Platinum-Based Regimens Are Active in Advanced Pediatric-Type Rhabdomyosarcoma in Adults and Depending on HMGB1 Expression

Round 1

Reviewer 1 Report

​Dear authors, I think the article is very interesting, and that it opens possibilities for future research. Here are some of my suggestions, which can be also debatable.

- introduction: it could be explained if BOMP-EPI is a standard treatment, because this is not clearly stated in the introduction. If this regimen is not a standard treatment, then how was offered to patients in all these years? Was it a part of clinical trials? How is this then a novel, alternative treatment, if it was used for all these years? Please explain.

- although this is a retrospective study, it includes genetic testing. It would be advisable to include ethics committee approval.

​- ​line 58: you state that OS is about 30% in the recurrent disease and then in line 118 you show that 70% of the patients died in about 2-3 years. Why is then BOMP-EPI clearly superior (written in abstract)?

-line 172: in results you state: One patient (10 %) showed complete response (CR), five patients (50%) with partial, response (PR), three patients (30%) with stable disease (SD) and one patient (10%) with progression, according to the RECIST response (Table 3). Then in the line 172 you state that ORR was 70% - from the results it is seen that 6 patients had the response (CR or PR). Could you explain these numbers or am I missing something? Also, it would be adviseable to compare this numbers with standard treatment with other regiments in order to compare outcomes.

- line 211: So in these cases, HMGB1 doesn’t seem to act binding and protecting adducts induced by cisplatin and avoiding its repair as has been described for other HMG proteins  - hard to understand. Is it possible to rephrase?

- I believe the strong point of your study is HMG - this should be emphasized, because it is interesting from translational medicine point of view. Because this is a small trial and retrospective it is hard to conclude that the treatment is very successful or better than the standard - it could be mentioned that the treatment shows potential, but the main emphasis should be on HMG

- there are some spelling mistakes. Do not use contractions like Don't

Author Response

Point 1. introduction: it could be explained if BOMP-EPI is a standard treatment, because this is not clearly stated in the introduction. If this regimen is not a standard treatment, then how was offered to patients in all these years? Was it a part of clinical trials? How is this then a novel, alternative treatment, if it was used for all these years? Please explain.

Response 1. Currently, there is not a standard treatment beyond the first line in the systemic therapy of rhabdomyosarcoma and there are no specific approved drugs for this disease. Thus, after relapses of disease, and in the absence of clinical trials, the treating physician has several available off-label options that can be chosen, based on toxicity profile and patient’s preferences as well. Platinum-based regimens have been tested in the pediatric population, however its value in adults is unknown.

In order to clarify the current text, we have modified the text in lines 66-73 in the introduction. 

Point 2. although this is a retrospective study, it includes genetic testing. It would be advisable to include ethics committee approval.

Response 2. “approved by the Biomedical Research Ethic Committee of Andalusia (protocol code GEI-SAR-2012-01, approved the 30th June 2016)” was included in the Institutional Review Board Statement.

Point 3. line 58: you state that OS is about 30% in the recurrent disease and then in line 118 you show that 70% of the patients died in about 2-3 years. Why is then BOMP-EPI clearly superior (written in abstract)?

Response 3. Outcome of adult patients with metastatic rhabdomyosarcoma is very poor. Systemic therapy in this context is palliative in the majority of patients and expected median overall survival is short. BOMP-EPI is active in terms of objective responses and in our series more than half of the patients had longer overall survival than the expected for this population. 

We have included a paragraph in lines 64-66 in order to better contextualize the problem and we have changed the word “clearly” from the abstract for “which seem” in order to smooth the affirmation given the retrospective nature of the study. 

Point 4. line 172: in results you state: One patient (10 %) showed complete response (CR), five patients (50%) with partial, response (PR), three patients (30%) with stable disease (SD) and one patient (10%) with progression, according to the RECIST response (Table 3). Then in the line 172 you state that ORR was 70% - from the results it is seen that 6 patients had the response (CR or PR). Could you explain these numbers or am I missing something? Also, it would be advisable to compare this numbers with standard treatment with other regiments in order to compare outcomes.

Response 4. Thank you for your observation. Indeed it is a typo and we have corrected it in the discussion. The correct figure for ORR is 60%

We have included a table with existing publications on second-line regimens for a better comparison.

Point 5. line 211: So in these cases, HMGB1 doesn’t seem to act binding and protecting adducts induced by cisplatin and avoiding its repair as has been described for other HMG proteins  - hard to understand. Is it possible to rephrase?

Response 5. “HMGB1 doesn’t seem to act binding and protecting adducts induced by cisplatin and avoiding its repair” was changed by “HMGB1 induces cisplatin resistance by mechanisms of cell autophagy or by binding to cisplatin adducts and protecting them from being repaired as has been described for other HMG proteins”

Point 6. I believe the strong point of your study is HMG - this should be emphasized, because it is interesting from translational medicine point of view. Because this is a small trial and retrospective it is hard to conclude that the treatment is very successful or better than the standard - it could be mentioned that the treatment shows potential, but the main emphasis should be on HMG

Response 6. Additional included experiments allow us to say: Our data also show HMGB1 downregulation enhances RMS sensitivity to cisplatin , indicating that low HMGB1 expression could be a predictive factor of benefit to therapy with cisplatin.

Point 7. there are some spelling mistakes. Do not use contractions like Don't

Response 7. Doesn’t was changed by does not

Reviewer 2 Report

Authors provied an original work on efficacy of cisplatine-base chemotherapy regiment for RMS. They also conduct an interressant ancillary study. Despite the sample is very low, theses result are relevant in this context of very rare disease. Please fin below some comments

For the clinical part :

Line 83 : Maybe it’s better to talk of AYA (adolescant and young adult) and adult to be more precise.

 

Line 104/105 : How many cycle did the three patients received the first time and for the rechallenge ?

For the biomarker study :

Table 4: I don’t think these results are significant. To assess predictive value of HMGB1 and 2 expression for response to cisplatin authors need to develop a KO cells model for these genes and try to induce overexpression with transfection to search for drug resistance in case of overexpression.

Furthermore, it would be interessant to test other platin drug such carboplatin and oxaliplatin to see if this mechanism is specific of cisplatin.

Author Response

Point 1. Line 83 : Maybe it’s better to talk of AYA (adolescant and young adult) and adult to be more precise.

Response 1.  These sentences were included in the introduction: “Age is a known prognostic factor, being the prognosis of adolescents, young adults (AYA) and adult patients poorer, with more than half of patients succumbing to the disease” and “This study aims to describe the activity of alternative platinum-based regimes (specifically BOMP/EPI) in AYA and adult patients with metastatic/relapsed RMS”

Point 2. Line 104/105 : How many cycle did the three patients received the first time and for the rechallenge ? 

Response 2. Three patients received rechallenge of BOMP-EPI at progression after stopping therapy while in response (two patients stopped therapy due to adverse effects after 2 and 3 cycles respectively and the third patient completed 6 cycles)

For the biomarker study :

Point 3. Table 4: I don’t think these results are significant. To assess predictive value of HMGB1 and 2 expression for response to cisplatin authors need to develop a KO cells model for these genes and try to induce overexpression with transfection to search for drug resistance in case of overexpression.Furthermore, it would be interessant to test other platin drug such carboplatin and oxaliplatin to see if this mechanism is specific of cisplatin. 

Response 3. Additional experiments of HMGB1 knockdown in the six RMS cell lines and in combination with cisplatin treatment have been included to assess the predictive value of HMGB1, the gene with better correlation with OS and PFS in this series. New research is necessary to test other platin drugs.

Reviewer 3 Report

In overall well written paper, however some minor corrections, but also connections to clinical treatment are asked.

Please correct a typo in line 63: in vitro.

There was also 17 years old patient. In the normal categorization she would not be counted as an adult. Please exclude or change the title of the paper.

In the table 1, does the clinical group mean stage?

There are extra lines in table 1 and 3, what is the meaning of those?

Although to the sarcoma specialists it is known what are the factors which make the risk group, for all of the readers it is not known. Please explain it shortly.

Please explain the reasons why some patients got this treatment already in the first line setting.

I am suprised if there were not any pulmonary toxicity. Please describe this.

What was the disease-free interval after the first line treatment in patients treated in the second or later line?

There are colour codes in Figures 2 and 3. Do they mean something? A explanation is missing. A response (or histology) would be meaningful to tell in these figures.

Check the line 157, I think there is missing a word "with".

What is the clinical meaning of the finding now or in the near future? Are these test easily done or available? For who should you choose this treatment= are the gene expression finding correlated with any clinically available factors?

Author Response

Point 1. Please correct a typo in line 63: in vitro.

Response 1. Invitro was changed by in vitro

Point 2. There was also 17 years old patient. In the normal categorization she would not be counted as an adult. Please exclude or change the title of the paper.

Response 2. In our country, management of patients from 16 years can be done in the context of adult Medical Oncology clinics. We have included a sentence explaining that the eligibility for this analysis includes all patients above 16 years at the moment of the diagnosis in the methods section. At the moment of treatment with BOMP-EPI, the youngest patient was 17.8 years, thus we have rounded up to 18 in the text.  

Point 3. In the table 1, does the clinical group mean stage?

Response 3. Not exactly. The group of the Intergroup Rhabdomyosarcoma Study (IRS) refers to the disease status after surgical resection. Group I: complete resection of primary tumor; Group II: microscopic residual disease; Group III: macroscopic residual disease; Group IV: metastatic disease at diagnosis.

We have changed the label in the table to IRS group and we have included definition on the legend. 

Point 4. There are extra lines in table 1 and 3, what is the meaning of those?

Response 4. Extra lines separate patients who received a second dose of BOMP-EPI. However they have been deleted because these patients are also marked with an asterisk.

Point 5. Although to the sarcoma specialists it is known what are the factors which make the risk group, for all of the readers it is not known. Please explain it shortly.

Response 5. Thank you for this comment. We have added included the main prognostic factors in the introduction

Point 6. Please explain the reasons why some patients got this treatment already in the first line setting.

Response 6. This series collects data from 1994, when the management of adult patients with metastatic rhabdomyosarcoma was much more heterogeneous. Indeed, the publications suggesting advantage also in the adult population from pediatric chemotherapy regimens were published after 2000.

Those patients managed in the first line with BOMP-EPI had metastatic disease at diagnosis, and were managed before 2000. Although the current first line therapy for them would be IVADo, at that time, BOMP-EPI was a reasonable alternative.

Point 7. I am surprised if there were not any pulmonary toxicity. Please describe this.

Response 7. No pulmonary toxicities were described in medical records for the analyzed patients. We have added in the toxicity section a sentence regarding pulmonary adverse events. 

Point 8. What was the disease-free interval after the first line treatment in patients treated in the second or later line?

Response 8. For the 7 patients that received BOMP-EPI in the second or further line, the estimated median disease free interval from initial diagnosis was 18.5 months (95% CI 6-31). We have implemented this information in the results section.

Point 9. There are colour codes in Figures 2 and 3. Do they mean something? A explanation is missing. A response (or histology) would be meaningful to tell in these figures.

Response 9. Each colour is a different tumor sample or tumor cell line. In the figure has been indicated that:

-RMS-03, RMS-07, RMS-08 and RMS-09 are alveolar rhabdomyosarcoma tumor samples, RMS-04, RMS-05 and RMS-06 are embryonal rhabdomyosarcoma tumor samples and RMS-10 is spindle cell rhabdomyosarcoma tumor sample.

-RH30, RH41 and RH28 are alveolar rhabdomyosarcoma cell lines and RD, A204 and Hs729T are embryonal rhabdomyosarcoma cell lines.

Point 10. Check the line 157, I think there is missing a word "with".

Response 10. mRNA-protein was changed by of mRNA with protein

Point 11. What is the clinical meaning of the finding now or in the near future? Are these test easily done or available? For who should you choose this treatment= are the gene expression finding correlated with any clinically available factors?

Response 11. The additional data indicate that patients with low HMGB1 expression level could be more sensitive to cisplatin treatment, so HMGB1 could be used as a predictive factor for selecting patients who could benefit from the BOMP-EPI treatment. The level of HMGB1, can be easily determined in the RNA extracted from the tumor sample by quantitative PCR or even could be assayed by immunohistochemistry using specific antibodies.

 

Reviewer 4 Report

The authors Hindi and colleagues investigate the role of bleomycin, vincristine, methotrexate, cisplatin alternating with etoposide, cisplatin, and ifosfamide (BOMP-EPI) in adult patients with relapsed/metastatic rhabdomyosarcoma (RMS). This retrospective study involved 10 patients affected by RMS who received a mean of 2.5 cycles of BOMP-EPI. The results showed 10% of complete response, 50% of partial response, 30% of stable disease and 10% of progression disease. All patients progressed with a median progression-free survival of 8.47 months and 70% of patients died with a median overall survival of 24.7 months. The authors conclude that BOMP-EPI represents a promising treatment for the management of adults metastatic RMS. Moreover the author investigate the expression of HMGB1, HMGB2 and HMGA2 genes and their potential involvement in chemoresistance observed in the case series and in RMS cell lines especially with cisplatin. In this regard the results showed a possible correlation  of HMGA2 and chemoresitance in the RMS cell lines. Finally the authors report that these observations deserve its exploration in a prospective study.

The manuscript is interesting since it focuses on rare entities. New insight are provided in terms of promising treatment and understanding of the RMS biology

The manuscript would benefit from the following

1.       Quality of the surgery should be included (R0,R1,R2).

2.       Diagnostic section including IHC analysis performed should be reported (MyoD1 positivity and other potential diagnostic biomarkers).

3.     The potential role of EMT-related genes, chemoresistance-relted genes including HMGA2 and chemotherapy as well as other potential biomarkers has been recently investigated in a case series of adult RMS. In this regard the following articles should be included in the manuscript for proper discussion:

·         Deciphering the Genomic Landscape and Pharmacological Profile of Uncommon Entities of Adult Rhabdomyosarcomas. Int J Mol Sci. 2021 Oct 26;22(21):11564. doi: 10.3390/ijms222111564. PMID: 34768995; PMCID: PMC8584142.

·         Identification of a novel RAB3IP-HMGA2 fusion transcript in an adult head and neck rhabdomyosarcoma. Oral Dis. 2022 Oct;28(7):2052-2054. doi: 10.1111/odi.14036. Epub 2021 Oct 10. PMID: 34592033.

·         Clinicopathologic and survival correlates of embryonal rhabdomyosarcoma driven by RAS/RAF mutations. Genes Chromosomes Cancer. 2022 Mar;61(3):131-137. doi: 10.1002/gcc.23010. Epub 2021 Nov 16. PMID: 34755412; PMCID: PMC8956004.

4.     The protocol number approved by Ethic Committee for that study should be included in the manuscript.

5.       Study limitations should be included

Major revision are requested

Author Response

Point 1. Quality of the surgery should be included (R0,R1,R2).

Response 1. Quality of the surgery for each patient was included in Table 1 (“Surgery” column)

 

Point 2. Diagnostic section including IHC analysis performed should be reported (MyoD1 positivity and other potential diagnostic biomarkers).

Response 2. Results of IHC analysis were included in Table 1 (“Pathology details” column)

 

Point 3. The potential role of EMT-related genes, chemoresistance-relted genes including HMGA2 and chemotherapy as well as other potential biomarkers has been recently investigated in a case series of adult RMS. In this regard the following articles should be included in the manuscript for proper discussion:

•         Deciphering the Genomic Landscape and Pharmacological Profile of Uncommon Entities of Adult Rhabdomyosarcomas. Int J Mol Sci. 2021 Oct 26;22(21):11564. doi: 10.3390/ijms222111564. PMID: 34768995; PMCID: PMC8584142.
•         Identification of a novel RAB3IP-HMGA2 fusion transcript in an adult head and neck rhabdomyosarcoma. Oral Dis. 2022 Oct;28(7):2052-2054. doi: 10.1111/odi.14036. Epub 2021 Oct 10. PMID: 34592033.
•         Clinicopathologic and survival correlates of embryonal rhabdomyosarcoma driven by RAS/RAF mutations. Genes Chromosomes Cancer. 2022 Mar;61(3):131-137. doi: 10.1002/gcc.23010. Epub 2021 Nov 16. PMID: 34755412; PMCID: PMC8956004.

Response 3. These papers have been included in the discussion.

 

Point 4. The protocol number approved by Ethic Committee for that study should be included in the manuscript. 

Response 4. “approved by the Biomedical Research Ethic Committee of Andalusia (protocol code GEI-SAR-2012-01, approved the 30th June 2016)” was included in the Institutional Review Board Statement.

 

Point 5. Study limitations should be included

Response 5. This sentence is at the beginning of the discussion: “Our series has some limitations, being its retrospective nature and its limited size the major issues”

Round 2

Reviewer 4 Report

The authors have adressed the requested corrections. Now the paper could be considered for publication