Familial hemophagocytic lymphohistiocytosis (FHL) is a fatal autosomal recessive immune disorder related to the dysfunction of cytotoxic, granule-mediated cell-death pathways. Prolonged activation of T and NK cells lacking the ability to kill target cells produces a high amount of cytokines, including interferon (IFN)-γ. This leads to the infiltration of activated macrophages and the production of interleukin (IL)-6, IL-18, and tumor necrosis factor (TNF)-α (1). Activation of T cells and macrophages plays a significant role in symptom development and organ damage in FHL. FHL type 3 (FHL3) is caused by a variant of the UNC13D gene encoding Munc13-4, which regulates the secretory process of cytotoxic granules of lymphocytes (2–4). In addition to the common clinical and laboratory findings, half of FHL3 patients present central nervous system (CNS) involvement and malignancy (4, 5). FHL is a fatal disease that requires early definitive diagnosis so that appropriate bridging therapy can be started until curative hematopoietic cell transplantation (HCT) is possible (4).

Immunomodulation of hemophagocytic lymphohistiocytosis (HLH) based on the pathophysiology and disease activity is warranted. However, specific cytokines that allow discrimination between FHL and secondary HLH have not been defined (6). Also, proteome analysis did not identify differential expression between primary and secondary HLH (7). Monitoring of serum cytokine profiles is effective in evaluating inflammatory status in cell therapies, infections, cancers, and auto-immune diseases (8). Time-course cytokine monitoring allows for appropriate selection and dosage of immunomodulators and is extremely important for understanding the pathogenesis of FHL. However, specific cytokines for monitoring disease activity have not been established in FHL patients.

FHL patients are predisposed to developing severe HLH even in utero or at birth, which can be fatal due to multiple organ failure, and often fail to survive until curative HCT (9–11). In the treatment strategy based on HLH-94/2004 protocols, HLH disease activity frequently recurs during steroid reduction and prolonged intervals of etoposide administration (12, 13). The HLH-2004 protocol has not improved pre-transplant patient mortality or improved survival (13), necessitating the need for novel therapeutic agents.

Ruxolitinib, a Janus kinase (JAK) inhibitor, has been used in the treatment of myelofibrosis, polycythemia vera, refractory acute and chronic graft-versus-host disease (GVHD), as well as COVID-19 acute respiratory distress syndrome and post-transplant idiopathic lung injury (14–19). Ruxolitinib is known to inhibit JAK1, JAK2, and signal transducer and activator of transcription (STAT) 3 pathways, thereby reducing downstream signaling and cytokine production, including IL-6 and IFN-γ (20–22). Reports of the use of ruxolitinib as an immunomodulator for FHL have been published (11, 23–29). However, the dose of ruxolitinib, combination of other therapies, and investigated cytokines were diversified.

In this case report, we describe an infant with FHL3 who underwent successful HCT following bridging therapy with ruxolitinib. The ruxolitinib dosage was adjusted based on monitored cytokine profiles.

In this case study, the dose adjustment of ruxolitinib, guided by cytokine monitoring, was an effective bridging therapy, and minimized the required dose of etoposide and DEX. This strategy also contributed to the decision of HCT timing with maintaining performance status. However, the cytokine levels of IL-18, CXCL9, sIL-2R, and sTNF-RII remained elevated above cutoff levels, suggesting that adequate doses of ruxolitinib combined with HLH94/2004-based therapy would be necessary for the treatment of FHL.

Past reports showed that cytokine levels, including IL-6 (cutoff level, 37.25 pg/mL), at initial diagnosis predict early death in children with HLH (40). The IL-6 level in our case was consistently below this cutoff ( Figure 1D ). The CXCL9/IL-6 ratio is discriminately elevated in HLH compared with systemic inflammatory response syndrome/sepsis (7). Similarly, we observed that serum CXCL9 which is IFN-γ-inducible CXCR3 ligands and reflecting amplification of IFN-γ signaling wathway (41), was consistently higher than the normal range throughout the clinical course in our case ( Figures 1C, D ). IL-18, sTNF-RII, and sIL-2R showed a similar trend to CXCL9 ( Figures 1C, E ). During the disease course, the patient had three episodes of fever without any apparent sign of infection, negativity in blood cultures, and the highest CRP level was 1.9 mg/dL. Thus, we monitored cytokines, which directly reflected FHL activity in our case. Further validation is warranted to determine whether these cytokine profiles are effective to monitor disease activities of FHL.

Effective salvage therapy has not been established for refractory FHL. A retrospective study using rabbit antithymocyte globulin (ATG) showed that one-third of patients relapsed after first course of ATG, and 19 of 38 patients who received HCT after first or second-line ATG have been cured. The median time between ATG therapy and HCT was 6 weeks (42). Alemtuzumab as salvage therapy showed partial responses and response was limited to two weeks (43). Both are not suitable salvage therapies for 1-3 months old infants who are required to wait for sufficient growth before HCT. Infectious complications and cytokine release syndrome are concerns on both therapies. Thus, alternative therapies are urgently required.

Hyperactivation of effector CD8+ T cells with overproduction of IFN-γ contributes to FHL pathogenesis (44). In a mouse model of FHL2, loss of IFN-γ receptors in hepatocytes reduced liver dysfunction, suggesting that a non-hematopoietic response is also crucial in developing FHL hepatitis (45). These findings suggest that targeting IFN-γ may be a promising treatment strategy. However, a recent clinical trial using the anti-IFN-γ monoclonal antibody emapalumab has not been successful (46). Despite the combination of emapalumab and dexamethasone, only 14 out of 34 cases (41%) survived without additional therapy. In addition, only 70% of patients in the emapalumab-treated group bridged to HCT, which was inferior to the 80% in the HLH-2004 study (13, 46, 47).

Ruxolitinib inhibits the JAK/STAT pathway, which is one of the main cytokine signaling pathways in T-cell activation and the maintenance of activation (22). The mouse FHL2 model suggested that ruxolitinib inhibits HLH through IFN-γ-dependent and independent pathways (48). Ruxolitinib acts not only on T cells, but also on human macrophages and inhibits the production of both IFN-γ and IL-6 (49). Our literature review showed that ruxolitinib was used for 13 FHL patients, including our case ( Table 1 ). In a recent single-arm study involving four FHL patients, three of them responded to ruxolitinib monotherapy. The study investigated 52 cases, including systemic autoinflammatory-associated HLH, chronic active Epstein-Barr virus infection (CAEBV), and EBV-HLH. The cytokine profiles, including sIL-2R, ferritin, and IFN-γ, of good responders normalized within two weeks (28), whereas non-responders required additional therapies. In the report, cytokine profiles of four patients with FHL were not shown (28). Cytokine profiles in our case and neonatal-onset FHL showed a non-responder pattern, suggesting that additional therapies are required for these cases (11). Except for one case without outcome information, 10 out of 12 cases were successfully managed by additional ruxolitinib administration ( Table 1 ). In recent a case of Griscelli syndrome type 2, ruxolitinib was used with HLH-94-based therapy for initial induction, and successfully led to HCT (29). Emapalumab and ruxolitinib combination therapy for a refractory CAEBV case has been reported (50). However, the dose of ruxolitinib, combination of other therapies, and investigated laboratory markers, including cytokines, were varied. The prospective clinical trial (NCT04551131) is crucial for determining response using 50mg/m²/day of ruxolitinib with dexamethasone for FHL. Selection of inflammatory markers and determining the appropriate cutoff value will be required in prospective studies.

Cytokine monitoring and trends of IL-18, CXCL9, sIL-2R, and sTNF-RII might reflect disease activity in FHL. Monitoring cytokine levels and administering ruxolitinib, combined with the HLH-94/2004 protocol or other recent target therapies, may be a potent bridging therapy for FHL patients.

The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the ethics boards of the Tokyo Medical and Dental University (TMDU) (G2019-004). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

DN and TI wrote the manuscript. Literature review: DN and TI. Measurement of cytokines: SK and MS. Acquisition and interpretation of data: DN, TI, NM, SK, DT, TK, MS, and HK. Supervision of the study: MT, KI, MK, MS, and TM. Conceptualization of the study and editing the manuscript: HK. All authors contributed to the article and approved the submitted version.

This work was supported in part by JSPS KAKENHI Grant Number (21H02878) and Takeda Science Foundation to TI and by the joint research grant with Medical and Biological Laboratories Co., Ltd (MBL) to MS. KAKENHI contributed an English editing fee. KAKENHI and Takeda Science Foundation contributed open access publication fee. The Joint research grant with MBL covered the reagents for cytokine monitoring. The funder, Medical and Biological Laboratories Co., Ltd, was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

We are grateful to Drs. Takayuki Miyamoto and Takahiro Yasumi for performing flow cytometry of Munc13-4. We thank the patient and his parents for participating in this study. We also thank the staff at the Department of Pediatrics, Tokyo Medical and Dental University. We thank Barbara Spathelf, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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