¹Onco Ematologia Pediatrica, Ospedale dei Bambini 'G Di Cristina', Palermo, Italy

²Oncoematologia Pediatrica, IRCCS Policlinico San Matteo, Università di Pavia, Italy

³Clinica Pediatrica, Università di Bari, Italy

⁴Biologia Generale e Genetica Medica, Università di Pavia, Italy

⁵Institut National de la Sante et de la Recherche Medicale (INSERM) U429, Hopital Necker Enfants Malades, Paris, France

Correspondence to: M Aricò, Onco Ematologia Pediatrica, Ospedale dei Bambini 'G Di Cristina', Palermo, Italy

Griscelli syndrome (GS) is a rare autosomal recessive disorder, characterized by pigmentary dilution of the skin and hair and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis (HLH). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment available for genetically induced HLH. Few cases of successful HSCT from a compatible donor have been reported in children with GS. We describe the first patient with GS cured with an allograft from a compatible unrelated bone marrow donor. We used a novel preparative regimen consisting of busulfan, thiotepa and fludarabine. The demonstrated curative effect of HSCT from an unrelated donor in a patient with genetically determined HLH also supports the use of a systematic diagnostic approach in these patients, in order to identify those with a worse prognosis and needing an urgent allograft in a timely manner.

Bone Marrow Transplantation (2002) 29, 995-998. DOI:10.1038/sj/bmt/1703567

Griscelli syndrome; hemophagocytic lymphohistiocytosis

Griscelli syndrome (GS) (OMIM 214450) is a rare autosomal recessive disorder that results in pigmentary dilution of the skin and hair, in the presence of large clumps of pigment in hair shafts and an accumulation of melanosomes in melanocytes.^1,2 Most patients with this disorder also develop an uncontrolled T lymphocyte and macrophage activation syndrome, known as hemophagocytic syndrome, or hemophagocytic lymophohistiocytosis (HLH), leading to death in the absence of allogeneic hematopoietic stem cell transplantation (HSCT).³ Early in life, some GS patients show severe neurologic impairment, without apparent immune abnormalities.⁴

First reports of this new clinical entity, distinct from Chediak-Higashi syndrome (OMIM 214500) (CHS), were provided simultaneously in 1978 by Griscelli et al¹ and by Siccardi et al.²

In 1997 Pastural et al⁵ found a homozygous C2332T truncating mutation of the gene encoding myosin VA (MYO5A) in a Turkish girl with GS. Other mutations of the same gene were reported in other cases. Recently, Pastural et al⁶ presented evidence suggesting the existence of a second locus associated with Griscelli syndrome in the 15q21 region, which is located less than 7.3 cm from the MYO5A gene. Mutations in RAB27A were found in 16 patients with GS.³ Unlike MYO5A, the GTP-binding protein RAB27A is involved in the control of immune regulation, and it seems to be a key effector of cytotoxic granule exocytosis, a pathway essential for immune homeostasis. All patients with RAB27A mutations, but none with the MYO5A mutation, developed HLH. In addition, RAB27A-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis, whereas MYO5A-defective T cells did not.

As mentioned above, GS is a very rare disease, with about 30 cases reported over the past 20 years;^{1,2,3,4,7,8,9,10,11} it is usually fatal within the first few years of life, due to HLH, which in most cases is triggered by common infections. We report a new case of GS in an Italian child cured after HSCT from a matched unrelated donor.

Case report

A male child was the only child born to healthy first cousins from Southern Italy. The pregnancy was uneventful. Family history failed to disclose any relevant medical problem. At the age of 5.5 months, the boy developed persistent fever. When first seen at the age of 7 months, he had fever, edema, hepatosplenomegaly and skin rash. His hairs and eyebrows were silvery-gray. Blood count showed leukopenia (2100/mm³, neutrophils 260/mm³), anemia (8.5 g/dl), thrombocytopenia (14 000/mm³); triglycerides were 363 mg/dl, fibrinogen 130 mg/dl, and ferritin 1040 ng/ml. Serum immunoglobulins were as follows: IgG 480 mg/dl (n.v. 351-919), IgA 7 mg/dl (n.v. 10-85), IgM 50 mg/dl (n.v. 38-204). Bone marrow aspiration showed increased numbers of monocytes with active erythrophagocytosis and no blast cells. Examination of the cerebro-spinal fluid (CSF) showed 17 cells/mm³ with no evidence of hemophagocytosis. On the basis of these findings, HLH was diagnosed. Natural killer (NK) activity was tested and found to be impaired, with 2%, 3% and 15% of lysis at the effector:target ratios of 10:1, 30:1, 100:1, respectively.

The child was treated with dexamethasone, etoposide and cyclosporine, according to the HLH-94 study,¹² with immediate response and stable disease control. A search for a suitable unrelated donor was started in the international registries of both bone marrow and cord blood donors.

The possibilities of CHS and GS were considered. Microscopic examination of the hairs showed unevenly clustered pigment in the medulla, characteristic of GS. Mutation analysis of the RAB27A gene showed a homozygous six base pair deletion (del 510 AAGCC) confirming the diagnosis of GS.

At the age of 16 months, treatment according to HLH-94 was withdrawn and the child underwent HSCT from an unrelated bone marrow donor. Donor and recipient were found to be completely identical using a high-resolution allelic technique for both HLA class I and class II antigens. Conditioning regimen before the allograft comprised busulfan (16 mg/kg) from days -9 to -6, thiotepa (10 mg/kg) on day -5, and fludarabine (40 mg/m²/day) i.v. from days -4 to -2. Graft-versus-host disease prophylaxis consisted of cyclosporine A, short-term methotrexate and anti-lymphocyte globulin (ALG, 10 mg/kg/day from days -4 to -2). The number of nucleated cells infused was 7.8 ´ 10⁸/kg of recipient body weight. Hematopoietic and lymphoid engraftment were documented by studying the genetic polymorphism of variable number of tandem repeat short DNA sequences (VNTRs).

Engraftment of donor cells was prompt. The child recovered an absolute neutrophil count more than 0.5 ´ 10⁹/l on day +14 after transplantation, whereas platelet engraftment (20 ´ 10⁹/l) was observed on day +21. The post-transplant course was complicated by an episode of sepsis from vancomycin-resistant Enterococcus faecium. Treatment with quinupristin/dalfopristin (7.5 mg/kg every 8 h) for 22 days and teicoplanin (10 mg/kg/day) for 12 days led to resolution of the clinical symptoms and disappearance of the organism from blood and stool.

Thirty months after HSCT, the patient is well, with complete donor chimerism and normal full blood cell counts; he is not receiving any immune suppression and has recovered normal function of the immune system (see also Table 1 for further details).

Discussion

Differential diagnosis between the different subtypes of HLH is crucial for appropriate and successful therapy. In particular, prompt identification of genetic disorders leading to HLH may confirm the indication for allogeneic HSCT as the only curative tool, while other less severe conditions should be identified as soon as possible in order to avoid potentially unnecessary and life-threatening treatments.¹³ Presence of silvery-gray hairs in patients with HLH should be considered a strong hallmark of CHS or GS.

Allogeneic HSCT is the only treatment proved to be potentially curative in genetically based HLH. We have been able to identify only five cases of GS in the literature treated with allogeneic HSCT, all from a family donor (Table 2).^7,9,14 In 1990 Schneider et al⁹ reported one consanguinous family in which four of seven children had GS. Three patients died before 20 months of age; in the fourth sibling, allogeneic HSCT was performed at 4 months of age. Two years later, the patient remained in excellent health.

In their report in 1994, Klein et al⁷ included three children with GS who were allografted. Two patients died during the immediate post-transplantation period of infectious complications, but one patient was cured and at the time of the report, the follow-up was 5 years. In 1999, Tezcan et al¹⁴ carried out an allogeneic HSCT from an HLA-identical sibling donor on a 4-year-old girl with GS who presented in accelerated phase with neurological manifestations. She was treated with etoposide, methylprednisolone and intrathecal methotrexate for 8 weeks and underwent BMT after receiving a conditioning regimen including ALG (rabbit, 10 mg/kg ´ 5 days), busulfan and cyclophosphamide. Engraftment occurred early and the post-transplant period was uneventful. Eighteen months after the allograft, she was well, with sustained engraftment and normal neurological examination except for minimal clonus.

To our knowledge, this is the first case of a successful allograft of haematopoietic progenitors from an unrelated HLA-matched donor reported in a patient with GS. Registries of unrelated donors have widened the possibility of performing allogeneic HSCT in these patients. However, the period of time needed for locating an unrelated stem cell donor can still unfavorably prejudice use of this treatment in children with GS. In this regard, stable disease control achieved with chemo-immunotherapy, can be extremely helpful in gaining the time needed to identify a suitable donor. Chemo-immunotherapy with dexamethasone, etoposide and cyclosporine according to the HLH-94 study¹² proved to be very effective in achieving disease control in our patient, as is the case in children with other forms of HLH. ATG has also been utilized successfully to induce remission of HLH in patients with GS.^7,15,16 Unrelated donor cord blood transplantation can represent a valid alternative option in offering a rapid allograft to patients with HLH, due to the more prompt availability of hematopoietic stem cells.¹⁷

Since graft rejection has been reported to be frequent in patients with HLH given a mismatched family donor allograft¹⁸ and the use of an unrelated donor per se can increase the risk of graft failure although it has also been successfully reported, we had concerns about the chances of obtaining sustained engraftment of donor cells. Thus, we decided to use a novel preparative regimen containing fludarabine and thiotepa instead of cyclophosphamide as the immunosuppressive agent.^{19,20,21,22,23,24,25} Terenzi et al have both documented that fludarabine can replace cyclophosphamide in pre-transplant conditioning therapy¹⁹ and that, in an experimental model of a fully mismatched transplant, enhanced engraftment of donor stem cells is achieved when thiotepa is added to the preparative regimen.²⁰ Moreover, previous clinical studies have shown that both thiotepa- and fludarabine-based preparative regimens are well tolerated, have limited extra-medullary toxicity and are currently used in patients who are not eligible for conventional myeloablative conditioning because of advanced age and/or poor performance status.^{21,22,23,24,25}

In conclusion, available data suggest that an HLH is likely to occur in GS patients with RAB27A mutations, making affected children candidates for an early allograft, even from an alternative donor. The demonstrated curative effect of HSCT even from alternative donors in patients with genetically determined HLH supports the need for a standardized diagnostic approach to HLH.

Acknowledgements

This work was partly supported by grants from: AIRC (Associazione Italiana Ricerca sul Cancro) (FL); CNR (Consiglio Nazionale delle Ricerche) (FL); MURST (Ministero dell'Università e della Ricerca Scientifica e Tecnologica) (FL); IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Policlinico S Matteo (FL and MA); Telethon (MA); 'Vaincre les maladies lysosomales (VML)' (GDSB).

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Table 1 Natural killer cell activity and immunological evaluation of the patient before and after allografting

Table 2 Reported cases of Griscelli syndrome treated with allogeneic HSCT