Cytokine release syndrome is not usually caused by secondary hemophagocytic lymphohistiocytosis in a cohort of 19 critically ill COVID-19 patients


 Background: Severe COVID-19 associated respiratory failure, poses the one challenge of our days. Assessment and treatment of COVID-19 associated hyperinflammation may be key to improve outcomes. It was speculated that in subgroups of patients secondary hemophagocytic lymphohistiocytosis (sHLH) or cytokine release syndrome (CRS) with features of macrophage activation syndrome might drive severe disease trajectories. If confirmed, profound immunosuppressive therapy would be a rationale treatment approach.Methods: Over a median observation period of 11 (IQR: 8; 16) days, 19 consecutive confirmed severe COVID-19-patients admitted to our intensive-care-unit were tested for presence of sHLH by two independent experts. HScores and 2004-HLH diagnostic criteria were assessed. Patients were grouped according to short-term clinical courses: discharge from ICU versus ongoing ARDS or death at time of analysis.Results: The median HScore at admission was 157 (IQR: 98;180), without the key clinical triad of HLH, i.e. progressive cytopenia, persistent fever and organomegaly. Independent expert chart review revealed the absence of sHLH in all cases. No patient reached more than 3/6 of modified HLH 2004 criteria. Nevertheless, patients presented hyperinflammation with peripheral neutrophilic signatures (neutrophil/lymphocyte-ratio>3.5). The latter best paralleled their short-term clinical courses, with declining relative neutrophil numbers prior to extubation (4.4, [IQR: 2.5;6.3]; n=8) versus those with unfavourable courses (7.6, [IQR: 5.2;31], n=9).Conclusion: Our study rules out virus induced sHLH as the leading cause of most severe-COVID-19 trajectories. Instead, an associated innate neutrophilic hyperinflammatory response or virus-associated-CRS appears dominant in patients with an unfavourable clinical course. Therapeutic implications are discussed.


Introduction
The SARS-CoV-2 globally poses medical and economic challenges. While epidemiologists and governments are buying time and capacity through various strategies, it is up to clinicians to optimize the treatment of severe COVID-19 1,2 . Various anti-viral drugs, including the polymerase-, or proteaseinhibitors and passive immunization attempts are currently undergoing clinical trial [3][4][5] .
Yet, in cases of serious COVID-19 with pneumonia and acute respiratory distress syndrome (ARDS), tocilizumab, a humanized monoclonal Interleukin-6-receptor antagonist, is administered in a phase II open label clinical trial (NCT04317092) to supress hyperin ammation that is assumed to cause fatal lung and multiorgan injury 6,7 . This is supported by cohort data from China, which report increased ferritin, and elevated TNF-α, IL-6 and IFN-γ levels in severe versus mild COVID-19-cases. Based on these preliminary data, cytokine release syndrome (CRS) or virus induced adult secondary hemophagocytic lymphohistocytosis (sHLH) have been proposed as underlying aetiology of severe COVID-19 7,8 . The latter is a clinical syndrome, characterized by massive systemic in ammation, persistent fever ares, hepatosplenomegaly, and severe cytopenia, which is rooted in hemophagocytic activity in bone marrow. It is a rare and often fatal clinical syndrome which can be observed in the context of malignancy, systemic autoimmunity, and viral infections such as Epstein-Barr virus 9 . Full blown sHLH in the latter context usually requires cytotoxic agents, i.e. etoposide, which is not to be used carelessly 9 .
The term CRS originally de nes drug toxicity, which can be observed subsequently to monoclonal antibody or adoptive T-cell therapies. The syndrome is rooted in massive aberrant B-, T-cell and monocyte activation and a systemic "cytokine storm". The clinical course is pleiotropic, with fever, skin rash, cytopenia, neurologic symptoms, coagulopathy, hypotension and eventually organ failure i.e. ARDS.
Application of tocilizumab usually resolves symptoms 10,11 . In the sense of an exuberant anti-viral innate immune response, elevated cytokine levels, fever and CRS-like symptoms, including organ injury have been reported in the context of viral infections, such as H5N1 or SARS-CoV-1 12,13 . To avoid confusion, we will refer to this with the term "virus-associated CRS".
Whether CRS, sHLH or both are present in severe COVID-19 profoundly impacts our understanding of the disease and impacts on therapeutic strategies. We therefore screened all consecutive 19 severe COVID-19-patients, that were admitted to our intensive care unit (ICU) for (a) CRS and (b) presence of sHLH, using the HScore, and 2004-HLH-diagnostic-criteria. Charts were reviewed by two independent HLHexperienced clinicians to rule in or out actual sHLH.

Results
Baseline characteristics, treatment and clinical course of severe COVID-19-cases 19 consecutive laboratory con rmed COVID-19-patients with typical chest X-ray (n=1) or CT-scan (n=18) were admitted to our ICU 14,15 . Overall, they were mostly male (89%), with a median age of 70 (IQR: 58; 78) years. All had at least one pre-existing comorbidity or history of organ failure, with arterial hypertension (58%) being the most frequent comorbidity.
Patients were treated in accordance with the "surviving sepsis campaign guidelines for severe COVID-19" 16 . All patients received intravenous broad-spectrum antibiotics and regular surveillance for superinfection. Two patients additionally received remdesivir within clinical trials. Most patients (89 %) showed rapid deterioration of oxygenation and were intubated after a median of 0 (IQR: 0;0) days.
Our approach resulted in clinical improvement of 47% of patients. After a median of 8.5 (IQR 8;12.5) days, eight patients were extubated, one patient was discharged without need for mechanical ventilation. The remaining cases were judged as unfavourable, due to prolonged weaning, progressive ARDS requiring organ support (n=7), or death (n=3) (see gure 1). No group differences (favourable versus unfavourable group) were detected with respect to time of observation, comorbidities, SOFA-Score ( . D-Dimers tended to increase in both groups (repeated-measure-ANOVA: p=0.13 or 0.2, gure 2h). Thus, an acute neutrophilic response rather than serum mediator kinetics paralleled the clinical course in our patients. This, lack of organomegaly and lymphopenia suggest that hyperin ammation is linked to innate immunity.

Assessment of virus induced HLH
Due to fever, hyperin ammation and high mortality, we asked whether patients with severe COVID-19disease and organ dysfunction suffer from virus induced secondary HLH.
Likewise, higher cut-off values for ferritin have been suggested 18,19 .
We therefore applied "modi ed" -"2004 HLH-diagnostic-criteria", which are considered positive in the presence of 4/6 of the following criteria: persistent fever, splenomegaly, persistent or progressive cytopenia, ferritin >10.000ng/ml, AST >50IU/l. As can be retrieved from table 3 only one patient from the unfavourable group reached the modi ed cut-off for serum ferritin. The sustained fever-and cytopeniacriteria were reached by 3 (16%) and 6 (32%) patients, respectively. 13 (68%) patients showed hypertriglyceridemia (table 3). Fever and cytopenia were judged in a strictly longitudinal manner. The remaining domains were identical to the HScore-domains (table 3), this resulted in 0/19 patients ful lling four of the "modi ed" -"2004-HLH-diagnostic-criteria".
In line with this, sHLH usually implies massive activation of T-cellular immunity, which can be assayed via elevated sIL-2R 20 . Although 5 (26%) of our patients showed values above 2000 U/ml, none exceeded the limit of 10000 IU/ml, which has high speci city for sHLH.
Most importantly, sHLH is a clinical syndrome, which is ultimately de ned by the assessment of the experienced clinician. We therefore had all case-charts (supplementary le 1) reviewed by experts. Both reached agreement, that the diagnostic criteria for classical sHLH were not ful lled in any of the presented cases. (table 3). However, CRS-like hyperin ammation associated with COVID-19 was assessed positive in 11 (58%) cases. 8 (42%) patients were de ned "uncertain" regarding the latter due to concomitant superinfection (table 3).
In summary, none of the patients showed evidence for secondary HLH within a median of 11 (8;16) days of observation after ICU-admission, despite the presence of ARDS, systemic hyperin ammation and eventually organ failure. Thus, virus-associated CRS but not sHLH is frequently associated with severe COVID-19.

Discussion
The SARS-CoV-2-pandemic is challenging. Based on SARS-CoV-1, an exuberant immune response has already been extrapolated as a major cause of lung injury, organ failure and mortality in COVID-19 21 . In fact, hyperin ammation is a hallmark of severe over moderate COVID-19 22 . This gave rise to the idea, that cytokine release syndrome (CRS) or even sHLH might drive severe COVID-19 7,23 . Anti-in ammatorycombination-studies are already underway (NCT04330638) before the anti-IL-6R-studies are completed, despite the fact that the exact pathomechanism of severe COVID-19 remains elusive.
Here we report on the clinical course and immunologic ndings of 19 consecutive severe COVID-19patients. We demonstrate that up to a median of 11 (8; 16) days post ICU-admission none of our patients showed classical evidence of sHLH, which practically excludes sHLH as an initial driver of severe COVID-19. We relied on various layers of evidence, including the screening for HLH using the HScore as suggested by Mehta P et al. only recent 7 . Apart from high HScores in individual cases, all patients lacked the classical clinical triad, i.e. persistent fever, organomegaly and progressive cytopenia 9,24 . As mentioned earlier the HScore must be interpreted with caution in severe COVID-19-patients, since ferritin, fever, cytopenia and even the bone marrow criterion ("hemophagocytosis") lose speci city in an ICUsetting where blood transfusions, drug-associated cytopenia, organ failure and sepsis are frequent events 18,[25][26][27] . Aside from hyperin ammation and coagulopathy, our patients lacked the classical immunephenotypical features usually associated with sHLH/MAS including hypo brinogenemia, expansion of CD8+ T cells and neutropenia 28,29 . Instead, we report rather low counts of CD8+ T cells, lymphopenia and relative neutrophilia. These observations agree with various reports from Wuhan 8,22 .
However, "absence of sHLH" is not contradictory to the hypothesis that virus-associated-CRS and hyperin ammation rather than viral replication determines the course of COVID-19 as some observations can hardly be explained by virus replication 22 . Cases from France indicate, that development of severe COVID-19-pneumonia can be accompanied by a decreasing viral load 30 . We and others nd that deterioration of oxygenation occurs with some delay, about 3 days after hospital admission accompanied by the peak in ammatory reaction 6 . Thereby, elevated in ammatory mediators, coagulopathic activity (D-Dimer) and elevated NLR were characteristics of the disease at onset of ARDS. Cumulatively, these data con rm the presence of a strong virus-associated CRS in SARS-CoV-2-infection, simultaneously to the onset of ARDS 6,22,31 . Our data adds to the current knowledge, that amongst severe COVID-19-patients, NLR and relative neutrophilia more than CRP paralleled the observed short-term clinical course. It is tempting to speculate that virus-associated CRS and the neutrophilic response are the main culprits in severe COVID-19-pneumonia, which occurs with a delay only when the "misguided antiviral-immune response" hits 23 . One might argue, that ARDS per se implies alveolar endothelial injury, complement-, neutrophil-activation, and NETosis and thus entails a systemic hyperin ammatory response [32][33][34] . Still, others have reported, that an increased NLR is apparent already before the onset of ARDS, and was an independent predictor for severe COVID-19 in 61 patients 31,35  Considering these data, combined anti-viral and anti-in ammatory treatment strategies are seemingly plausible options to improve outcomes. Although we could not nd an association of serum IL-6 with the clinical course of our patients and although experimental data indicate potentially harmful effects of IL-6R blockade in experimental lung injury there have been occasional reports of therapeutic success with tocilizumab in COVID-19 23,36 . In addition, both IL-6 and IL-1 were associated with outcomes in ARDS decades ago 37 . Still, we believe that a sense of proportion is required here.
In the absence of sHLH, cytoreductive drugs, i.e. etoposide, are not indicated. Further, we advocate caution when applying combined cytokine-directed treatment protocols in this vulnerable patient population. Our patients are elderly, carry comorbidities and are not immune to bacterial superinfections.
The latter was also observed in a larger cohort 6 . After all, 9/19 patients improved and overcame viral-CRS by standard supportive care. Thus, treatment within clinical trials should be prioritized when using experimental immunomodulation. Moreover, short acting substances, e.g. IL-1R-blockade with anakinra for which positive data exist from other neutrophilic hyperin ammatory conditions such as Still's disease with ARDS and septic shock with coagulopathy, deserves special consideration, as it allows the therapy to be quickly de-escalated 38,39 . Also, Ruxolutinib a tyrosine kinase inhibitor interfering with Jak-STAT dependent cytokine signalling seems appealing (NCT 04338958). If neutrophils prove to be causally involved, colchicine could be another short-acting and cost-effective option for emerging countries, (NCT04322682) 40 .
Our short-term observational study has limitations. 2 patients were transferred to our ward more than 48 hours after external ICU admission, thus datasets for sIL-2R and ferritin were not complete. We can indeed exclude sHLH as a common underlying pathology in severe COVID-19 with ARDS. However, we cannot eliminate the possibility that some patients will develop secondary HLH later on, e.g. as a result of bacterial sepsis or prolonged viral-CRS 41 . Moreover, the judgment of the (un)favourable disease-courses based on clinical criteria holds risk for bias. Nevertheless, all patients classi ed as favourable were permanently discharged from ICU at time of manuscript preparation. Further, in clinical reality, therapeutic decisions will likely also depend on similar clinical assessments. Yet, if immune-modulatory drugs meet our expectations, our observations raise the questions of (a) which (sub)groups should be treated and (b) at what timepoint, to not endanger patients with self-limiting courses by overtreatment.

Study design and patient characteristics and clinical course
This observational cohort study is an interims analysis of the multi-centre register of "COVID-19-register to document cases of secondary hemophagocytic lymphohistiocytosis" NCT04347460. All patients, their families or legal guardians gave written and informed consent. The study was approved by the local ethics committee of the Klinikum rechts der Isar of the Technical University of Munich (Ref. 161/20 S) and accordance with the declaration of Helsinki.
Patients' characteristics were entered based on interviews with patients or families and the clinic's medial record system. BMI, Horovitz Index (HI) and sequential-organ-failure-assessment-(SOFA)-Score were calculated on admission as body weight [kg]/height² [m²], paO2/FiO2 or using the online-calculator https://mdcalc.com/sequential-organ-failure-assessment-sofa-score, respectively. Clinical data were independently reviewed by 2 researchers.
Severe COVID-19, shock and ARDS were de ned according to the National Health Commission of China's and the World Health Organization's interims papers de nition, respectively 42,43 . As observation periods were limited, favourable spontaneous clinical course of severe COVID-19-patients were de ned by either "no need for intubation" or "was extubated" at time of analysis versus those who were in need of mechanical ventilation, or in need of extracorporeal lung assist devices, prolonged weaning, or had died at time of analysis.
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Scores and assessment for HLH
The HScore was calculated at time of ICU-admission as described by Fardet et al. (see also table 4, 17 ). Yet, in an ICU-setting only a cut-off value for ferritin ≥10.000 ng/ml demonstrated su cient sensitivity in these patients 18 . The bone marrow biopsy criterion was further shown to have poor speci city for HLH among seriously ill patients 44 . Thus, the pediatric 2004-HLH criteria, were modi ed according to centre speci c cut-offs, and a cut-off for ferritin of 10.000 ng/ml was used ("modi ed 2004-HLH criteria"; table 4). Chart reviews to determine actual presence of sHLH were independently performed by L.G. M.P. and LR.P., the latter two having at least ten years of experience in diagnosis and treatment of secondary HLH.
Laboratory work and diagnostic procedures PCR-testing for SARS-CoV2 was performed from tracheal specimens. 18/19 patients had positive results.
Statistics: Was performed using IBM SPSS Version 23. We report median and interquartile range (IQR) or counts and percent of total (%) as applicable. Group comparisons were done using ANOVA, Mann-Whitney-U-, Kruskal-Wallis-, repeated measure paired ANOVA or paired-Wilcoxon-rank-test as applicable.

Declarations
Acknowledgments: We thank all patients and their families. Further, special thanks to nurses, doctors and all other clinical staff involved in the treatment of these patients.

Author contributions:
GL designed the study, did data collection, data analysis, data interpretation, wrote the manuscript, was involved in treatment of the patients and is part of HLH-registry founders. PM was involved design, data collection, data analysis, preparation of gures, data interpretation, reviewed the manuscript, provided expert judgement and is part of HLH-registry founders. QB did data collection, data analysis and preparation of gures, and critically reviewed the manuscript. LP analyzed the data, critically reviewed the manuscript and provided expert clinical judgment. HS was responsible for laboratory data assessment and analysis. RMS did data collection, data analysis, and assisted writing the manuscript. CS was involved in treatment of patients, and is part of HLH-registry founders, provided critical input for the manuscript. UH, RS, HA did data interpretation and provided critical input for the manuscript. TL and WH are taking care of the patients, are part of HLH-registry founders, provided critical input and reviewed the manuscript. CS is head of the founder of the HLH-registry, was responsible for ethics approval, did data analysis, trial registration, data interpretation and was responsible to obtain informed consent statements. GL and PM contributed equally to this work and share correspondence, WH and CS also contributed equally  Groups: "favourable" versus "unfavourable" clinical group -group medians are only reported for the favourable group.* Comorbidities were defined as follows: Pulmonary disease: Asthma, chronic obstructive pulmonary disease or fibrotic lung disease; Cardiovascular disease (CVD): Pulmonary artery embolism, peripheral arterial occlusive disease, history of lung oedema; Atrial fibrillation (AF); Liver disease: history of gastrointestinal bleeding, liver-cirrhosis or pancreatitis; ** prior immunosuppression included recent history of stem-cell transplantation, intake of cyclosporine and low dose steroids. Abbreviation: Advanced organ support (ADVOS), acute kidney injury -transient / requiring dialysis (AKI), Aspartate-aminotransferase (AST), chronic kidney disease (CKD), extracorporeal membrane oxygenation (ECMO), not applicable (n.a.), sustained low efficiency dialysis (SLED), Sepsis-related organ failure assessment score (SOFA) Wallis-test. We report median and IQR only for the favourable group (n=9) at time of admission. ** Serum ferritin and sIL-2R receptor were assayed after a median of 2 days (missing data n=1) post admission (ADM) and 4 days later -we report peak values. We report median and interquartile range (IQR) or frequencies as counts and percent of total. ANOVA was used for group comparisons: favourable versus unfavourable clinical course. Abbreviations: Interleukin (IL), C-reactive-protein (CRP), neutrophil/lymphocyte-ratio (NLR), soluble Interleukin 2 receptor (sIL-2R)  * We used centre specific cut-offs for definition of hypo-fibrinogenaemia, hypertriglyceridemia, leucopenia and neutropenia. In addition the ferritin criterion was modified within the adjusted 2004 HLH-diagnostic guidelines to a cut off of ≥ 10.000 ng/ml, according to better specificity in ICU-patients 18 . The HScore is calculated as a sum of points -see Fardet et al. 17 . The sum-score can be transformed into a probability score of HLH -however, this has never been validated for assessment of HLH in an ICU-setting. Abbreviations: Aspartate-aminotransferase (AST), Haemoglobin (Hgb), triglycerides (TAG)   Immunologic parameters, neutrophilia and IL-6, in relation to clinical course; Differential blood counts and in ammatory parameters were assessed (from left to right) at ICU-admission, at day 4-7, when patients were still intubated and prior to extubation if applicable. Otherwise (unfavourable group) blood counts and in ammatory parameters were selected from a comparable timepoint (median of 10 days).