Features of hemophagocytic lymphohistiocytosis in the intensive care unit (ICU) : a 260-patient retrospective analysis

Adult hemophagocytic lymphohistiocytosis (HLH) is highly lethal in the ICU. The diagnostic and therapeutic emergency that HLH represents is compounded by its unknown pathophysiological mechanisms. Here, we report on a large cohort of adult-acquired HLH in the ICU. We analyzed prognostic factors associated with mortality to dene the diagnostic and therapeutic challenges in this specic population, “hemophagocytosis”, “hemophagocytic “macrophage-activation


Introduction
Adult hemophagocytic lymphohistiocytosis (HLH) can be de ned as the most extreme form of the in ammatory process continuum. Imbalances in or a failure of feedback between pro-and anti-in ammatory pathways in response to a trigger lead to uncontrolled macrophage/monocyte and lymphocyte activation and proliferation [1]. A sustained cytokine storm may complicate HLH syndrome, ultimately leading to multiorgan dysfunction (MODS) [2,3]. Diagnostic criteria for HLH include clinical parameters (fever, adenopathy, splenomegaly, hepatomegaly) and biological variables (cytopenia, hyperferritinemia, hypertriglyceridemia, hypo brinogenemia), although these criteria are non speci c and may be inappropriate, as they are extrapolated from the pediatric population [4]. Pediatric primary HLH is mostly caused by genetic mutations in genes involved in lymphocyte cytotoxicity [5][6][7][8]. In contrast, the analysis of adult HLH has highlighted the absence of a lymphocyte cytotoxicity defect [9].
As adult-onset HLH constitutes a post-trigger in ammatory process, pathophysiological clusters can be described, mirroring their etiology [10,11]: infection-associated HLH (bacteria, Herpesviridae, mycobacteria, parasites, fungi), malignancy-associated HLH, and systemic auto-immune/in ammatorydisease-associated HLH (systemic erythematosus lupus, juvenile chronic arthritis, adult Still disease), the latter still de ned as macrophage-activation syndrome [1,10]. Careful etiological diagnosis must be performed promptly, as it has a large in uence on the prognosis and de nes treatment [12].
Understanding the pathophysiology underlying HLH is the key to cytokine-storm and/or cellularproliferation targeted therapy [13,14].
The diagnosis of HLH in ICU patients is often di cult, presenting as "hyper-in ammatory sepsis" [15]. Sepsis is often described as the trigger for HLH in ICU patients [16,17]. Without any obvious etiology, such as cancer, the diagnosis of acquired HLH leads to uncertainty concerning the decision to initiate speci c treatment. Therapy for ICU-acquired adult HLH mainly involves standard organ support, thorough etiological screening, and urgent treatment of the HLH trigger (infection, auto-immune underlying condition, malignant process). "Speci c" HLH therapies (corticosteroids, polyvalent immunoglobulins, anti-cytokine therapies, etoposide) are often solely administered in the severest cases of ICU-acquired adult HLH. Targeted therapy is based on the HLH-2004 protocol [18][19]. However, acquired adult HLH and primitive pediatric HLH are two different diseases, with non-comparable pathophysiology [9]. In addition, systematic immunosuppressive therapy on fragile ICU patients could aggravate their condition and expose them to a high risk of infection [20].
We aimed to describe the epidemiological, clinical, and biological characteristics of ICU-acquired HLH in adults according to etiological cluster to better understand the disease and aid in its rapid diagnosis and delivery of appropriate treatment. By de ning the prognostic factors associated with ICU mortality, we focus on patients for whom their condition constitutes a diagnostic and therapeutic emergency.

Methods
We performed a retrospective observational multi-institutional study for patients admitted to the ICU's of the European Hospital, Timone University Hospital, and Nord University Hospital and the Onco-Hematological ICU of the Paoli-Calmette Institute, in Marseille, France between January 2010 and June 2020. The medical information department (DIM) of each hospital provided the cohort using the following keywords: "hemophagocytosis", "hemophagocytic lymphohistiocytosis", "macrophageactivation syndrome" and "hyperferritinemia". Only adult patients with ICU-acquired HLH were included among the selected les. An HLH diagnosis required ful lment of at least 4 of 8 HLH-2004 criteria (95% sensitivity and 93.6% speci city for ICU adult patients [21]) and/or an HScore > 169 (93% sensitivity and 86% speci city [22]). This study was communicated to the Commission on Data Processing and Freedom representative of each center and approved by APHM ethic committee (#2019 − 316). Immunosuppression was reported for stem-cell or solid-organ transplantation, solid-organ cancer, hematological disease, chemotherapy administration within six months prior to ICU admission, human immunode ciency virus (HIV) or acquired immunode ciency syndrome (AIDS), and long-term immunosuppressive treatment (including steroids). Severity was assessed using the Sepsis-related Organ Failure Assessment (SOFA) score, ranging from 0 to 24, with higher scores indicating a higher severity of organ failure [23], at ICU admission and again at HLH diagnosis (de ned as the day of bone-marrow aspiration or ferritin measurement). The Delta SOFA score was obtained by subtracting the SOFA score at HLH diagnosis from that at ICU admission.
Sepsis-related HLH (extracellular bacteria and fungi) was distinguished from intracellular bacterial infection-related HLH, as the latter leads to different immunological and pathophysiological patterns [24]. In addition, intracellular infections are rarely associated with sepsis and are believed to lead to a more speci c prognosis. Epstein-Barr Virus (EBV)-associated HLH was retained for patients with a concomitant positive serum EBV polymerase chain reaction (PCR) and HLH diagnosis. Malignancy-related HLH concerned patients who were newly diagnosed with cancer or who relapsed at HLH diagnosis, without concomitant infection. None of the reported cases of HLH were attributed to systemic or auto-immune disease.
After describing the overall population, we compared populations according to their main etiology using the Chi² test (or Fisher exact test) for qualitative variables and analysis of variance (ANOVA -corrected using the Welch method if the variances were inequal between groups) for quantitative variables, followed by a Bonferoni post-hoc test.
We analyzed ICU mortality risk factors using univariate and multivariate logistic regression analyses. A 0.20 alpha threshold of signi cance in univariate analysis was set. Backward variable elimination was then performed to determine factors signi cantly associated with ICU mortality in multivariate analysis, using a 0.05 threshold for the p-value. The main etiology was forced into the model. The Hosmer-Lemeshow test was then used to evaluate the goodness of t of the model. Quantitative variables are described using medians, with the 25% and 75% interquartile ranges (IQR 25%-75%), and qualitative variables using frequencies and percentages. All statistical analyses were performed using SAS 9.4 software. A p value < 0.05 was considered signi cant.

Discussion
We report on one of the largest cohorts of ICU-acquired adult HLH [25]. Our retrospective ndings need to be con rmed by further studies, as the number of HLH diagnoses could have been underestimated (potentially incomplete medical coding) or overestimated (low speci city of HLH-2004 and HScore criteria). In addition, retrospective etiological diagnosis can be di cult, as various concomitant etiologies can lead to HLH. Our reported cases are representative of the ICU population and are in accordance with the literature. We con rmed the association between acquired HLH and a poor prognosis, frequent multiorgan failure, and a propensity for a background of immunosuppression. The high ICU mortality rate varied from 50 to 60%, in accordance with both the adult and pediatric literature [25,26]. Mortality risk factors included age and the severity of organ failure assessed by the SOFA score [27][28][29]. The worsening of organ failure prior to the HLH diagnosis led to a worse prognosis (Delta SOFA). The Delta SOFA slightly correlated with a longer time interval between ICU admission and HLH diagnosis (Rho = 0.19, p = 0.002).
This nding underscores the necessity of a prompt diagnosis and immediate treatment, although ICUacquired HLH is still widely underdiagnosed according to the literature [30]. Persistent fever that is refractory to antibiotics, pancytopenia, major hyperferritinemia, or unexplained chemical hepatitis should lead practitioners to screen patients for HLH [31][32][33]. Although variable and non-speci c, bone-marrow hemophagocytosis is still a hallmark HLH criterion [34][35][36] and was associated with a higher ICU mortality rate in our study. Among biological factors, non regenerative profound anemia and hypo brinogenemia have been reported to be independent predictors of a poor outcome [27][28][29]37]. The severity of cytopenia and coagulopathy have been shown to correlate with both the prognosis and TNF-a and IFN-g-mediated cytokine storm ares [2,3]. High ferritin levels were not signi cantly associated with ICU mortality, contrary to the literature [27,33]. Ferritinemia is a re ection of macrophage activation and the IL-1b/IL-18 signaling pathway and is a reliable HLH biomarker for disease follow-up and monitoring, although the 500 µg/L threshold currently used has been shown to have poor sensitivity and speci city in the ICU [38][39][40][41][42].
In our study, mortality was not signi cantly associated with HLH etiology. However, reported mortality was higher for patients with underlying lymphoma or intracellular infection (60%) than for those idiopathic or sepsis-related HLH (50-55%). None of our patients presented with iatrogenic HLH following the administration of CAR T-cells or other immunotherapy [43]. Aggressive lymphoma further worsened the poor outcome due to the exacerbation of organ failure [44,45]. Patients admitted to the ICU with intracellular infections (mycobacteria, pneumocystosis, Herpesviridae) tended to have a greater mortality rate due to underlying severe comorbidities and/or immunosuppression.
We expected the prognosis to differ according to HLH physiopathology. Lymphocytes can initiate HLH and cytokine storms (lymphoid HLH). The pathophysiology of EBV-associated HLH implies an immune defect that leads to lymphoproliferation [46,47]. The involvement and proliferation of a tumor clone was evident in the pathogenesis of lymphoma-associated HLH. However, lymphoproliferation was not encountered in sepsis-associated HLH. Such patients show enhanced lymphopenia and tissue lymphocyte apoptosis [48,49]. Hence, sepsis-associated HLH would imply a myeloid-induced cytokine storm (myeloid cell-associated HLH). Toll Like Receptors have been shown to sustain stimulation in murine models and human in ammasome gain-of-function genetic mutations have been associated with HLH [50][51][52][53][54][55]. Myeloid versus lymphoid onset thus illustrates two separate pathophysiological mechanisms, highlighting the necessity of better-targeted therapies. Anti-lymphoproliferative treatments, such as etoposide and ciclosporin A, would solely be of interest in lymphoma or EBV-associated HLH, alone or associated with an etiological treatment (rituximab for EBV, speci c chemotherapy for lymphoma). These therapies should not be used in sepsis-related HLH. The most severe and hyperin ammatory cases of sepsis-associated HLH could bene t from therapies that target cytokine storms, such as anakinra (anti-IL-1), tocilizumab (anti-IL-6), and/or ruxolitinib (anti-JAK2) [16,56,57].
Our study did not reveal any differences in the prognosis according to the speci c administered HLH treatment. However, the number of treated patients, especially those treated with ciclosporin A, etoposide, and intravenous immunoglobulin (IVIG), was too low to show any statistical pattern. As IVIG administration is safer than HLH immunosuppressive therapies, its use in non-malignancy-related HLH needs to be studied in a prospective-controlled trial [58]. Based on the literature, patients with malignancyassociated HLH and EBV-associated HLH should receive early targeted therapy [29,59]. For other HLH etiologies, the use of steroids has been debated but does not appear to provide any bene t [60]. The recently approved anti-IFN-gamma monoclonal antibody (emapalumab) is strictly applicable to only primary HLH and cannot be extended to ICU-acquired HLH [61].

Conclusion
Still underdiagnosed, hemophagocytic lymphohistiocytosis was associated with a 50 to 60% mortality rate in the ICU, reaching 70% at the six-month follow-up. The prognosis worsens with the severity of organ failure and cytokine storms. An often non-speci c clinical and/or biological set of arguments, above all for immunosuppressed patients, should lead practitioners to search for HLH. Thorough and rapid etiological screening is an absolute priority as it leads to rapid selection of appropriate targeted therapy.
Patients with HLH due to lymphoproliferative triggers would likely bene t from anti-lymphocyte proliferation treatment (etoposide), whereas myeloid cell-associated HLH, such as that encountered in sepsis, should probably be treated with anti-cytokine storm therapies on a case-by-case basis. Clinical trials must be developed to con rm these hypotheses.

Declarations
Ethical Approval and Consent to participate This retrospective study was communicated to the Commission on Data Processing and Freedom representative of each center and approved by APHM ethic committee (#2019-316).

Consent for publication
Written informed consent was not obtained for publication of these data. Consent to publish was not applicable for this study.

Availability of supporting data
All data can be requested from Dr Amandine BICHON, email Amandine.BICHON@ap-hm.fr Competing interests