Mutational Landscape of Patients Referred for Elevated Hemoglobin Level

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

We are thankful to the reviewer for a thorough review of our study and historical perspective on the changing definition for PV. We appreciate the perspective and attempted to address all the suggestions in the revised manuscript.  

"Please see the attachment."

Author Response File: Author Response.docx

Reviewer 2 Report

The issue of JAK2 negative erythrocytosis is a common one for hematologists, and is an area that needs additional investigation.  The authors aimed to test for mutations in a panel of 40 myeloid genes in an unselected population of 529 patients with erythrocytosis.  Secondarily, they aimed to test for the same myeloid mutations as modifiers of JAK2-mutated erythrocytosis within the sample. 

The authors found that in the JAK2-negative cohort (n=471), 7% of the individuals harbored an ASCO Tier I/II myeloid mutation, which was similar in incidence and mutation distribution to clonal hematopoiesis in the general population. 

Major Issues:

1. There is no report the total number of Tier III variants (which would be of interest).  Agree with the author that Tier III variants in SH2B3 found in 4% of JAK2-negative patients are clinically relevant and need additional investigation given the known link with SH2B3 and erythrocytosis.  

2. I do not understand the relevance of the included table reviewing mutations in JAK2-negative erythrocytosis.  Most of the genes in the table were not included in the Oncomine panel used by the investigators.  

3. There is no mention of how the authors determined secondary erythrocytosis.  Would also be relevant to have a breakdown of the underlying causes of erythrocytosis and distribution of myeloid mutations, given reported increased incidence in smoking and COPD. 

4. Would be of interest to see the peripheral blood counts, serum erythropoietin level, and clinical characteristics of the JAK2-negative individuals with and without myeloid mutations. 

5. Oncomine myeloid panel does not sequence the entire JAK2 gene.  This should be mentioned specifically in the text.  Are there other publications looking at mutations outside of JAK2 exons 12-15?

Overall, I believe this is a very clinically relevant topic, but the current manuscript merely reports the prevalence of common myeloid mutations in individuals undergoing evaluation for erythrocytosis.  There is incomplete characterization of the individuals with erythrocytosis with a myeloid mutation versus those without.  The data regarding additional myeloid mutations found in JAK2 positive erythrocytosis seems extraneous to the manuscript as does the table reporting mutations (most familial) in JAK2 negative erythrocytosis.  

Author Response

We are thankful to the reviewer for a thorough review of our study. We appreciate the perspective and attempted to address the suggestions provided below in the revised manuscript.  

 "Please see the attachment."

Author Response File: Author Response.docx

Reviewer 3 Report

In the current study the authors investigated the incidence of myeloid mutations other than the common JAK2 mutations in unselected patients referred for elevated hemoglobin levels (using cut-off levels requested for the diagnosis of polycythemia vera by the 2016 WHO classification of myeloid neoplasms).

The topic is interesting and the study is overall well conducted and written.

Moreover, there are many limits and concerns:

1)      In the Introduction the authors underline the importance of high-throughput Next Generation Sequencing (NGS) analysis to study and eventually detect multiple gene mutations in neoplasms. On the other hand, it is important the role of highly sensitive quantitative PCR based methods for diagnosis purpose (as droplet digital PCR, for instance). I recommend to underline also this aspect.   

2)      In Materials and Methods, you stated about “patient selection criteria” and NGS assays (as per previously published methods); however, I strongly recommend to declare how MPN diagnosis (PV in particular) were done or excluded (according to 2016 WHO criteria?). In this case it is pivotal the role of bone marrow histopathology, which is not mentioned. BM biopsy was performed in all the patient? Please, clarify this aspect, which is crucial.

3)      The paragraph 2.3 “Literature review “does not make sense alone; I recommend to merge with the previous 2.2. as it constitutes a continuum to define the role of mutations.

4)      I suggest to implement Table 1 data with leukocytes, hematocrit, platelet count, EPO levels (if available). The latter is important for PV diagnosis as a minor criterion. Moreover, I strongly suggest to implement table 2 with the same data for JAK2 positive and JAK2 negative patients.  

5)      Secondary erythrocytosis develops secondary to disorders that cause tissue hypoxia, inappropriately increased erythropoietin production, or increased sensitivity to erythropoietin… What about your patients? This topic is crucial, must be clearly clarified. Moreover, you used gene tests to exclude congenital erythrocytosis? If not specify why.  

 

6)      Review of the literature (Table3) is not so pertinent. I suggest to delete. Polycythemia vera and secondary erythrocytosis (including also congenital erythrocytosis) are different diagnoses and this table in your study is quietly confusing.

 

7)      In discussion you stated that “Whether the combination of SFRS2, ASLX1 and IDH2 mutations, in the absence of a JAK2 mutation, is sufficient to confer the MPN phenotype or describes a unique MPN is uncertain and warrants further study. Both patients with JAK2-negative PV had typical features of myeloproliferative disorders including splenomegaly, and required repeated phlebotomy and hydroxyurea therapy”. You don’t mention bone marrow histopathology, a major criterion in MPN diagnosis! You may have a triple-negative myelofibrosis without splenomegaly…how you diagnose that?

Author Response

We are thankful to the reviewer for a thorough review of our study. We appreciate the perspective and attempted to address the suggestions provided below in the revised manuscript.  

"Please see the attachment." 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have responded to my comments appropriately but on line 206 the citation for strict blood count criteria should also reference the British Society of Haematology criteria independently of the WHO criteria as Wouters et al. did.

Reviewer 2 Report

Thank you for incorporating the feedback.  Y

Reviewer 3 Report

Dear Authors, 

after your revisions, the paper is accettable for publication.