key: cord-0724016-oh3pmz0w
authors: Kritselis, Michael; Yambayev, Ilyas; Prilutskiy, Andrey; Shevtsov, Artem; Vadlamudi, Charitha; Zheng, Hanqiao; Elsadwai, Murad; Ma, Lina; Aniskovich, Emily; Kataria, Yachana; Higgins, Sara; Sarita-Reyes, Carmen; Zuo, Tao; Zhao, Qing; Quillen, Karen; Burks, Eric J.
title: Distinctive Pseudopalisaded Histiocytic Hyperplasia Characterizes the Transition of Exudative to Proliferative Phase of Diffuse Alveolar Damage in Patients Dying of COVID-19
date: 2021-07-14
journal: Hum Pathol
DOI: 10.1016/j.humpath.2021.06.008
sha: 5989f24ff978933a709ca651a3cc382da42b75bc
doc_id: 724016
cord_uid: oh3pmz0w

OBJECTIVES: Severe COVID-19 results in a glucocorticoid responsive form of acute respiratory distress (ARDS)/diffuse alveolar damage (DAD). Herein we compare the immunopathology of lung tissue procured at autopsy in patients dying of SARS-CoV-2 with those dying of DAD prior to the COVID-19 pandemic. METHODS: Autopsy gross and microscopic features stratified by duration of illness in twelve patients who tested positive for SARS-CoV-2 viral RNA as well as seven patients dying of DAD prior to the COVID-19 pandemic were evaluated with multiplex (5-plex: CD4, CD8, CD68, CD20, AE1/AE3) and SARS-CoV immunohistochemistry to characterize the immunopathologic stages of DAD. RESULTS: We observed a distinctive pseudopalisaded histiocytic hyperplasia interposed between the exudative and proliferative phase of COVID-19 associated DAD which was most pronounced at the fourth week from symptom onset. Pulmonary macrothrombi were seen predominantly in cases with pseudopalisaded histiocytic hyperplasia and/or proliferative phase DAD. Neither pseudopalisaded histiocytic hyperplasia nor pulmonary macrothrombi were seen in non-COVID-19 DAD cases, whereas microthrombi were common in DAD regardless of etiology. CONCLUSION: The inflammatory pattern of pseudopalisaded histiocytic hyperplasia may represent the distinctive immunopathology associated with the dexamethasone responsive form of DAD seen in severe COVID-19.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 155 million worldwide and led to over 3.2 million deaths at the time of this writing. [1] The disease course is highly variable, ranging from asymptomatic carrier [2] to severe viral pneumonia with acute respiratory distress syndrome (ARDS). Subsets of patients with severe illness have viral sepsis [3] , cytokine storm [4] and occasionally meet clinicopathologic criteria for hemophagocytic lymphohistiocytosis (HLH). [5] Part of the spectrum in disease course has been linked to inborn errors in the type I interferon immune pathway [6] as well as the development of neutralizing autoantibodies against type I interferons in a subset of patients. [7] These findings would suggest that immunopathologic differences might be observed between asymptomatic individuals and those with severe COVID-19 disease; a hypothesis which is supported by a randomized control trial in which dexamethasone improved outcome in COVID-19 patients requiring respiratory support. [8] Since the onset of the pandemic, there have been several autopsy series describing the histopathologic features of patients dying with COVID-19. [9] [10] [11] [12] [13] Early results showed typical features of diffuse alveolar damage (DAD) [14, 15] and an apparent increase in pulmonary thrombi. [16, 17] More recently a timeline of the usual histopathologic phases of DAD as they progress from exudative, proliferative, and fibrotic stages has been proposed [18] with in situ viral detection generally limited to the exudative phase of disease. [19] Concordant with these observations, RNA sequencing data have shown marked differences between the immune response in early vs. late COVID-19 DAD which was supported by single-plex J o u r n a l P r e -p r o o f immunohistochemistry in subsets of cases. [20] In this study, we seek to (1) 

After obtaining consent from next of kin, autopsy was performed on twelve consecutive patients testing positive for SARS-CoV-2 nucleic acid on antemortem upper respiratory swab between April 15 and July 24, 2020. Chart review of the electronic health record was performed to determine age, sex, race, date of symptom onset, date of hospitalization, date and types of treatment, C-reactive protein (CRP) and D-dimer levels throughout the course of hospitalization. Diagnostic SARS-CoV-2 testing was performed by RT-PCR of nasopharyngeal swabs in a CLIA-certified laboratory during hospital admission. D-dimer assay was serially monitored throughout the hospital course using the IL D-dimer HS assay (Instrumentation Laboratory, Bedford, MA, USA) which is a quantitative immunoturbimetric assay reported in Ddimer units (DDU). IgG antibody analyses were performed by the clinical pathology laboratory at Boston Medical Center (BMC). Serum samples were run on the Abbott Architect i2000 Instrument using the Abbott SARS-CoV-2 IgG assay per the manufacturer's instructions (SARS-CoV-2 IgG; Abbott Laboratories, Abbott Park, IL). This assay is a chemiluminescent microparticle immunoassay (CMIA) for detection of IgG antibody in human serum against the SARS-CoV-2 nucleoprotein. Samples were interpreted as positive (index value >= 1.4) or negative (index value < 1.4) based on the index values reported by the instrument. Qualitative results were used in the analyses. Cases 1 to 4 have been separately reported as manifesting features of SARS-CoV-2 associated hemophagocytic lymphohistiocytosis. [5] The autopsies were performed in a negative pressure room using Personal Protective Equipment (PPE) including N95 mask and powered air purifying respirator (PAPR). Harvested J o u r n a l P r e -p r o o f organs were thinly sliced, photographed, and fixed for 24 hours in 10% neutral buffered formalin (NBF). Four tissue blocks were routinely taken from each lobe of lung (20 total) and fixed for an additional 6-12 hours in 10% NBF before processing. Seven patients dying of diffuse alveolar damage preceding the pandemic (2007-2019) were identified having FFPE lung blocks (median, 2 per case) available to serve as controls. Hematoxylin and Eosin (H&E) stained sections were prepared and reviewed by two anatomic pathologists (MK and EB). Reviewers were not blinded to the cause of DAD.

Immunohistochemistry was performed using freshly cut 5 µm thick FFPE tissue sections from a lung tissue block most representative of the overall stage of DAD for the individual patient. The proportion of blocks chosen from right vs. left lobes was similar (2:1) between the symptomatic COVID-19 and control group. Single-plex immunohistochemistry was performed on a Ventana Benchmark Ultra (Roche, Tucson, AZ) using a rabbit polyclonal antibody against the SARS-CoV Nucleocapsid protein (NB100-56576: Novus Biologicals, Centennial, CO) incubated for 36 minutes at 1:400 dilution after heat induced epitope retrieval for 32 minutes at 95° C using alkaline buffer (CC1, #950-124, Ventana). Slides were visualized with Optiview detection (#760-700, Ventana). Multiplex immunohistochemistry was performed using the Ventana Discovery-Ultra platform (Roche, Tucson, AZ) using pre-dilute antibodies directed against CD8 (clone: Teal, #760-239. Primary antibody inactivation for the multiplex reactions were carried out using temperature induced denaturation for 8 minutes at 95° C with CC2 buffer (#950-123, Ventana).

Internal and on-slide external controls (Tonsil/Appendix) were examined for each slide and judged satisfactory before interpretation.

The Institutional Review Board reviewed this study and waived jurisdiction.

J o u r n a l P r e -p r o o f

Clinical features of SARS-CoV-2 positive patients are summarized in Table 1 . Twelve patients tested positive by PCR on nasopharyngeal swabs prior to death. Ten of these were symptomatic from COVID-19 prior to death while 2 patients were asymptomatic, dying of unrelated causes.

Symptomatic patients were older (median age 69.5 years) than those who were asymptomatic (median age 51.5 years), and 8/10 (80%) self-identified as black. Seven patients received biologic agents as part of their treatment prior to death. Anakinra, a human interleukin-1 receptor antagonist, was administered to 2 patients who received 6 doses of 100 mg IV over 3 days which was completed 9 and 85 days prior to death. Sarilumab, a human monoclonal interleukin-6 inhibitor, was administered to 5 patients as a single dose of 200 mg IV which was completed a median of 20 days (range 2-29 days) prior to death. Seven patients dying of diffuse alveolar damage preceding the pandemic (2007-2019) were identified having FFPE tissue available to serve as controls. The median age was 55 years with a male to female ratio of 2:5 and a minority who were black (2 of 7). There were diverse and overlapping disease states associated with DAD in these patients including cancer (pheochromocytoma, esophageal, and lung carcinomas) undergoing multiple treatments (surgery, radiation, chemotherapy), immune disorders (SLE, HLH, and AIDS), and infections (cryptococcal meningitis, Pneumocystis pneumonia, and fungal sepsis).

J o u r n a l P r e -p r o o f Two patients (case 1 and 2) died of COVID-19 after the first week of symptom onset (6-8 days) without mechanical ventilation. Both patients had dyspnea and hypoxia but were not intubated due to advanced age and co-morbidities. The lungs were heavy in both patients (right 750-950

gm & left 512-750 gm). Figure 1 shows the gross and histologic findings. Grossly the lungs showed patchy areas of consolidation which were edematous and firm. Histologic findings were that of early exudative phase of DAD, being dominated by alveolar edema, pneumocyte sloughing and foci of early hyaline membrane formation. One of the two cases showed focal squamous metaplasia. SARS immunohistochemistry was positive multifocally in aggregates of epithelioid cells in both patients. Multiplex immunohistochemistry showed numerous CD68 macrophages and CD4 positive T-cells admixed with sloughed AE1/AE3 positive pneumocytes but only scattered CD8 positive T-cells and rare CD20 positive B-cells. (Table 1) . Control patients dying with DAD prior to COVID-19 pandemic were compared to patients dying of SARS-CoV-2, matching them by time from intubation. The tempo of histologic and immunophenotypic changes were similar with the exception that none of the control cases exhibited the pseudopalisaded histiocytic hyperplasia seen most prominently at the fourth week post symptom onset in those dying of SARS-CoV-2. Figure 5 shows the gross and histologic features. The gross appearance was congested and red but without discrete foci of consolidation. Histologically, the interstitium showed mild expansion of loose fibrous tissue, dilated capillaries, and minimal chronic inflammation. No other hallmarks of interstitial lung disease such as collagenous fibrosis, microcystic honeycombing, traction bronchiectasis or vascular changes were observed. Multiplex immunohistochemistry showed scattered interstitial lymphocytes with an admixture of CD4 and CD8 positive cells. AE1/AE3 highlighted an intact layer of pneumocytes and CD68 positive histiocytes had decreased to near normal levels. SARS immunohistochemistry was negative.

Two patients tested positive for SARS-CoV-2 by RT-PCR on admission and on the day preceding death with no COVID symptoms. Both patients were also positive for IgG and IgM SARS-CoV-2 antibodies in their serum within 3 days of death indicating the asymptomatic period was long enough to mount an immune response against the virus. In one case (case 11) the cause of death was spontaneous tumor lysis associated with metastatic cancer while the other patient (Table 1 ) but values were generally higher in those with macrothrombi (median 29,941 ng/mL DDU) compared to those without (median 6258 ng/mL DDU). Among patients with macrothrombi, only one patient had extrapulmonary thrombosis manifesting as basal ganglia stroke (case 3) and this patient also had the highest D-dimer level (69,000 ng/mL DDU). Areas of pulmonary hemorrhage and infarct were occasionally observed adjacent to macrothrombi ( Figure 7A ).

J o u r n a l P r e -p r o o f

Herein we describe the gross, histologic, and immunopathologic pulmonary features in a series of 10 patients dying at differing time points following symptomatic COVID-19 and compare these with 7 patients dying from DAD/ARDS prior to the COVID-19 pandemic. We further observe 2 patients dying from other causes with asymptomatic SARS-CoV-2 infection. We establish an immunopathologic time sequence and note a unique inflammatory pattern characterized by a pseudopalisaded histiocytic hyperplasia developing between the exudative and proliferative phases of DAD in SARS-CoV-2 but not in control cases of DAD.

Diffuse alveolar damage histologically has sequential exudative, proliferative and fibrotic phases. [21] The exudative phase is characterized by cell mediated destruction of the alveolar/endothelial structure leading to leakage of plasma and cellular content into the interstitium and air space. We observed these features in those dying at 6-18 days from symptom onset, in particular in those dying without mechanical ventilation. Subsequently the protein and cellular debris organize into hyaline membranes which was most confluent at the third week post-symptom onset. This is followed by the proliferative phase of DAD characterized by a pneumocytic hyperplasia, attempting to restore the epithelial layer, and a myofibroblastic proliferation within the interstitium with morphologic features typical of connective tissue repair. The proliferative phase was observed most prominently in week 4-5 post symptom onset and only in those who had received mechanical ventilation, usually for at least 3 weeks duration. Finally, a fibrotic phase inconsistently develops as a consequence of failure to resorb collagen deposited during the proliferative phase leading to chronic interstitial J o u r n a l P r e -p r o o f fibrosis. We observed only a single patient who had recovered from severe COVID-19 respiratory infection, 3-months after symptom onset and 1-month since mechanical ventilation, who then died of non-pulmonary causes. Lung weights had returned to near normal and there was only minimal residual interstitial expansion of loose fibrous tissue and scattered chronic inflammatory cells. Typical hallmarks of interstitial lung disease (collagenous fibrosis, microcystic honeycombing, traction bronchiectasis, vascular changes) were not observed. [22] The timing of these morphologic phases in SARS-CoV-2 associated DAD are in keeping with the timeline proposed in a recent systematic review of pathologic findings from 131 post-mortem lung samples taken from patients dying from COVID-19. [18] Using multiplex immunohistochemistry, we were able to characterize the inflammatory 

It is interesting in the context of this distinctive inflammatory pattern that the RECOVERY collaborative group showed dexamethasone reduced 28-day mortality in patient with COVID-19 who required mechanical ventilation when given after the first week of illness but had no effect in patients not receiving respiratory support. [8] The authors of this study suggested that the stage of disease in which therapy was initiated was likely dominated by immunopathological elements, with active viral replication only playing a secondary role. To this point, we observed maximal immunohistochemical viral detection in the preceding exudative phase with limited to absent staining upon advancement to proliferative phase, a finding confirmed by other groups as well. [19] As such, the pseudopalisaded histiocytic hyperplasia observed in our series correlates well with the timing of the observed mortality benefits demonstrated in the RECOVERY dexamethasone trial, corresponding to a time when in situ viral detection is diminishing. None of the patients in our series received dexamethasone as they presented prior to the results of the RECOVERY trial. Current clinical guidelines recommend glucocorticoids be administered to patients with early, moderate to severe ARDS and those precipitated by a steroid responsive process. [27] Glucocorticoids are generally not recommended for patients with mild ARDS, late-stage disease (beyond 14 days) [28] , or in patients whose ARDS was precipitated by a viral infection such as influenza as their use has been associated with worse outcomes. [29, 30] A more recent meta-analysis, however, is challenging these guidelines, suggesting that corticosteroids probably reduces mortality in patients with ARDS regardless of whether COVID-19 was the inciting agent. [31] A further confounding factor in our conclusion is that 4 of the 5 patients with the pseudopalisaded histiocytic reaction were also treated with sarilumab a median of 20-days prior to death to J o u r n a l P r e -p r o o f manage cytokine release syndrome. [25] Sarilumab is a monoclonal antibody inhibitor of IL-6 which is produced by innate immune cells, such as monocytes/macrophages, as part of the host defense against infectious agents [26] and thus we cannot exclude that the pseudopalisaded histiocytic reaction is a result of IL-6 inhibition in these patients. It will be interesting to see if the pattern of pseudopalisaded histiocytic hyperplasia becomes less frequent in post-mortem samples following the routine use of dexamethasone rather than sarilumb in hospitalized patients requiring respiratory support.

Intriguingly, two patients dying of causes unrelated to COVID-19 tested positive for SARS-CoV-2 by PCR and serology, but showed either no lung involvement, or in one case limited pulmonary involvement (~5% of parenchyma) but with similar immunopathologic features of early exudative phase DAD. A concept of regional alveolar damage (RAD) was proposed 3 decades ago to describe such histologic findings, however patients with RAD all developed respiratory failure with 9 of the 10 patients requiring mechanical ventilation. [32] Our single patient showing regional alveolar damage as well as the other patient without such findings both tested positive by RT-PCR at admission and prior to death and also showed evidence of immune response with Sars-CoV-2 IgG and IgM antibodies detected in serum prior to death. Thus, their findings cannot be merely explained as an early pre-symptomatic phase of infection. It remains unclear why some patients develop severe and fatal COVID-19 while others can be asymptomatic carriers. Advanced age and co-morbid conditions (obesity, hypertension, diabetes, chronic heart and kidney disease) are more frequent in fatal COVID cases. A subset of patients with severe/fatal disease have been linked to inborn errors in the type I interferon J o u r n a l P r e -p r o o f immune pathway [6] as well as the development of neutralizing autoantibodies against type I interferons. [7] An association between immune variations and co-morbid conditions has also been proposed. [33] Aside from DAD, much attention has been given to the thrombosis associated with COVID-19. [16, 34, 35] Microthrombi within the alveolus is a common finding in DAD from any etiology and the frequency of this finding is likely proportional to the number of tissue sections sampled. We observed microthrombi in 60% of patients dying of COVID-19 and in 43% of non-SARS-CoV-2 associated DAD controls. Given that the number of tissue blocks sampled among the SARS-CoV-2 cases was 10-fold greater than the non-SARS-CoV-2 controls, this mild difference may not be significant. A similar frequency of pulmonary microthrombi between cases of DAD related and unrelated to COVID-19 has been reported in another autopsy series as well. [36] Conversely, macrothrombi were observed in 40% of COVID-19 associated DAD but in none of the asymptomatic SARS-CoV-2 cases or non-COVID-19 DAD controls. This finding is in keeping with other large COVID-19 autopsy studies in which large vessel thrombi were observed in 20-42% cases. [11, 13] In our series, macrothrombi were observed more frequently in those dying the fourth to fifth week from symptom onset (third to fourth week of ventilation) usually in association with pseudopalisaded histiocytic hyperplasia and/or proliferative phase DAD as compared to microthrombi which were observed in all phases of DAD. Similar observations of the timing of macrothrombi were seen in a larger multi-institutional autopsy cohort [13] and a clinical cohort where those not improving on mechanical ventilation were evaluated by pulmonary angiography. [34] Consistent with clinical studies, we observed J o u r n a l P r e -p r o o f macrothrombi in those with the highest D-dimer values. [35] The specifics of COVID-19 induced coagulopathy remain complex with cytokine-induced overexpression of tissue factor, endothelial dysfunction leading to loss of antithrombotic phenotype, and overall hypoxia and stasis being the leading mechanistic hypothesis. [37] What is clear is that the degree of inflammation appears to correlate strongly with coagulopathy, with D-dimer levels showing strong correlation with other inflammatory markers such as CRP, ESR, ferritin and procalcitonin. [35] It is possible that the pseudopalisaded histiocytic hyperplasia is a marker of an inflammatory phase of disease prone to accentuate the coagulopathy of COVID-19. Of note, inflammation associated coagulopathy is not specific to SARS-CoV-2 as it has also been described in critically ill patients with H1N1 and SARS-CoV-1. [38] Randomized control trials to determine the optimal use of anticoagulation in COVID-19 patients are ongoing.

Several limitations must be considered in the interpretation of our findings. First, our sample size in both COVID-19 and the non-COVID-19 control cases is small. Second, the control group suffered from a variety of immune disorders (cancer, AIDS, autoimmune diseases) which were not frequent in our COVID-19 cases. Third, the control cases in our series were a median of 2 decades younger than our COVID-19 cases and thus we cannot assess the role of age related immunosenescence contributing to the immunopathology in our COVID-19 cohort. Finally, as already stated, we cannot exclude that IL-6 inhibition due to treatment with sarilumab resulted in the pseudopalisaded histiocytic reaction. 

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