Protein expression signature distinguishes early from late stage diffuse alveolar damage

In a recent study posted to medRxiv* preprint server, researchers identified a protein expression signature that can distinguish early diffuse alveolar damage (DAD) from late DAD in coronavirus disease 2019 (COVID-19) patients.

Background

To date, the COVID-19 pandemic has resulted in more than 646.5 million cases and 6.6 million deaths globally, despite the introduction of vaccines against the causal agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). DAD is a primary histological characteristic of fatal COVID-19. Nevertheless, the molecular and cellular pathways underlying the progression from the early exudative phase of DAD (EDAD) to the late organizing phase (ODAD) are unclear.

About the study

In the present study, researchers applied digital spatial profiling (DSP) to identify differences in protein expression changes between regions identified as EDAD or ODAD in a cohort of COVID-19 autopsy cases in the United Kingdom. Lung tissue specimens from 27 deceased COVID-19 patients were collected from biobanks at the Imperial College London, the University of Edinburgh, and the University of Newcastle.

Subjects were selected based on the histological evidence of DAD without additional complications related to pneumonia or cardiac failure. For spatial profiling, 4 μm-thick formalin-fixed paraffin-embedded (FFPE) sections were used. Slides were stained with cluster of differentiation 68 (CD68) and CD3 as morphological markers and with a panel of 68 oligonucleotide-conjugated antibodies.

A pathologist identified the regions of interest (ROIs) based on hematoxylin and eosin (H & E)-stained serial sections. Regions of approximately 600 μm² reflective of EDAD, ODAD, or mixed (MDAD) phenotype were identified. Digital counts indicative of protein expression were analyzed using dimensionality reduction, class discrimination methods, and linear mixed modeling.

Findings

Most selected subjects were male (22) and Caucasian (20); seven were Black, Asian, or ethnic minority. The researchers examined 194 ROIs, which included 122 regions with EDAD, 22 with MDAD, and 50 with ODAD. Principal component analysis (PCA) revealed the segregation of each type of DAD, with PC1 and PC2 accounting for 41.4% of the variance.

Next, the authors applied partial least-squares (PLS) regression to identify those responsible for group segregation. Eight proteins were identified that largely reproduced what was noted with PCA and were used for classifying ROIs in a PLS linear discriminate analysis with leave-one-patient-out (LOPO) cross-validation. This yielded 93% predictive accuracy for EDAD and 80% for ODAD.

MDAD ROIs were misclassified, likely reflecting the transitional nature and heterogeneity of the pathology. Linear mixed modeling identified 11 targets that distinguish EDAD from ODAD. Similarly, MDAD was distinguished from ODAD, whereas proteins did not significantly differ between MDAD and EDAD.

Taken together, the findings suggested that a protein expression signature comprising granzyme B (GZMB), arginase 1 (ARG1), CD127, indoleamine 2,3-dioxygenase 1 (IDO1), proline-rich Akt substrate of 40 kDa phosphorylated at T246 [phospho-PRAS40 (T246)], Ki67, and V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) could distinguish EDAD ROIs from ODAD ROIs in COVID-19 patients.

Conclusions

The researchers reported a core protein expression signature distinguishing the early phase of DAD from the late stages. The identified proteins have essential functions in immune and inflammatory responses but have not been previously investigated in relation to the progression of DAD. The study’s limitations include the small cohort precluding sub-group analysis based on gender, age, COVID-19 duration, and place of death.

Notably, DSP cannot distinguish between many cells with low expression of the target(s) and a few cells with high expression. Moreover, it could not be ruled out whether patients had different forms of DAD in regions not sampled in this study. Further investigations are warranted to determine the immunotherapeutic potential of these proteins in modulating DAD progression. 

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.