Lines 35-36 - The part about subtypes can be confusing for readers. I would revise the statement. Also, similar mention has been made in the abstract (lines 13-14).

A1. Thank you for your comment. We agree with the comments. Accordingly, we rewrote the relevant sentence in the revised manuscript.

Q2. Line 56 - "the blood vessel containing the vein" - I would refine this statement. You can use either blood vessel or vein to be more specific.

A2. We agree with the suggestion; accordingly, we rewrote the sentence in the revised manuscript, as shown below:

Typically, uterine leiomyomas are benign tumors that do not invade the blood vessel containing the vein.

Q3. Lines 66-68 - How did you come to conclusion? Is there any evidence to back this statement?

A3. Thank you for your comment. In uterine leiomyosarcoma, a malignant tumor, lymphatic metastasis is rare (frequency ≤ 10%), whereas hematogenous metastasis is common [5,6]. Although typically, uterine leiomyomas are benign tumors that do not invade the veins, intravenous leiomyomatosis invades the veins. Therefore, there may be similarities between the physiological activities of unique intracellular factors of intravenous leiomyomatosis and the hematogenous metastatic potential of uterine leiomyosarcoma.

We are merely stating the possible oncological similarities between the physiological features of intravenous leiomyomatosis and hematogenous metastases in leiomyosarcoma.

Q4. Line - related to reference 7 - CD133 predominantly was shown to be a reliable marker than CD44. Also authors used this in colorectal cancer cell lines. Is there any reasoning behind being able to extrapolate this to uterine leiomyomas?

A4. Thank you for your comment. Cancer stem cells play important roles in the formation and growth of cancer and are associated with metastasis and recurrence. Previous studies have indicated that CD13, CD44, and CD133 are markers for cancer stem cells. Moreover, in MSCs, CD105 (SH2), CD73, CD44, CD90, CD71, and Stro-1 are known molecular biomarkers as are the adhesion molecules CD106, CD166, and CD29. A comprehensive examination of these reports suggests that CD44 is an appropriate marker of uterine mesenchymal tumor stem cells.

Previously, we performed a side population method using surface markers of CD133 to isolate uterine leiomyosarcoma stem-like cells. Evidently, we isolated uterine leiomyosarcoma stem-like cells using the surface markers of CD133. However, the yield of uterine leiomyosarcoma stem-like cells was slightly lower. Therefore, in this experiment, we performed a positive fraction method using a surface marker of CD44 to isolate stem-like cells of uterine leiomyosarcoma. We isolated stem-like cells of uterine leiomyosarcoma using the surface markers of CD44. Either CD44 or CD133 cell surface markers can be used to isolate uterine leiomyosarcoma stem-like cells.

Q5. Lines 177-186 - Section 2.5 (Ethical approval and consent to participate) seems to be a repetition of section 2.2 (lines 113-123).

A5. Thank you for your comment. As suggested by the reviewer, we have removed “2.5 subsection” and revised it to “2.2 subsection.”

Q6. Lines 191-193 - I would change the statement to 'Recently, we treated a patient with intravenous leiomyomatosis (IVL). Even in IVL, we noticed increased tumor growth/burden in the vein.' 

A6. Thank you for your comment. In line with the suggestion, we rewrote the relevant sentence in the revised manuscript, as shown below:

Recently, we treated a patient with intravenous leiomyomatosis (Supplementary material 2). Even in intravenous leiomyomatosis, we noticed increased tumor growth/burden in the vein.

Q7. Line 194 - Any citation to back this statement of 'distance metastasis leading to antitumor agent resistance' would be helpful. If not, I would remove this.

A7. Thank you for your comment. In line with your suggestion, we added the citation and the following reference in the revised manuscript:

Vinogradov S, Wei X. Cancer stem cells and drug resistance: the potential of nanomedicine. Nanomedicine (London, England). 2012;7(4):597-615. doi: 10.2217/nnm.12.22.

This paper states that human cancers emerge from cancer stem cells (CSCs), which are resistant to cancer chemotherapeutic agents, radiation, and cell death. Furthermore, much evidence shows that CSCs cause tumor initiation, progression, metastasis, and cancer recurrence.

Q8. Table 1 --> Leiomyoma --> Ordinary has been misspelled as 'ordinally' and potential has been misspelled as 'potentialy'

A8. Thank you for your comment. We are grateful to the reviewer for checking the details. In line with the suggestion, we revised Table 1.

Q9. Lines 407-408 - I would reconsider this statement. One cannot presume the "oncological nature" of IVL. Rather I would use a phrase like "pattern of growth" or "proliferation".

A9. Thank you for your comment. We are grateful to the reviewer for checking the details. We agree with your comment. Accordingly, we rewrote the sentence in the revised manuscript, as shown below:

Therefore, the oncological pattern of the growth nature of intravenous leiomyomatosis is similar to the oncological characteristics of uterine leiomyosarcoma.

Q10. Lines 410-413. IVL behaves in a quasi-malignant way due to its vascular invasion potential. Because it is not expressing Cyclin E and Ki-67, I do not believe one can presume that IVL carries a poor prognosis. I agree with the statement that they behave different to malignant tumors.

A10. Thank you for your comment. We are grateful to the reviewer for checking the details. In our clinical research with a large cohort, poor-prognosis uterine mesenchymal tumors, i.e., uterine leiomyosarcoma, have high positive rates for cyclin E and Ki-67. Therefore, we believe that the high positive rates of cyclin E and Ki-67 are prognostic markers for uterine mesenchymal tumors. Intravenous leiomyomatosis has a low positive rate for cyclin E and Ki-67, suggesting that intravenous leiomyomatosis may not be a malignant tumor with a poor prognosis.

As per the suggestion of the reviewer, we added the following sentence in the revised manuscript:

In our clinical research with a large cohort, poor-prognosis uterine mesenchymal tumors, i.e., uterine leiomyosarcoma, have high positive rates for cyclin E and Ki-67. Therefore, we believe that the high positive rates of cyclin E and Ki-67 are prognostic markers for uterine mesenchymal tumors.

Q11. Figure 5: I would provide references for the statement regarding cytotoxic effect of celecoxib.

Line 428 --> resistance to antitumor agents. Given tumor growth driven by CD44 positivity we can conclude that CD44 has a role to play in the proliferation of IVL. However, I do not think it is safe to extrapolate this finding to antitumor resistance.

A11. Thank you for your comment. We are grateful to the reviewer for checking the details. Accordingly, we added the following sentence in the revised manuscript:

However, CD44-positive mesenchymal tumor stem-like cells may be sensitive to antitumor agents because of their low positive rates for cyclin E and Ki-67.

We also added the following reference in the revised manuscript:

Huang C, Chen Y, Liu H, Yang J, Song X, Zhao J, He N, et al. Celecoxib targets breast cancer stem cells by inhibiting the synthesis of prostaglandin E(2) and down-regulating the Wnt pathway activity. Oncotarget. 2017;8(70):115254-115269. doi: 10.18632/oncotarget.23250.

 

Author Response File: Author Response.pdf