Stephan Ladisch

HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis

In HLH‐94, the first prospective international treatment study for hemophagocytic lymphohistiocytosis (HLH), diagnosis was based on five criteria (fever, splenomegaly, bicytopenia, hypertriglyceridemia and/or hypofibrinogenemia, and hemophagocytosis). In HLH‐2004 three additional criteria are introduced; low/absent NK‐cell‐activity, hyperferritinemia, and high‐soluble interleukin‐2‐receptor levels. Altogether five of these eight criteria must be fulfilled, unless family history or molecular diagnosis is consistent with HLH. HLH‐2004 chemo‐immunotherapy includes etoposide, dexamethasone, cyclosporine A upfront and, in selected patients, intrathecal therapy with methotrexate and corticosteroids. Subsequent hematopoietic stem cell transplantation (HSCT) is recommended for patients with familial disease or molecular diagnosis, and patients with severe and persistent, or reactivated, disease. In order to hopefully further improve diagnosis, therapy and biological understanding, participation in HLH studies is encouraged. Pediatr Blood Cancer 2007;48:124–131.

Chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the HLH-94 treatment protocol

Hemophagocytic lymphohistiocytosis (HLH) used to have a dismal prognosis. We report the final results of HLH-94, the largest prospective diagnostic/therapeutic HLH study so far. The treatment includes immunosuppressive and cytotoxic therapy aiming at clinical remission, followed by HSCT in patients with familial, persistent, or recurrent disease. Altogether, 249 patients fulfilled inclusion criteria and started HLH-94 therapy (July 1994-December 2003); 227 (91%) were followed-up for ≥ 5 years. At 6.2 years median follow-up, estimated 5-year probability of survival was 54% ± 6%. Seventy-two patients (29%) died before HSCT, 64 within 1 year, 97% of whom had active disease. In 124 patients who underwent HSCT, 5-year survival was 66 ± 8%; tendency to increased survival (P = .064) in patients with nonactive disease at HSCT. Patients with familial disease had a 5-year survival of 50% ± 13%; none survived without HSCT. Patients deceased during the first 2 months more often had jaundice, edema, and elevated creatinine. Forty-nine patients (20%) were alive without signs of HLH activity and off-therapy > 1-year without HSCT; they presented at older age (P < .001), were more often female (P = .011), and less often had CNS disease (P < .001) or hepatomegaly (P = .007). To conclude, HLH-94 chemoimmunotherapy has considerably improved outcome in HLH. Collaborative efforts are needed to further reduce early mortality, HSCT-related mortality, and neurologic late effects.

Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) poses major therapeutic challenges, and the primary inherited form, familial haemophagocytic lymphohistiocytosis (FHL), is usually fatal. We evaluated, including Cox regression analysis, survival in 86 children (29 familial) that received HLH‐94‐therapy (etoposide, dexamethasone, ciclosporin) followed by allogeneic stem cell transplantation (SCT) between 1995 and 2000. The overall estimated 3‐year‐survival post‐SCT was 64% [confidence interval (CI)u2003=u2003±10%] (nu2003=u200386); 71u2003±u200318% in those patients with a matched related donor (MRD, nu2003=u200324), 70u2003±u200316% with a matched unrelated donor (MUD, nu2003=u200333), 50u2003±u200324% with a family haploidentical donor (haploidentical, nu2003=u200316), and 54u2003±u200327% with a mismatched unrelated donor (MMUD, nu2003=u200313). After adjustment for potential confounding factors, estimated odds ratios (OR) for mortality were 1·93 (CIu2003=0·61–6·19) for MUD, 3·31 (1·02–10·76) for haploidentical, and 3·01 (0·91–9·97) for MMUD, compared with MRD. In children with active disease after 2‐months of therapy (nu2003=u200343) the OR was 2·75 (1·26–5·99), compared with inactive disease (nu2003=u200343). In children with active disease at SCT (nu2003=u200337), the OR was 1·80 (0·80–4·06) compared with inactive disease (nu2003=u200349), after adjustment for disease activity at 2‐months. Mortality was predominantly transplant‐related. Most HLH patients survived SCT using MRD or MUD, and survival with partially mismatched donors was also acceptable. Patients that responded well to initial pretransplant‐induction therapy fared best, but some persisting HLH activity should not automatically preclude performing SCT.

Frequency and spectrum of central nervous system involvement in 193 children with haemophagocytic lymphohistiocytosis

Haemophagocytic lymphohistiocytosis (HLH) may cause meningoencephalitis and significant neurological sequelae. We examined the relationship between neurological symptoms and cerebrospinal fluid (CSF) at diagnosis, and long‐term outcome, in all children enroled in the HLH‐94‐study prior to July 1, 2003, for whom information on CSF at diagnosis was available (nu2003=u2003193). Patients were divided into four groups: (i) normal CSF (cells/protein) and no neurological symptoms (nu2003=u200371); (ii) normal CSF but neurological symptoms (nu2003=u200321); (iii) abnormal CSF but no symptoms (nu2003=u200350); and (iv) abnormal CSF with neurological symptoms (nu2003=u200351). At diagnosis, neurological symptoms were reported in 72/193 (37%) (seizuresu2003=u200323); abnormal CSF in 101/193 (52%), and either or both in 122/193 (63%). Altogether 16/107 (15%) survivors had neurological sequelae at follow‐up (median 5·3u2003years). Multivariate hazard ratios (HR) for mortality were 0·98 [95% confidence interval (CI)u2003=u20030·42–2·31], 1·52 (0·82–2·82) and 2·05 (1·13–3·72) for groups 2–4, compared with group 1. Moreover, sequelae were more frequent in group 4 (7/21, 33%) compared to groups 1–3 (9/86, 10%) (Pu2003=u20030·015). Patients with abnormal CSF at diagnosis had significantly increased mortality [HRu2003=u20031·78 (95% CIu2003=u20031·08–2·92), Pu2003=u20030·023]. Thus, a substantial proportion of HLH survivors suffer neurological sequelae, and children with abnormal CSF have increased risk of mortality and neurological sequelae. Prompt treatment of HLH at onset or relapse may reduce these complications.

Risk factors for diabetes insipidus in langerhans cell histiocytosis

Diabetes insipidus (DI) is the most frequent central nervous system (CNS)‐related permanent consequence in Langerhans cell histiocytosis (LCH), which mostly requires life‐long hormone replacement therapy. In an attempt to define the population at risk for DI, 1,741 patients with LCH registered on the trials DALHX 83 and DALHX 90, LCH I and LCH II were studied.

Therapy prolongation improves outcome in multisystem Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH)-III tested risk-adjusted, intensified, longer treatment of multisystem LCH (MS-LCH), for which optimal therapy has been elusive. Stratified by risk organ involvement (high [RO+] or low [RO-] risk groups), > 400 patients were randomized. RO+ patients received 1 to 2 six-week courses of vinblastine+prednisone (Arm A) or vinblastine + prednisone + methotrexate (Arm B). Response triggered milder continuation therapy with the same combinations, plus 6-mercaptopurine, for 12 months total treatment. 6/12-week response rates (mean, 71%) and 5-year survival (84%) and reactivation rates (27%) were similar in both arms. Notably, historical comparisons revealed survival superior to that of identically stratified RO+ patients treated for 6 months in predecessor trials LCH-I (62%) or LCH-II (69%, P < .001), and lower 5-year reactivation rates than in LCH-I (55%) or LCH-II (44%, P < .001). RO- patients received vinblastine+prednisone throughout. Response by 6 weeks triggered randomization to 6 or 12 months total treatment. Significantly lower 5-year reactivation rates characterized the 12-month Arm D (37%) compared with 6-month Arm C (54%, P = .03) or to 6-month schedules in LCH-I (52%) and LCH-II (48%, P < .001). Thus, prolonging treatment decreased RO- patient reactivations in LCH-III, and although methotrexate added no benefit, RO+ patient survival and reactivation rates have substantially improved in the 3 sequential trials. (Trial No. NCT00276757 www.ClinicalTrials.gov).

Immunosuppression by human gangliosides: I. Relationship of carbohydrate structure to the inhibition of T cell responses

Causes of cellular immunodeficiency frequently associated with cancer remain poorly understood. One possible mechanism is tumor cell membrane shedding of immunosuppressive molecules, such as the sialic acid-containing glycosphingolipids, gangliosides. To explore this interesting hypothesis and establish structure-activity relationships, we examined the effects of a series of highly purified human gangliosides on T cell function. In all, ten individual molecular species of two major biosynthetic pathways were compared for their ability to inhibit human T cell proliferative responses. They include GM1, GD1a, GD1b, and GT1b (the predominant normal brain species), and GM4, GM3, GM2, GD3, GD2 and GQ1b. Strikingly, each HPLC-purified molecule, from the simplest monosialoganglioside to the most complex polysialoganglioside, had potent inhibitory activity; even the ganglioside with the most elemental carbohydrate structure (GM4, one sialic acid linked to a monosaccharide) strongly inhibits T cell proliferative responses to tetanus toxoid (ID90 = 1.5 microM). The data also reveal a complex interplay between elements of oligosaccharide structure in determining immunosuppressive activity. Sialic acid is critical to maximal activity, and (i) immunosuppression is most potent in gangliosides containing a terminal sialic acid. (ii) Total desialylation almost abolishes activity and (iii) partial alteration (lactone formation) reduces activity. (iv) Activity is generally but not always higher with higher numbers of sialic acid residues/molecule, and (v) some larger neutral glycosphingolipids retain measurable immunosuppressive activity. Overall, the potent inhibition by gangliosides supports the hypothesis that shedding of these molecules by tumors creates a highly immunosuppressive microenvironment around the tumor, thereby inhibiting the function of infiltrating host leukocytes and contributing to diminished T cell responses in cancer.

Immunodeficiency in familial erythrophagocytic lymphohistiocytosis.

4 children with familial erythrophagocytic lymphohistiocytosis and hyperlipidaemia were found to have a previously unrecognised immunological deficiency syndrome which included defects in both humoral and cellular immunity and a plasma inhibitor of in-vitro lymphocyte blastogenesis. The inhibitory activity was proportional to the increase in the triglyceride concentration in the patients plasma. Immunological deficiency, to which hyperlipidaemia may be a contributing factor, appears to be a significant feature of familial erythrophagocytic lymphohistiocytosis.

Cellular Gangliosides Promote Growth Factor-induced Proliferation of Fibroblasts

Cell surface gangliosides have been proposed as modulators of transmembrane signaling. In this study, we used two complementary approaches to investigate the function of cellular gangliosides in the response of mammalian fibroblasts to growth factors. First, inhibition of glucosyl ceramide synthase by a new specific inhibitor ofd-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl (glucosylceramide synthase), which depletes cellular gangliosides at a concentration of 1 μm without causing an increase in ceramide levels, blocked epidermal growth factor-stimulated proliferation of fibroblasts. Similarly, responses to several other growth factors that activate receptor tyrosine kinases, including fibroblast growth factor, insulin-like growth factor-I, and platelet-derived growth factor, were inhibited by 50–100%. Conversely, enrichment of cellular gangliosides by preincubation of the mouse and human fibroblasts with exogenously added gangliosides enhanced growth factor-elicited cell proliferation. Novel findings of this study, distinguishing it from previous similar studies, include differential effects of preincubation versus continuous incubation of cells with gangliosides on growth factor-dependent cell proliferation and the growth factor-like action of NeuNAc α2–3Galβ1–3GalNAcβ1–4(NeuNAcα2–3)Galβ1–4Glcβ1–1Cer when cells are pretreated with the ganglioside.

Glucosylceramide synthase inhibition enhances vincristine-induced cytotoxicity.

As a strategy to enhance tumor cell sensitivity to vincristine, we tested whether modulation of sphingolipid metabolism would alter vincristine cytotoxicity since this is linked to accumulation of the intermediate metabolite, ceramide. We blocked ceramide metabolism in a series of variably vincristine‐resistant cell lines derived from CCRF‐CEM leukemia cells using an inhibitor of glucosylceramide synthase, DL‐threo‐phenyl‐2‐hexadecanoylamino‐3‐pyrrolidino‐1‐propanol (PPPP). PPPP alone (1.0 μM), while nearly completely blocking glucosylceramide synthesis, was not toxic and did not increase cellular ceramide levels. Vincristine alone was toxic, caused apoptosis or programmed cell death (PCD) and caused an elevation in ceramide levels. Strikingly, the combination of PPPP and vincristine resulted in a further increase, over that of vincristine alone, of (i) cellular ceramide concentration, (ii) cytotoxicity associated with PCD and (iii) G2/M cell‐cycle arrest. PPPP had no effect on P‐glycoprotein expression or function. We conclude that vincristine cytotoxicity occurs in part through a ceramide‐dependent mechanism, resulting in both G2/M block as well as PCD, and that the blockade of glucosylceramide synthase, in itself not toxic, causes augmented accumulation of ceramide resulting from vincristine exposure, which in turn maximizes ceramide‐dependent, vincristine‐induced cytotoxicity. Inhibition of glucosylceramide synthesis may be a means of circumventing drug resistance by enhancing signaling through a cell‐death pathway.