The Current State of Donor-Derived Cell-Free DNA Use in Allograft Monitoring in Kidney Transplantation

Round 1

Reviewer 1 Report

Thank you for giving me the opportunity to review this manuscript. Transplantation from hepatitis C positive donors to hepatitis C negative donors has not been a novel issue. However, there's not been much data on comparison of hepatitis C positive donors to hepatitis C negative recipients versus hepatitis C positive donors to hepatitis C positive recipients due to the fact that both of these recipients are different from each other. Hepatitis C positive recipients are usually with chronic hepatitis C virus for a long period of time ( with plus/minus treatment history)  vs hepatitis C negative recipients who received a kidney from hepatitis C positive donor and develop +/- acute hepatitis C . From medical standpoint,  I don't understand from the introduction that how can we compare these two groups and what benefit can be achieved. 

Also previously there has been a lot of literature on hepatitis C negative recipients receiving the kidneys from hepatitis C positive donors w.r.t hepatitis C medications DAA because some of the centers get DAA as prophylaxis therapy whereas other centers get the DAA  once recipients develop hepatitis C viremia. And I don't see any comment in the script about this major fact which can in turn overall impact the graft survival or overall recipient survival.

I think we need to clarify the hypothesis of this study and the rationale to build that hypothesis. There are too many things that are addressed here as hypothesis /objectives and there is less understanding about the rationale to create this hypothesis and create this comparison

And also, the researchers have tried to focus on COVID pandemic era which I don't understand that how could affect the transplantation from hepatitis C positive donors to hepatitis C positive recipients or hepatitis C positive donors to hepatitis C negative recipients other than the generalized reduction in the volume of the transplantation. I think we need better clarification of all these objectives.

 Figure one has shown different percentages and to my understanding researchers are trying to create a percentage of the two groups from the total number of transplants that were picked up for the study. We need to clarify the breakup of these percentages.

  Appreciate the hard work of the researchers but need some clarifications and strong statements.

Author Response

I apologize for the confusion, but it seems as though this may be a review for a different paper as it describes transplantation from hepatitis C positive donors. This topic was not covered in our submission: The current state of donor-derived cell-free DNA use in allograft monitoring in kidney transplantation.

If there is something I can do to help with this confusion, please let me know.

Thank you.

Reviewer 2 Report

Background 

This paper analyses the current state of Donor-Derived Cell-Free DNA (ddcfDNA) and its role as a non-invasive genetic marker of graft injury and rejection. In the first part of the paper, a detailed history of ddcfDNA’s development until its actual practical use is well exposed. 

In US, there are three commercially available ddcfDNA tests for clinical use showing a better negative predictive value for AMCR compared to TCR (Table 1); we suggest moving “figure 1” and the explanation of why ddcfDNA is more sensitive for AMCR (pages 7-8) in the same section of table 1, to give reason of data displayed in the table. 

Current uses and limitations 

The primary use of ddcfDNA in clinical practice is to rule out rejection if suspected and avoid potentially unnecessary biopsy; detection of ddcfDNA has a high negative predictive value rather than positive, because its levels might be modified by other non-rejection pathologies such as bacterial and viral infections (BKV nephropathy) or high BMI levels. 

The concept is clear; it would be useful to make a revision of any other factor (described in literature) that can modify ddcfDNA levels rather than rejection, and the pathophysiology at its basis. These data could be reported in a table. This would make this section more complete and would help clinicians in their decision-making at the time of the application of the test. 

Future Directions

Main concern is to define the optimal use of ddcfDNA levels by:

-       Monitoring the response to treatment for chronic rejection

-       Facilitating the transition of immunosuppression regimens

-       Identifying states of graft immune quiescence

Each of these points is well developed and gives an overview of the possible applications of the test. 

Referee’s conclusion

This paper offers a clear view of the experience on ddcfDNA up until now, giving a critical and accurate description of the negative predictive value of this test and of its possible utility in clinical practice.  We suggest adding more information in section “limitations to current use” through a more accurate examination of various factors that can modify ddcfDNA levels. 

Author Response

Reviewer Comments and Suggestions for Authors

“Background 

This paper analyses the current state of Donor-Derived Cell-Free DNA (ddcfDNA) and its role as a non-invasive genetic marker of graft injury and rejection. In the first part of the paper, a detailed history of ddcfDNA’s development until its actual practical use is well exposed. 

In US, there are three commercially available ddcfDNA tests for clinical use showing a better negative predictive value for AMCR compared to TCR (Table 1); we suggest moving “figure 1” and the explanation of why ddcfDNA is more sensitive for AMCR (pages 7-8) in the same section of table 1, to give reason of data displayed in the table. 

1. Figure 1 with explanation for ddcfDNA sensitivity to AMCR has been moved to follow Table 1. Further explanation is provided with possible mechanisms that influence the test’s sensitivity to AMCR.

Current uses and limitations 

The primary use of ddcfDNA in clinical practice is to rule out rejection if suspected and avoid potentially unnecessary biopsy; detection of ddcfDNA has a high negative predictive value rather than positive, because its levels might be modified by other non-rejection pathologies such as bacterial and viral infections (BKV nephropathy) or high BMI levels. 

The concept is clear; it would be useful to make a revision of any other factor (described in literature) that can modify ddcfDNA levels rather than rejection, and the pathophysiology at its basis. These data could be reported in a table. This would make this section more complete and would help clinicians in their decision-making at the time of the application of the test. 

1. We reviewed the “limitations to current use” portion and agree that non-rejection mediated processes need to be further discussed.
   1. Infection and ddcfDNA
      1. We conducted a literature review regarding the influence of BK viremia on ddcfDNA levels. Based on current published data, no definitive processes have been described that precipitate increased ddcfDNA in BK viremia. However, cellular injury seems to be necessary in most patients with elevation in ddcfDNA.
      2. New addition to manuscript – Lines 173 to 186
         1. “In a retrospective study from 2020, Goussous et al. illustrate instances of elevations in ddcfDNA with concomitant BK viremia, however the incidence was not high enough for statistical significance [27]. Kant et al. further investigated the effect of BK viremia and simultaneous rejection on the levels of ddcfDNA. Though positive correlation was demonstrated between BK viremia and ddcfDNA levels from a small cohort of 10 patients, there was no significant effect of rejection in patients with BK viremia on ddcfDNA level [28]. While cellular injury is deemed to be the perpetrator behind elevation in ddcfDNA, there is currently not enough research to delineate the pathophysiology behind nonrejection mediated insults that effect ddcfDNA levels.”
      3. Obesity and ddcfDNA
         1. There is scant number of studies that have explored the effect of obesity in ddcfDNA levels in patients with or without rejection. Our reference includes a retrospective study by Sureshkumar in 2020 (see below). Without doubt, more modifiable patient factors need to be evaluated. Furthermore, the pathophysiology behind these mechanisms are not well defined at this time and would require further investigation.
         2. New addition to manuscript – Lines 192 to 196
            1. Sureshkumar et al. in 2020 conducted a retrospective study that evaluated the influence of obesity on ddcfDNA levels. While morbid obesity is noted to have an inverse relationship with ddcfDNA levels ( R = 0.29, R² = 0.089, P = 0.001), the interaction with increased adipose tissue and ddcfDNA is not well understood.

Future Directions

Main concern is to define the optimal use of ddcfDNA levels by:

• Monitoring the response to treatment for chronic rejection
• Facilitating the transition of immunosuppression regimens
• Identifying states of graft immune quiescence

Each of these points is well developed and gives an overview of the possible applications of the test. 

1. Thank you for your comments. No further action requested.

Referee’s conclusion

This paper offers a clear view of the experience on ddcfDNA up until now, giving a critical and accurate description of the negative predictive value of this test and of its possible utility in clinical practice.  We suggest adding more information in section “limitations to current use” through a more accurate examination of various factors that can modify ddcfDNA levels.”

1. Please see responses under “Current use and limitations”.

We appreciate your comments and providing us the opportunity to bolster our review paper under your guidance.