Positron Emission Tomography Can Support the Diagnosis of Dialysis-Related Amyloidosis

Round 1

Reviewer 1 Report

In this study, the authors suggest that PET-FDG is a potentially useful tool to support the clinical diagnosis of DRA. The study presents some limitations, which are, however, extensively discussed in the Discussion section. In addition, I have the following comments:

·         It is not clear if histology evidence of DRA is available in patients who had surgery for carpal tunnel. Please clarify.

·         The most controversial point is explaining why sites of amyloid deposition should be metabolically more active than the surrounding tissue. Inflammation is not a typical local features in sites of amyloid deposition in most systemic amyloidoses. The authors should cite reports indicating that b2-m deposition triggers local inflammation, or down-tone the considerations regarding local inflammation in DRA.

·         Given that this is a retrospective study, in which PET was performed to explore co-morbidities, could the authors exclude with high confidence other possible reasons for increased metabolic activity in joints of these patients, such as paraneoplastic phenomena? Please discuss.

·         page 7, line 305: (5ure 6) instead of figure 6

·         page 7, line 327 gold standard diagnosis instead of gold standard dialysis

Author Response

Thank you very much for your comments and advise to review our paper.

Please find the revised version of the paper (uploaded manuscript) and the answers to your comments, in the following lines:

Comments:
In this study, the authors suggest that PET-FDG is a potentially useful tool to support the clinical diagnosis of DRA. The study presents some limitations, which are, however, extensively discussed in the Discussion section. In addition, I have the following comments:

1.        It is not clear if histology evidence of DRA is available in patients who had surgery for carpal tunnel. Please clarify.

Answer: This was available in 5/7 cases; this is an importtant point and was better clarified as follows: . 

The following sentences were added to clarify this important issue.

in the introduction:
Its occurrence in patients on chronic dialysis is considered as linked to DRA, unless otherwise proved (36-37).

in the methods:

Carpal tunnel syndrome was considered a proxy for dialysis-related amyloidosis, excluding cases in which diagnosis antedated dialysis start. In keeping with the indications of the Japanese school, formal histological demonstration of Beta-2 amyloid deposits was not required for definition; however, DRA was confirmed in all cases for which morphologic analysis results were available (47).

in the results: 

In this cohort, seven patients underwent surgery for a carpal tunnel. Morphological confirmation of the presence of Beta2 amyloidosis in patients with carpal tunnel was available in 5/7 cases (in one case the intervention was too old to be tracked in the electronic records and the morphologic description was not available, and in another one it was performed in a different setting; both presented evidence of destructive spondilo-arthropathy, another acknowledged hallmark of DRA).

2. Comment:

·         The most controversial point is explaining why sites of amyloid deposition should be metabolically more active than the surrounding tissue. Inflammation is not a typical local features in sites of amyloid deposition in most systemic amyloidoses. The authors should cite reports indicating that b2-m deposition triggers local inflammation, or down-tone the considerations regarding local inflammation in DRA.

Thank you for this comment.

Indeed,local (as well as systemic) inflammation is a crucial element in the developement of DRA, for still non completely elucidated reasons.

This was further underlined in  the introduction, and several references were added. WE extensively modified the introduction to highlight this important point:

The main element in the pathogenesis of DRA is linked by Beta-2 microglobulin deposition; Beta-2 microglobulinis a polypeptide of 11.8 kilo-Daltons, is physiologically present on the surface of most nucleated cells and in most biological fluids, including blood, urine and synovial fluid; it forms the Beta chain of the human leukocyte antigen class I molecule (HLA-1), and is characterized by a beta pleated structure. The normal production rate has been estimated as between 2 and 2.5 mg/Kg/day, and its removal is mainly by glomerular filtration and tubular breakdown. As a consequence, its level increases in chronic kidney disease (CKD), as it is the case of most “middle molecules” whose toxicity explains many of the signs and symptoms of advanced CKD (17-23).

While virtually all organs and tissues may be involved, the deposits have a preference for joints, muscles and bone, and amyloid-related arthropathy continues to afflict patients on long-term dialysis (8-25).

Beta-2 microglobulin, in native or modified, with partially proteolysis, oxidation or conjugation with advanced glycation end products (AGEs), is the key universal component of the deposits, but it is not the only one, and the relative contribution of other substances, including serum amyloid component P, apolipoprotein E or glycosaminoglycand, is likely to play a role in the different clinical manifestations of the disease. One particular characteristic of DRA is its link with inflammation. At the local level, Beta-2 microglobulin, and even more the modified forms, including AGE-Beta-2 microglobulin, are capable of directly enhancing bone reabsorption via the induction of several lymphokynes, including interleukin 1 and 6 (IL-1, IL-6) and tumor necrosis factor (TNF). Furthermore, Beta-2 microglobulin may by itself act as a growth factor [18-21]. A specific affinity for collagen has been reported, in vivo and in vitro; this may explain the fact that the first clinical manifestations usually involve collagen-rich tissues, like joints (20-23).

The link with systemic inflammation is likewise important: systemic inflammation enhances Beta-2 microglobulin production, and DRA has been reported as more frequent in patients with inflammatory diseases, or treated with non-biocompatible dialysis membrane or non-ultrapure water, and indeed, improvement in water quality, together with the advances in the dialysis techniques and supplies, is considered as of pivotal importance in reducing the incidence, or at least delaying the clinical onset of DRA (22-35).

 3. Comment

·         Given that this is a retrospective study, in which PET was performed to explore co-morbidities, could the authors exclude with high confidence other possible reasons for increased metabolic activity in joints of these patients, such as paraneoplastic phenomena? Please discuss.

Answer, thank you for the keen comment: we added the following sentence in the limitations:

Furthermore, even if joint involvement suggestive of a metastatic disease was not found in dialysis patients tested in the presence of active neoplasia, a paraneoplastic arthropathy couldn’t be formally excluded. The interpretation that systemic inflammation favors Beta-2-amyloid deposition, which we consider more likely, needs formal confirmation in further studies.

4. errors

·         page 7, line 305: (5ure 6) instead of figure 6

·         page 7, line 327 gold standard diagnosis instead of gold standard dialysis

thanks, we corrected. 

Reviewer 2 Report

In this manuscript the authors investigated the potential of PET in the diagnosis of amyloid deposits in dialysis patients. The manuscript is appropriately organized and the experiments are carefully described. Please address some questions:

1) No supplementary information was found by the reviewer despite the fact that the authors refer several time to SI tables and figures in the text.
2) Data presentation is not clear: tables and figures are combined together (figure 3) as one figure without clear panel labelling. In addition, some figures (graph + table) are separated to different pages (pages 12 and 13, and pages 15 and 16) that makes difficult to follow the results.
3) Page 8 line 305 typo in the reference to the figure.
4) No Y-axes label on figs. 5 and 6.
5) Please add information regarding methods that have been used to observe scans presented on the fig. 1 for better readability of the figure.

Author Response

Thank you for the interest in our manuscript and for the time dedicated at improving it.

We are now answering to your requests, as follows:

1) No supplementary information was found by the reviewer despite the fact that the authors refer several time to SI tables and figures in the text.

This is absolutely true; we did not find this part of the manuscript on the site, even if we had uploaded it; since however, authors do not see the final formatting in the MDPI site we detected this problem only now. We therefore now included the supplemental material in the submitted manuscript

2) Data presentation is not clear: tables and figures are combined together (figure 3) as one figure without clear panel labelling. In addition, some figures (graph + table) are separated to different pages (pages 12 and 13, and pages 15 and 16) that makes difficult to follow the results.

4) No Y-axes label on figs. 5 and 6.

Once more, these are probably formatting problems: in our versions the figures are one per page, and seem rather clear; We'll tag this to the editors, to try to understand what happened in this regard.

We modified again the format, with one figure or table per page, to allow easier reading of the figures and tables.

Please be aware that the figures and the tables will be moved in the text in the final version, by the editorial team (which is sometimes quite a problem for the authors), and that in any case this is not the final layout.

3) Page 8 line 305 typo in the reference to the figure. OK (we hope we found this, since the original formatting may have been lost)

5) Please add information regarding methods that have been used to observe scans presented on the fig. 1 for better readability of the figure.

thanks for this precision, we added the following sentence: 

Whole-body emission scans acquired beginning 60 minutes after the intravenous injection of FDG (2.5MBq/kg). Images are combined with CT scan (voltage 130-150 kV, tube current 56-90mAs) The image reconstruction was performed as a 3D reconstruction of FOV iterative algorithms (50 cm, image matrix size: 128 × 128). All viewing of co-registered images was performed with dedicated Keosys imaging reading software.  

Round 2

Reviewer 2 Report

The reviewer believes that the manuscript has been significantly improved and now can be accepted for publication in JCM.