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International Journal of

HEMATOLOGY

Review Article

Advances in the Management of Hemophagocytic Lymphohistiocytosis

Shinsaku Imashuku

Kyoto City Institute of Health and Environmental Sciences, Kyoto, Japan

Received November 18, 1999; received in revised form December 6, 1999; accepted December 14, 1999

Abstract

Hemophagocytic lymphohistiocytosis (HLH) is a prototype of the hemophagocytic syndrome and occurs most often in chil- dren. Progress in cytokine research has now made it possible to show that HLH occurs as a consequence of uncontrolled, dys- regulated cellular immune reactivity caused by a number of different underlying diseases. Three major risk groups of HLH can be identified: (1) familial HLH (FHL), (2) Epstein-Barr virus–associated HLH (EBV-HLH), and (3) life-threatening infection- associated or underlying disease–unknown HLH in infancy. Diagnostic criteria now exist that allow the differential diagnosis of these groups, which is important because distinct therapeutic measures are advised for each group. FHL patients require imme- diate application of immunochemotherapy with a core combination of corticosteroids and etoposide together with monitoring of central nervous system disease by early and repeated magnetic resonance imaging of the brain, followed by timely stem cell transplantation (SCT). EBV-HLH should also be treated with a combination of corticosteroids and etoposide. Aggressive or relapsed cases should be treated with cyclosporin A and, if necessary, with more intensive chemotherapy, such as that used for non-Hodgkin’s lymphoma. SCT may also be needed in these refractory cases. In cases of herpes simplex virus, adenovirus 7, and other pathogen-undetermined HLH in early infancy, it is of great importance to administer appropriate antiviral or antibacter- ial agents. The most important point to make regarding HLH treatment is that the underlying cause of HLH must be promptly established to enable the rapid application of the appropriate therapy. Currently, 30% to 40% of HLH cases have a poor out- come. It is necessary for hematologists to cooperate with specialists in other fields so that early diagnosis, which is critical for improvements in outcome, can be made. Int J Hematol. 2000;72:1-11. © 2000 The Japanese Society of Hematology

Key words: Hemophagocytic lymphohistiocytosis; hemophagocytic syndrome; cyclosporin A; Epstein-Barr virus; cytokine

1. Introduction

Hemophagocytic syndrome is associated with viral, bacte- rial, and fungal infections, lymphomas or other malignant dis- eases, autoimmune diseases, and metabolic diseases.Thus, the diseases underlying this syndrome vary widely [1-5]. As shown in Figure 1, the occurrence of hemophagocytosis can thus be considered a disease marker akin to a float used in line fishing: movement of the float may simply be due to the biting of a small fish (benign reactive disease); on the other hand, it may indicate the presence of a large, fierce fish (neo- plastic malignant disease).

Correspondence and reprint requests: Shinsaku Imashuku, MD, Kyoto City Institute of Health and Environmental Sciences, 1-2 Higashi-Takada-cho, Mibu, Nakagyo-ku, Kyoto 604-8845, Japan; fax: 81-75-311-3232 (e-mail: shinim95@mbox.kyoto-inet.or.jp).

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Patient age can help determine the underlying disorder that leads to hemophagocytosis. Hemophagocytic lymphohistiocy- tosis (HLH), a prototype of hemophagocytic syndrome, occurs most often in childhood. There are familial (FHL) and nonfa- milial forms of HLH [6], also classified as primary and sec- ondary HLH, respectively [1,2]. HLH has been diagnosed not only by hematologists but also by neonatologists, infectious disease specialists, rheumatologists, oncologists, transplant spe- cialists, and physicians in many other clinical fields. HLH com- monly presents as persistent unexplained fever, cytopenia, hepatic dysfunction, hepatosplenomegaly, hypofibrinogene- mia, and/or hypertriglyceridemia, and as hemophagocytosis in bone marrow, spleen, and lymph nodes [1-3]. HLH may be caused by an immune dysfunction where hypercytokinemia, produced by activated or clonally proliferating T cells or nat- ural killer (NK) cells and activated macrophages, develops into reactive hemophagocytosis. This review discusses the recent changes made in the management of HLH patients due to an improved understanding of the disease [7-9].

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Imashuku / International Journal of Hematology 72 (2000) 1-11

/ International Journal of Hematology 72 (2000) 1-11 Figure 1. The phenomenon of hemophagocytosis can be

Figure 1. The phenomenon of hemophagocytosis can be likened to a float used in line fishing. Movement of the float could be due to either a good small fish (benign reactive disease) or a large, fierce fish (neoplastic malignant disease) biting the hook. IAHS indicates infection-associated hemophagocytic syndrome; VAHS, virus-associated hemophagocytic syndrome; MAHS, malignancy-associated hemophagocytic syndrome; LAHS, lymphoma-associated hemophagocytic syndrome.

2. Identification of Three Major Risk Groups of HLH

Currently, pediatric HLH patients in Japan have an acute death rate of 16% and a 3-year survival rate of about 60% [8]. As analyzed for patients registered in the international

HLH-94 protocol study in Japan, the causes of death in 31 fatal cases of 109 HLH cases (Table 1) indicate that there are

3 major risk groups of childhood HLH (summarized in Table

2). The first group consists of FHL cases [10], and the second group consists of severe and aggressive HLH ensuing from Epstein-Barr virus infection (EBV-HLH).The third group is a mixture of patients in early infancy with HLH associated with either a life-threatening infection or an unknown underlying disease. It is often difficult, however, to distinguish between familial (primary) and nonfamilial (secondary) disease. The time of death after disease onset varies in the

3 groups. Although death occurs late in FHL, in EBV-HLH death can occur either early or late. Rapid deaths are caused by opportunistic infections, whereas late deaths are the result of a refractory disease associated with cytogenetic abnormalities [11,12]. In the third HLH group, death is the most rapid; therefore, early diagnosis of the underlying dis- ease, followed by systemic care administering appropriate antiviral or antibacterial agents, is essential for these patients. For cases in which hematopoietic stem cell trans- plantation (SCT) is the only measure leading to a cure [10,13], delays in performing the transplantation is another risk factor that can lead to a poor outcome.

2.1. Familial Hemophagocytic Lymphohistiocytosis

The first step in diagnosing FHL is to determine whether there is a familial link. Genetic studies have suggested that FHL is associated with genes on chromosomes 10q21-22 [14] or 9q21.3-22 [15]. Another study has shown that whereas the SHP-1 (protein tyrosine phosphatase) gene is

intact,Tyk2/SHP-1 interaction is reduced in FHL cases [16]. At the time of this writing, 1 of the pathogenetic genes or gene products associated with FHL has been identified [14a]; however, the assessment of a familial connection still depends on the analysis of family history and on the pres- ence of characteristic clinical indicators such as low NK cell activity [17]. In an analysis of 122 FHL cases, Arico et al [10] showed that the estimated 5-year survival rate was only 10.1% for patients treated with immunochemotherapy alone and 66.0% for patients who had allogeneic bone marrow transplantations (BMTs). In contrast, we found in a Japanese study of 82 cases of pediatric HLH patients that the 4-year survival rate was 57.2% for familial inheritance-unknown (FIU) cases treated with only immunochemotherapy (with the exception of 1 case in which the patient also received BMT) [8]. The significant difference in survival rates of FHL and FIU patients treated with only immunochemotherapy indicates that allogeneic SCTs and/or BMTs are indispensable in the treatment of FHL patients [10,18] but not in the treatment of FIU patients. In FHL cases, central nervous system (CNS) disease frequently occurs [19]. This occurrence is a characteristic of FHL and, unless a SCT or BMT is performed as soon as pos- sible, such CNS lesions, which are refractory to systemic and intrathecal chemo-immunosuppressive therapy, will progress and result in neuro-developmental deterioration and fatal out- come [20,21].Therefore, the key to obtaining a better progno- sis for FHL patients is the immediate application of intensive immunochemotherapy together with monitoring of the CNS by early and repeated brain magnetic resonance imaging (MRI), followed by timely SCT or BMT.

2.2. Severe and Aggressive EBV-HLH

EBV-HLH has characteristic clinical features [22] and is easy to diagnose because we can directly detect the presence of EBV by molecular and serologic methods. Reports of fatal EBV-HLH cases have been found in the literature [22-24]. We have previously estimated, based on our own experiences and a survey of the literature, that mortality in EBV-HLH is

Table 1.

Number of Fatal Cases Among 109 Registered HLH Cases*

Disease

n

FHL† FHL highly suspected‡ EBV-HLH§ Underlying disease–unknown infantile HLH§,|| Adenovirus 7/neonatal HSV-HLH§,¶

6

2

12

8

3

*HLH indicates hemophagocytic lymphohistiocytosis; FHL, familial HLH; EBV-HLH, Epstein-Barr virus–associated HLH; HSV-HLH, herpes simplex virus HLH. †Stem cell transplantation was not performed in 5 of the 6 cases. ‡Diagnosed from the central nervous system disease characteristic of but not exclusive to FHL. §No family history could be demonstrated. ||Disease onset at ages ranging from 1.5 months to 7 months; 5 of the 8 patients died within 8 weeks of onset. ¶All patients died within 3 weeks of disease onset.

Management of HLH

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Table 2.

Treatment and Outcome of the 3 Major Risk Groups in HLH*

Major Risk Groups

Initial Management (Response)

Subsequent Course and Management

Outcome (Mortality, %)

FHL

Cs/VP-16

Progressive CNS disease and poor outcome if SCT is not promptly performed

If refractory, NHL-type or HD-type chemotherapy, and if necessary, SCT recommended None#

No SCT (90)† SCT (34)† 21/51 (41)‡

EBV-HLH

(good) ±PE/ET, Cs/VP-16 ± CsA (good)§ Antiviral agents Cs/CsA(?)¶ ± VP-16 (poor)

Life-threatening

infantile HLH

Adenovirus 7 pneumonia (18)** Neonatal HSV infection (57)†† Other infantile severe infection (variable)

*HLH indicates hemophagocytic lymphohistiocytosis; FHL, familial HLH; Cs, corticosteroid; VP-16, etoposide; CNS, central nervous system; SCT, hematopoietic stem cell transplantation; EBV-HLH, Epstein-Barr virus HLH; PE/ET, plasma exchange/exchange transfusion; CsA, cyclosporin A; NHL, non-Hodgkin’s lymphoma; HD, Hodgkin’s disease; HSV, herpes simplex virus. †Arico et al [10]. §Poor outcome in refractory cases; however, well-planned systemic therapy ± SCT produced a better outcome [9]. ‡Imashuku [22]. Poor outcome in neutropenic cases complicated by fungal or bacterial infections. ¶The effect of CsA has not been well evaluated. #First-aid life saving is most important in this group. **Takahashi et al [38]. ††Jacobs [31].

around 41% (Table 2) [22]. This figure may, however, be somewhat higher than the actual incidence because cases with poor outcome rather than cases with good prognoses tend to be preferentially reported in the literature. We recently reported that the majority of patients with EBV-HLH attain remission following treatment with the combination of corticosteroids and etoposide (VP-16) [9]. However, the relapse rate after stopping treatment at 8 weeks was approximately 30%. The majority of relapse cases can be effectively treated with cyclosporin A (CsA), but some need more intensive chemotherapy, such as that used for non- Hodgkin’s lymphoma. In some relapse cases, SCTs or BMTs must also be performed to further control the disease [9]. Although immunochemotherapy is generally effective, analy- sis of the fatal cases in Table 1 and of the literature indicates that some patients suffered from rapid infections of fulminant neutropenia-associated bacteria or fungus [12,25,26] and died owing to active disease (or later due to refractory disease).

2.3. Life-Threatening Infection-Associated or Underlying Disease–Unknown Hemophagocytic Lymphohistiocytosis in Infancy

We have estimated that the rate of acute death (ie, within 2 months after onset of disease) in pediatric HLH cases is 16.0%. The majority of these deaths occur in infantile HLH cases, and the remaining deaths are due to fatal EBV-HLH in older children [8].The exact cause of death in the majority of infantile HLH cases remains to be determined. Some inborn metabolic disease may be hidden. Although the prognosis is good for secondary non–EBV-HLH in older children [24,27], such non–EBV-HLH cases are often severe and fatal in early infancy [28-31]. Severe life-threatening infections seem to be rare in FHL, but it is possible that these are FHL cases that neither demonstrate the clinical markers for FHL nor show a family history of HLH. It is well known that severe viral (her- pes simples virus [HSV]-1, HSV-2, coxsackie B, human her- pesvirus [HHV]-6) or bacterial infections that occur at birth

or soon after can cause disseminated multiorgan disease with fulminant liver failure and disseminated intravascular coagu- lation as well as hemophagocytosis [28-34]. Consequently, we believe that infantile HLH is most probably caused by severe infectious diseases and that in most cases the presence of the etiological pathogen(s) has simply not been determined or detected premortem. One pathogen that has recently been implicated in infan- tile HLH is adenovirus 7. Infection of young children (about 40% under 1 year of age) with adenovirus 7 has increased since 1995 in Japan and has recently been associated with hemophagocytosis, a severe clinical course, and poor progno- sis with a mortality rate of 18% (Table 2) [35-39]. Early and accurate diagnostic measures and the prompt introduction of the appropriate therapy are indispensable for HSV-, aden- ovirus 7–, and other pathogen-associated HLH cases in early infancy. The combination of corticosteroids and VP-16 appears to be ineffective in treating severely affected infan- tile HLH patients; therefore, it is of great importance to administer appropriate antibiotics/antiviral agents to the patient and to test the effect of introducing, in a timely man- ner, immunosuppressants such as CsA.

2.4. Delays in Finding a Suitable Donor for Stem Cell and Bone Marrow Transplantations

For FHL or refractory EBV-HLH cases, SCT or BMT should be performed in a timely manner. As shown in Table 1, we have had several patients who died because SCT could not be performed because of the lack of suitable donors. In practice, it takes longer than 6 months to find and obtain HLA-matched marrow cells from an unrelated donor using the Japan Marrow Donor Program. Cord blood, in con- trast, is more readily available and has been successfully employed by Tanaka et al [40] in the therapy of an FHL case. Thus, for HLH cases that need SCTs quickly, cord blood may constitute a better source of stem cells than bone marrow when no HLA-matched sibling donor is available.

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Imashuku / International Journal of Hematology 72 (2000) 1-11

/ International Journal of Hematology 72 (2000) 1-11 Figure 2. Typical images of large granular lymphocytes

Figure 2. Typical images of large granular lymphocytes (LGLs), promonocytoid cells, and hemophagocytes found in peripheral blood or bone marrow smears from hemophagocytic lymphohistiocytosis (HLH) patients. a. Blastic LGLs. b. Mature LGLs. c. Promonocytoid cells. d. Hemophagocyte. (Original magnification ×500)

3. Biomarkers Useful for the Establishment of Appropriate Treatment Strategies and as Prognostic Indicators

3.1. Immune Cell Morphology, Function, and Cell-Surface Markers

The diagnosis of HLH patients requires the careful assessment of peripheral blood and bone marrow smears. Characterizing lymphocyte as well as monocyte-macrophage morphology helps to identify the underlying disorders. We routinely classify the lymphocytes into small agranular lym- phocytes, mature large granular lymphocytes (mature LGL), and LGL with nucleoli and basophilic cytoplasm (blastic LGL). The monocyte-macrophages are classified into mono- cytes, promonocytoid/monoblastoid cells, and hemophago- cytes (Figures 2 and 3). The presence of hemophagocytes is critical for the diagnosis of HLH, but we found that for dif- ferential diagnosis, studying the morphology of lymphocytes is more important. Figure 3 illustrates this type of analysis of bone marrow smears taken at disease onset. The patterns designated as types A and C in Figure 3, characterized by LGL proliferation, account for 36% and 10% of patients, respectively. Thus, approximately 50% of pediatric HLH cases are characterized by LGL proliferation. The pattern designated as type B in Figure 3, characterized by prolifera- tion of promonocytoid cells, constitutes 15.4% of HLH cases. Proliferation of mature or blastic LGL (types A and C) is a characteristic in both FHL and EBV-HLH, whereas prolif- eration of promonocytoid cells (type B) is associated with HSV- or adenovirus-related HLH [41]. In the remaining 43.6% of cases, bone marrow smears at disease onset showed the presence of hemophagocytes only and did not provide any additional clues regarding possible underlying diseases. Bone marrow smears can also help in the diagnosis of autoimmune (collagen) disease-related or lymphoma-associ- ated hemophagocytic syndrome.

Patients with FHL exhibit abnormalities in certain immunologic functions (eg, persistently low or absent NK cell activity), and this knowledge can be used for diagnosis and to determine the underlying disease [17,42,43]; however, in nonfamilial HLH, immunological functions appear com- petent. The expansion of certain peripheral blood mononu- clear cell (PBMC) subsets, characterized by particular cell surface markers, can also be informative in the differential diagnosis of HLH. When we performed a comparative study of familial and FIU-HLH cases, we found that the CD3 + , CD3 + HLA-DR + , and CD45RO + cell subsets occurred signi- ficantly more frequently in FHL than in FIU, although het- erogeneity was noted [43]. Another study also showed that EBV-HLH could be differentiated from non–EBV-HLH by

that EBV-HLH could be differentiated from non–EBV-HLH by Figure 3. Distribution of large granular lymphocytes

Figure 3. Distribution of large granular lymphocytes (LGLs), promonocytoid cells (promono), and hemophagocytes (hemophago) in the bone marrow of hemophagocytic lymphohistiocytosis (HLH) patients, and patterns permitting the differential diagnosis of HLH sub- groups. Values in the ordinate indicate percentages in the myelogram. Type A: proliferation of blastic (major) and mature LGLs, a pattern typ- ically found in Epstein-Barr virus–associated HLH, accounting for 36% of cases examined; type B: proliferation of promonocytoid cells, a pat- tern associated with neonatal herpes simplex virus or adenovirus 7 infection–associated cases, accounting for 15.4% of cases examined; type C: proliferation of mature (major) and blastic LGLs, a pattern typ- ically found in familial HLH, accounting for 10.0% of cases examined; and type D: nonspecific pattern, accounting for the remaining 43.6%. bl-LGL indicates blastic LGL.

Management of HLH

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Table 3.

Useful Biomarkers for the Establishment of Treatment Strategies and as Prognostic Indicators*

Study

Material

Biomarkers

Cell morphology

PB (BM) smear

LGLs (mature, blastic) Promonocytoid cells Hemophagocytes Marker (CD3 + ,CD56 + ,CD19 + ) NK-cell activity EBV genome by PCR, Southern blot TCR rearrangement (β, γ, δ) Cytogenetics Cytokines Serology

Marker/cell function

PBMC (BMMC)

EBV genome/clonality

PBMC (BMMC)

Cytokine/serology

Sera

*PB indicates peripheral blood; BM, bone marrow; LGL, large granular lymphocyte; MC, mononuclear cells; NK, natural killer; EBV, Epstein-Barr virus; PCR, polymerase chain reaction; TCR, T-cell receptor.

the significantly increased frequency of CD3 + HLA-DR + cells [24]. Furthermore, NK cell–type EBV-HLH (character-

ized by NK-cell proliferation), whose prognosis is worse than

T cell–type HLH (typified by T-cell proliferation), could be

detected by expansion of the CD3 CD56 + subset. An atypi- cal CD3 + CD4 low cell population has been found in fatal EBV infections, but the significance of this remains to be deter- mined [44]. These data together suggest that assessment of bone marrow lymphocyte and monocyte-macrophage cell morphology, NK-cell activity, and PBMC subsets is useful for the prompt differential diagnosis of HLH.

3.2. Measurements of EBV Involvement in HLH

More than half of the pediatric HLH cases in Japan may be associated with EBV infection. EBV-HLH can be suspected from LGL proliferation in peripheral blood and/or bone marrow smears, from an increase in the CD3 + HLA-DR + cell subset, or from an augmented T helper (Th) 1 cytokine response, and diagnosed by detection of the EBV genome by polymerase chain reaction (PCR), South- ern blotting, or serology. EBV is associated with both famil- ial and nonfamilial HLH [45,46]. EBV-HLH develops after

primary exposure to the virus, at virus reactivation, or at the terminal stage of chronic active Epstein-Barr virus infection (CAEBV). In CAEBV, it was recently found that clinical pictures include severe mosquito bite hypersensitivity, NK- cell leukemia, and hydroa vacciniforme–like eruptions [47]. EBV-HLH is a subtle disease with a clinical course ranging from mild/self-limiting to severe/aggressive and fatal. The prognosis of EBV-HLH that develops in the terminal phase

of CAEBV is particularly poor [48].

Improving the prognosis of these cases requires a better understanding of how EBV virulence and host immunity can lead to the development of HLH. To date, we know that the majority of EBV-HLH cases are characterized by mono- or oligoclonal proliferation of EBV-infected NK or T cells. Further, hypercytokinemia and apoptosis via the Fas (CD95)/Fas ligand system are commonly involved in aggres-

sive clinical courses in patients with EBV-HLH. In addition,

a genetic predisposition for severe fatal EBV infection has been found in X-linked lymphoproliferative disease (XLP) cases, where the SAP (DSHP/SH2DIA) gene was found to

play a critical role [49-51]. However, whether there is a genetic predisposition to severe disease in non-XLP or spo- radic and fatal nonfamilial EBV infection–associated dis- eases remains to be determined. Novel techniques such as real-time PCR, which permits the easy quantification of EBV genome copy numbers in lymphocytes, serum, and plasma [52-55], make it possible to determine whether the viral load of EBV also contributes to disease severity. This quantitative method may also be useful in monitoring patients’ clinical responses to therapy. The proliferating cells incorporating the EBV genome (T or NK or B cells) may be associated with the clinical fea- tures and responses to therapy in EBV-related diseases [56], and mutations in the EBV genome may contribute to disease severity. EBVs with mutant LMP1 (latent membrane protein 1) have been associated with various EBV-related disorders including EBV-HLH [57-59]. Thus, delineation of the risk factors for severe disease (eg, the genetic predispo- sition of the host, the viral load, and viral mutations) may permit us to subgroup EBV-HLH cases more precisely into high- and low-risk categories.

3.3. Clonality of Immune Cell Proliferation in HLH

In recent years, concern has grown over the lethal potential of clonal proliferation in virus-associated HLH, particularly when EBV is involved [60-64]. NK- and T-cell clonal prolifer- ation in HLH can be measured using a variety of biological materials and methods such as karyotypic analysis, assessment of T-cell receptor (TCR) rearrangements, and study of the ter- minal repeats of the EBV genome [65] (Table 3). Ishii et al [46] also developed a meticulous method of measuring pref- erential variable region β (Vβ) usage by T cells to study the degree of clonality in HLH. Although case reports of clonal HLH have accumulated in recent years [60-64,66], the impact of T- or NK-cell clonality on the outcome for patients with HLH remains to be determined; thus, we recently studied this issue using 3 of the techniques described earlier [11]. Of the 32 HLH cases studied, 22 were EBV-clonal, 15 were TCR-clonal, and 7 were cytogenetically clonal. All 7 cases with cytogeneti- cally abnormal clones were found to be fatal, with a 3-year survival rate, by Kaplan-Meier analysis, of only 14%. In con- trast, the 3-year survival rate of the 22 EBV-clonal HLH cases,

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Imashuku / International Journal of Hematology 72 (2000) 1-11

/ International Journal of Hematology 72 (2000) 1-11 Figure 4. Schematic illustration of how to utilize

Figure 4. Schematic illustration of how to utilize cytokine data to determine the underlying disease after onset and for establishing a future treatment plan. Note that the limit of 8 weeks (8W) needed for treatment has been set by the international HLH-94 protocol. HLH indicates hemophagocytic lymphohistiocytosis.

3 of whom also had cytogenetic abnormalities, was 64% and the survival rate of the 15 TCR-clonal cases was 53%. Our observation suggests that cytogenetically abnormal HLH cases present an extremely high risk of fatality.

3.4. Serum Cytokines in HLH

HLH is associated with hypercytokinemia characterized by a bias toward Th1 cytokines. This hypercytokinemia has been demonstrated to cause the basic pathophysiology of HLH [67-71]. Measuring the type and the degree of hyper- cytokinemia might permit differential diagnosis and assess- ment of risk factors [27,68,69]. We have found that the serum of older children with EBV-HLH contains signifi- cantly high levels of both a classic Th1 cytokine (inter- feron-γ [IFN-γ]) and soluble interleukin-2 receptor (sIL-2R); sIL-2R is released upon Th1 cell activation [24]. In the dis- ease, EBV by itself and IFN-γ affect the survival of leuko- cytes [72,73]. The serum of infantile HLH patients infected with HSV or adenovirus has higher levels of IL-6 and/or macrophage colony-stimulating factor, and there was only a slight increase in sIL-2R levels in these patients [27,37]. In fact, cytokine production may differ between neonatal and adult lymphocytes [74]. These observations suggest that in EBV-HLH, primarily T and/or NK cells are being activated, whereas in infantile HSV, adenovirus-infected HLH mono- cytes are the predominant cytokine producers. Although cytokine data are not immediately available for the first week of treatment, they have a retrospective value in that they can help identify the underlying diseases and thus may assist in determining future treatment policies (Figure 4). In a current protocol for pediatric HLH [7], we provide physi- cians with treatment suggestions based on the patient’s cytokine data. If the cytokine data indicate a low risk, the patient can be taken off treatment at or before 8 weeks of treatment. However, more careful observation of the patient is needed if there are extremely high levels of serum cytokines such as sIL-2R (>10,000 U/mL) and IFN-γ (>100 U/mL) at disease onset.

4. Treatment Strategies

Historically, patients with HLH have been treated with corticosteroids, intravenous immunoglobulin (IVIg), VP-16, or a combination of these drugs. However, it remains difficult to determine exactly how each HLH case should be treated. Not all pediatric patients whose clinical signs and symptoms and laboratory data are compatible with the diagnosis of HLH should have immediate immunochemotherapy. Symp- tomatic therapy alone is successful in the majority of infec- tion-associated HLH cases except for EBV-HLH [24] in apparently immunocompetent older children. IVIg therapy has been reported to be beneficial in some low-risk cases but not in others [75-78], whereas VP-16 has been documented to be effective for HLH in general [79]. We propose here a treatment strategy for the 3 high-risk HLH groups as categorized in section 2. The same treat- ment strategy is employed for FHL and EBV-HLH cases (Table 2) because both exhibit LGL proliferation and are believed to be clonal diseases [9,46]. In designing this strat- egy, a treatment protocol (proposed by the Histiocyte Soci- ety) consisting of VP-16, dexamethasone, and cyclosporine followed by SCT or BMT, has been particularly helpful [7,9]. As mentioned above, the establishment of more spe- cific therapies for EBV-HLH must await future risk-based subclassification of this group. The treatment strategy for nonfamilial infantile HLH cases seems to be different from that for FHL and EBV-HLH (Figure 5) because the clinical course is so rapid and aggressive.

4.1. Treatment of FHL and EBV-HLH Cases

4.1.1. Correction of Basal Pathological Conditions

Therapeutic plasma exchange (PE) (plasmapheresis) or exchange transfusion (ET), IVIg, and corticosteroids have been the first-choice treatment regimens most commonly

have been the first-choice treatment regimens most commonly Figure 5. Treatment plan for high-risk familial

Figure 5. Treatment plan for high-risk familial hemophagocytic lym- phohistiocytosis (FHL) and Epstein-Barr virus–associated hemophago- cytic lymphohistiocytosis (EBV-HLH) patients. Bone marrow trans- plantation (BMT) or stem cell transplantation (SCT) is necessary in all cases of primary HLH (FHL) and in refractory cases of secondary HLH. PR/NR indicates partial remission/no remission; CR, complete remission; Dexa, dexamethasone;VP-16, etoposide; CsA, cyclosporin A.

Management of HLH

7

employed in severe HLH cases. In particular, PE or ET is used to correct the tendency to bleed and to control hyper- cytokinemia [80-82]. We believe that prompt continuous infusion of CsA (3 mg/kg per day, for several days) may help alleviate the cytokine storm in severely affected patients.

4.1.2. Core Combination of Corticosteroids and

Etoposide

Currently, the most common treatment for HLH is a core combination of corticosteroids and VP-16, as sug-

gested in the international HLH-94 protocol [7,9]. This therapy aims to eventually eradicate the proliferating

T and NK cells and activated macrophages and thus result

in a cure for EBV-HLH. We propose that initial therapy should be maintained for 8 weeks (Figure 5). In a previous treatment report on EBV-HLH, we found that 1 of 17 cases responded successfully to a regimen in the absence of VP-16 [9]. It may therefore be possible to design a proto- col in the future that consists of immunosuppressive drugs but lacks VP-16 and that can still lead to remission and prevention of a fatal outcome in EBV-HLH.

4.1.3. CsA and Antithymocyte Globulin Therapy

CsA has been found to be effective in controlling various cytokine-related pathological conditions [83-88]. CsA effi- ciently and rapidly suppresses the cytokine storm caused by

dysregulated T cells and activated macrophages; thus, CsA is a key drug in the acute phase as well as maintenance therapy of the international HLH-94 protocol [7]. We have also reported that introducing CsA treatment effectively sup- ports neutrophil recovery during the acute phase of HLH in severely neutropenic patients [89]. In addition, antithymo- cyte globulin (ATG) therapy with or without corticosteroids

is another effective regimen for HLH [90,91]. Perel et al [90]

reported that ATG had a dramatic effect in a pediatric case

of refractory EBV-HLH in which the patient had been heav-

ily treated with chemotherapy.

4.1.4. Care of CNS Disease

complete remission status of CNS and systemic disease sub- sequently did well and showed normal neurological func- tions and cognitive development. Shuper et al [95] also reported an FHL patient who showed gradual neurodevel- opmental normalization after BMT at 5 months of age. These results indicate the potential for BMT to reverse neu- rodevelopmental deterioration in HLH and also indicate the importance of both early diagnosis of CNS disease and prompt introduction of SCT or BMT.

4.1.5. Strategy for Refractory Disease

4.1.5.1. Intensive Chemotherapy

For cases not responding to the initial combination of cor-

ticosteroids and VP-16, the first choice of treatments is to add CsA if it has not been administered previously. CsA has been proven to be effective in both FHL and nonfamilial EBV- HLH [9,91,100], as well as in LGL-proliferating diseases such

as LGL leukemia/lymphoma [84,101]. Other choices of ther-

apy for refractory cases are various multiagent chemothera- pies, particularly a combination of ACOP (CHOP) (adri- amycin or cyclophosphamide plus doxorubicin, vincristine, and prednisone) used for non-Hodgkin’s lymphoma (NHL- type chemotherapy), or a combination of ACOPP and ABVD regimens (adriamycin, cyclophosphamide, vincristine, prednisolone, and procarbazine, plus adriamycin, bleomycin, vindesine, and dacarbazine) used for Hodgkin’s disease (HD- type chemotherapy), or high-dose cytosine arabinoside, or cyclophosphamide plus VP-16 [9,102,103]. A combination of CsA with multiagent chemotherapy is considered to be the most effective therapy and in some cases even good enough for a cure without SCT; however, other cases eventually require SCT or BMT. More recently, Obama et al [104] reported that L-asparaginase induced complete remission in EBV-positive, multidrug-resistant cutaneous T-cell lym- phoma, suggesting that this drug may be useful in treating refractory EBV-HLH.

4.1.5.2. Hematopoietic Stem Cell Transplantation

Clinical neurological symptoms in HLH patients include seizures, coma, brain stem symptoms, or ataxia. Although

Myeloablative chemotherapy and subsequent SCT or BMT are the most acceptable treatment regimens for FHL and therapy-resistant nonfamilial HLH cases [13]. Familial

routine cerebrospinal fluid cytology is commonly used for

as

well as nonfamilial HLH cases have been successfully

the early diagnosis of CNS disease, brain MRI is a much more sensitive method for the detection of presymptomatic

treated with SCT/BMT from various stem cell sources, including allogeneic related or unrelated bone marrow

CNS lesions. CNS disease has been documented in FHL [19- 21,92-95] as well as in nonfamilial cases such as EBV-HLH [96,97] and rotavirus infection–associated HLH [98]. Typical neuropathological findings in FHL include lymphohistio-

[13,105-107], peripheral blood stem cells (PBSCs) [108], autologous PBSCs [109], haploidentical stem cells [110], and cord blood [40,111]. Jabado et al [112] recently reported that BMT was successful even when the donor was nonidentical

cytic infiltration of the leptomenges and perivascular spaces.

in

HLA. As discussed above, however, cord blood seems to

Further, calcification and necrotic lesions particularly in the

be

a safer and a more easily available alternative stem cell

putamen, internal capsule, thalamus, and dentate nucleus

source for emergency SCT, as reported by Tanaka et al [40].

have been described [99].

A

busulfan/VP-16/cyclophosphamide conditioning regimen

Once CNS disease develops, its management is trouble-

is

commonly used for FHL-SCT, whereas for refractory

some. The outcome of patients treated by systemic and intrathecal chemotherapy and/or immunosuppressive agents has been reported to be poor [19]. However, in the same

EBV-HLH cases, a regimen containing TBI has been employed. We reviewed the FHL and nonfamilial Japanese HLH cases treated with SCT or BMT and found that 12 of

study, 7 of 9 patients who were treated with BMT in the first

the 17 recipients were currently alive and well [13].

8

Imashuku / International Journal of Hematology 72 (2000) 1-11

Table 4.

Treatment Strategies for High-Risk HLH Cases*

FHL and EBV-HLH Correct basal pathological conditions Immunochemotherapy Plasma exchange and/or exchange transfusion, CsA Combination of corticosteroids/VP-16 ± CsA Care of opportunistic infectious complications due to neutropenia Care of central nervous system disease Strategy for refractory disease Intensive chemotherapy Stem cell transplantation Life-threatening infantile HLH Early diagnosis of triggering factor(s)† Prompt introduction of anti-infectious agent(s) Possible early CsA application with corticosteroids

*HLH indicates hemophagocytic lymphohistiocytosis; FHL, familial HLH; EBV-HLH, Epstein-Barr virus HLH; CsA, cyclosporin A; VP-16, etoposide; The international HLH-94 protocol initially consists of dex- amethasone/VP-16 for induction (for 8 weeks), followed by dexam- ethasone/VP-16/CsA for maintenance therapy in unresolved cases [7] (see Figure 5). †In the majority of infantile HLH cases, the exact cause has not been clarified.

4.1.6. Treatment of High-Risk Infantile and Other HLH Cases

For high-risk HLH in infancy, such as neonatal HSV- associated or severe adenovirus 7 infection–associated cases, development of better therapeutic measures is essential. Refined treatments that contain CsA remain to be tested. The high mortality of these cases may be reduced by early and accurate diagnosis, by detection of multiorgan failure as early as possible, and by the prompt introduction of anti- infectious agents (summarized in Table 4).The application of VP-16 in these cases seems to be questionable. Autoimmune disease (collagen disease)–related hemo- phagocytic syndrome (CAHS) is a category distinct from HLH [4,113] and it occurs in, among other diseases, systemic lupus erythematosus, juvenile rheumatoid arthritis, der- matomyositis, and cytophagic histiocytic panniculitis (CHP) [86,88,113-117]. Hemophagocytosis develops either at the onset of an autoimmune disease or during disease treat- ment. In the latter case, it is often difficult to determine whether an intervening infection or insufficient control of the disease is responsible. It is important to distinguish CAHS from HLH cases. Once a diagnosis is established, corticosteroids or CsA can be used. There is no need to administer VP-16. For better control of the disease, we rec- ommend a continuous infusion of CsA (3 mg/kg per day for one week) followed by oral CsA (6 mg/kg per day) together with corticosteroids and, if necessary, oral methotrexate weekly or a nonsteroid anti-inflammatory drug.

5. Problems Regarding Therapy-Related Malignancy

Etoposide is one of the key drugs in the treatment of HLH but it has also been linked with the development of therapy-

related secondary myeloid leukemia (t-AML). Sporadic cases of t-AML have been documented to date [118-120],and Kitazawa et al reported on a patient with EBV-HLH who had been treated with the international HLH-94 protocol and developed t-AML 3 years later (personal communication). Careful follow-up of HLH patients treated with VP-16 is required. It has also been reported that acute lymphoid leukemia (ALL) and HLH can occur simultaneously [121] and that ALL developed 6 months following the treatment of HLH [122], suggesting some common pathogenetic mecha- nisms in the development of lymphoid leukemia and HLH.

6. Conclusions

Over the past decade, a great deal of information regard- ing the treatment of HLH has accumulated. It is now possi- ble to determine whether the HLH patient falls into a high- risk group and to know which therapy to apply. Pinpointing the underlying disease is critical because different therapeu- tic measures, such as immunochemotherapy or SCT/BMT, are required for some risk groups but not for others. The most important point to make here is that the most appro- priate treatment strategy must be given to each HLH case without delay. The rapid diagnosis required in these cases will require close cooperation between hematologists and physicians in other fields.

Acknowledgments

The author thanks the members of the Japan Society of Pediatric Hematology who participated in the cooper- ative study on HLH cases treated according to the inter- national HLH-94 protocol. Yasuko Hashimoto is also gratefully acknowledged for her assistance in the prepa- ration of this review.

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