Systemic and Nodular Hyperinflammation in a patient with Refractory Familial Hemophagocytic Lymphohistiocytosis 2

Abstract

Familial hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome resulting from defective cytotoxicity. A previously healthy 3 month old female presented with fever, irritability, abdominal distention, and tachypnea. She ultimately met all eight HLH-2004 diagnostic criteria, accompanied by elevated CXCL9. Initial empiric anti-inflammatory treatment included anakinra and IVIg, which stabilized ferritin and cytopenias. She had molecular and genetic confirmation of perforin deficiency and was started on dexamethasone and etoposide per HLH-94. She clinically improved, though CXCL9 and sIL-2Ra remained elevated. She was readmitted at week 8 for relapsed HLH without clear trigger and HLH-94 induction therapy was reinitiated. Her systemic HLH symptoms failed to respond and she soon developed symptomatic CNS HLH. She was incidentally found to have multifocal lung and kidney nodules, which were sterile and consisted largely of histiocytes and activated, oligoclonal CD8 T-cells. The patient had a laboratory response to salvage therapy with alemtuzumab and emapalumab, but progressive neurologic decline led to withdrawal of care. This report highlights HLH foci manifest as pulmonary/renal nodules, demonstrates the utility of monitoring an array of HLH biomarkers, and suggests possible benefit of earlier salvage therapy.

Introduction:

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome first described in 1939 in children found to have fever, systemic inflammation, coagulopathy, and histologic hemophagocytosis¹, and later with dramatic hyperferritinemia. A genetic link to perforin-deficiency was first reported in 1999², and since that time accumulating genetic and functional observations have established severe impairment of granule-mediated killing by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells as the mechanism of Familial HLH (FHL). Iterative clinical trials demonstrated a potentially curative role for allogeneic hematopoietic stem cell transplantation (HSCT).³ The presence of this syndrome in the context of infection, malignancy, or rheumatic diseases has been dubbed reactive or secondary HLH in patients of all ages^3,4. In addition to ferritin, HLH-related biomarkers include soluble IL-2 receptor (sIL-2RA/sCD25); the inflammasome activated cytokine IL-18 (most strongly associated with Macrophage Activation Syndrome); and several IFNg-induced molecules (the purine analog neopterin, the chemokine CXCL9, and the soluble inhibitor IL-18 Binding Protein, IL-18BP)^5,6. This report focuses on several unique but instructive features of an infant with ultimately fatal perforin-deficiency and FHL-2.

Case Report & Methods:

An African-American female presented at 3 months of age with fever, irritability, abdominal distention, and increased work of breathing. She had benign and noncontributory birth, past medical, and family histories.

She was found to have transaminitis, pancytopenia, hyperferritinemia, elevated lactate, hypofibrinogenemia, and hepatosplenomegaly. Infectious evaluation (including stool and blood CMV and EBV, and blood adenovirus, enterovirus, HSV, and HIV) was unrevealing. HLH-related functional testing and biomarkers at presentation supported an HLH diagnosis with extremely elevated CXCL9 and soluble IL-2 receptor alpha (sIL-2Ra, figure 1). Bone marrow was normocellular with trilineage hematopoiesis, prominent hemophagocytosis, and no evidence of malignancy. She met all eight HLH-04 criteria (table 1), and was diagnosed with HLH. ³ MRI of the brain on hospital day (HD) 8 did not show evidence of enhancement, ventriculomegaly, or edema indicative of CNS HLH⁷. Analysis of CSF on HD9 was normal.

Figure 1: Longitudinal Biomarkers of HLH.

Key laboratory features of HLH and concomitant treatments throughout this patient’s hospital course. The y-axis represents the Log₁₀ fold-change from the upper limit of normal (ferritin, AST, c-reactive protein (CRP), soluble IL-2 receptor alpha (IL-2Ra), IL-18, IL-18 binding protein (IL-18BP), CXCL9) or lower limit of normal (platelet count, PLT).

Table 1:

HLH-04 criteria and biomarkers

HLH Criterion/Biomarker	Values at presentation	Values at readmission
1. A molecular diagnosis consistent with HLH	homozygous PRF1 (c.50del, p.Leu17Arg_fs*34)
2. Diagnostic criteria for HLH fulfilled
Fever	present	present
Splenomegaly	present	present
Cytopenias (affecting ≥ 2 of 3 lineages in the peripheral blood): Hemoglobin <9 g/dL Platelets <100 109/L Neutrophils <1.0 109/L	6.5	7.8
	18 109	95 109
	0.19 109	0.22 109
Hypertriglyceridemia and/or hypofibrinogenemia: Fasting triglycerides ≥ 3.0 mmol/L (i.e., ≥ 265 mg/dl) Fibrinogen ≤ 1.5 g/L	Triglycerides not obtained	Triglycerides not obtained
	1.36	3.70
Hemophagocytosis in bone marrow or spleen or lymph nodes	Present (bone marrow)
Low or absent NK-cell activity	Undetectable NK cell function, absent perforin expression
Ferritin ≥ 500 ng/mL	5741.9	1,507.5*
Soluble IL-2R alpha (a.k.a. soluble CD25) (622–1619 pg/mL)	12,828	11,545
CRP (< 0.29 mg/dL)	1.45	0.92
IL-18 (< 540 pg/mL)	14,356	10,355.23
IL-18 BP (3000–9000 pg/mL)	59,181	33,718
CXCL9/MIG (< 121 pg/mL)	6,299	3,013
Neopterin (CSF) (< 16.5 nmol/L)	Not obtained	32.9

^*peaked at 6524ng/mL later in readmission, see Figure 2

Eventually, perforin-deficiency and FHL-2 were confirmed by the absence of perforin expression by flow cytometry, as well as the observation of a homozygous early frameshift mutation in PRF1 (table 1). Additionally, she was found to have heterozygous variants of uncertain significance (VUS) in LYST (p.His333Tyr) and NLRC4 (p.Asn273Tyr) deemed not clinically actionable.

Prior to formal diagnosis of FHL, she received anakinra and IVIg beginning HD2, which coincided with improvement of AST and stabilization of ferritin and cytopenias. She was subsequently transitioned to dexamethasone (HD9) and etoposide (HD15) per HLH-94. She tolerated therapy initiation well and was discharged home on HD44. She was readmitted during week 8 of induction therapy for recurrent fever. Repeat infectious workup was negative, her symptoms were attributed to relapse of HLH (Table 1), and she was restarted on HLH-94 induction therapy.

CT scans performed on HD89 as part of her infectious work-up and in preparation for HSCT were notable for new focal nodular consolidations in the bilateral lungs (largest 1.1 × 1.1cm), and kidneys (largest 1.8 × 1.5cm, supplemental figure 1). She had no pulmonary or renal symptoms at this time. Biopsies of both the pulmonary and renal lesions revealed similar findings: extensive infiltration of proliferating (60% Ki67+) T-cells with strong CD8 predominance and numerous CD68+CD163+ histiocytes (figure 2). T-cell receptor clonality assessment identified oligoclonality, with a single TCRbeta chain clone and two TCRgamma chain clones, all in a polyclonal background. Specific stains for infection (including EBV) and malignancy were negative.

Figure 2. Bone marrow and lung biopsies identified active foci of HLH activity.

A) Bone marrow smear demonstrating red blood cells and nucleated cells within macrophages (H&E,×400);

B) The Lung lesion at low power (H&E, ×50) shows an extensive proliferation of inflammatory cells replacing the lung parenchyma;

C) Higher power view at the lung lesion (H&E, ×200) demonstrating numerous macrophages and lymphocytes;

D) CD163 immunohistochemical stain highlighting the macrophages (×200) E) CD4 and

F) CD8 immunohistochemical stains highlighting the T-cells with low CD4/CD8 ratio (×200).

Re-induction of HLH-94 failed to improve her clinical or laboratory signs of HLH (Figure 1), and on HD100 brain MRI showed extensive and global parenchymal enhancement, multifocal restricted diffusion, and generalized atrophy consistent with CNS HLH (supplemental figure 2), and intrathecal methotrexate and anakinra were added. Given her poor response to re-induction therapy, she received alemtuzumab on HD106. However, her neurologic status continued to worsen and ferritin remained elevated, and IFNg blockade with emapalumab was initiated on HD121 (figure 1).

Despite some improvement in ferritin, platelet count, and sIL-2Ra, her neurologic status progressively declined and her course was additionally complicated by respiratory failure, acute kidney injury, hypertension, feeding tube dependency, and pneumatosis coli. Infectious evaluations remained negative. Repeat brain MRI on HD169 showed worsening volume loss, restricted diffusion, and hemorrhagic conversion in the periventricular regions and cerebellum (supplementary figure 2). Given her neurologic decline and smoldering HLH disease activity despite aggressive medical therapies, she was redirected to comfort care and passed away on HD189.

Discussion:

This tragic case of an infant with profound perforin deficiency and FHL2 illustrates a few points relevant to care of severe HLH. First, her onset was stabilized with anti-inflammatories like IVIg and anakinra. The practice of early inflammatory stabilization in patients with suspected HLH is used increasingly for its possible temporizing effect. However, of critical importance, these therapies may prevent decompensation, but they may also provide a false sense of security and delay initiation of more definitive therapies. This patient received dexamethasone on HD9 and etoposide on HD15; more study is needed to determine how even small delays of treatment initiation might affect long-term outcomes. Little about her presentation presaged the challenging course that awaited her, and better prognostic indicators are sorely needed. However, her sIL-2Ra remained >4-fold above the upper limit of normal even with clinical improvement, suggesting ongoing subclinical HLH.

Second, her disease flared toward the end of her first induction cycle without a clear triggering event. This relapse proved refractory to re-induction of HLH-94. During re-induction her sIL-2R and CXCL9 still failed to improve, and her total IL-18 levels rose without a similar degree of elevation of IL-18BP. Without any evidence of infection, and despite potent anti-inflammatory and immunosuppressive treatment, what mechanism of disease continued to drive her HLH activity?

It is possible that part of the answer resided in her pulmonary and renal nodules (Supplemental Figure 1 and Figure 1). Such nodules, composed of activated and proliferating oligoclonal (largely CD8) T cells and histiocytes, are consistent with the expected pathology of HLH but, to our knowledge, have not been described previously in HLH. Despite negative work-up and broad prophylaxis, these nodules could nevertheless reflect the interaction of infection and immune dysregulation at the heart of FHL. Occult lymphoma is also a consideration, but FHL2 is not known to be a risk factor for lymphoma and these lesions developed while on etoposide-based chemotherapy. The observation of these nodules thus remains an intriguing association in this patient with refractory biomarkers and recurrent FHL2.

Finally, after nearly 30 days of re-induction therapy, we initiated salvage therapy. The patient demonstrated an impressive laboratory response to alemtuzumab and ultimately emapalumab (FDA approved only a few months prior to the patient’s relapse), with normalization of ferritin, sIL-2Ra, and thrombocytopenia. It seems likely that both salvage therapies contributed to this laboratory improvement (Figure 1). These interventions were ultimately fruitless in the face of such long-standing immunopathology. This case supports continuous monitoring of HLH biomarkers even in the face of clinical improvement. Likewise, it raises the possibility that etoposide re-induction may be contraindicated. To provide the best chance for prompt and safe allogeneic transplant, patients who flare (or simply fail to fully normalize HLH biomarkers during induction) may benefit from immediate use of therapies like emapalumab or alemtuzumab in a “treat-to-target” approach that more heavily weighs HLH biomarkers.

HLH highlights the complex interplay between environmental factors, genetics, and immune response, with uncontrolled immune response due to defective cytotoxicity leading to the clinical spectrum of disease that is HLH. Our case demonstrates a unique presentation of FHL-2 with a relapse characterized by refractory inflammation including chronically elevated IL-2Ra and CXCL9, highly elevated IL-18, and sterile pulmonary and renal nodules. Further study is essential to understand the varied clinical features of FHL.

Abbreviations:

HLH

Hemophagocytic lymphohistiocytosis

FHL

Familial hemophagocytic lymphohistiocytosis

HSCT

hematopoietic stem cell transplantation

VUS

variants of uncertain significance

NK

Natural killer cell

CTL

cytotoxic T lymphocyte

sIL2-R

Soluble interleukin-2 receptor

IL-18BP

Interleukin-18 Binding Protein

CNS

Central nervous system

CSF

Cerebrospinal fluid

MRI

Magnetic resonance imaging

IVIG

Intravenous immunoglobulin

HD

Hospital Day