Challenges and Advances in the Detection of Leukemic Blasts in Cerebrospinal Fluid in Pediatric Acute Lymphoblastic Leukemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review reports on the important matter but there are several issues to be raised to improve the value of the manuscript.

Major comments:

1. Table 1 is inconsistent, some traumatic tap subcategories are duplicated (eg CNS2b appears twice, CNS2c is inconsistent). Please clarify which classification system Table 1 uses (eg COG vs other), because the CNS1 definition in the table (≤5 WBC/µL) differs from the footnote stating COG CNS1 is defined regardless of WBC count
2. Rephrase “cytomorphology is the gold standard” to reflect it as the historical method with limited sensitivity, and position flow as the key adjunct for low cellularity CSF.
3. The auhors should strengthen the flow section with a short, standardized practice framework covering preanalytics, time-to-analysis, acquisition expectations (minimum events/thresholds…), explicit handling of traumatic taps as a major pitfall, antibody panel guidance, and a more cautious interpretation of “flow-only positivity” (true CNS disease vs contamination vs borderline LoD).
4. Please clarify what is, regarding PCR/ddPCR/NGS/cfDNA/miRNA, research-only vs clinically used and the value of the methods over FACS
5. Standardize how sensitivity is expressed (eg you are mixing % and 10^x without explanation, so in table there is eg 1%-5% for FISH and 10-6 for ddPCR -difficult to follow and compare 
6. The NCCN CNS lymphoma flow recommendation is peripheral to pediatric ALL and would advise to remove it or explicitly justify as a methodological analogy.
7. Please add a simple integrated diagnostic pathway (cytology/TdT + flow + molecular..) for common scenarios such as equivocal cytology, traumatic tap, and relapse surveillance.

Minor comments:

1. Typos: cable - capable, optimally performance - optimal performance, therefor - therefore, cytoplastic - cytoplasmic etc…
2. Standardize µL (avoid µl/µL mix), WBCs/µL vs cells/µL….
3. Please use consistent terminology (qPCR for DNA targets, RT-qPCR for RNA/fusion transcripts) rather than mixed qPCR/qRT-PCR phrasing
4. Reference formatting should be fixed
5. Would be useful for the readership for the graphical abstract to explicitly mention low CSF volume and need for rapid processing, and the simple summary could maybe say that combining cytology + flow improves sensitivity in low-cellularity CSF

Author Response

Comment 1: Table 1 is inconsistent, some traumatic tap subcategories are duplicated (eg CNS2b appears twice, CNS2c is inconsistent). Please clarify which classification system Table 1 uses (eg COG vs other), because the CNS1 definition in the table (≤5 WBC/µL) differs from the footnote stating COG CNS1 is defined regardless of WBC count

Response 1: This table was designed to merge the current COG classification with the European and St Jude classifications. For this reason, we included notations below the table to highlight the differences between systems.  To improve clarity and readability for readers, we have now revised the presentation by splitting it into Table 1A and Table 1B to separate the two classification systems.

Comment 2: Rephrase “cytomorphology is the gold standard” to reflect it as the historical method with limited sensitivity, and position flow as the key adjunct for low cellularity CSF.

Response 2: I added the term “historical” in front of gold standard and added another sentence after this: “This paradigm may be subject to future refinement as more sensitive and objective diagnostic modalities are developed and validated”.

Comment 3: The authors should strengthen the flow section with a short, standardized practice framework covering preanalytics, time-to-analysis, acquisition expectations (minimum events/thresholds…), explicit handling of traumatic taps as a major pitfall, antibody panel guidance, and a more cautious interpretation of “flow-only positivity” (true CNS disease vs contamination vs borderline LoD).

Response 3: We added appropriate preanalytics and time to analysis such as minimal volume 1-3ml, within 1-2 hours, into the manuscript. Related comments were addressed by rewriting the whole section under the flow cytometry.

 Regarding “flow-only positivity”, as stated in lines 177-178, “Large cohort studies confirm that any detectable CSF blasts, regardless of WBC count, correlate with poorer outcomes, supporting risk-adapted intensification of CNS-directed therapy” and lines 188-189 “Patients with negative or indeterminate cytology but positive flow cytometry findings may therefore benefit from therapeutic intervention”.

Comment 4: Please clarify what is, regarding PCR/ddPCR/NGS/cfDNA/miRNA, research-only vs clinically used and the value of the methods over FACS

Response 4: For qPCR, we added the sentence: “qPCR is often used clinically alongside flow cytometry to enhance the detection of occult CNS involvement to refine risk stratification and diagnose relapses”.

For ddPCR, we included: “Although the ddPCR technique remains in the clinical study phase, it is expected to be adopted into routine clinical practice in the near future due to its significant advantages.”

For NGS, we added: “NGS is increasingly utilized employed a high-sensitivity adjunctive tool for the detection and monitoring of CNS leukemia, particularly in cases where conventional methods, such as cytomorphology and flow cytometry, yield inconclusive results. Achievement of NGS-negative status in the CNS compartment has been strongly associated with superior event-free and overall survival. While still under clinical investigation, NGS is already being implemented in practice at several institutions”.

Comment 5: Standardize how sensitivity is expressed (eg you are mixing % and 10^x without explanation, so in table there is eg 1%-5% for FISH and 10-6 for ddPCR -difficult to follow and compare 

Response 5: The expression of sensitivity has been revised as recommended. For clinical applicability and to align with routine practice, when the level is approximately 1%, we have retained both expressions in the manuscript.

Comment 6: The NCCN CNS lymphoma flow recommendation is peripheral to pediatric ALL and would advise to remove it or explicitly justify as a methodological analogy.

Response 6: Actually, it should be corrected as “to improve the sensitivity of detecting CNS involvement in hematologic malignancies”.

Comment 7: Please add a simple integrated diagnostic pathway (cytology/TdT + flow + molecular..) for common scenarios such as equivocal cytology, traumatic tap, and relapse surveillance.

Response 7: We have added two algorithms in the Conclusion section: one for routine clinical conditions and one specifically for cases with equivocal cytology. These algorithms are designed to address scenarios such as traumatic tap and relapse surveillance in the respective conditions.

Minor comments:

1. Typos: cable - capable, optimally performance - optimal performance, therefor - therefore, cytoplastic - cytoplasmic etc…

Response: these typos are corrected.

1. Standardize µL (avoid µl/µL mix), WBCs/µL vs cells/µL….

Response: It is all unified as “WBCs/µL”.

1. Please use consistent terminology (qPCR for DNA targets, RT-qPCR for RNA/fusion transcripts) rather than mixed qPCR/qRT-PCR phrasing

Response:  The terminology regarding qPCR is unified in the context. Thank you for pointing out this.

1. Reference formatting should be fixed

Response: The references were fixed and reformatted by Endnote.

1. Would be useful for the readership for the graphical abstract to explicitly mention low CSF volume and need for rapid processing, and the simple summary could maybe say that combining cytology + flow improves sensitivity in low-cellularity CSF

 Response: We have added two algorithms: one for routine conditions and one specifically for cases with equivocal cytology.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-written narrative review covering current approaches to detecting leukaemic blasts in CSF in paediatric ALL. The overview of cytomorphology, flow cytometry, and molecular approaches is accurate and appropriately referenced, and the manuscript clearly explains why sensitive CNS detection remains clinically important. The sections on flow cytometry and molecular MRD techniques are particularly useful, as they bring together literature that is often dispersed across pathology and clinical oncology sources. Overall, the manuscript reads as a solid synthesis of the field rather than an argument for a specific diagnostic approach.

I think the paper would benefit from a few targeted revisions before publication.

First, the manuscript occasionally reads as a catalogue of technologies rather than a clinical review. A short integrative section explaining how these methods are actually used together in practice would strengthen the paper. For example, when flow cytometry should be considered routine versus confirmatory, or where molecular assays realistically fit today, would help anchor the review in clinical decision-making.

Second, the conclusion mentions the need for standardised CSF evaluation protocols, which is an important point, but it currently feels a bit brief. Expanding this discussion, even just outlining what standardisation might look like in practice, would give the review a stronger closing argument. Specifically, the review could address the practical hurdles of sample stability, such as the time-sensitive nature of CSF processing or the use of stabilising reagents, which are often the biggest barriers to harmonising these protocols across different institutions.

Third, the sections on ddPCR, miRNA biomarkers, and NGS are informative but could be tightened. At present they read somewhat like parallel summaries of emerging technologies rather than a single forward-looking perspective. A clearer distinction between techniques that are clinically implemented, those used in specialised centres, and those still primarily research-based would improve the narrative.

Finally, there are a few minor language edits needed throughout (e.g., “cytoplastic” → “cytoplasmic”, “cable of detecting” → “capable of detecting”), but nothing substantial.

Overall, this is a clear and clinically relevant review that should be suitable for publication after revision.

Author Response

Comment 1:  First, the manuscript occasionally reads as a catalogue of technologies rather than a clinical review. A short integrative section explaining how these methods are actually used together in practice would strengthen the paper. For example, when flow cytometry should be considered routine versus confirmatory, or where molecular assays realistically fit today, would help anchor the review in clinical decision-making.

Response 1: Regards flow cytometry, this method is further modified in lines 168-188. When cytomorphology is confounded by clinical conditions, flow cytometry can be used as confirmatory. However,  the NCCN recommends routine use flow cytometry in combination with cytomorphology to detect CNS involvement. We added the sentence: “In line with these recommendations, many academic institutions have adopted this combined approach in routine clinical practice” and have revised the flow cytometry section for improved readability.

For qPCR, we added the sentence: “qPCR is often used clinically alongside flow cytometry to enhance the detection of occult CNS involvement to refine risk stratification and diagnose relapses”.

For ddPCR, we included: “Although the ddPCR technique remains in the clinical study phase, it is expected to be adopted into routine clinical practice in the near future due to its significant advantages.”

For NGS, we added: “NGS is increasingly utilized employed a high-sensitivity adjunctive tool for the detection and monitoring of CNS leukemia, particularly in cases where conventional methods, such as cytomorphology and flow cytometry, yield inconclusive results. Achievement of NGS-negative status in the CNS compartment has been strongly associated with superior event-free and overall survival. While still under clinical investigation, NGS is already being implemented in practice at several institutions”.

Comment 2: Second, the conclusion mentions the need for standardised CSF evaluation protocols, which is an important point, but it currently feels a bit brief. Expanding this discussion, even just outlining what standardisation might look like in practice, would give the review a stronger closing argument. Specifically, the review could address the practical hurdles of sample stability, such as the time-sensitive nature of CSF processing or the use of stabilising reagents, which are often the biggest barriers to harmonising these protocols across different institutions.

Response 2: We added two algorithms to direct clinicians on how to apply the protocol across different institutions.

Comment 3: Third, the sections on ddPCR, miRNA biomarkers, and NGS are informative but could be tightened. At present they read somewhat like parallel summaries of emerging technologies rather than a single forward-looking perspective. A clearer distinction between techniques that are clinically implemented, those used in specialised centres, and those still primarily research-based would improve the narrative.

Response 3: As stated above as response to the first comment.

Comment 4: Finally, there are a few minor language edits needed throughout (e.g., “cytoplastic” → “cytoplasmic”, “cable of detecting” → “capable of detecting”), but nothing substantial.

Response 4: These typos are corrected.