Increased Expression of CD169 on Monocytes in Adult-Onset Kikuchi–Fujimoto Disease

Round 1

Reviewer 1 Report

The manuscript by Giacomo Malipiero et al describes two cases of Kikuchi-Fujimoto disease diagnosed in young adult men of Indian origin, years after their migration to Italia. Each case is reported with much detail and a long but exiting discussion argues for the use of CD169 cell surface marker at the surface of circulating monocytes as a potential biomarker that would facilitate disease recognition. The usefulness of such a marker remains to be validated.

As CD169 expression on monocytes is the central feature of the manuscript, it would be useful to provide a precise description of the flow method used to detect it. The gating strategy of each of the two cases might be illustrated.

Is CD169 expressed on classical, intermediate and non-classical monocyte subsets or only on classical monocytes? As MFI was calculated, results might be shown. Together, figures showing in more details how CD169+ monocytes were identified and quantifies are needed.

Even though there is no cohort analysis as the disease is rare, another flaw of the manuscript is the lack of positive controls. The authors demonstrate but do not show (and should do it) the absence of / low level of labelling of healthy donor monocytes with the selected antibody.  They did not identify other pathological situations that could induce or not CD169 expression on monocytes (positive controls) such as viral infections, autoimmune / inflammatory disease or lymphomas, such positive controls would be useful

Did the authors explore CD169 expression on CD68+ histiocytes?

What does “activated” monocyte mean (page 9, line 341)?

Line 351, the suggestion is to remove any mention of "specificity" as there is no chance that this test will be really specific of a given disease or subtype and, as mentioned, there is no demonstration that CD169 segregates inflammatory from malignant lymphoproliferation.

Line 359, there is no proof that CD169 is involved in disease pathogenesis. The suggestion is that it could be a biomarker that helps to suspect the diagnosis, pending its absence of expression in malignant lymphoma that needs to be checked.

Author Response

As CD169 expression on monocytes is the central feature of the manuscript, it would be useful to provide a precise description of the flow method used to detect it. The gating strategy of each of the two cases might be illustrated.

As suggested, we revised the description of the flow cytometry protocol and gating strategy and modified the title of the paragraph (as also required by the other Reviewer). Figure 1 was updated to include these pieces of information.

 

Is CD169 expressed on classical, intermediate and non-classical monocytes subsets or only on classical monocytes? As MFI was calculated results might be shown. Together, figures showing in more details how monocytes were identified and quantified are needed.

It is known from literature that CD169 expression can be upregulated in different monocyte subsets. Namely, CD169 has been also explored in classical and intermediate monocytes in a very recent study by Sakamura et al. on pediatric KFD patients, that we discussed in the revised version of our manuscript and added to the references. According to Sakamura et al., CD169 expression was upregulated in both subsets of monocytes. Unfortunately, our gating strategy did not sub-classify monocytes (classic-intermediate, non-classic). Anyway, we provided additional information in the text (Methods) and Figure 1 to show how monocytes were gated and how MFI was calculated.

 

Even though there is no cohort analysis as the disease is rare, another flaw of the manuscript is the lack of positive controls. The authors demonstrate but do not show (and should do it) the absence of/low level of labeling of healthy donor monocytes with the selected antibody. They did not identify other pathological situations that could induce or not CD169 expression on monocytes (positive controls) such as viral infections, autoimmune/inflammatory disease or lymphomas, such positive control would be useful.

Thank you for this valuable suggestion. To provide a reference range for evaluating MFI results in our 2 KFD patients, we relied on the reference range and thresholds provided by the work of Bedin et al. [20] and also verified the validity of this range in a group of healthy control subjects (as specified in Methods under “Myeloid activation test”). To address Reviewer’s issue and improve the validity of this reference range, we also provide in the revised version additional analyses in a positive control cohort of COVID-19 patients, a condition associated with CD169 over-expression on monocytes. Unfortunately, data from treatment-naïve lymphoma patients were not available and could not be added to the analysis. Results of this analysis are reported in Methods section and displayed in Figure 1.

 

Did the authors explore CD169 expression on CD68+ histiocytes?

Indeed, CD169 status in KFD CD68⁺-histiocytes would be of great value to explore an ontogenetic and pathophysiological link between circulating monocytes and tissue histiocytes. Unfortunately, we could not perform such an analysis since an anti-CD169 antibody was not available for tissue immunochemistry at our Institution.

 

What does activated monocyte mean?

 “Activated monocyte” generally indicate that monocytes express activation markers on their surface upon stimulation, namely HLA-DR, co-stimulatory receptors (and CD169 in our cases). We admit that the term is operatively not well defined in medical literature and besides the research field and removed it from the manuscript. Anyway, we added a brief paragraph in the discussion to describe CD169 function and why it is considered a monocyte activation marker.

 

Line 351, the suggestion is to remove any mention for specificity as there is no chance that this test will be really specific of a given disease or subtype and as mentioned, there is no demonstration that CD169 segregates inflammatory from malignant lymphoproliferation.

We totally agree with the Reviewer. While we acknowledge that the specificity of CD169 expression for KFD is relatively low and should not be used as a diagnostic marker per se, we would also like to point out that one of the main clinical differential diagnosis of KFD is malignant lymphoproliferative disease, for which increased CD169 expression on circulating monocytes has not been clearly reported in medical literature. While CD169 expression in lymphoma patients will be one of our main research topic for the next future, we believe that CD169 expression on KFD and other rare inflammatory diseases is worth the effort, as long as evidence is not yet available. We revised the discussion in order to clarify this issue and give a better perception of the limits of this test.

 

Line 359, there is no proof that CD169 is involved in disease pathogenesis. The suggestion is that it could be a biomarker that helps to suspect the diagnosis, pending its absence of expression in malignant lymphoma  that needs to be checked.

Indeed we did not provide any evidence that CD169 is involved in disease pathogenesis and, by this time, we suggest that it could be only a potential diagnostic/prognostic marker, proven that its expression is not increased in lymphoma or other malignant diseases. Anyway, generally speaking, there is some evidence that CD169-histiocytes are functionally involved in CD8+ T-cells activation, as also reported in the discussion. We modified the manuscript discussion in order to make the point clearer and briefly summarize the known functions of CD169 on mononuclear cells.

Reviewer 2 Report

I read the manuscript, hemato-2409939, which is a case report of two cases diagnosed as Kikuchi-Fujimoto disease (KFD).  You suggest the increased levels of CD169 expression on the surface of monocyte in peripheral blood to be used as a reference of diagnosis and disease monitoring of KFD.

 

Major

 

1.     The biggest problem of KFD is that it is difficult to distinguish from other inflammatory disorders because it has no unique finding as you mention in Abstract.  Because the increased level of CD169 expression on the surface of monocytes in the peripheral blood of KFD has been reported before (Clinical Immunology 250 (2023) 109329) and there are reports of increased levels of CD169 expression in other inflammatory disorders, I do not see the value of validating CD169 expression level to be used in diagnosis.  It would have been valuable to investigate if it is useful in monitoring the disease, or in another words if it is associated with the severity of the disease. 

2.     You also need to present the figure showing the flow cytometry of CD169 expression in case 2 also.

3.     I suggest you to simplify the explanations and the descriptions throughout the entire manuscript.  For example, the first three paragraphs of Discussion explain KFD too detailed as if it were a review article.  I suggest you to focus on CD169 related matters in one paragraph.

 

Minor

1.     “2.1 Myleloid Activation Test” in Materials and Methods is better titled “CD169 expression”

2.     What is the control in determining the increased level of CD169 expression on the surface of monocytes?  Is it of healthy donors? Or is it of patients of other disorders?

Author Response

Major Point 1

We agree we the Reviewer and revised the discussion in order to clarify this issue and give a better perception of the limits of the test. While we acknowledge that the specificity of CD169 expression for KFD is relatively low and should not be used as a diagnostic marker per se, we would also like to point out that one of the main clinical differential diagnosis of KFD is lymphoma, for which increased CD169 expression on circulating monocytes has not been clearly reported in medical literature. In the suspicion of lymphoma/leukemia, CD169 hyper-expression on monocytes, reflecting innate immunity activation, might prove to be a useful biomarker that help differentiate at least benign versus malignant lymphoproliferation. Indeed, we acknowledge that additional validation is required and CD169 expression in lymphoma patients should be systematically investigated. Moreover, follow-up data were not available to monitor disease response to therapy and the low number of patients prevented us from establishing a correlation between disease severity and CD169-MFI. We discussed these limitations in the discussion.

We noticed that CD169 expression on monocytes in KFD was recently reported by Sakamura et al. (Clinical Immunolgy 250, 2023). We added this study to the discussion. Anyway we mark that our cases report on two adult patients, one of whom also affected by an autoimmune inflammatory condition. Moreover, our flow cytometry protocol is different in that we normalized CD169 expression on monocytes to lymphocytes expression in order to provide a standardized measurement. That allowed us to compare our results to those of Bougoin et al. [20-21].

We did not proposed this manuscript as a validation study of CD169 expression in KFD diagnosis nor to obtain diagnostic specificity/sensitivity values. As a case report, we wanted to provide useful information about the expression of this marker in a rare condition and discuss the potential involvement of the innate immune system in this benign lymphoproliferative and potentially autoimmune condition. We admit that most of the discussion is speculative but nonetheless we believe that case reports like these  can also provide useful hints for future systematic clinical research.

 

Major Point 2

Figure 1 was enriched with CD169 expression analysis of case 2. Eventually, Figure 1 includes monocytes gating strategy, results in healthy individuals and in a cohort of COVID-19 patients and results in our 2 cases.

 

Major Point 3

As suggested, we simplified the discussion to include the major revisions that were required. Paragraphs of the text that were considered not-contributory to the discussion were deleted and replaced by discussion of the new issues that emerged during the revision process: a brief paragraph on CD169 pathophysiology was included, the work of Sakamura et al. was added and specificity limitations of the CD169 assays were highlighted.

 

Minor Point 1

We agree that “myeloid activation test” is a misnomer and changed the title of the paragraph to “Flow cytometry evaluation of monocyte CD169 expression”, as suggested. “Activated monocyte” generally indicate that monocytes express activation markers on their surface upon stimulation, namely HLA-DR, co-stimulatory receptors (and CD169 in our cases). We admit that the term is operatively not well defined in medical literature and besides the research field and removed it from the manuscript. Anyway, we added a brief paragraph in the discussion to describe CD169 function and why it is considered a monocyte activation marker.

 

Minor point 2

Thank you for this valuable suggestion. To provide a reference range for evaluating MFI results in our 2 KFD patients, we relied on the reference range and thresholds provided by the work of Bedinet al. [20] and also verified the validity of this range in a group of healthy control subjects (as specified in Methods under “Myeloid activation test”). To address Reviewer’s issue and improve the validity of this reference range, we also provide in the revised version additional analyses in a positive control cohort of COVID-19 patients, a condition associated with CD169 over-expression on monocytes. Unfortunately, data from treatment-naïve lymphoma patients were not available and could not be added to the analysis. Results of this analysis are reported in Methods section and displayed in Figure 1.

Round 2

Reviewer 1 Report

The authors took into account the suggestions made in the first review. No additional suggestion.

Author Response

Thank for your interest and your time.  

Reviewer 2 Report

The main problem with KFD is that it is difficult to distinguish it from other inflammatory diseases due to the lack of characteristic findings. However, since the expression of CD169 on the surface of monocytes has already been reported to be found in other inflammatory diseases, we do not consider it to be of diagnostic value.

Author Response

We have accepted the reviewers's recommendations and we have removed all inference to the diagnostic value of the test.