[Author's Reply to the Reviews] Usage of anti-NPM1 antibodies for diagnostics and research of acute myeloid leukemia: a systematic review

Letter to the Reviewers

I thank the reviewers for their detailed and helpful comments. I revised the manuscript, accordingly, taking into account all comments. In general, many paragraphs were revised to make the article more readable; unnecessary details and speculations were omitted. The usage of strict formal and scientific language was ensured. Specifically, the abstract is now more concise and meets the word count requirements. Especially in the introduction several citations were added to provide sources for any facts mentioned. Additionally the link of the antibody usage and more personalized medicines was established more clearly. In the methods part a time frame of studies admitted was added and  the selection process was described in more detail. All comments by the reviewers are addressed in italic on the original letters written by the reviewers. I will remain at your disposal for further questions and clarifications and look forward to hearing from you.

Best regards,

Maximilian Zuleeg

Responses to Review Nr. 1

MJ: It seems to me that the statement "If mutated, it [NPM1] is one of the most common molecular causes for acute myeloid leukemia (AML)." is imprecise, since it is noted in section 1.3 that it is not clear whether mutation of NPM1 actually leads to a higher growth rate and thus could be considered a causal cause of AML, so to speak, or whether AML is merely associated with mutation of NPM1. I would rather put it as in 1.3 that NPM1 mutations have been found in many AML patients and therefore represent a potential biomarker for AML disease.

Point was addressed. It is now specified that the NPM1 mutation is found in adult AML rather than causing it. 

MN: Somewhat lost in the detailed description of the applications of anti-NPM1 antibodies in general is what exactly is the analytical question of the study. As I understood it, the research question regards the comparison of anti-NPM1 antibodies with other diagnostic method for NPM1 mutations. This could be stated explicitly in the abstract.

The research goal is to systematically analyze the current usage and potentials of anti-NPM1 antibodies in diagnostics and research around AML. This is specified in the abstract. The details which are mentioned later were omitted and the abstract was cleaned up to be more concise. 

Introduction 

MN: The phrase "Additionally different publications show that NPM1 interacts with different tumor suppressors and proto-oncogenes, such as Adenosine diphosphate-Ribosylation Factor (ARF)." Implies several publications supporting the statement, but only one is cited. This contradiction could be easily resolved by a specific reference to the cited study or wording in the passive.

Citations were added.

MJ: References are missing for the first 5, fact-filled sentences of section 1.2. 

Citations were added 

MN: "In particular, for NPM1-mutated AML, there is a clear lack of development of more specific targeted therapies." I feel there is a lack of basis for this sentence from section 1.3. For example, in the paper cited shortly thereafter [16], it is noted that: "NPM1 mutation status did not influence treatment effectiveness".

Exactly that is the problem: the MRC AML 15 trial revealed that a personalized medicine did not work better on NPM1 mutated AML even though NPM1 mutated AML often presents itself with CD33 overexpression. It was therefore expected that the addition Gemtuzumab should increase treatment effectiveness. 

If NPM1 mutated AML patients need specific therapies, I miss the reference on this. Also, the sentence mentions target therapies, but the rest of the paper does not mention any paper that would have been about NPM1 mutant proteins as a therapy target. Rather, it is primarily about the role of anti-NPM1 antibodies in diagnostics, and thus the weakly documented reference to therapy seems inappropriate to me.

The point of the antibodies in research is the unveiling of the leukemic lineage so that AML cell populations may be targeted more effectively. The summary and conclusions part puts more emphasis on this point now. 

MN: Section 1.4 could be introduced in more detail, e.g. by not only writing "different types of antibodies", but by specifically distinguishing between polyclonal and monoclonal antibodies and mentioning that the antibodies in the class are again distinguished by the respective epitopes and whether they attack wild type or mutant. In its current version, the section is somewhat content-poor.

More content was added to the section. 

Results  

MN: In section 3.2, some references are missing in the second part. For example, in "Additionally, NPM1 mutations with aberrant cytoplasmic localization coupled with fluorescent proteins were detected by immunohistochemistry during different studies." several studies are mentioned, where it is not clear whether these are the studies mentioned in the following part of the paper or if further studies are meant. I also miss a reference for the description of the fluorescence detection of the antibodies, although it seems to be accurate to me.

Point specified, citation added. 

MN: "The scientific community however has been looking for additional methods for diagnostics and research of NPM1 mutations." This is quoted without reference, although it does not seem self-evident to me. Why do we actually need anti-NPM1 antibodies in AML diagnostics when we already have a well-functioning gold standard, PCR? As indicated in the discussion, it seems to me to be a matter of cost, speed, and better spatial resolution with which to better describe tumor heterogeneity. The disadvantages of PCR could be mentioned earlier in the introduction (with references) so that it is clearer why alternative methods are being sought in the first place.

The reasoning and a citation were added. 

MN: What is the "most common" (Section 3.3) mutation that Gruszka et al. targeted?

Point was addressed. 

MN: Is there a reason why Gruszka et al. used an Fc block protocol (section 3.3) and Tan et al. did not? And if so, is this information relevant to the comparison of the antibodies? This is not revisited in the paper and therefore should not be mentioned here if it has no further relevance.

Statement was removed. 

MN: I see a similar problem in section 3.4, as it is not clear what relevance the normalization of mean fluorescence intensity by Du Pisani et al. has compared to the non-normalized parameter.

The comparison was removed, now it is just stated that the normalized MFI was used to distinguish NPM1 mutational status. 

MN: Since in section 3.4 a chronological classification of the work of the two working groups is made (by which the use of different antibody kits is justified), it seems to me to make sense for the sake of clarity to include the year numbers also in the text, so that one does not have to take a look in the bibliography.

Year numbers were included. 

 MN: The abbreviation MFI for "mean fluorescence intensity" is used without being introduced before.

Abbreviation is now introduced. 

Discussion 

MN: The sentence "The defining factor of AML is the presence of 20% or more blasts in the bone marrow or in the peripheral blood." (Section 4.1) in my opinion fits better in the introduction, where AML has already been introduced as a clinical picture, but should in any case be backed up with a source.

Sentence was removed because in the case of NPM1 associated AML the presence of the NPM1 mutation itself is enough to diagnose AML. 

MN: "The other publications [...]" (section 4.1) is too vaguely worded. Since there are only 2 others, I would name them specifically.

Publications were mentioned specifically. 

MJ: The second to last paragraph in section 4.1 falls a bit out of the line of argument and belongs for me rather in the introduction. The fact that detection of NPM1 mutations can spare cytogenetic diagnostics is a good motivation for improving NPM1 mutation diagnostics, but in my eyes does not play a role for the central question of the paper, whether anti-NPM1 antibodies are suitable for mutation diagnostics.

Paragraph was removed for the sake of clarity since economic aspects of the antibody usage is then highlighted in the following paragraph. 

Conclusion 

MN: The theory that the two most promising antibodies of the considered research groups are no longer commercially available because the results were not reproducible, I consider to be speculative and to be irrelevant in the conclusion with regard to the research question.

Speculation was removed. 

Responses to Review Nr. 2

Major Issues 

It would be highly eligible if the inclusion and exclusion criteria would be further specified. There are several systematic flow charts, decision trees, etc., which allow the reader to fully understand the criteria used to determine the „publication´s reliability, completeness, and relevance“ (s. point 2.2 in paper).

Selection process was specified. 

It would also be necessary to state the period in which papers were included in the review. Currently, there is only the time of literature review stated and not the time frame in which studies were admitted.

Time frame was added. 

Minor Issues 

In the Discussion it would be desirable to specify on what ground the Abcam antibody should not be used, please include numbers.

Specification and numbers were added.