Adult secondary hemophagocytic lymphohistiocytosis

Abstract Background Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive inflammation and tissue destruction due to abnormal immune activation. HLH carries a very high mortality, and while delays in patients’ presentation to hospital, time to suspicion of HLH, investigation, and initiation of therapy all play a part, mortality remains high even with timely diagnosis and treatment. Classical manifestations of HLH include persistent fever, cytopenias, and liver dysfunction. Case presentation We present four cases of secondary HLH, highlighting the demographic and clinical characteristics of these patients, underlying triggers (including systemic lupus erythematosus, lymphoproliferative disorders, and leishmaniasis), together with challenges associated with the diagnosis and treatment of this rare disorder and a brief review of literature. Conclusion HLH has protean manifestations and requires a high index of suspicion as it can be a great clinical masquerader. Mortality due to multiorgan failure is often high even with early recognition and treatment.


METHODS AND MATERIALS
We conducted a retrospective review of the patient charts of four patients, who were diagnosed with HLH at the Aga Khan University Hospital, Nairobi between 2018 and 2019. The diagnosis of HLH was based on the HLH-2004 guidelines that require five of eight criteria to qualify for a diagnosis of HLH.

Case A
A 30-year-old male patient was referred to our outpatient department following treatment for neutropenic sepsis and bone marrow failure at a peripheral facility. He had been unwell for 2 months prior, with fatigue, unexplained weight loss, and fevers as the predominant symptoms. There had been severe anemia necessitating blood transfusion, easy bruising, and evidence of mucosal hemorrhage, with gum bleeding and frank haematuria.
Initial assessment revealed temperature of 39 • C, heart rate (HR) of 80/minute, respiratory rate (RR) of 20/minute, and blood pressure (BP) of 90/60 mm Hg. The physical examination was significant for hepatosplenomegaly. Initial investigations at time of admission are summarized in Table 1.
A computerized tomography (CT) scan of the chest and abdomen revealed features of pulmonary edema, scattered ground glass opacities, and gross splenomegaly of 20 cm.
Despite broad-spectrum antibiotic therapy, the patient remained febrile. Blood cultures were unrevealing for any microbial growth.

Case B
A 47-year-old woman presented with a 2-week history of generalized body weakness. She had been diagnosed with and treated for malaria at a peripheral facility. She also had new onset acute kidney injury, which had been attributed to malaria. Initial assessment at our facility revealed temperature of 38.5 • C, HR 100/min, RR 20/min, and BP 155/89 mm Hg. Her abdominal examination was significant for splenomegaly. Initial investigations at presentation are shown in Table 2.
During the initial workup for anemia, ferritin was noted to be markedly elevated at 1794 ng/mL, which prompted further workup for  Four days into her admission, and after the first dose of methylprednisolone and red cell transfusion, she suffered a tonic clonic seizure, suffered a cardiac arrest, and died despite prolonged resuscitation attempt.
Results of the autoimmune screen were obtained 1 day later, and gave positive ANA in a speckled pattern, negative double-stranded DNA (dsDNA), while the extractable nuclear antigen was positive for both anti-Sm and anti-RNP, clinching the diagnosis in this case to be HLH in a patient with untreated SLE.
Transient confusion while on the ward coupled with a seizure prior to death are highly suggestive of CNS HLH, which carries a dire prognosis.

SUMMARY OF RESULTS
The age of the patients ranged from 25 to 47 years (male to female ratio 1:1). Fever of undetermined origin, cytopenias, hyperferritinemia, and either splenomegaly or hepatosplenomegaly were prominent features at presentation in each of the cases. All patients met the modified HLH diagnostic criteria according to the HLH-2004 guidelines that require five out of eight criteria to qualify for a diagnosis of HLH. The clinical and laboratory characteristics are summarized in Table 5. and NK cell testing are not available in our country hence were not tested.
A secondary cause of the HLH was identified in all of the patients; two had previously undiagnosed and untreated SLE, one visceral leishmaniasis and the fourth classic EBV-driven Hodgkin's lymphoma.

DISCUSSION
HLH is a syndrome of excessive inflammation and tissue destruction due to abnormal immune activation. HLH is on the spectrum of macrophage activating syndromes, in which over activation of macrophages leads not only to exaggerated cytokine production, but also to hyperferritinemia, in the release of iron from macrophages, and hemophagocytosis of blood cells [5][6][7][8].
The sustained activation of macrophages, NK cells, and cytotoxic T cells leads to excessive cytokine production. The persistent production of cytokines, including interferon gamma, tumor necrosis factor alpha, interleukins (ILs) such as IL-6, IL-10, and IL-12, and the soluble IL-2 receptor, further recruits additional inflammatory cells, in a vicious cycle, leading to a cytokine storm. This cytokine storm is the primary mediator of tissue damage [9].
Macrophages are a storage reservoir of ferritin, and this accounts for high ferritin levels in HLH [10]. Recorded peak ferritin levels greater than 10 000 g/L was found to have a 90% sensitivity and 96% specificity for HLH during a study done at the Texas Children's Hospital [11].
Therefore, when the clinical and laboratory features are suggestive, a serum ferritin of >10 000 g/L strongly supports a diagnosis of HLH [2].
This was certainly reflected in three of four cases of this series, where ferritin, initially performed in assessment of anemia, unexpectedly gave hyperferritinemia > 10 000 g/L, immediately triggering investigations for HLH.
The most common presentation of HLH as per the HLH-2004 study included fever, splenomegaly, bicytopenia, hypertriglyceridemia or hypofibrinogenemia, hemophagocytosis, ferritin > 500 mcg/L, low or absent NK cell activity, and soluble CD25 elevation [12]. As summarized above, all four patients described in this series presented with fever, splenomegaly, hyperferritinemia, and cytopenias.
In the early stages of HLH, hemophagocytosis might not be seen in the bone marrow. This and the patchy nature of this phenomenon in bone marrow samples mean that its absence does not negate HLH as the diagnosis. The incidence of bone marrow involvement varies between 25% and 100% [13] and bone marrow analysis for presence of hemophagocytosis has a sensitivity of around 60% [14]. One of the patients (Case B) did not have hemophagocytosis on the bone marrow, but did have an excess of histiocytes within the bone marrow. This probably corresponds with the patient having the "mildest" and possibly earliest form of HLH in the cohort and hence being picked up at the earlier than the other cases.
The overall mortality of HLH is high, varying between 26.5 and 74.8%, and depends on the patient characteristics, underlying precipitating conditions, and stage of disease [15]. This was also seen in our case series, which reported a high mortality rate, with the only survivor having been diagnosed at a relatively early stage of the disease course, and remaining in good health to date.

Diagnostic criteria
The diagnostic criteria is based on the HLH-2004 trial [12,16]. To make a diagnosis, at least five out of eight findings are required as highlighted in Tables 6 and 7

Triggers of HLH
In all four cases, a trigger for HLH was identified, but in only one of these cases, characterised by early patient presentation, timely clinical suspicion, work up and treatment resulted in a favourable patient outcome. Macrophage activation has been reported to be triggered by infections, most commonly viral infections such as EBV [20][21][22], and has also been associated with leishmaniasis and CMV [22]. Malignancies, most commonly lymphoproliferative, are also recognized to be activators of the immune system as are autoimmune conditions.
Although a seemingly heterogeneous group of conditions, the disorders mentioned above all have the ability to activate the immune system and lead to a macrophage activating syndrome typical of HLH, characteristic of the disordered, exaggerated response, and its resultant tissue damage.

Treatment
Therapy is based on the HLH-94 protocol [23,24]. The standard treatment is corticosteroids, to which etoposide is added in cases which do not initially respond, and in which no other cause is found for the HLH, along with identification and treatment of any underlying cause. Rituximab is recommended in EBV-driven HLH, with the mechanism of action being eradication of the EBV reservoir through clearance of B cells [15,19]. For central nervous system disease, intrathecal methotrexate and hydrocortisone are added to the induction therapy [24]. In patients not showing signs of improvement, the above therapy is continued while planning for allogeneic hematopoietic cell transplantation in suitable cases.
Patient A had EBV-driven classical Hodgkin's lymphoma but the diagnosis was only available postmortem. Additionally, he had been unwell for a number of months prior to presentation. Despite initiating etoposide and methylprednisolone within days of presentation at our center, he had a rapid clinical deterioration and died. Rituximab therapy would have been a recommendation in his case, had EBV PCR been performed and found positive [15,19]. HLH associated with lymphomas tends to have a higher mortality compared to HLH due to infections and autoimmune conditions [22]. Worsening cytopenias and cholestasis are predictive of death in HLH, and a rapid drop in ferritin levels after initiation of treatment is associated with improved outcome [25].

CONCLUSION
Diagnosis of HLH presents a diagnostic challenge to clinicians due to the nonspecific signs and symptoms that portend a wide array of differential diagnosis. Therefore, a high index of suspicion is warranted especially when a patient presents with unremitting fevers and cytopenias.
In our series, an unexpectedly high ferritin, which in each case was performed early in course of admission to investigate anemia, resulted in early suspicion of the condition and relatively early appropriate investigations and treatment for HLH.
However, despite benefitting from an early diagnosis and initiation of therapy for HLH, vis-a-vis their admission to our facility, three of these patients presented with already established macrophage activation, HLH, and features of organ failure. Therefore, the HLH-directed treatment, though appropriate, proved futile in these cases.
The mortality of patients with HLH is very high and delay in diagnosis confers the greatest risk to a fatal outcome, as shown in this series where the time to presentation ranged from 2 weeks to 2 months.
This case series highlights the difficulty in obtaining a good outcome in HLH, even when the diagnosis is suspected early on. Additionally, HLH was not suspected at peripheral facilities, probably due to a combination of factors: (a) it is uncommon and shares features with sepsis, which is far more common; (b) ferritin level, which was markedly elevated and prompted a search for HLH at our center, is not widely available at peripheral facilities and can be costly.
It cannot be stressed enough that a high index of suspicion is warranted, when faced with a patient with a triad of fever, cytopenias, and organomegaly, which is unexplained, and that checking a ferritin level in this case may highlight unexplained hyperferritinemia and lead to appropriate investigations for HLH. The patients described here were diagnosed with HLH promptly within our service, partly because we were able to quickly discount alternative diagnoses as treatment of these had already been initiated with poor outcome at peripheral facilities.
A multidisciplinary approach, including early consultations with a haematologist, either remotely or in person is essential to promptly investigate and initiate appropriate therapy, which is relatively accessible. Prompt, lifesaving and timely treatment, is likely to have to be initiated before all test results have been returned, hence relying on a consensus of clinical and laboratory features compatible with the diagnosis, but not yet diagnostic of it.