Imaging techniques, such as CT and 18F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) have been traditionally used in diagnosis to exclude an underlying malignancy. These imaging techniques can also be useful in raising HLH suspicion in the context of FUOs by showing an inflammatory disease involving bone marrow, lymph nodes and the spleen (47,48). Moreover, some 18F-FDG PET/CT metabolic parameters can help identify the etiology of sHLH in children (for example, malignancy and EBV-HLH), and provide directions for further inspection and information about the prognosis (47-49).

A high sCD25/ferritin ratio has been observed in malignancy-associated HLH (M-HLH) secondary to a lymphoma (50) and in HLH secondary to multisystem Langerhans cell histiocytosis (51), and may represent a novel useful marker for the malignancy-associated subset of sHLH (50,51).

A Chinese group suggested that using the T helper (Th)1/Th2 cytokine profile (dosage of sCD25/IFN-γ signature, TNF, IL-10, IL-6, IL-4 and IL-2 on cytometric bead array technique) for early quick diagnosis of HLH. This has demonstrated that a significant increase of IFN-γ and IL-10 and a slight increase of IL-6 is an early, specific and prognostic cytokine pattern for childhood HLH (52). More recently, to discriminate FHL from sHLH, lower IL-4 and IFN-γ levels have been demonstrated to more likely indicate primary HLH (53). This quick cytokine profile seems also useful in discriminating HLH vs. sepsis (54), and to identify EBV-HLH (55), so that a combined approach HLH2004 and Th1/Th2 profile is currently available.

Other new diagnostic tools are being developed, even though they are currently used in preclinical or research settings. Some relevant examples include studies on IFNγ and its ‘signature’ (CXCL9 and CXCL10) as serum biomarkers of disease activity in both primary HLH (pHLH) and sHLH (56), or as predictors of predominant liver involvement in sHLH (57), or studies on soluble TNF-like weak inducer of apoptosis selective elevation in HLH patients (58) and studies on secretory sphingomyelinase upregulation in HLH, with the C16 ceramide:sphingosine ratio determining a poorer prognosis (59).

Genetic analysis of polymorphisms of the TNF-α promoter (60) or of the interferon regulatory factor 5 gene (61) have identified variants that increase susceptibility to sHLH/MAS-HLH.

Determination of the NK-cell dysfunction type (type 1-4) and of the underlying genetic defect has been used to guide HSCT indication (62) and to estimate the risk of progression to acute leukemia (63).

Finally, targeted sequencing of FHL genes appears to be insufficient to identify pathogenic mechanisms in the majority of patients with HLH, and whole exome sequencing (WES) is increasingly used. WES is rapidly expanding the range of causal mutations, with a double sided effect: It accelerates diagnosis in a subset of patients, but has redefined numerous other cases of secondary HLH as primary HLH (54). Furthermore,a proved mutation in HLH-related gene cannot exclude the coexistence of an underlying malignancy (41).

Infection-associated HLH represents a challenge in classification. This is because infectious diseases can trigger HLH in both primary and secondary forms, and because septic shock can either be a presentation of HLH syndrome or may mime it, with great implications in therapeutic approach. A thorough infectious screening is thus highly recommended when facing an HLH syndrome (7,8).

Among the case reports the present study reviewed, the most frequent infectious triggers were viruses, such as the herpetic viruses, dengue virus, Crimean Congo hemorrhagic fever virus, Eastern equine encephalitis virus, hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), adenovirus, parvovirus B19, measles, influenza virus and severe acute respiratory syndrome coronavirus 2 (Sars-Cov2).

EBV has been frequently reported from all across the world, and especially from the Asian Countries (37,65-73). The reason for this geographical heterogeneity may involve a higher virulence in EBV viral strains circulating in Asia (74), or a different immune predisposition in the Asian patients, but this issue has not been clarified yet. EBV typically targets B cells, but in a subset of patients with EBV-HLH, of prevalent Asian origin again, it infects T or NK cells leading to oligoclonal or monoclonal proliferation and massive cytokine production (69,75). In extreme cases, EBV-HLH clinical picture can be difficult to differentiate from T cell lymphoproliferative disorder (69). EBV-HLH is usually quite aggressive, with a frequent involvement of CNS. Once EBV is demonstrated by serology tests or molecular biology methods, a targeted approach is recommended (74,76). Clinical scores to differentiate low risk vs. high-risk patients have been proposed (76), but have not achieved conclusive results yet. Targeted treatment is discussed later in this review.

Cytomegalovirus (CMV) is typical of newborns and immunocompromised patients (72,73,77-81) and so is the varicella zoster virus (82). Dengue virus plays a significant role in tropical countries (73,83-90). Tick-borne diseases include the Crimean-Congo hemorrhagic fever nairovirus (91,92) and the Eastern equine encephalitis virus (93). Sporadic case reports include HLH related to HAV (90,94), HBV (95), HIV (89,96), Adenovirus (72,89,97,98), Parvovirus B19(72), Measles (88) and Influenza virus (99-101).

Sars-Cov2 infection, causative of the recent COVID19 pandemic, has been reported to also be a HLH trigger, mostly in adults (102,103). In 2021, a Mexican group reported the unusual case of a 7-years old boy that developed a severe inflammatory syndrome triggered by Sars-Cov-2 and Dengue virus coinfection (104). The authors diagnosed both multisystemic inflammatory syndrome in children (MIS-C) for the presence of coronary involvement and HLH for fulfilling the HLH 2004 criteria. The patient required mechanical ventilation and maximal support treatment for 2 weeks, but completely recovered. The authors agree that it is a very unusual presentation of HLH. In 2022, an Iranian group described the case of an 8-year-old boy who developed MIS-C with heart failure requiring heart transplantation (105). Liver and bone marrow biopsy, together with typical laboratory findings, fulfilled HLH04 diagnostic criteria. He was initially treated with intravenous immunoglobulins (IVIGs) and steroids, followed by heart transplantation and the consequent heavy immunosuppression. Unfortunately, the patient died 20 days after surgery. A third case, regarding a female newborn birthed from a SARS-CoV-2-positive mother via cesarean section at 35 weeks of gestation, was reported from Jordan in 2022(106). The newborn tested positive for SARS-CoV-2 on the first day after birth and progressively developed typical HLH syndrome from the 6th day of life. The patient died on day 51 from severe respiratory failure.

Bacterial infections associated to HLH have frequently been reported in tropical countries and include Brucella (72,107-109), Salmonella enteritidis (73,90,110), Tuberculosis (88,90,111), sepsis by streptococcal infection [group B streptococcus (112) and Streptococcus suis (113)], by Listeria (72) or without identified pathogen (90). Orientia tsutsugamushi, the causing agent of scrub typhus, has been reported in India (73,89,90) and Korea (114-117). Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, has been reported from the US (118-122). A case of S. pneumoniae 23A, serotype that is not included in the pneumococcal 13-valent conjugated vaccine (PCV-13) has been reported from Japan (123).

HLH in kidney transplant recipients has been associated with Ehrlichiosis in one case (121), and with Bartonella henselae in one other case (124). A case associated with Serratia marcescens has been reported in a preterm newborn (125).

Fungal infection-associated HLH has been reported in immunocompromised hosts (being treated for aplastic anemia and preB-acute lymphoblastic leukemia, respectively), caused by Trichosporon asahii (126).

Leishmania (71,72,127-135) and Plasmodium (90) [falciparum (136) and vivax (137)] have been reported as parasitic triggers of HLH.

M-HLH represents another challenging category, because it can be the initial manifestation of an underlying malignancy, it may develop during therapy as the consequence of chemotherapy or of treatment-induced mutations [treatment-induced HLH (Ch-HLH)], or it can develop as a consequence of an intercurrent infection (138-141).

HLH as a manifestation of underlying malignancy. The excess of proinflammatory cytokines produced by activated T cells infiltrating or surrounding the tumor or by the neoplastic T-cells in T/NK cell lymphoma is likely to cause M-HLH (1,38). In rare cases, the diagnosis of HLH anticipates that of malignancy by several weeks (138,142).

M-HLH is common in adults, where it accounts for ~50% of HLH cases (15), while it is significantly rarer in children, with a prevalence of 8-11% (138,143).

Lymphomas, though relatively uncommon at pediatric age, are frequently reported in association with HLH (138). Among the cases we reviewed in the present study, HLH has been associated with Hodgkin lymphoma (73,121,140,144), anaplastic large cell lymphoma (73,145,146), peripheral T-cell lymphoma (145,147), post-transplant lymphoproliferative disorder-lymphoma (148), subcutaneous panniculitis-like T-cell lymphoma (71) extranodal NK/T cell lymphoma, hepatosplenic T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood (145). In addition, HLH and lymphoma can be alternative diagnoses as well (149), and extreme caution is required in differentiating the two conditions before starting steroid therapy.

Acute leukemia (both myeloblastic and lymphoblastic) presenting as HLH syndrome has also been reported (72,121,138-141,145).

Among the cases we reviewed in the present study, HLH developed on therapy or after treatment for juvenile myelomonocytic leukemia (150), acute monocytic leukemia (151), Langherhans cell histiocytosis (152) and solid tumors [Wilms tumor (153), neuroblastoma (72,154), rhabdomyosarcoma (140)]. Langerhans cell histiocytosis has been associated to HLH at presentation, during therapy or as a consequence of viral infection (140,142,155).

The immunosuppression induced by treatment frequently causes viral infections or reactivations, that in turn can trigger HLH. Among the cases reviewed, viral reactivation included EBV, CMV, respiratory syncytial virus, BK virus, human herpes virus 6, adenovirus and parvovirus B19 (141,156).

HLH syndrome occurring in the context of rheumatological disorders has been commonly referred to as MAS (157). While traditionally considered separate entities that share common features, it is now clear that they should be viewed as the same disease, regardless of differences in presentation and treatment (64). However, the detailed description of MAS-HLH is beyond the scope of the present work.

Various emerging therapies, such as chimeric antigen receptor T cells (CAR-T cells) and Blinatumomab, have been associated with CRS (29). CRS shows major overlaps with HLH, so numerous authors classify it as a form of sHLH (158-160); some other authors, conversely, restrict the diagnosis of HLH following CAR-T infusion (carHLH) to the cases in which a severe CRS is associated to the typical HLH laboratory findings, often including hemophagocitosis (161,162).

CAR-T cells were officially licensed for use in children and young adults by the U.S. Food and Drug Administration in 2017 for the treatment of refractory B-cell acute lymphoblastic leukemia (163). CRS is a common severe adverse reaction following such therapies, especially in the acute phase (29). It probably results from the high amount of IFN-γ and IL-6 produced by the constitutionally activated CAR T cells (164) or by the subsequently activated macrophages (165-167) and by the targeted tumor cell lysis (168). Development of carHLH seems associated with pre-infusion NK lymphopenia (although it is not associated with impaired NK function) that is further amplified by treatment (161).

The St. Jude Children's Research Hospital group recently described a cohort of 27 pediatric patients treated with CD-19 CAR-T cells (162): 12/27 patients developed CRS alone, while four progressed to carHLH despite appropriate therapy. The carHLH subgroup showed higher and more sustained inflammation parameters and a poorer antileukemic response and survival. Blinatumomab, as well, has been associated with CRS and/or HLH in adults (29) and children (169).

Patients treated with T-activating therapies should be closely monitored for HLH, in order to optimize diagnosis and therapeutic approach.

Additional iatrogenic causes of cytokine storm include rituximab (170), gene therapies, immune checkpoint inhibitors, allogenic HSCT and heart transplantation (29).

HLH has been described in the context o HSCT (171,172) and, more rarely, in kidney (124,165) and liver (173-175) transplant recipients. Post-transplant HLH (PT-HLH) might be triggered by a combination of tissue damage, immunosuppressive therapy (176), alloimmune response, residual malignancies or by infections (171). Viral infections or reactivations, such as EBV and CMV but also gastrointestinal viruses, represent the most frequent trigger, but bacterial and fungal infections have also occasionally been described (124,165,172,177-179).

HLH post-HSCT (post-HSCT HLH) can occur within the first 30 days after transplantation (early onset) or later (late onset) (171). Late onset is usually related to infectious events, while for early onset the causes are not fully understood. In some cases the triggering factor may be the residual disease (171). PT-HLH diagnosis is extremely challenging because of the complex clinical and laboratory picture of the affected patients, for whom specific diagnostic criteria do not exist yet, and a high suspicion rate is essential (180).

In early onset post-HSCT HLH, aspects of differential diagnosis may occur with engraftment syndrome and acute graft-vs.-host disease (171). The treatment of PT-HLH is also particularly complex, since we are dealing with immunosuppressed patients, often with transplant-related toxicity (171). In EBV-related forms, the use of rituximab plays an important role (37,181).

HLH has recently been described in the context of febrile infection related epilepsy syndrome (FIRES) (182), an epileptic encephalopathy characterized by refractory status epilepticus following unspecific febrile illnesses. The pathophysiology of FIRES has not been completely understood, but it likely to depend, once more, on dysfunctional activation of the innate immune system (182). Moreover, though rare, this association stresses the need of screening for patients with HLH presenting with severe CNS symptoms.

A curious case of HLH following spider bite (Loxosceles reclusa) has been reported from the US (183).

Drug reaction with eosinophilia and systemic symptoms induced by vancomycin, carbamazepine and levetiracetam has been described as an occasional trigger of HLH (72).

Among the cases reviewed in the present study, several patients were diagnosed with sHLH/HLH syndrome in the context of an inherited condition associated with metabolic diseases, such as Wolman's disease (184), galactosemia, Gaucher disease (185), Niemann-Pick disease, methylmalonic acidemia and propionic acidemia (186). Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (187), type 1 glycogen storage disease (188), lysosomal acid lipase deficiency (189), mucopolysaccharidosis-plus syndrome (190).

These patients fulfilled the HLH 2004 criteria, but it is possible that some HLH features, such as splenomegaly and cytopenias, were caused by the metabolic disease itself rather than by immune hyperactivation (184-190). On the other hand, it is possible that metabolites accumulation activated macrophages and triggered a proper sHLH. The link between metabolic disorders and HLH needs to be clarified, but an extensive screening for underlying metabolic diseases should be performed in patients presenting with HLH (184,185).

Since a neat distinction between FHL and sHLH is often impossible in the clinical setting, the HLH 94/04 protocol (7,193) suggests a pragmatic approach to HSCT indication, which remains a mainstay of therapy.

HLH 94 induction chemotherapy is based on steroids and etoposide administration for 8 weeks with or without intrathecal methotrexate, followed by maintenance with cyclosporine A (CSA), etoposide and dexamethasone pulses while waiting for HSCT (193). Chemotherapy might be suspended after 8 weeks of treatment once clinical remission is obtained and a clear genetic base is excluded (193). Otherwise, if a clear genetic base is proved, or in case of reactivation, HSCT is recommended (193).

Treatment intensification was proposed in 2004, but failed to improve the outcome (6). The Histiocyte Society thus recommends HLH-94 as the standard of care (7).

The heavy mortality rate before HSCT (7) and the significant toxicities of chemotherapy urge the clinicians to find new treatments (6). This is especially true in patients with sHLH, whose prognosis widely varies from a 55% survival at 3 years with the standard protocols (193) to 100% survival in specific conditions (such as Leishmania-triggered HLH when treated appropriately) (135).

The ongoing recognition of the central role played by cytokines suggests a treatment shift towards targeted therapies. Whether these drugs can eradicate HLH alone or require the addition of etoposide remains to be clarified, as well as how they are best used [including doses, drug combinations and use in adults/children (6).

Emapalumab (NI-0501; Novimmune SA), an anti-IFN-γ monoclonal antibody has been used successfully in pediatric patients with refractory or previously untreated pHLH (194,195).

The most recent clinical trials focus on hybrid immunotherapy (ATG + etoposide + dexamethasone; NCT01104025), monoclonal antibodies such as Alemtuzumab (196) (NCT02472054) and tyrosine-kinase inhibitors such as ruxolitinib (197) (NCT04999878; NCT04120090; NCT04551131; NCT03795909; ChiCTR2000029977) and zanubrutinib (NCT05320575). Studies on selectively cytokine-targeting molecules include Tocilizumab (198) (NCT02007239), Anakinra (NCT02780583) and Emapalumab/NI-0501(195) (NCT05001737; NCT03311854; NCT03312751; NCT01818492). In the subset of EBV-HLH patients, the ongoing clinical trials focus on Rituximab (NCT05384743), anti CD20 monoclonal antibody Sintilimab and Lenalidomide (NCT05258136), Tabelecleucel (NCT04554914), programmed cell death protein 1 antibody alone (NCT05039580) or in association with Lenalidomide (NCT04084626).

In intensive care settings, nonspecific physical therapies showed interesting results, at least in the acute phase (199,200). If a massive release of cytokines/a cytokine storm is responsible for organ damage, blood purification techniques can blunt the process with a rapid non-selective effect, potentially translating into survival benefit (199). These techniques include plasma exchange, which has been successfully applied in medium resource contexts (such as Turkey and India) (201,202), and hemofiltration combined with highly immune adsorbent cartridges (119,191,200). HLH recommendations have recognized blood purification as a potential salvage treatment in adults (203). Moreover, these non-specific therapies might be combined with targeted therapies (204), but do not replace appropriate treatment of the underlying trigger of HLH, especially in patients with refractory disease (205).

The most recent consensus guidelines on recognition, diagnosis and management of HLH in critically ill children (46) stress the importance of aggressive treatment. Steroids, in particular, should not be delayed as part of first-line treatment in different protocols (HLH-94 and ATG-based protocols) and are effective in both primary and secondary HLH.

A Canadian group reported nine cases of pediatric liver transplantation in acute liver failure secondary to sHLH, suggesting that HLH might not be an absolute contraindication to transplantation and instead play a role in a very restricted subgroup of patients (19).

Treatment of infection-associated HLH deserves specific considerations according to the underlying agent, since some forms respond to anti-infectious treatment [namely Leishmania (131-134), Ehrlichiosis (122), scrub typhus (89,90) and other tropical diseases-associated forms], while reaction to some other pathogens displays intermediate features and benefits of standard aggressive immunosuppression.

Several studies (131,132,134,206) have reported complete response of Leishmania-associated HLH to amphotericin B therapy +/- steroids, with an excellent prognosis [3 year overall survival rate 24% in pHLH vs. 100% in visceral leishmaniasis-associated HLH; P<0.001; 2021 Iranian cohort of 60 children (133)]. A Chinese retrospective study (135) reported good results with antiparasitic therapy associated with chemotherapy according to the HLH94 protocol in patients with visceral leishmaniosis-HLH; however, the cohort includes children and adults and it is impossible to discriminate whether treatment intensification depends on age.

Human monocytic ehrlichiosis, can also trigger an HLH form that has proved to respond to antimicrobial therapy alone (doxycycline) (122).

Scrub typhus, dengue fever and tropical infections in general respond to steroids alone or to regimens without cytotoxic drugs in cases with mild to moderate disease activity (89,90).

The hepatitis-associated HLH cases reviewed in the present study have experienced complete recovery on antiviral therapy and ruxolitinib in the case of a patient with HBV (95) and after IVIG-infusion in the cases of patients with HAV (94).

A patient developing HLH after HIV diagnosis has required both antiretroviral therapy and chemotherapy according to HLH94 protocol (96).

The Crimean Congo hemorrhagic fever-associated HLH has been resolved using antiviral therapy combined with IVIG therapy and plasma exchange (91).

The case of EBV-HLH is more complex. EBV-HLH standard treatment is by the HLH94 protocol, and early initiation of etoposide-based regimens improves survival in severe EBV-HLH (8,74,207). Chemotherapy can be combined with targeted therapies such as Rituximab (37,181) or Ruxolitinib (208). Treatment intensification with the L-DEP regimen (PEG-asparaginase in combination with liposomal doxorubicin, etoposide and high-dose methylprednisolone) has been reported as salvage therapy and bridge to alloHSCT for children with refractory disease (209). Blood purification techniques (plasma exchange and continuous renal replacement therapy) have proved to be safe and effective in addition to standard chemotherapy in severe cases (199). On the other hand, patients presenting with mild-to-moderate disease activity may only require immunomodulating therapies, such as IVIGs, CSA and steroids (69). A Japanese group (210) described a cohort of 22 patients aged 6 months to 41 years in which >60% recovered after immunotherapy (IVIG, CSA 6 mg/kg/day, prednisone); they suggest that early immunotherapy may modulate T-cell activation and reduce the chance of unnecessary chemotherapy. Sporadic cases of spontaneous resolution have been reported: An American group (68) reported the cases of two adolescents who fulfilled the HLH2004 diagnostic criteria in the context of an acute EBV infection. In both cases, treatment was planned according to HLH94 protocol, but the patients spontaneously improved before the initiation of therapy. The authors recommend caution in starting aggressive treatment, especially when patients experience mild-to-moderate symptoms. These differences in treatment approaches and outcomes underline the need for evidence-based risk stratification criteria in patients with EBV-HLH.

Malignancy-triggered forms should be distinguished from the forms that occur after chemotherapy (Ch-HLH) to optimize the therapeutic approach.

Ch-HLH. Ch-HLH is often caused by infections related to immunosuppression, and benefits of anti-infectious therapy, steroids and IVIGs.

HLH as a manifestation of underlying malignancy. M-HLH is associated with increased mortality and the early detection of malignancy can favorably influence the prognosis. Treatment is not homogeneous, and can address the underlying malignancy first, be focused on HLH or a combination of the two (41,138,145,211-213). The published series refer to small numbers and suggest an individualized therapeutic approach. Aggressive HLH-directed therapy at the onset of malignancy-associated HLH can delay or complicate anticancer treatment (39,41).

MAS-HLH usually responds to steroid therapy or to anakinra (recombinant human IL-1 receptor antagonist), especially when given in the early course of therapy (214,215). The present study refers to specific international guidelines for more detailed considerations on MAS-HLH (157,216,217).

The cytokine release syndrome following CAR-T or Blinatumomab administration usually responds to Tocilizumab, steroids, Anakinra or to a combination of such molecules (29,165-167,169). Whether carHLH should be differentiated from a severe form of CRS is still an open question, and a more precise definition of both conditions is essential in order to guide therapeutic choices in the most complex cases (162,218).

Post-transplant HLH requires prompt and aggressive treatment because of the high risk of graft failure, but no international consensus exists on treatment protocol. Reported therapeutic approaches range from corticosteroids, IVIGs and low-dose etoposide (219-221) to standard HLH94 protocol with eventual rescue HSCT (172). Ruxolitinib was recently reported as salvage therapy in 2 children, with alternating results (222). Outcome is unclear, usually compromised by graft failure.