Measurable Residual Disease in Acute Myeloid Leukemia Using Flow Cytometry: A Review of Where We Are and Where We Are Going

Round 1

Reviewer 1 Report

This review article provides an insightful perspective on the measurable residual disease (MRD) assessment in AML and the associated prognostic values. The article is well structured and the discussion covers the major areas relevant to the topic. The literature citations are relatively up-to-date, but it would be better to include some from 2020. Below are some major comments:

1. The authors should elaborate on the different choices of cell surface marker used for measurement of MRD and their prognostic values, which should be summarized in a table.
2. While the sensitivity issue was commented in the molecular methods such as qPCR and NGS, it has not been discussed in the flow cytometric methods.
3. How the sample quality and the preparation method affect the detection of MRD by flow cytometry should be discussed.
4. The authors stated that 60% of AML patients will have a suitable molecular marker for PCR-based MRD analysis, which is based on the calculation from the patients with fusion genes or NPM1 mutation. In fact, there are many other studies using other combinations of genetic markers, e.g. DNMT3A, IDH1/2 mutations, to monitor MRD. Therefore, it seems that the 60% claim by the authors has under-estimated the applicability of PCR-based methods.
5. The column “strategy” in Table 1 is confusing. I suggest the authors construct (i) a table that compares the advantages and disadvantages of different methods in MRD analysis, e.g. qPCR, NGS, MFC-LAIP, MFC-DfN, etc., and (ii) a table focuses on the summary of various MRD studies using MFC method (with a column for sample type and surface marker choice) and the associated prognostic outcomes.
6. In Chapter 5, the authors cited that including the parameter of LSC can further improve the prognosis by comparing the MRDneg/LSCneg with the MRDpos/LSCpos samples. However, such claim should be demonstrated by the comparison between MRDpos/LSCpos to MRDpos/LSCneg samples.

Minor comments:

1. Line 235, clarify the “two methods”.
2. Line 239, what are the surface markers measured by the “10 colour MFC”.

Author Response

Reviewer 1 comments

This review article provides an insightful perspective on the measurable residual disease (MRD) assessment in AML and the associated prognostic values. The article is well structured and the discussion covers the major areas relevant to the topic. The literature citations are relatively up-to-date, but it would be better to include some from 2020. Below are some major comments:

1. The authors should elaborate on the different choices of cell surface marker used for measurement of MRD and their prognostic values, which should be summarized in a table.

Recommendations for cell surface markers are now outlined in paragraph from line 180. We have included a table on methodologies of MFC for MRD assessment (Table 2) and included some of the surface markers used in the assessment (Not all papers specified the exact combination of markers). Markers associated with adverse prognosis are included in lines 111-115. We have also included the surface markers recommended by the ELN to monitor MRD (line 185)

2. While the sensitivity issue was commented in the molecular methods such as qPCR and NGS, it has not been discussed in the flow cytometric methods.

The issues regarding MFC sensitivity has been added (from line 176)

3. How the sample quality and the preparation method affect the detection of MRD by flow cytometry should be discussed.

The importance of a good quality sample, interpretation and use of standardised protocols has been added (from line 190). The issues of viable events and cutoffs is mentioned from line 195 and reduced sensitivity with significant haemodilution of the marrow from line 199.

4. The authors stated that 60% of AML patients will have a suitable molecular marker for PCR-based MRD analysis, which is based on the calculation from the patients with fusion genes or NPM1 mutation. In fact, there are many other studies using other combinations of genetic markers, e.g. DNMT3A, IDH1/2 mutations, to monitor MRD. Therefore, it seems that the 60% claim by the authors has under-estimated the applicability of PCR-based methods.

This statement has been qualified that 60% corresponds to widely available qPCR methods but that MRD techniques for further molecular targets are being developed and we would expect this to increase (line 125). We have discussed the issue of using DNMT3A and FLT3 for MRD analysis in the paragraph starting line 137.

5. The column “strategy” in Table 1 is confusing. I suggest the authors construct (i) a table that compares the advantages and disadvantages of different methods in MRD analysis, e.g. qPCR, NGS, MFC-LAIP, MFC-DfN, etc., and (ii) a table focuses on the summary of various MRD studies using MFC method (with a column for sample type and surface marker choice) and the associated prognostic outcomes.

We have now included two tables to make it simplified – table 1 comparing MRD methodologies (MFC vs PCR vs NGS) and another (Table 2) summarising MFC studies using different MFC techniques as you have suggested

6. In Chapter 5, the authors cited that including the parameter of LSC can further improve the prognosis by comparing the MRDneg/LSCneg with the MRDpos/LSCpos samples. However, such claim should be demonstrated by the comparison between MRDpos/LSCpos to MRDpos/LSCneg samples.

We have added results from both the MRDneg/LSCpos group, which is worse than both MRDneg/LSCneg and MRDpos/LSCneg, from line 299. This shows the added prognostic information LSC interrogation gives to the overall MRD analysis.

Minor comments:

1. Line 235, clarify the “two methods”.

This has been clarified – studies using MFC combined with either NGS or qPCR (from line 244)

1. Line 239, what are the surface markers measured by the “10 colour MFC”.

We have changed this to 3 tube, 10 colour flow cytometry, using surface markers approximately consistent with the ELN MRD recommendations (see above)

See attached file

Author Response File: Author Response.pdf

Reviewer 2 Report

In the paper “Measurable residual disease in acute myeloid leukemia using flow cytometry: a review of where we are and where we are going” by Dix and coworkers is submitted to Journal of Clinical Medicine. The paper describes the use of measurable residual disease (MRD) in acute myeloid leukemia (AML). The authors describe how MRD is being incorporated into prognostic models and can be a powerful predictor of relapse. They focus mainly on multiparametric flow cytometry (MFC) for identification of MRD.

The paper is well written, and the topic is interesting, although other papers also have discussed these features recently.

In general, I miss two things:

1. A greater discussion and comparation of using molecular markers compared to MFC in detecting flow. Although I am aware all patients do not have molecular markers which can be used, I think a better discussion and comparison could be highlighted in this paper, parable also including a table. This is also investigated in the cited paper by Jongen-Lavrencic and coworkers.

2. A greater discussion of the advantage of detecting MRD. Although the use of MRD is well established in ALL and APL, this is still not so straight forward for AML. I think a better discussion of these features will improve this manuscript.

Minor:

Name of genes and fusion gens should be written in italic.

Author Response

Reviewer 2 comments

The paper is well written, and the topic is interesting, although other papers also have discussed these features recently.

In general, I miss two things:

1. A greater discussion and comparation of using molecular markers compared to MFC in detecting flow. Although I am aware all patients do not have molecular markers which can be used, I think a better discussion and comparison could be highlighted in this paper, parable also including a table. This is also investigated in the cited paper by Jongen-Lavrencic and coworkers.

Further discussion comparing concordance of MFC and molecular (PCR and NGS) analyses has been added from line 241. A table comparing molecular methods and flow has also been added (Table 1), including papers that have used both MFC and molecular methods.

1. A greater discussion of the advantage of detecting MRD. Although the use of MRD is well established in ALL and APL, this is still not so straight forward for AML. I think a better discussion of these features will improve this manuscript.

Discussion about how the difficulties of incorporating this information into treatment and outcome situations has been expanded (paragraph starting from line 264).

Minor: Name of genes and fusion gens should be written in italic.

Genes and Fusion gene names have been changed to italic.

See attached file

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

My main concerns have been addressed. The paper is suitable for publication.