Pathogenesis of haemophagocytic lymphohistiocytosis

Haemophagocytic lymphohistiocytosis (HLH) is a rare, fatal disease of early infancy. The familial form of this disease was originally described by Farquhar and Claireaux (1952) and overlooked for several years. Janka (1983) detailed the main features of familial HLH in a comprehensive clinical review. Autosomal recessive inheritance has been demonstrated for familial HLH (Gencik et al, 1984). As only a limited proportion of cases have documented familial recurrence, despite similar presenting features and outcome, the term HLH is commonly used to indicate the disease in both familial or apparently sporadic cases. Its incidence has been estimated to be 0·12 per 100 000 children in a retrospective Swedish study (Henter et al, 1991a). A similar incidence was later found in a British study (cited in Henter et al, 1998). In a recent Italian study, the estimate of the incidence of HLH in the whole country over the last 25 years was 0·006 per 100 000 children per year, but in the Southern part of Italy the disease was three times more frequent than in the Northern and Central areas. Owing to the improvement in detecting HLH cases, the observed frequency of the disease has increased over time, reaching the value of 0·118 per 100 000 children per year in the last period (1990–99) (unpublished observations). The main features of HLH include persistent fever and hepatosplenomegaly. Central nervous system involvement varies (ranging from irritability to overt meningoencephalopathy), and lymphadenopathy and skin rash may also be observed (Janka, 1983; Aricòet al, 1996, Haddad et al, 1997; Henter et al, 1998). Evidence of anaemia and thrombocytopenia may suggest the existence of acute leukaemia which can easily be ruled out by bone marrow examination. The above described clinical picture, the absence of malignant cells together with the possible association with active haemophagocytosis (which may be already evident at presentation or at repeated examinations during the disease course, in the bone marrow or in the spleen, lymph nodes or cerebrospinal fluid cells; Favara, 1989; Ost et al, 1998) are highly suggestive of HLH. Biochemical alterations largely overlap with those characteristic of the macrophage hyperactivation syndrome (Ravelli et al, 1996) and include hypertriglyceridaemia, hypofibrinogenaemia, hyponatraemia, elevated ferritin and lactase dehydrogenase (LDH) levels. Cerebrospinal fluid (CSF) pleocytosis is common. Plasma levels of soluble interleukin 2 receptor (CD25) are also increased (Komp et al, 1989; Henter et al, 1991b). Clinical and laboratory markers of associated infection were reported in 50 of the 122 (41%) children reported to the HLH registry. Of these, 25 had a positive family history of HLH and their features were not significantly different from the other 25 non-familial ones, or from the 72 children (34 familial) without evidence of associated infection at presentation. The spectrum of reported infectious agents mirrored that of common pathogens, including cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, parvovirus, hepatitis B virus, herpes and Coxsackie (Aricòet al, 1996; Henter et al, 1993). Histological examination of involved organs typically shows infiltration by histiocytes and lymphocytes, with haemophagocytosis (Akima Favara, 1989; Ost et al, 1998); spleen biopsy may also provide evidence of haemophagocytosis, but because of possible complications it should be applied with caution; liver biopsy is often less informative, showing a hepatitis-like lymphocytic infiltration. Thus, repeated bone marrow aspirations appear to be the best approach for the documentation of haemophagocytosis. Differently from the more common Langerhans' cell histiocytosis, bone and adjacent soft tissues are not involved in HLH (Favara et al, 1997; Aricò Fischer et al, 1985). On this basis, the HLH study group of the Histiocyte Society started an international prospective therapeutic trial named HLH-94 (Henter et al, 1997). The preliminary results of this study showed that combined immunochemotherapy with dexamethasone, etoposide and cyclosporine was effective in achieving control of the disease manifestations within a few weeks, and also in prolonging the survival (J. I. Henter personal communication). The increased incidence documented in Italy, together with current availability of such an effective treatment, probably resulted in the marked reduction of cases of HLH who died in an early stage of the disease owing to uncontrolled progression. This may provide an opportunity for a patient in good clinical condition to proceed to bone marrow transplantation (BMT). After an initial report by Fischer et al (1986), BMT from matched related donors was successfully applied (Todo et al, 1990; Blanche et al, 1991). As most patients lack a familial donor, alternative sources for haemopoietic stem cells are necessary, including bone marrow (Baker et al, 1997; Jabado et al, 1997; Durken et al, 1999) or cord blood (Schwinger et al, 1998; Tanaka et al, 1998; Imashuku et al, 1999a) derived from matched unrelated donors. At present, BMT remains the treatment of choice for HLH and the only potentially curative one, as suggested also by evidence of restored natural killer (NK) cell activity in patients in long-term remission. The diagnosis of HLH is often difficult and, in the past, it frequently could not be established before the patient's death. The main problem has been the lack of a specific marker for the disease, in which the clinical picture is non-specific and mostly suggestive of disseminated infection or haematological malignancy. When details about more than one familial case are available, as for the 15 families described in the Registry study, the age at the disease onset was similar in the first and the second case (median, 2·8 versus 2·4 months; range, 0–54 months versus 2–64 months respectively), while the time required to diagnosis was longer in the first case, as documented by the older age at diagnosis (median, 8·9 months versus 4·9 months; range, 2–64 versus 0–42 respectively) (Aricòet al, 1996). Therefore, the Histiocyte Society developed a set of diagnostic criteria to help clinicians identify, on purely clinical grounds, children with this obscure but very severe disorder (Henter et al, 1991c). Yet such criteria, although useful to identify patients with suspected HLH, are not specific enough; they are characteristic of a macrophage activation syndrome which may also be observed in different pathological conditions, including infection by viruses or other agents. Remarkably, this picture may develop not only in apparently healthy children, without any family history, but also in children with constitutional or acquired immune deficiency (Aricòet al, 1999a) or in patients with juvenile rheumatoid arthritis (McPeake et al, 1993; Ravelli et al, 1996; Stephan Janka et al, 1998). Visceral leishmaniasis has been reported in association with a clinical picture of HLH and should be carefully investigated even outside endemic areas (Gagnaire et al, 2000). Differential diagnosis between these various conditions has obvious prognostic and therapeutic implications. Reports of the sporadic, infection-associated cases led to a belief that familial cases, apparently not associated with infection(s), could be those with a more serious outcome. The sporadic cases associated with viral infection were thought to be characterized by a better prognosis. As a consequence, specific HLH-directed treatment was considered unnecessary in this second group. We have learned, however, that this may not be always the case for several reasons. First, scarcity of large families, especially in western countries, may frequently hamper the ability to recognize the familial nature of the disease; second, viral infections have often been documented in familial cases (Henter et al, 1993; Aricòet al, 1996). In Japan, the propensity to discriminate between sporadic EBV-associated HLH and documented familial cases may have contributed to the tendency to spare the non-familial cases from specific therapy, which appears to have resulted in an excess mortality (Imashuku et al, 1999b). It is possible that similar behaviour, although unreported, has also been applied in other geographical areas. Furthermore, the clinical picture of HLH in children may vary considerably. Thus, some patients develop the disease soon after birth and present with persistent spiking fever, massive hepatosplenomegaly, severe cytopenia (anaemia, progressive thrombocytopenia and, later, also neutropenia) and nervous alterations, which range from irritability to seizures or even a rapidly progressive encephalopathy (Henter nevertheless, this picture may be quite difficult to document and in about half of cases it is evident only at follow-up examination (Aricòet al, 1996). Thus, although it represents the eponym of the disease, the diagnostic value of haemophagocytosis cannot be overemphasized. The clinical course of HLH may be very aggressive and sometimes initial treatment may be necessary to prevent early fatalities, even though the diagnostic work-up has not been completed (Aricòet al, 1996). In some cases, fever and/or splenomegaly may precede the onset of some of the biochemical alterations, so that the diagnostic criteria may not be fulfilled in the initial stage (Henter unpublished observations). It may be hypothesized that the extensive use of dexamethasone may contribute to prevent central nervous system dissemination, clearly documented by necropsies of patients who died with progressive or fulminant disease (Akima Kletzel et al, 1986; Kaneko et al, 1995) have not been confirmed in larger studies (Aricòet al, 1996). Hasle et al (1996) reported one case of HLH associated with constitutional inversion of chromosome 9, but additional studies showed that the reported breakpoints were not linked to the disease gene (Aricòet al, 1999b), as also reported by Ohadi et al (1999). Linkage analysis using homozygosity mapping in four inbred HLH families of Pakistani descent isolated a putative disease gene to an approximately 7·8-cm region between markers D9S1867 and D9S1790 at 9q21.3–22 (Ohadi et al, 1999). Furthermore, linkage analysis of a group of 17 families with HLH revealed mapping of a locus linked to HLH in the proximal region of the long arm of chromosome 10 in the 10q21–22 region in 10 families but not in the remaining seven, providing evidence for genetic heterogeneity in HLH (Dufourcq-Lagelouse et al, 1999). While no further cases of HLH linked to the 9q21.3–22 locus have been reported, recently Stepp et al (1999) identified nine different mutations, three nonsense and six missense, in the two coding exons of the perforin 1 gene (PRF1) in a group of eight unrelated patients, providing the first evidence for a disease entity related to PRF1. Four novel mutations (Goransdotter et al, 2001), together with a further six (Clementi et al, 2001), have recently been demonstrated in patients with HLH of different geographical origins. Thus, at present PRF1 mutations may be considered the most frequent genetic defect underlying HLH. Although all six patients of Turkish origin with PRF1 mutations shared the same mutation, patients from Southern Italy showed a range of different PRF1 mutations, suggesting that HLH may have a common founder effect among Turkish but not Italian patients (Graham et al, 2000; Clementi et al, 2001; unpublished observations). In their original report, Farquhar Aricòet al, 1996). Thus, it has been suggested that viruses may play a role as non-specific triggers of HLH. But why does the clinical picture of the infection degenerate into HLH? Children with HLH appear to be unable to control some infections; thus, they try to overcome them by an uncontrolled inflammatory response with sustained hyperactivation of T cells and macrophages. Hyperproduction of cytokines such as tumour necrosis factor alpha (TNF-α), interleukin 1 (IL-1), IL-6 and interferon gamma (IFN-γ) account for part of the clinical manifestation, as they may lead to severe tissue damage (Henter et al, 1991b). Hyperactivated lymphocytes and macrophages (Hansmann et al, 1989; Burgio et al, 1990) disseminate around the body and, together with haemophagocytosis, account for the eponym of HLH. of these cells is as a of or cell has not been In one study that the in of lymphocytes from eight patients to etoposide or to was ruled out et al, 1999). an immune defect in HLH in the T cell or the macrophages for many years et al, 1989; et al, et al, 1993). with infections suggested that HLH was to a immune deficiency et al, evidence of in HLH has been confirmed et al, 1984; Aricòet al, et al, of absence of natural killer (NK) cell activity by the blood lymphocytes of the patients has to the to control viral infections and has been used by some clinicians as a further diagnostic cells have long been as cells Although their ability to cells has been for many the involved in their have only recently been main the of cells of or cells from The main by cells is the of the et al, 1996). cells recognize that In these different for Therefore, while killer recognize receptor 1 has a and the of which is on the of that lack the an that frequently in and virus are by cells et al, A of different of with different the body to the or absence of different on the cell et al, 1996, Thus, the of cells may be at different with and clinical that are not In to a activation cells large of cytokines including factor and which a effect in immune and as as in cell Thus, for of to macrophage activation and may induce the response of cells It is evident that activation may the of response and of cytokines a recent report on showed that cells are of in response to et al, 2000). of the immune response may occur two main The first is spontaneous of the response by the In infection triggers a immune including and of Once the cells are the for immune response is and further is not The alternative to the immune response is that of an In this case, specific the response on a different activation of T The of the receptor may be considered a good to this the receptor the is one of and in the receptor the the is to the and induce et al, 2000). This is confirmed by evidence that of a But which The most is that the of the the the receptor has a with the This may the of and, thus, the response to the et al, 2000). is a protein that is in lymphocytes but also in macrophages and other bone marrow et al, et al, et al, 1998). Its main role in the is to form in the membrane of cells this perforin is in that may be as cell perforin is into the membrane and to form that may lead to of the Once the cell membrane has been by the of and other are to the The for cell is by et al, which may the cell the B was recently demonstrated entering cells in a using the factor receptor et al, 2000). Furthermore, in a for lymphocytes of A and B but these their and were et al, that both perforin and contribute to that control the and of in et al, 2001). cell were shown to have of tumour cells and of tumour and et al, 2000). Furthermore, in a prospective study of observed for with activity were found to be at a of providing evidence for natural et al, 2000). cell is thought to be by a that the involvement of perforin is may not be et al, 1999) and this may in persistent of T This might represent an additional which perforin may the immune In patients with HLH, as as in other related conditions such as and et al, et al, the to may the of viral the between the virus and the may be and in different the response is and cells are rapidly and the infection is owing to virus the is less viral infection may be owing to the of specific This further results in virus Thus, the a viral infection is in the of the infection and, the patient's clinical in children with PRF1 (or mutations they are by a Although T of to activation and the cells to the cells and, thus, the of this persistent activation results in the of large of including and both to represent two macrophage The sustained macrophage activation and to the of activation results in tissue infiltration and the of levels of and which play a role in tissue damage and the clinical It is that the characteristic of and the same pathological and clinical a different in children with disease a condition that may be from HLH in some et al, 2000; Aricòet al, 2001). with are unable to control Epstein-Barr virus infections as a of a of the which an of an et al, 2000). of the cell defect in HLH. In the suggest that a perforin defect results in HLH, a rapidly fatal disease, owing to ability of these patients to control viral infections. of the virus, together with a inflammatory results in extensive and disseminated tissue damage that ultimately to and in the perforin gene to perforin account for the clinical picture in a proportion of patients with HLH et al, et al, 2001; unpublished observations). the not the of a which can only be a larger of mutations are The role of other involved in such as should be carefully in the remaining patients, the majority of are for the of an alternative underlying It appears that the Pakistani patients in linkage to chromosome was found may represent an of HLH. evidence of families in which HLH may have a very despite an evident additional genetic heterogeneity (Allen et al, 2001). The of using activity as a marker of the disease, in the of familial is et al, 1998). The for a better of the genetic in families with HLH is in to provide to the and to treatment and genetic including of the of HLH may the curative potential of Furthermore, evidence of long-term remission from HLH after of BMT et al, as as an remission in two patients acute BMT (unpublished suggest that immune and may play different in immune and the At present, BMT remains the only curative treatment but a better of the and the underlying genetic could the to alternative therapeutic including possible gene therapy of HLH. This was supported in part by the Italy, and Italy and from the The are to for help in