Phosphorylated S6 ribosomal protein expression by immunohistochemistry correlates with de novo donor-specific HLA antibodies in lung allograft recipients

https://doi.org/10.1016/j.healun.2021.06.021Get rights and content

Background

Per the ISHLT 2016 definition, a C4d-positive lung biopsy is required to meet criteria for definite antibody-mediated rejection (AMR). Unfortunately, C4d has poor sensitivity and specificity, and low inter-rater reliability. Phosphorylated S6 ribosomal protein (p-S6RP) expressed via the mTOR pathway has been shown to be a biomarker of AMR and correlates with donor-specific antibodies (DSA) in heart allografts. However, p-S6RP immunohistochemistry (IHC) in the setting of pulmonary AMR has yet to be evaluated. We sought to determine whether p-S6RP IHC performed on lung biopsies correlates with de novo DSA.

Methods

IHC for p-S6RP performed on 26 biopsies from lung transplant recipients with de novo HLA DSA (DSA+) and 28 biopsies from patients with no DSA (DSA-) were evaluated by 3 pathologists who independently scored the degree of alveolar macrophage and pneumocyte staining. Staining in ≥50% of the biopsy as determined by at least 2 pathologists was considered positive.

Results

Twenty-one (81%) DSA+ biopsies stained positive for p-S6RP in pneumocytes and 21 (81%) in macrophages. Six DSA- biopsies (21%) stained positive for p-S6RP in pneumocytes, 6 (21%) were positive in macrophages. Pneumocyte p-S6RP staining was 81% sensitive and 79% specific for DSA. Macrophage staining showed the same sensitivity and specificity but with lower inter-rater agreement (κ = 0.53 vs 0.68).

Conclusions

This study demonstrates a positive relationship between de novo DSA and p-S6RP expression in pneumocytes and macrophages using IHC. p-S6RP is relatively sensitive and specific, and has superior inter-rater reliability compared to C4d.

Introduction

Antibody-mediated rejection (AMR) following lung transplantation is a controversial entity. Prior to 2016, a published consensus definition of pulmonary AMR did not exist.1 Yet, studies had demonstrated a relationship between circulating anti-human leukocyte antigen (HLA) antibodies and acute rejection, lymphocytic bronchiolitis, and bronchiolitis obliterans syndrome .2, 3, 4 Furthermore, several studies had demonstrated poor outcomes in patients with antibodies to donor specific HLA molecules.5, 6, 7 Currently, few would dispute the existence of pulmonary AMR. Nevertheless, pulmonary AMR continues to be a challenging diagnosis to render.

Per the International Society for Heart and Lung Transplantation (ISHLT) 2016 criteria1, the diagnoses of definite clinical or subclinical pulmonary AMR require the presence of donor specific antibodies (DSA), histologic evidence of AMR, and immunopathologic evidence of AMR, in the form of C4d immunohistochemistry (IHC) or immunofluorescence. Unfortunately, histologic features of AMR are not specific8, 9, 10 and C4d IHC is insensitive, positive in only about 20% to 40% of suspected cases.11, 12, 13 C4d inter-rater reliability is unacceptably poor—kappa values reported in the literature range from 0.14 to 0.4.8,13 Furthermore, there is growing acceptance that graft injury may occur via complement-independent antibody-mediated mechanisms (ie, C4d-negative AMR).14, 15, 16 It is clear that with our current toolkit, the utility of transbronchial biopsy to confirm or exclude a diagnosis of AMR is lacking.

It has been demonstrated that DSA-HLA interactions signal endothelial cell proliferation via the mTOR pathway.17,18 One member of this signaling cascade is the S6 ribosomal protein, which becomes phosphorylated following ligation of either class I or class II HLA molecules on the surface of endothelial cells. A phospho-specific antibody to this protein (p-S6RP) has been shown to be an effective biomarker of cardiac AMR and DSA status in endomyocardial biopsies.19,20 However, the utility of p-S6RP IHC in the setting of pulmonary AMR has yet to be elucidated.

Preliminary studies from our laboratory have demonstrated a positive relationship between pneumocyte and alveolar macrophage expression of p-S6RP and the presence of circulating DSA. Therefore, we sought to determine whether p-S6RP IHC performed on lung allograft biopsies can distinguish patients with and without circulating DSA, and to assess inter-rater reliability. Although this does not directly address whether p-S6RP expression is indicative of definite AMR, it may be a useful alternative to C4d to increase certainty alongside clinical, serologic, and histologic data.

Section snippets

Patient and biopsy selection

The study was approved by the UCLA institutional review board and is in compliance with the ISHLT Ethics statement. Using our institution's lung transplant immunogenetics registry, we identified 26 patients who developed de novo HLA class I or class II DSA between January of 2007 and July of 2016 and retrieved archival tissue from the most contemporaneous transbronchial biopsy. We then selected biopsies from 28 sex and age matched controls with post-transplant serologic tests that were negative

Study cohort and demographics

The patient characteristics and pathologic findings are presented in Table 1. All 54 samples were forceps biopsies obtained via bronchoscopy as part of each patient's protocol post-transplant surveillance or in the setting of clinical concern for rejection or infection. The median interval between biopsy and HLA antibody testing was 2 days in the DSA+ group (0-82; interquartile range = 8) and 14 days in the DSA- group (0-81; interquartile range = 34).

DSA results

One DSA+ patient had only HLA class I DSA,

The significance of p-S6RP staining

The results confirm a positive relationship between p-S6RP expression in pneumocytes and macrophages and the presence of DSA. When a cutoff of 50% of the biopsy is applied, p-S6RP staining in pneumocytes was 81% sensitive and 79% specific for de novo HLA DSA. Macrophage staining showed equal sensitivity and specificity but had lower inter-rater reliability (κ = 0.53 vs 0.68).

The true specificity of p-S6RP IHC for AMR remains undetermined, because only HLA DSA testing was performed. It is

Conclusion

In summary, this study demonstrates a positive relationship between p-S6RP expression in pneumocytes and macrophages and de novo circulating DSA. p-S6RP is relatively sensitive and specific, and has superior inter-rater reliability compared to C4d. p-S6RP positivity may potentially serve as immunopathologic evidence of pulmonary AMR in cases where AMR is suspected but C4d is negative. Further investigation is needed to address whether p-S6RP IHC should be included as an additional criterion in

Disclosure statement

The authors have no conflicts of interest or financial relationships to disclose.

Authors contribution: Brian Cone – study design, interpretation of pathology, preparation of the manuscript; Jennifer Zhang – study design, interpretation of immunogenetics data, preparation of the manuscript; Rebecca Sosa – interpretation of immunogenetics data, preparation of the manuscript; Fiorella Calabrese – interpretation of pathology, preparation of the manuscript; Elaine Reed – study design, interpretation

Acknowledgements

Michael C. Fishbein, MD, for reviewing the immunohistochemistry.

This manuscript was supported in part by NIAID 1R01AI135201 to EFR and GAF.

References (38)

Cited by (0)

View full text