Intrachromosomal Amplification of Chromosome 21 (iAMP21) Impacts Event-Free Survival but Not Overall Survival Among Pediatric Patients with Acute Lymphoblastic Leukemia: A Single-Center Experience Using an Asparaginase-Intensified Spanish Regimen

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is interesting, clear an well-written.

It is a pity PCR-MRD was not available? What year was it intruduced in the centre?

Also some stats are negative bacause the numbr of positive patients was very low.

Author Response

1) It is a pity PCR-MRD was not avalilable? What year wass introduced in the centre?

Dear reviewer, thank you for taking your time for this review and for your comments and seggestions.

PCR-MRD is yet not available at our centre. We have performed this study (centralized in another lab) in a few patents diagnosed with Philadelphia-positive ALL and KMT2A-rearranged ALL. No patents with KMT2A-rearranged ad only 2 Philadelphia-positive ALL cases were included in this series; none of them had PCR-MRD moitoring.

We are currently working for the opening of the Alltogether trial as well as the next IntReALL trial in our institution, and we hope we can offer PCR-MRD monitoring to all ALL patients starting during the nexrt weeks or months.

We agree that adding a more sensitive method such as PCR for MRD response assessment might provide aditional information for an adequate risk-response allocation of patients.

2) Also some stats are negative because the number of positive patients was very low.

Thank you for this comment. We completely agree. Sample size is an important limitation as stated in the discussion section

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the prognostic impact of iAMP21 in pediatric acute lymphoblastic leukemia (ALL) patients. Patients are treated via the Spanish “SEHOP-PETHEMA 2013” protocol, which includes asparaginase-intensified therapy. The authors retrospectively evaluate 89 patients and compare outcomes between iAMP21-positive and non-iAMP21 patients, with an added molecular characterization of the iAMP21 subgroup.

The study supports the international trend that iAMP21 should be considered a high-risk feature, irrespective of early treatment response and also shows the potential benefit of salvage therapy with immunotherapy and SCT.

The findings are statistically and clinically sound, especially the independent impact of iAMP21 and MRD ≥1% at day 33 on EFS and the molecular findings for NRAS, PAX5, SH2B3 mutations align with other iAMP21 studies and improves biological significance.

Findings are clearly presented, iAMP21 was found to significantly worsen event-free survival (EFS) (25% vs. 85.6%, p = 0.001), but had no impact on overall survival (OS), as all affected patients were successfully salvaged. Also, multivariate analysis confirms iAMP21 as an independent prognostic factor for poor EFS.

Study is methodologically sound, as FISH is used to identify iAMP21 and it is consistent with established definitions. Also, applied statistical analyses (Kaplan-Meier, Cox regression, Fine & Gray) are appropriate.

The inclusion of MLPA and NGS data from iAMP21-positive patients provides deeper insight into co-occurring genetic alterations (e.g., NRAS, PAX5, SH2B3, TP53), which could be useful for future treatment strategies.  

Size of sample group is very small, as only 4 patients with iAMP21 were identified. This small number limits statistical power even though the observed differences were significant. Additionally, the study highlights that iAMP21-positive patients had good early MRD responses, yet relapsed. This raises questions about the adequacy of using MRD alone for risk stratification in this subgroup. Also, the lack of routine asparaginase activity monitoring is a limitation.

The text should be modified, because the manuscript includes long dense paragraphs with some repetition. The presentation would benefit from tighter writing and improved figure/table clarity.

This is a valuable contribution to pediatric leukemia research. While limited by its size, the findings underscore the clinical importance of recognizing iAMP21 early and adjusting treatment intensity accordingly. The study advocates for protocol revisions to incorporate iAMP21 into high-risk stratification. With minor modifications, it would be valuable reference for all working in pediatric leukemia.

Author Response

1) Size of sample group is very small, as only 4 patients with iAMP21 were identified. This small numbers limits statistical power even though the observed differences were significant.

Dear reviewer, thank you for taking your time for this review and for your comments and suggestions.

We completely agree, and this is mentioned in the text as one of the main drawbacks of the study. Since iAMP21 is quite uncommon among pediatric (and adult) paptients with ALL, it woul be worthy to expand the study to a Spanish nation-wide level, and we are planning to purpose this aim to our national coordinators.

2) Additionally, the study highlights tha iAMP21-positive patients had good early MRD responses, yet relapsed. This raises questions about the adequacy of using MRD alone for risk stratification in this subgroup.

Thank you for this comment. Infact, this was one of the main objectives of our study since, currently most of the alternative high-risk genetic subtypes frequently loss their prognostic impact after an adequate risk.stratification according to MRD early response and subsequent treatment intensification. To this regard, iAMP21 seems to represent somehow an exception.

3) The text should be modified, because the ,manuscript includes long dense paragraphs with some repitition. The presentation would benefit from tighter writing and improve figure/table clarity.

Thank you for this suggestion. In order to accomplish with this recommendation, some changes have been made in table 1, 2 and 3 in order to try to help the reading; moreover, the text within the discussion section regarding reported results from previous studies have been modified deleting some parts of the poriginal text.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

In this single-center retrospective study the authors present their experience in the treatment of pediatric patients with ALL and intrachromosomal amplification of chromosome 21. The paper is well written, the cases are clearly described, the secondary genetic abnormalities have been studied, the discussion is adequate. The outcome of this small group of patients agrees with what has already been described in the literature on larger series.

The retrospective genetic analysis of patient 1 shows a loss-of-function FANCA pathogenic variant. This data is worth discussing, also considering the possible repercussions on the HSCT conditioning. Was there any phenotypic alteration? Was a family study done?

Minor point. lines 488-490: unclear

Author Response

1) The retrospective genetic analysis of patient1 shows loss-of-function FANCA pathogenic variant. This data is worth discussing, also considering the possible repercussions on the HSCT conditioning. Was there any phenotypic  alteration? Was a family study done?

Dear reviewer, thank you for taking your time for this review and for your cooments and suggestions.

This FANCA variant was presetn with a 46% VAF and, accordingly, was interpreted as a somatic variant or a possible heterozygous germline alteration provided that there were no clinical findings suggestive of Fanconi anemia phenotype. Thus, treatment plan, including conditioning regimnen before stem cell transplantation, was not modified. This comment has been adeed now in tyhe revised manuscript.

We are finding variant alterations in different genes which sometimes are related to cancer-predispositionb syndromes; in this situation, we are regerring this patients for genetic counseling after leukemia treatment is finished.

2) Minor point. lines 488-490: unclear.

Thanks for this comment. We have made a change in this sentence in order to try to make it easier to read (please see the new draft)

 

Author Response File: Author Response.docx