MANAGEMENT AND OUTCOME OF PATIENTS WITH LANGERHANS CELL HISTIOCYTOSIS AND SINGLE-BONE CNS-RISK LESIONS: A MULTI-INSTITUTIONAL RETROSPECTIVE DESCRIPTIVE STUDY Oussama Abla1, Sheila Weitzman1, Deepakbabu Chellapandian1, Nour Abuhadra1, Anne-Sophie Carret2, Itziar Astigarraga3, Rima Jubran4, Tiffany Chang4, Barbara Degar5, Karen Mandel6, Cor van den Bos7, Mark Belletrutti8, David Dix9, Johann Visser10, James Whitlock1, Furqan Shaikh1 Hospital for Sick Children, University of Toronto, Toronto, ON Canada; 2CHU SainteJustine/Universite´ de Montre´al, Montreal, QC Canada; 3Hospital Universitario Cruces, Barakaldo, Bizkaia, Spain; 4Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA USA; 5Dana-Farber Cancer Institute, Boston, MA USA; 6 Children’s Hospital of Eastern Ontario, Ottawa, ON Canada; 7Emma Children’s Hospital, Academic Medical Center, Amsterdam, Netherlands; 8Stollery Children’s Hospital, Edmonton, AB Canada; 9British Columbia’s Children’s Hospital, Vancouver, BC Canada; 10 Leicester Children’s Hospital, Leicester, United Kingdom

mutation in STX- 11 gene FHLH type 4. He received matched allogeneic bone marrow transplant 4 monthsafter his diagnosis. He has improved significantly clinically. Conclusion: The physicians should be aware of the neurological presentation at the onset of FHL.



Purpose: There is an increased risk of adverse events such as reactivation, diabetes insipidus (DI) and neurodegeneration (ND) among children diagnosed with Langerhans cell histiocytosis (LCH) and single-bone CNS-risk lesions. However, it is unknown whether the addition of chemotherapy or radiotherapy changes outcomes in these patients. The purpose of this study was to compare event-free survival (EFS) for patients treated with systemic therapy (chemotherapy or chemo-radiotherapy) versus local therapy such as biopsy, curettage and/or intralesional steroids. Methods: A retrospective analysis of 93 patients from ten centers in Canada (34), USA (31) and Europe (28) was performed. We calculated EFS and overall survival (OS). Cox multivariable regression analysis was performed adjusting for treatment group, specific craniofacial site (eye, ear, oral and cranial fossa) and degree of intracranial extension. Results: 59 patients received systemic and 34 patients received local therapy. Median age was 4.08 years (0.46 to 17.03 years) with a median follow up of 4.1 years. Number of events in the local therapy group was: reactivation ¼ 5, DI ¼ 0, ND ¼ 0 and death ¼ 1; while events in the systemic therapy group were: reactivation ¼ 11, DI ¼ 4, ND ¼ 1 and death ¼ 1. The 5-year EFS and OS was 85% (SE 6%) and 95% (SE 5%) in the local therapy group and 80% (SE 5%) and 98% (SE 2%) in the systemic therapy group. There was no statistical significance among either group with regards to EFS (p ¼ 0.26) and OS (p ¼ 0.78). On multivariable analysis, there was again no significant difference among the two treatment groups after adjusting for site and extension (HR ¼ 2.17, 95%CI ¼ 0.72 to 6.53; p ¼ 0.17). Conclusion: Although our findings are limited by a small sample size, these data suggest that chemo-radiotherapy does not reduce the risk of reactivation or late events as compared to less aggressive local control measures.


Anthony M. Alvarado, Reuben Antony, Mohamad M. Al-Rahawan University of Illinois College of Medicine at Peoria, Peoria, IL USA Introduction: Cutaneous Langerhans cell histiocytosis (LCH), often a diagnostic and therapeutic challenge, may manifest as either single or multiple organ LCH. Herein we describe our experience using pamidronate in treating recurrent cutaneous LCH in a pediatric patient. Case Report: An 18-year-old male formerly treated for histologically proven disseminated LCH presented with an inguinal rash resembling a preexisting rash at the time of his initial LCH diagnosis. Initial presentation for pituitary involvement at age 13 was treated with focal radiotherapy (9 Gy). At age 15 he presented with pelvic bone pain and an inguinal rash with biopsy confirmed bony LCH. As per the LCH-III protocol, he received vinblastine and prednisone for oneyear. Ninemonths later he reported posterior neck pain that was PET scan confirmed skull base LCH reactivation; subsequently treated using radiotherapy (7 Gy). A few months later, his inguinal rash recurred without activation in any other site. Topical and systemic antifungal therapy served ineffective. A skin biopsy displayed evolving epitheliotropic T-cell dyscrasia with background aggregation of atypical Langerhans cells. Narrow band phototherapy provided minimal improvement. We initiated monthly intravenous pamidronate (1mg/kg) for 1year as per Morimoto, A. et al. [Pediatric Blood Cancer. 2011 Jan;56(1):110–5]. Initial improvement prevailed at week 16 of treatment with only residual skin erythema persisting at week 24. Skin integrity continually improved with normalization by 44weeks. To date (5 months off pamidronate), his inguinal skin remains normal, without additional intervention, and there is no evidence of LCH reactivation at other previously affected sites. Conclusion: While the efficacy of bisphosphonates in reducing complications and pain of skeletal LCH is well documented there are only a few reports regarding pamidronate use for cutaneous LCH. We suspect pamidronate mediates tissue repair by suppressing and inhibiting recruitment of osteoclast-like histiocytes. Further investigation of bisphosphonate use in targeting pediatric LCH skin lesions is warranted.

AP3d DEFICIENCY DEFINES A NEW TYPE OF HERMANSKY PUDLAK SYNDROME Sandra Ammann1,2, Ansgar Schulz3, Ingeborg Kra¨geloh-Mann4, Martin Scho¨ning4, Horst von Bernuth5, Udo zur Stadt6, Kai Lehmberg6, Stephan Ehl1, Hans-Christian Hennies7

Muayad Alali, Shahrukh Shamshad, Kayyali Husam, Bayoumy Mahamed, Mohammed Saeed Bin Mahfoodh, Abosoudah Ibraheem King Fisal Specialist Hospital & Research Center, Pediatric Oncology, Jeddah, Saudi Arabia


Center for Chronic Immunodeficiency (CCI), University Medical Center Freiburg, Germany; Faculty of Biology, University Freiburg, Germany 3Department of Pediatrics, University Medical Center Ulm, Germany; 4Department of Pediatrics, University Medical Center Tu¨bingen, Germany; 5Department of Pediatrics, University Medical Center Charite´ Berlin, Germany; 6Center for Diagnostic, University Medical Center Hamburg Eppendorf, Germany; 7Pediatric Hematology and Oncology, University Medical Center Hamburg Eppendorf, Germany; 8Cologne Center for Genomics, University of Cologne, Germany


Purpose: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disorder of immune dysregulation characterized by proliferation and infiltration of hyperactivated macrophages and T lymphocytes in multiple organs. Predominant neurologic manifestations at presentation are rarely encountered in FHL. Case Report: We report a 2-year-7-month-old previously healthy Saudi boy who presented with 6 weeks history of intermittent fever, irritability and cerebellar dysfunction. MRI brain showed marked enlargement of cerebellum and obstructive hydrocephalus requiring an urgent ventriculoperitoneal shunt. Transient mild pancytopenia and hepatic dysfunction were also present. Diagnosis of hemophagocytic lymphohistiocytosis was difficult due to remitting course and absence of hemophagocytes in two bone marrow biopsies. Treatment with dexamethasone, etoposide and cyclosporine (HLH-2004 protocol) was initiated based on strong clinical suspicion. Diagnosis was later confirmed on genetic testing which showed

 C 2015 Wiley Periodicals, Inc. DOI 10.1002/pbc.25505 Published online in Wiley Online Library (wileyonlinelibrary.com).

Purpose: Hermansky-Pudlak syndrome (HPS) is a genetically heterogenous disease with albinism and a bleeding disorder. HPS2 is associated with neutropenia, impaired NK and T cell degranulation and cytotoxicity, leading to a mild risk of developing HLH. We present a patient with oculocutaneous albinism, mild facial dysmorphism, hip dysplasia and reduced hearing. The patient had chronic neutropenia, frequent airway infections and persistent hepatosplenomegaly. The presentation resembled Hermansky-Pudlak-Syndrome Type 2



(HPS2), but additionally the patient had severe psychomotor retardation with generalized seizures. Methods: Degranulation of NK and T cells was measured by CD107a surface expression via flow cytometry and cytotoxicity was analyzed by radioactive 51Cr release assay. Genetic analysis was done bySanger sequencing and whole exomesequencing (WES). Results: Consistent with the suspected diagnosis of HPS2, NK and T cell degranulation and cytotoxicity were absent. However, genetic analysis revealed no mutations in AP3B1 (HPS2) or other genes associated with immunodeficiency and albinism. WES identified a homozygous late frame-shift mutation in AP3D1 leading to a premature stop codon. A natural mouse pigmentation mutant with an AP3D1 null mutation (Mocha Mice) shows the platelet storage pool deficiency characteristic of HPS and shares the neurological and hematological phenotype of our patient. AP3d is a subunit of the heterotetramer adaptor-protein 3 (AP3) complex existing in a ubiquitously expressed form and a primarily neuronal expressed form. The AP3d subunit is essential for both forms, whereas AP3b3A affected in HPS2 is substituted by AP3b3B in the neuron-specific heterotetramer, explaining the severe neurological phenotype of our patient compared to HPS2 patients. Conclusion: AP3d deficiency causes a new variant of HPS in humans, which we propose to classify as HPS2b.

T-CELLS ARE NOT REQUIRED FOR THE PATHOGENESIS OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IN HUMANS Sandra Ammann1, Sebastian Bode1,2, Kai Lehmberg3, Stephan Ehl1,2 for the GPOH, the Histiocyte Society and the IEWP of the EBMT 1

Centre of Chronic Immunodeficiency, University Medical Centre Freiburg, Germany; Centre for Pediatrics and Adolescent Medicine, University Medical Centre Freiburg, Germany; 3Department of Haematology and Oncology, Children’s Hospital, University of Hamburg, Germany


Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of severe hyperinflammation. Impaired release of perforin-containing granules by T-/NK-cells is a key factor in hereditary (1˚) HLH. Mouse models of 1˚ HLH indicate that T-cells and IFN-g are essential for disease pathogenesis. Whether T-/NK-cells are required for other forms of HLH in humans is unclear. Methods: Primary immunodeficiencies (PID) provide an excellent opportunity to study the role of immune cells and mediators in immune-mediated diseases. We collected the experiences in the treatment of patients with PID, except cytotoxicity disorders or XLP, who developed HLH from the GPOH, the Histiocyte Society and the IEWP of the EBMTand added cases from the literature. Results: 46 patients with PID had at least one episode of HLH. 14 patients had severe combined immunodeficiency (9 with < 100 T-cells), 11 had partial T-cell deficiencies. Most of these HLH episodes were triggered by viruses. 11 patients had HLH in the context of chronic granulomatous disease, mainly associated with bacterial infection. 10 patients had other ID. Patients with T-cell deficiency showed significantly lower soluble IL-2 receptor (sCD25) levels and higher ferritin levels than patients with cytotoxicity defects. Lower sCD25 levels were also observed in patients with infection-induced 2˚ HLH compared to patients with 1˚ HLH. Discussion: Our results are consistent with the concept of a T-cell dependent and a T-cell independent pathway of HLH pathogenesis. T-cells appear crucial for 1˚ HLH, but are not required for the development of HLH in general and appear less active in many patients with 2˚ HLH. Conclusion: HLH is a heterogeneous syndrome representing a common endpoint of different pathways of pathogenesis. T-cell directed therapy might not appropriate for all patients with HLH.

TREATMENT OF RELAPSED AND PROGRESSIVE LANGERHANS CELL HISTIOCYTOSIS WITH CLADARABINR (2-CdA) BASED REGIMEN; EXPERIENCE FROM CHILDREN CANCER HOSPITAL, KARACHI, PAKISTAN Muhammad Shamvil Ashraf, Muhammad Rafie Raza Children Cancer Hospital, Karachi, Pakistan Purpose: To assess the safety and efficacy of reduced dose 2-CdA for treatment of relapsed and progressive LCH at our institution. Method: Since 2009 we have started treating relapsed and progressive Langerhans cell histiocytosis with 2-CdA based regimen. Seven children (6 female, 1 male), age range 1–5 years have been treated so far. 2 had multisystem disease while 5 had cranio-facial multifocal bone lesions with diabetes insipidus. Salvage was given in 3 with reactivation on treatment, 3 with relapsed after treatment and one with induction failure. The treatment consisted of 4 courses of 2-CdA 5mg/m2  5 days at 3 weekly intervals followed by 24 months maintenance with vinblastin, prednisolne and oral 6 MP in 3 cases while in 4 cases courses of 2-CdA were combined with Ara –C 150mg/m2  5 days. The disease reassessment was done after 4 courses. Result: All children are in remission with a median follow- up of 23 months (2–42 months). There was no toxic death and, apart from myelosuppression and febrile neutropenia, there were no major adverse effects. Conclusion: Reduced dose 2-CdA containing regimen can be given in oncology centers with constrained resources. It is well tolerated with no toxic death in ourseries of 7 patients. All

Pediatr Blood Cancer DOI 10.1002/pbc

children are in remission. Longer follow up is needed to assess the long term survival in these patients.

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS COMPLICATING ADULT PATIENTS WITH HIV INFECTION Itziar Astigarraga1, Susana Garcia-Obregon1, Koldo Agirrebengoa2, nez5, Amaia Balerdi3, Juan Carlos Garcia-Ruiz3, Beatriz Diaz4, Maria Jose Nu~ Pilar Miralles6, Juana Gil7 1 Servicio de Pediatrı´a, BioCruces Health Research Institute, Hospital Universitario Cruces, Barakaldo, Bizkaia, Spain; 2Servicio de Enfermedades Infecciosas, BioCruces Health Research Institute, Hospital Universitario Cruces, Barakaldo, Bizkaia, Spain; 3Servicio de Hematologı´a, BioCruces Health Research Institute, Hospital Universitario Cruces, Barakaldo, Bizkaia, Spain; 4Servicio Medicina Interna, Unidad de Enfermedades infecciosas, Hospital Universitario La Paz, Madrid, Spain; 5Servicio Medicina Interna, Unidad de Enfermedades infecciosas, Hospital Clı´nico San Carlos, Madrid, Spain; 6Servicio de Enfermedades Infecciosas, Hospital General Universitario, Madrid, Spain; 7Servicio de Inmunologı´a Clı´nica, Hospital General Universitario, Madrid, Spain

Purpose: To differentiate hemophagocytic lymphohistiocytosis (HLH) and severe presentations of HIVinfection is a diagnostic challenge in adults. Most HLH cases appear in advanced stages and associated concomitant infections or malignancy. The purpose of the study is to describe the characteristics of HLH in adult patients with a recent HIV infection diagnosis. Methods: Analysis of medical charts of HIV-1- infected adult patients with reactive HLH from 3 Spanish hospitals in the last 10 years. Review of clinical, laboratory and radiological findings of 4 HLH-VIH cases. Results: In 2003, a 20-year-old-man had HLH caused by acute HIV infection alone (stage B3) and recovered spontaneously. Until 2012 no other cases were identified. The second patient was a 33-year-old-man who suffered a severe HLH course that responded to HLH-94protocol. Apart from HIV, only a parasite (Babesia divergens) and the initiation of highly active antiretroviral therapy (HAART) were found as triggers. Due to life-threatening reactivations, splenectomy and allogenic HSCT were performed. In 2014, a 40-year-old-man suffered HLH with CNS lesions after 1 month of HAART and responded to HS-HLH-94 protocol and rituximab due to concomitant EBV infection. The last patient was diagnosed with severe HLH, CMV infection and Hodgkin disease in the first year of HAART and treated with HS-HLH-94 protocol and chemotherapy. All 4 patients are alive. Conclusion: In this smallcohort of patients we observed that HLH could be associated with acute primary HIV infection, so it should be included in the differential diagnosis of HLH, mainly in young adults. In HIV patients, HLH might be triggered by viral or parasitic acute infection, reactivation of chronic EBV and CMV viruses, malignancies or the initiation of HAART. Physicians’ awareness about HLH in adult patients is increasing so more life-threatening cases are diagnosed and treated properly with a better prognosis.

A RARE COMPLICATION OF PARANEOPLASTIC AUTOIMMUNE DISORDER IN LANGERHANS CELL HISTIOCYTOSIS Thandar Aung, Christopher Dardis St Joseph Hospital Center (Barrow Neurology Institute) Phoenix, AZ, USA Purpose: We report a case of Langerhans cell histiocytosis (LCH) in a 64-year-old man who presented with symptoms and signs of CNS involvement, including dysfunction of the pituitary gland and brainstem. Brain lesions were biopsied,which showed neurodegenerative lesions. Case Report: A 64-year-old right-handed Hispanic male presented with two years history of vague symptoms including fatigue, weight loss, loss of libido, personality changes and possible seizure like events. At that time, he was diagnosed with diabetes insipidus, hypothyroidism and severe depression. His initial examination was remarkable for ocular bobbing and right central facial nerve palsy. Patient was also found to have right greater than left quadriparesis with spasticity and upgoing toe. All routine blood tests were negative apart from fluctuatingserum sodium level. We eventually performed lumbar puncture. All CSF tests were negative except minimally elevated white count with lymphocytes and macrophages. Complete paraneoplastic panel was negative. CT scan showed lytic bone lesions. MRI was remarkable for T2 hyperintense lesions in the left frontal lobe, right mesial temporal lobe and brainstem which were minimally enhancing. Hospital course is complicated by status epilepticus, septic shock and pulmonary embolism. Left frontal bone lesion biopsy showed positive immunohistochemistry stained for CD1a and S100 which led to diagnosis of LCH. A second biopsy was performed at right temporal lesion which showed reactive astrocytes with eosinophils, which was a neurodegenerative lesion. Patient eventually improved with IV immunoglobulin and Cladribine. Discussion: Grois et al in 2005 described three types of neuropathology of LCH. Type one are the typical circumscribed granulomas. Type two are infiltrating granulomas with profound Tcell dominated inflammation and severe neurodegneration. Type three are only neurodegenerative lesions without LC and typically affecting brainstem and cerebellum. The presence of inflammation without LC has led to the view that these lesions are autoimmune in origin. In our case, temporal lobe lesions are of the second type and brainstem lesions are typical of the third type. Neurodegeneration associated with this condition is a rare complication. There is

HISTIOCYTE SOCIETY 30th ANNUAL MEETING ABSTRACTS currently no specific treatment of this complication. CD1a antibiodies and treatment along the lines of T-cell mediated paraneoplastic disorders have been tried.




Division of Rheumatology, The Children’s Hospital of Philadelphia, Philadelphia, PA USA; Department of Pathology, Alfred I. duPont Hospital for Children, Wilmington, DE USA; Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA USA

Purpose: Familial hemophagocytic lymphohistiocytosis (FHL) is thought to be driven by an excess of anti-viral CD8þ T cells and their overproduction of interferon-gamma (IFNg). However, although viral infections are often the trigger for FHL, the proximal immunologic events leading to this overactive immune response are incompletely understood. Recent work has suggested that signaling by interleukin-33 (IL-33), an alarmin, is necessary for mounting complete viral antigen-specific CD8þ T cell responses. In this study, we sought to determine whether blockade ofIL-33 signaling in the perforin knock-out (PKO) murine model of viral induced FHL would sufficiently limit the CD8þ T cell response to prevent the development of FHL. Methods: PKO mice were infected i.p. with 2  10^5 PFU LCMV-Armstrong and received 150 mg i.p. of either IL-33 receptor (IL-33R)-blocking antibody or isotype control every 2–3 days, beginning on day 3 post-infection. Mice were monitored for weight loss, survival, complete blood count, serum cytokines, spleen and liver histology, LCMV titers, frequency of antigen-specific CD8þ T cells and T cell cytokine production. Results: LCMV-infected PKO mice receiving IL-33R blocking antibody (IL-33RB) showed reduced weight loss (P ¼ 0.0170) and highly reduced mortality (HR ¼ 0.25, P ¼ 0.0605). IL33R blockade reduced levels of serum IFNg 16-fold (P ¼ 0.0005) and lowered frequencies of IFNg-producing CD8þ T cells (P ¼ 0.0003). Additionally, IL-33RB mice had reduced hepatic parenchymal damage, although leukopenia, anemia and thrombocytopenia were not improved. Despite the reduced inflammation in IL-33RB mice, they maintained similar frequencies of LCMV-specific CD8þ T cells compared to isotype-treated controls and showed equivalent titers of LCMV in the spleen. Conclusion: IL-33R blockade improved morbidity and mortality in a mouse model of FHL without reducing the antiviral response. Our results suggest that blockade of IL-33 signaling may be a viable treatment strategy for patients with FHL.





Claudia Bracaglia , Ivan Caiello , Kathy de Graaf , Giovanni D’Ario , Florence Guilhot2, Walter Ferlin2, Lidia Meli1, Giusi Prencipe1, Sergio Davı`3, Grant Schulert4, Angelo Ravelli3, Alexei Grom4,Cristina De Min2, Fabrizio De Benedetti1 1 Division of Rheumatology, Department of Pediatric Medicine, IRCCS Ospedale Pediatrico Bambino Gesu`, Rome, Italy; 2Novimmmune SA, Plan-les-Ouates, Geneva, Switzerland; 3 University of Genoa, Istituto Giannina Gaslini, Genoa, Italy; 4Division of Pediatric Rheumatology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH USA

Purpose: IFNg is the pivotal mediator in murine models of primary HLH. Given the similarities between primary and secondary (sHLH), including MAS, we analyzed IFNg levels in patients with sJIA and MAS and evaluated the pathogenic role of IFNg in a murine MAS model. Methods: We measured levels of IFNg, IFNg-related chemokines (CXCL9, CXCL10, CXLC11) and IL-6 in patients with sHLH (n ¼ 14), and in patients with sJIA (n ¼ 54) of whom 20 had MAS at sampling using the Luminex multiplexing assay and evaluated their relation to disease activity. The effect of the anti-IFNg antibody XMG1.2 was assessed in IL-6 transgenic (IL6TG) mice in which a MAS-like syndrome leading to death is triggered by TLR ligands (Strippoli, Arthritis Rheum 2012). An LPS LD50 (5 mg/gr body weight) was used, as a trigger for MAS, followed 10 hours later by administration of 100 mg/gr of XMG1.2. Results: Levels of IFNg and of IFNg-related chemokines [median pg/ml(IQR)] were markedly elevated in active MAS and active sHLH, with no significant differences between active sHLH [IFNg 34.7(23.9–170.1); CXCL9 33598(3083–127687); CXCL10 4420(799.7– 8226); CXCL11 1327(189–2000)] and active MAS [IFNg 15.4(5.1–52.6); CXCL9 13392 (2163–35452); CXCL10 1612(424.8–4309); CXCL11 564.8(197.5–1007)]. Levels in active sJIA without MAS at sampling [IFNg 4.88(3.2–8.7); CXCL9 836.5(470.9–2505); CXCL10 307.3(198.9–693.7); CXCL11 121.7(62–197.1)] were lower (all p-values 5 at inclusion and BRAFV600E mutation. Here, we report the first case. Methods: Patient received vemurafenib at a dose of 30 mg/kg/d orally twice daily for an investigational period of 4 weeks. In case of progression between week 2 and week 4, the patient will receive 2-CdA/AraC. Results: LCH was diagnosed in a 2-month-old girl with rashes since birth. Soon after diagnosis, vinblastine/steroid based induction was started because of disease progression (DAS ¼ 5) and extension (skin, bones, nodes, spleen, hematological dysfunction). After 2 vinblastine/steroid inductions, DAS was still at 5. A course of 2-CdA monotherapy (5mg/ m2 x 5 days) was performed but between D21-D28, the disease frankly progressed (DAS ¼ 7), indicating a salvage therapy approach. As BRAFV600E mutation was detected, the patient was eligible for the study and vemurafenib was started after informed parents’ consent. At day 7, the DAS decreased at 3 and at day 14 the DAS was 0, with dramatic improvement on all anatomic soft tissue lesions (size reduction between 50 and 70%). The patient was discharged at day 20. No side effect was observed in exception of a grade 2 skin rash.

Pediatr Blood Cancer DOI 10.1002/pbc

Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel Purpose: Craniofacial and intracranial Langerhans cell histiocytosis (LCH) is a common site of the disease either as a presenting site or as a site of LCH reactivation. Intracranial LCH can be solitary or an extension from the calvarium with a soft tissue mass that can be surprisingly large despite few clinical symptoms. The calvarium and facial bones are the most frequent sites, however, base of skull lesions can occur as well. The clivus is an infrequent site of LCH and its manifestations and clinical course are not well documented. Methods: Four patients with LCH of the clivus are documented. The clinical course, radiologic manifestations and response to treatment are described. Results: Four patients with LCH of the clivus were treated in our hospital during the past 5 years. The age at diagnosis varied from 5–18 years. In three patients the clivus was the primary site of disease and the fourth a reactivation. The presenting symptom was abducens nerve palsy with headache and/or torticollis in two patients, neck pain in the third and the fourth had a frontal bone mass with no symptoms from the skull base lesion. 3/4 had multifocal bone disease; one had pulmonary disease as well. A biopsy of skull base was performed uneventfully in three patients. All lesions displayed a good clinical and radiologic response to vinblastine and prednisone treatment. The pulmonary lesions did not resolve and were complicated by pneumothorax. 6th nerve palsies resolved in the first weeks of therapy. Conclusions: The occurrence of unexplained torticollis, neck pain or 6th nerve palsy in a child can be the primary manifestation of clival LCH. A good response to treatment with vinblastine and prednisone is documented. Our small series showed a high rate (75%) of multifocal bone disease.

PEDIATRIC LANGERHANS CELL HISTIOCYTOSIS, EXPERIENCE OF NATIONAL CANCER INSTITUTE, CAIRO UNIVERSITY Emad Ebeid, Iman Attia, Sayed Abdel Hamid, Rana Hamdy National Cancer Institute, Cairo University, Cairo, Egypt Purpose: Langerhans cell histiocytosis (LCH) is a rare disease that can occur at any age and usually presents with skin rash or painful bony lesions. Depending on the site and extent of



disease, treatment may include surgery, radiation therapy or oral, topical and intravenous medications. Our study aimed at evaluating the demographics, disease characteristics and treatment outcome for pediatric LCH patients treated at National Cancer Institute, Cairo University. Methods: This retrospective study included 36 eligible patients with established diagnosis of LCH during the period from January 2008 to June 2013. Patients were treated according to LCH-III protocol. They were followed up until December 2013. Results: Among the 36 patients, 26 were males (53%); median age at diagnosis was 3 years (range 0.6–18 years). Patients were divided into high risk (HR) group having one or more risk organ involved (15/36 patients, 41.6%) and low risk (LR) group (21/36, 58.4%). Median follow up period was 27.2 months (range 2.9 to 70.6 months). The 3-year overall survival (OS) and disease-free survival (DFS) rates were 97.2% and 81.3% respectively. There was no significant difference between HR and LR patients regarding survival rates (3-year OS 93.75% versus 100% respectively, 3-year DFS 83% and 79.6% respectively), 14/36 patients (38.9%) had CNS risk lesions with 3-year DFS of 92.9%, 5/14 patients(35.7%) had diabetes insipidus. Presence of CNS risk lesions was significantly associated with risk organ involvement (P value ¼ 0.004) and multiple systems affection (P value ¼ 0.006). Conclusions: The outcome of patients in this study is favorable for a developing country with limited resources. CNS risk lesions were significantly associated with risk organ involvement and multiple systems affection.

A NON DEFINITIVE CASE OF ERDHEIM CHESTER DISEASE Brenda Ernst, John Camoriano, Thomas Colby Mayo Clinic Arizona, Scottsdale, AZ USA Purpose: Erdheim-Chester disease (ECD) is a rare systemic non-Langerhans histiocytosis that can present in a myriad of ways. This review will discuss the frequency of clinical presentations of ECD in the literature. Further discussion will include the role of diagnostic concordance for a rare disease of which the pathologic findings are distinctive but by no means unique and must be interpreted in context. Methods: ECD is historically a morphologic diagnosis that corresponds within the appropriate clinical context. Despite distinct radiographic findings, most cases are still diagnosed by the pathologist identification of foamy appearing non-Langerhans’ cell histiocytes. There have been roughly 500 cases described in the literature of which the diagnosis of ECD has been suspected based on mixed clinical presentations and imaging with final diagnosis established by biopsy. The frequency of this disorder presenting with a classical clinical and radiographic pattern but without diagnostic tissue is unknown and problematic since biopsy confirmation is part of the standard of care. Results: Owing to its rarity, ECD remains a difficult clinical and pathologic diagnosis which relies upon the collaborative involvement of imaging, pathology and clinicians. To date there remains no strict consensus for establishing an ECD diagnosis or the initiation of therapy. We describe a case identified through imaging, with several supportive clinical correlates in the absence of serologic or pathologic confirmation by traditional methods, although the pathologic changes seen in biopsy material were consistent with the diagnosis. Conclusion: This case illustrates a clinical-radiographic presentation of ECD in absence of a pathological diagnosis by tissue biopsy despite extensive efforts to secure diagnostic tissue. We argue that theconstellation of classic radiographic and clinical findings may be adequate to tender the diagnosis of ECD given its unique and unusual findings and acknowledge this challenges the classic mandate for tissue diagnosis within hematology and oncology.

Conclusions: This is the first report of cases seen at the NIH CC. Enrollment is active. The disease affected participants in many different ways, with bone pain, retroperitoneal fibrosis and diabetes insipidus among the common findings. Treatments varied, but survival improved in this cohort. Molecular testing of affected tissues must be performed routinely since therapies are becoming available. It is still early to give conclusions regarding the natural history of ECD.

OUTCOME OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS UNDERGOING ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION USING (FLUDARABINE/MELPHALAN/ATG) CONDITIONING: SINGLE CENTER EXPERIENCE Abousoudah Faisal Ibraheem, Alali MuayadMamdooh, Muhammad Matloob Alam, Bayoumy Mohamed Salaheldin King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of excessive immune activation. The introduction of hematopoietic stem cell transplantation (HSCT) has dramatically improved HLH patients’ survival, however, the optimal conditioning regimen for HLH is still debated. Methods: We retrospectively reviewed the outcome of 10 HLH patients who underwent allogeneic HSCT from a matched family donor using reduced intensity conditioning (RIC) comprised of Fludarabine/Melphalan/ATG at King Faisal Specialist Hospital and Research Center-Jeddah between January 2010 and March 2014. Results: There were 7 (70%) males. The median age at presentation was 19 months (range 0.18–7.2 years); the median time to HSCT was 5 months (range 0.19–0.8 years). Family history of HLH was positive in (60%), consanguinity was present in (70%). Genetic mutations were found in (80%) of patients, 2 cases (STX11), one case (UNC13D), one case (STXBP2), 2 cases of Chediak-Higashi (LYST) and one case of XLP1 (SH2D1A). Central nervous system disease was present in (30%) of cases. Bone marrow hemophagocytosis was documented at diagnosis in (70%) of cases. HLH activity prior to HSCT was controlled in all patients but one. Unfortunately, she succumbed to her disease. The average CD34 dose was 7  106/kg. All patients engrafted, the median time of neutrophil and platelet recovery were at day 14, 17 respectively. Acute graft-versus-host disease (GVHD) was observed in (30%), all were between grades II to III, and chronic GVHD was in (10%). CMV reactivation occurred in (50%). Post-transplantation mixed chimerism was observed in (30%). There was no transplant related mortality. The overall and event free survivals after HSCT were (90%). Conclusion: The outcome of Fludarabine/Melphalan/ATG conditioning in this cohort of patients shows promising results. However, large cohort multi-center prospective studies are needed to validate effectiveness and long-term survival with this regimen.

BRAF MUTATION IN CHILDHOOD LCH CORRELATES WITH MORE AGGRESSIVE DISEASE ACTIVITY AND EXTENSION Sebastien He´ritier1,2, Mohamed Barkaoui1, Anne Moreau3, Vale´rie Rigau4, Fre´de´rique Dijoud5, Catherine Chassagne-Cle´ment6, Fre´de´ric Charlotte7, Karima Mokhtari8, Sabah Boudjemaa9, Aurore Coulomb9, Michel Peuchmaur10, Marie-Christine Copin11, Sylvie Fraitag12, Thierry Molina12, Eric Jeziorski13, Caroline Thomas14, Julien Haroche15, Mathilde de Menthon16, Jean-Francois Emile2,17, Jean Donadieu1,2 Centre de re´fe´rence des Histiocytoses, Hoˆpital Trousseau, APHP, Paris, France; 2EA4340 Biomarqueurs en Cance´rologie et Onco-He´matologie, Universite´ de Versailles-SaintQuentin-en-Yvelines, Versailles, France; 3Service d’Anatomie et Cytologie Pathologiques, Hoˆpital Hoˆtel Dieu, CHU Nantes, France; 4Laboratoire d’Anatomie et de Cytologie Pathologique, CHU Gui de Chauliac-Saint Eloi, Montpellier, France; 5Centre de Pathologie et de Neuropathologie, CHU Lyon-Est, France; 6De´partement de Biopathologie, Centre Le´on Berard, Lyon, France; 7Service Central d’Anatomie et de Cytologie Pathologiques, CHU Pitie´-Salpeˆtrie`re, APHP, Paris, France; 8Service de Neuropathologie R. Escourolle, CHU Pitie´-Salpeˆtrie`re, APHP, Paris, France; 9Service d’Anatomie et de Cytologie Pathologiques, Hoˆpital Trousseau, APHP, Paris, France; 10Service d’Anatomie et de Cytologie Pathologiques, CHU Robert Debre´, APHP, Paris, France; 11Institut de Pathologie, Centre Oscar Lambret, Lille, France; 12Service d’Anatomie et de Cytologie Pathologiques, CHU Necker-Enfants Malades, APHP, Paris, France; 13Service de pe´diatrie III, CHU Arnaud de Villeneuve, Montpellier, France; 14Service d’he´mato oncologie Pe´diatrie, CHU de Nantes, France; 15Service de me´decine Interne, CHU Pitie´-Salpeˆtrie`re, APHP, Paris, France; 16 Service de me´decine Interne, CHU Saint Louis, APHP, Paris, France; 17Laboratoire de Pathologie, CHU Ambroise Pare´, APHP, Boulogne-Billancourt, France 1

ERDHEIM CHESTER DISEASE: CLINICAL CHARACTERISTICS OF 40 CASES SEEN AT THE NIH CLINICAL CENTER Juvianee Estrada-Veras, Jhonell Alvarado-Enriquez, Kevin O’Brien, William Gahl National Human Genome Research Institute, National Institute of Health, Bethesda, MD USA Purpose: ECD is a rare NLCH. The clinical characteristics of ECD range from asymptomatic to multisystemic involvement affecting bones, kidneys, retroperitoneal space and brain. BRAF V600E and NRAS Q61R mutations have been recently identified in affected tissue. After diagnosis, the disease can progress rapidly causing fatal outcomes. The diagnosis of ECD relies upon imaging studies and specific pathologic findings, i.e., fibrosis and infiltration of the affected tissues with foamy histiocytes, lymphocytes and plasma cells. Immunohistochemistry reveals cells positive for CD68 and negative for CD1a and S-100. Alphainterferon, imatinib, anakinra, cladribine and, recently, BRAF inhibitors are proposed treatments. CNS involvement is an outcome predictor for increased morbidity and mortality. In recent years, research and awareness have improved significantly, but the natural history is still an area of active research. Methods: 46 patients were evaluated at the NIHCC under NHGRI study 11-HG-0207 “Clinical and Basic Investigations into Erdheim-Chester disease” between 2011 and 2014. Body imaging, laboratory testing and multidisciplinary specialty evaluations were performed. Results: The preliminary analysis showed bone involvement 91%, right AV groove mass 76%, diabetes insipidus 46%, retro-orbital infiltration 44%, retroperitoneal 48%, Lung-Pleura 43%, CNS 42%, among others. Rare findings included splenic cyst, mesentery ischemia and portal hypertension. BRAF V600E mutation has been detected in 47% and NRAS in 0%. Treatments included Interferon alpha 20%, Peg-Interferon alpha 17%, Cladribine 11%, Imatinib 4%, Anakinra 13%, Vemurafenib 7% and others.

Pediatr Blood Cancer DOI 10.1002/pbc

Purpose: In 2010, BRAF mutation has been reported in half of the patients with Langerhans cell histiocytosis (LCH). So far, no clear correlation between BRAF mutations and clinical features has been identified. Methods: 125 children (age T), 1 patient in UNC13D (c.2782C > T, c. 64C > T), 2 patients in STX11 (c.829A > G, c. 70G > A,), 8 patients in STXBP2 (c.387C > T, c.661G > A, c.7954C > T, c.1246 þ 29G > A, c.124710C > T, c.1356 þ 18A > G, c.1356 þ 77A > G, c.1538 þ 28C > T, c.153947G > A, c.1576A > G, c.1696 þ 20A > G, c.1696 þ 28G > C, c.169726T > G), 1 patient in SH2D1A (c.-346C > T), 2 patients in BIRC4 (c.1099 þ 264G > C, c 12A > G) and 1 patient in Rab27A (c 14C > T); all of them are known polymorphisms. Conclusion: Variants in HLH-related genes may coexist with infections, cancer and other inflammatory conditions and contribute to disease pathogenesis in late-onset patients.



Sebastien He´ritier3,12, Loı¨c Le Guennec1, Caroline Decaix2, Jean Donadieu3, Maria Santiago-Ribeiro4, Nadine Martin-Duverneuil5, Richard Levy2, Daniel Delgadillo1, Aure´lie Kas6, Aure´lie Drier5, Laurent Magy7, Eleonore Bayen8, Khe Hoang-Xuan1,9,10,11, Ahmed Idbaih1,9,10,11 1

AP-HP, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Service de neurologie 2-Mazarin, Paris, France; 2AP-HP, Hoˆpital Saint-Antoine, Service de neurologie, Paris, France; 3AP-HP, Hoˆpital Trousseau, Service d’oncologie he´matologie pe´diatrique, Paris, France; 4CHU Tours, Service de me´decine nucle´aire, Tours, France; 5AP-HP, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Service de neuroradiologie, Paris, France; 6AP-HP, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Service de me´decine nucle´aire, Paris, France; 7CHU Limoges, Hoˆpital Dupuytren, Service de neurologie, Limoges, France; 8AP-HP, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Service de me´decine physique et de re´adaptation, Paris, France; 9Universite´ Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de la Moelle Epinie`re (CRICM), UMRS 975, Paris, France; 10Inserm U 975, Paris, France; 11CNRS, UMR 7225, Paris, France; 12 EA4340 Biomarqueurs en Cance´rologie et Onco-He´matologie, Universite´ de VersaillesSaint-Quentin-en-Yvelines, Versailles, France Purpose: Clinical spectrum of cognitive troubles complicating neurodegenerative Langerhans cell histiocytosis (ND-LCH) is poorly known. The aim of this study is to evaluate cognitive functions in ND-LCH. Methods: The cognitive functions of a series of eight adult patients (seven males and one female; mean age: 26 years IQ: 25–75; range: 20–33) suffering from clinical and/or radiological ND-LCH were evaluated using the following tests: (i) forward/backward digit and spatial span tasks of the WAIS-R scale and the Corsi block task, (ii) the French version of the free and cued selective reminding test, (iii) verbal fluency tests, (iv) the Frontal Assessment Battery -FAB-, (v) backward measurement of the verbal and visuospatial memories of the WAIS-R scale, (vi) the Rey complex figure test, (vii) the Trail Making Tests A and B, (viii) digit symbol and symbol search of the WAIS-IV scale and (viii) the Stroop test. Results: Episodic (i.e. autobiographical or personal) memory free recall, categorical verbal fluency, phonological verbal fluency, visuospatial processing skills, attention, speed of processing and sensitivity to interference were impaired in ND-LCH patients. In contrast, verbal and visuospatial short-term memories (i.e. immediate memories or forward span tasks) were preserved in all patients. Conclusion: Adult ND-LCH patients suffer from a severe but dissociated dysexecutive syndrome, mostly affecting executive strategies and relatively sparing short-term memory. Our study supports the need of assessing executive functions using comprehensive cognitive evaluation in ND-LCH patients for early diagnosis.

Claudio Giraudo1, Waldo A. Spessott1, Maria L. Sanmillan1, Margaret E. McCormick1, Nishant Patel2, Joyce Villanueva3, Kejian Zhang4, Kim E. Nichols2 1 Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia & University of Pennsylvania, Philadelphia, PA USA; 2Department of Pediatrics, Division of Oncology, The Children’s Hospital of Philadelphia & University of Pennsylvania, Philadelphia, PA USA; 3Division of Bone Marrow Transplant and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics University of Cincinnati College of Medicine, Cincinnati, OH USA;4Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics University of Cincinnati College of Medicine, Cincinnati, OH USA

Purpose: To characterize the cellular and molecular defects leading to HLH in patients bearing a monoallelic STXBP2 mutation (194 G > A; R65Q). Methods: CD8þ T and NK cells were isolated by negative selection from PBMCs from normal healthy controls or from the patient, who exhibited classical features of HLH. STXBB2 protein levels were analyzed by Western blotting. The physical interaction between STXBP2 and STX11 was assessed by immunoprecipitation (IP). The cytolytic activity of CTLs and NK cells was evaluated using a non-radioactive target cell-killing assay. The liposome-liposome in-vitro fusion assay was used to assess the function of STXBP2. Results: STXBP2R65Q retained its ability to interact with and stabilize Syntaxin-11 expression. However, endogenous expression of STXBP2R65Q in FHL cells and transfection in normal control CTL and NK cells significantly diminishes CTL and NK cell cytotoxicity. This effect could not be restored by the addition IL-2. Microscopy studies in patient cells showed that lytic granule formation and polarization was not affected. Mechanistic studies revealed that STXBP2R65Q competes with the wild type form and hinders membrane fusion by arresting late steps of SNARE complex formation. Conclusion: This study demonstrates for the first time a dominant negative mode of action of an HLH-associated genetic mutation. R65Q mutation does not influence the levels of STXBP2 protein, nor does it impair its ability to bind to and stabilize STX11. In contrast, the R65Q mutation inhibited the ability of WT STXBP2 to bind to SNARE complexes and promote membrane fusion. These studies highlight the clinical and biological importance of monoallelic FHL gene mutations and emphasize the need for in vitro biochemical, microscopic and functional assays to investigate the clinical relevance of other known and/or novel heterozygous HLH-associated.

RECURRENT RAS AND PIK3CA MUTATIONS IN ERDHEIM-CHESTER DISEASE AND HISTIOCYTIC SARCOMA DOES GENETIC SUSCEPTIBILITY CONTRIBUTE TO LATE-ONSET PRESENTATION OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS? Juana Gil Herrera1, Marı´a Alonso-Martı´nez1, Itziar Astigarraga2, ´ lvarez-Riego1, Lydia Martin-Martı´n1, Irene Martı´nez-Rı´o1, Ramon Urrea1, Olga A Paloma Francos1, Carmen Rodrı´guez-Sainz1, Susana Garcı´a-Obregon2, Eduardo Ferna´ndez-Cruz1 Hospital General Universitario “Gregorio Mara~non”, Madrid, Spain; 2Hospital Universitario de Cruces, Bilbao, Spain


Purpose: Late presentations of hemophagocytic lymphohistiocytosis (HLH) associated to hypomorphic missense and splice site mutations in PRF1, UNC13D, STXBP2, SH2D1A and BIRC4 genes are being increasingly reported. We aimed to study the genetic background in a series of late-onset patients referred to our hospital for HLH diagnosis. Methods: 20 HLH patients (12 male, 8 female) older than 12 years of age were tested because of suspected genetic disease from 2006 to 2014. Perforin and granzyme B expression, quantitation of CD107a on NK-cell, and CD45 isoforms were measured by flow cytometry (BD Biosciences). NK-cell cytotoxicity was analyzed using Cr51 labelled-K562 cells. DNA was obtained from peripheral blood samples; PRF1, UNC13D, STX11, STXBP2, SH2D1A, BIRC4, RAB27A and CD45 genes were amplified and direct, bi-directional sequencing was performed (3130  1 Genetic Analyzer, Applied Biosystems). Results: HLH was associated to B-cell lymphoma (n ¼ 2), T-cell lymphoma (n ¼ 1), leukemia (n ¼ 1), HIV (n ¼ 3), EBV (n ¼ 2), HSV (n ¼ 1), CMV (n ¼ 1), tuberculosis infection (n ¼ 1) and inflammatory bowel disease (n ¼ 1); no trigger was found in 7 cases. 3/20 patients underwent allogenic SCT transplantation. 6 (30%) patients died, 13 remain alive and 1 has

Pediatr Blood Cancer DOI 10.1002/pbc

Julien Haroche1, Eli L. Diamond2, Jean-Franc¸ois Emile3, Zofia He´lias-Rodzewicz3, Fleur Cohen-Aubart1, Fre´de´ric Charlotte4, David M. Hyman5, Raajit Rampal6, Minal Patel6, Chezi Ganzel7, Shlomzion Aumann8, Gladwys Faucher3, Catherine Le Gall3, Karen Leroy9, Magali Colombat10, Jean-Emmanuel Kahn11, Salim Trad12, Phlippe Nizard13, Jean Donadieu14, Vale´rie Taly13, Zahir Amoura1, Omar Abdel-Wahab8 1 Department of Internal Medicine & French Reference Center for Rare Autoimmune & Systemic Diseases, Pitie´-Salpeˆtrie`re Hospital, APHP, Paris, France; 2Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY USA; 3Pathology Department, Ambroise Pare´ Hospital, APHP, Boulogne, France; 4Department of Pathology, Hospital Pitie´-Salpeˆtrie`re, APHP, Paris, France; 5Experimental Therapeutics Unit, Memorial Sloan Kettering Cancer Center, New York, NY USA; 6Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY USA 7Department of Hematology, Shaare Zedek Medical Center, Jerusalem, Israel; 8Human Oncology and Pathogenesis Program and Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY USA; 9 Department of Pathology, Hospital Henri Mondor, APHP, Cre´teil, France; 10Department of Pathology, Foch Hospital, Suresne, France; 11Department of Internal Medicine, Foch Hospital, Suresne, France; 12Department of Internal Medicine, Ambroise Pare´ Hospital, APHP, Boulogne, France; 13Universite´ Paris Sorbonne Cite´, INSERM UMR-S1147, Paris, France; 14Department of Pediatrics & French Reference Center for Langerhans Cell Histiocytosis, Trousseau Hospital, APHP, Paris, France

Purpose: Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH) frequently harbor BRAFV600E mutations and have been found to respond to BRAF inhibition in pilot



studies. An oncogenic mutation in NRAS(Q61R) has been identified in a single BRAFwildtype ECD patient, but the true incidence of BRAF V600E mutations in ECD is uncertain as is the recurrence of other mutations affecting MAP kinase and PI3K/AKT signaling pathways remains unknown. Methods: From a cohort of 80 ECD patients, 25 ECD and 4 histiocytic sarcoma (HS) patients whose tissue was found to be BRAF p.V600 wildtype were analyzed for mutations in BRAF, N/K/HRAS, AKT1 and PIK3CA using allele-specific real time PCR, next-generation sequencing of lesional DNA, Sequenom mass spectrometric genotyping and multiplex picodroplet digital PCR. Results: An additional 8 ECD patients were identified as being BRAFV600E mutant. Of the BRAF p.V600 wildtype patients, 3 were found to have NRAS mutations (NRASG12D, NRASQ61K, NRASQ61R) and an additional 3 were found to have PIK3CA mutations (PIK3CAE542K, PIK3CAE545K, PIK3CAA1046T). A fourth PIK3CA mutation was also detected in 1/9 BRAF mutated case (PIK3CAH1047R). Furthermore, genetic analysis of CD14þ cells purified by double-FACS sorting of peripheral blood mononuclear cells from an NRASQ61R mutant ECD patient clearly revealed the presence of the NRAS mutation in CD14þ cells but not in CD3þ cells. Conclusion: Overall, using multiple methodologies to detect mutant BRAF, we identified a BRAFV600E mutational frequency of 57.5% (46/80) in ECD. Identification of recurrent NRAS mutations in BRAF wildtype ECD patients affirms the central role of RAS/RAF/MEK/ ERK as a major common pathway in these histiocytoses. Furthermore, the finding of recurrent NRAS and PIK3CA mutations also newly implicates the importance of PI3K/AKT/mTOR signaling in ECD. Detection of an NRAS mutation in peripheral blood mononuclear cells further suggests that the histiocytic proliferations in ECD are derived from genetically aberrant circulating myeloid hematopoietic cells.

THE NOTCH SIGNALING PATHWAY IN LCH Caroline Hutter1,2, Gunhild Jug2, Ingrid Simonitsch-Klupp3, Heinrich Kovar2, Milen Minkov1,4, Wolfgang Holter1,2 1 St. Anna Children’s Hospital, Department of Pediatrics, Medical University of Vienna, Austria; 2Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria; 3Clinical Institute of Pathology, Medical University of Vienna, Austria; 4 Krankenanstalt Rudolfstiftung, Department of Pediatrics, Vienna, Austria

Purpose: The Notch pathway is a highly conserved signaling pathway and aberrantly activated Notch can act as an oncogene in hematologic malignancies. We have previously shown that the Jagged-Notch pathway is active in Langerhans cell histiocytosis (LCH) lesions and that in vitro the Notch ligand Jagged2 can induce the differentiation of monocytes into CD1aþ CD207þ cells reminiscent of LCH cells. The goal of this project was to investigate the impact of Notch signaling on cellular pathways in LCH. Methods: Monocytes were isolated from buffy coats of healthy donors and plated onto dishes coated with recombinant human Jagged2. After 5–7 days cells were harvested and analyzed for CD1a and CD207 expression. The induction of LCH-relevant genes by Jagged2 was determined using Taqman analysis. IF and IHC of LCH biopsies were used for validation. Results: In addition to the induction of CD1a and CD207, we observe the expression of LCHcharacteristic genes upon differentiation of monocytes in the presence of recombinant Jagged2, corroborating earlier results obtained using Jagged2 expressing feeder cells. Among these genes we identified IL7RA, an IL7 receptor subunit that is implicated in lymphoid leukemia as well as myeloproliferative disease and highly expressed in LCH. We also provide evidence that Jagged2 induces the TNF receptor family member RANK, which is expressed in LCH and could be involved inthe systemic increase of T-regs. Conclusion: Our findings corroborate the view that Jagged2 signaling can induce features of LCH on monocytes and identified new promising target genes of the Notch pathway. From a clinician’s perspective these results are interesting and warrant further investigation because targeting the Notch pathway might be a feasible treatment approach: Notch signaling is active in the majority of LCH patients and since Notch inhibitors are currently evaluated in clinical trials, they would also be available for the treatment of LCH.

MANIPULATING THE DNA DAMAGE RESPONSE FOR TARGETING PATHOLOGIC T CELLS IN HLH Michael Jordan, Scott Millen, Catherine Terrell Cincinnati Children’s Hospital Medical Center, Cincinnati, OH USA Purpose: Better ways of halting pathologic immune activation in patients with HLH are needed. Current approaches (HLH94/2004, ATG-based) may be associated with dosedependent marrow suppression, prolonged immune suppression and/or inadequate efficacy for many patients. Clinical and preclinical studies have illustrated how T cells are at the heart of HLH pathogenesis and recent work from our group has demonstrated that etoposide alleviates HLH by selectively killing activated T cells. Biochemically, etoposide acts by damaging DNA and activating the cellular DNA damage response (DDR). A variety of new drugs are under development for oncologic indications that allow one to directly manipulate the DDR without primarily damaging DNA. However, their immune-modulating qualities remain largely unexplored. Methods: We examined non-genotoxic activators of p53, inhibitors of endogenous DNA repair mechanisms and other agents effecting the DDR for their ability to selectively kill

Pediatr Blood Cancer DOI 10.1002/pbc

activated T cells in vitro and treat HLH in vivo using the common model of HLH- LCMVinfected perforin-deficient mice. Results: We have found that several DDR-based strategies may either potentiate etoposide (allowing substantial dose decreases) or may substitute entirely for etoposide (and other DNA damaging drugs) for the selective targeting of activated T cells and treatment of experimental HLH. The therapeutic qualities and relative toxicity advantages of these strategies will be presented in detail. Conclusion: New pharmacologic methods for manipulating the DDR hold promise for improving treatment for patients with HLH and other immunopathologic disorders. Supported by a grant from the Histiocytosis Association.

EXPLORATION OF THE IL-23/IL-17A AXIS IN LANGERHANS CELL HISTIOCYTOSIS Egle Kvedaraite1,2, Magda Lourda1,2, Puran Chen2, De´sire´e Gavhed1, Mattias Svensson2, Jan-Inge Henter1 1 Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden; 2Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

Purpose: Langerhans cell histiocytosis (LCH) is a disease of unknown etiology. It manifests as a single or multisystem organ disease that may be fatal, sometimes involving lifethreatening pulmonary involvement. In LCH, interleukin (IL)-17A has been suggested to mediate tissue pathology and we recently demonstrated the presence of IL-17A producing monocytes in peripheral blood of LCH patients. Importantly, the IL-17A expression levels in peripheral blood monocytes correlated with disease activity. The underlying cause contributing to IL-17A production in LCH remains to be identified. However, IL-23 is a proinflammatory cytokine that promotes the development and maintenance of IL-17mediated inflammation. Therefore, our goal is to further explore the role of IL-23 and IL-17A in LCH associated tissue pathology using innovative organotypic models of human skin and lung. Methods: By analyzing patients’ tissue biopsies in combination with implanting blood monocytes from LCH patients and controls into human organotypic models of skin and lung, we will explore the crosstalk between monocyte-derived phagocyte cells (CD68 positive) and tissue specific fibroblasts (CD90 positive) and epithelial cells (EpCam positive) with respect to IL-23/IL-17A production and tissue pathology. Results: Preliminary investigations of LCH skin tissue biopsies revealed extensive infiltrates of HLA-DRþ and CD68þ cells in LCH skin lesions. Moreover, comparative examination of LCH and control skin biopsies using immunofluorescence confocal microscopy revealed increased epithelial production of IL-23 in LCH skin lesions compared to control skin. Conclusion: Our results suggested that epithelial cells may play an important role in IL-23/IL17A mediated pathology associated with LCH. Furthermore, by using our organotypic models of human skin and lung tissues we will have a unique opportunity to dissect cellular and molecular mechanisms of IL-23/IL17A-mediated tissue pathology in LCH.


Center of Chronic Immunodeficiency, University Medical Centre Freiburg, Germany; Center for Pediatrics and Adolescent Medicine, University Medical Centre Freiburg, Germany; 3Pediatric Hematology and Oncology, University Medical Center, Hamburg, Germany; 4Center for Diagnostic, University Medical Center, Hamburg-Eppendorf, D-20251 Hamburg, Germany


Background: Leishmania is the most common protozoal trigger of hemophagocytic lymphohistiocytosis (HLH). Even though infrequent innon-endemic areas, visceral leishmaniasis (VL)-induced HLH is an important differential diagnosis. Liposomal amphtotericin B (L-Amb) is the treatment of choice to treat VL. Methods: We retrospectively analyzed clinical and laboratory features, diagnostic procedures and outcome of treatment of 13 HLH patients with imported VL, reported to the German HLH reference center (1999–2012). Special emphasis was put on diagnostic clues and pitfalls as well as neurological symptoms. Results: The spectrum of presentations was indistinguishable from patients with primary HLH or with secondary HLH triggered by other infectious pathogens. Eight patients were 4 years from end of treatment. Conclusion: Pulmonary LCH is very uncommon in children but needs to be considered in all with skin and bone involvement. Lung disease does not usually cause mortality or long term morbidity in young children possibly due to ability of alveoli to repair.

UNC13D MUTATIONS ARE THE MOST COMMON CAUSE OF FHLH IN POLAND Iwona Malinowska1, Aleksandra Jasinska2, Bianka Tesi3, Yenan Bryceson3, Katarzyna Popko4, Wojciech Młynarski3, Wanda Badowska5, Jacek Wachowiak6, Walentyna Balwierz7, Jerzy Kowalczyk8, Mariusz Wysocki9, Lucyna Kapuscinska-Maciejka10, Tomasz Urasinski11, Maria Wieczorek12, Graz˙yna Sobol-Milejska13, Krzysztof Kalwak14 1 Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw, Warsaw, Poland; 2Department of Pediatrics, Hematology, Oncology and Diabetology, Medical University of Lodz, Lodz, Poland; 3Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden; 4Department of Laboratory Medicine and Pediatric Immunology, Medical University of Warsaw, Warsaw, Poland; 5Department of Pediatrics, Hematology and Oncology, Olsztyn, Poland; 6Department of Pediatric Oncology,



Hematology and Transplantology, Poznan University of Medical Sciences, Poznan, Poland; 7 Department of Pediatric Oncology, Hematology Jagiellonian University Medical College, Krakow, Poland; 8Department of Pediatric Hematology, Oncology and Transplantology, Lublin, Poland; 9Department of Pediatrics, Hematology and Oncology, Bydgosz, Poland; 10 Department of Pediatrics, Hematology and Oncology, Gdansk, Poland; 11Department of Pediatrics, Hematology and Oncology, Szczecin, Poland; 12Department of Pediatrics, Hematology and Oncology, Chorzow, Poland; 13Department of Pediatrics, Hematology and Oncology, Katowice, Poland; 14Department of Pediatric Transplantology, Hematology, Oncology, Wroclaw, Poland Purpose: Data obtained from different populations have shown ethnic differences in the mutation type and frequency of FHLH (Familial Hemophagocytic Lymphohistiocytosis). The incidence of FHLH in Swedish children is 1.2 cases per million per year, but it is believed that these figures are strongly underestimated. An increasing number of FHLH cases is recognized in Poland. Nevertheless, FHLH is still diagnosed in Polish pediatric population less frequently (0.82/million/year) than in Western Europe, with a reasonable number of patients thought to be undiagnosed. There has been no published data on the genetic background of FHLH in Poland. We investigated the molecular genetics of FHLH among Polish pediatric population. Methods: Thirty patients (10 girls and 20 boys) fulfilling the HLH-2004 criteria, treated at 12 Polish pediatric hematology/oncology centers, were included in the study. According to clinical indications, molecular analysis of PRF1, UNC13D, STXBP2, SH2D1A and BIRC4 was performed with the use of direct DNA sequencing. Results: Median age at diagnosis was 3 2/12 years (range from 1/12 to 17 years). Genetic background of HLH was identified in 7/30 patients. One patient had SH2D1A nonsense mutation (Arg55X), consistent with XLP1 diagnosis, and one BIRC4 missense mutation (Glu219Lys) - XLP2 diagnosis. Five out of seven patients from 6 families had UNC13D mutations: 3 patients from 2 families were homozygous for a splice site mutation (c.753 þ 1G > T), 1 patient was homozygous for a deletion (R782delGGAG), and 1 patient had compound heterozygous nonsense and deletion mutation (R256X þ R782delGGAG). No mutations in PRF1, STXBP2 or STX11 mutation were found. Conclusions: UNC13D mutations are the most common cause of FHLH in Poland.

MAPPING THE BRAFV600E HEMATOPOIETIC CLONE OF ADULT MULTISYSTEM LANGERHANS CELL HISTIOCYTOSIS Paul Milne1, Matthew Collin1, Venetia Bigley1, Chris M. Bacon2, Naomi McGovern1, Kevin Windebank2, Carl Allen3, Kenneth McClain3, Muzlifah Haniffa1 1 Human Dendritic Cell Lab, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom; 2Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom; 3Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX USA

BRAFV600E mutation is associated with Langerhans cell histiocytosis (LCH) and Erdheim Chester disease (ECD). Detection of BRAFV600E in blood or bone marrow may provide new insights into mechanisms of pathogenesis. Among 14 adult patients including 6 with BRAFV600Eþ LCH, BRAFV600E was detected in the blood and bone marrow of 2 patients with active multi-system LCH but not in patients with single system LCH or unmutated BRAF. 95% of BRAFV600E alleles were mapped to HLA-DRþ lineage- mononuclear cells and 95–98% of these occurred in monocytes, including CD14 þ CD16þ intermediate and CD16þ non-classical monocytes. Myeloid dendritic cells, contributed A) and splice site mutation (c.227– 1G > A) in MVK coding MK. Conclusion: The phenotypic features of severe MK deficiency can include antenatal hydrops, hepato-splenomegaly, jaundice, fever, rash, lymphadeno-pathy and anemia, which are coincident with symptoms of HLH. MK deficiency may lurk in fetal onset familial HLH.

CLINICAL PRESENTATION AND OUTCOME IN CHILDREN WITH LCH UNDER THE AGE OF 24 MONTHS Maria Moschovi1, Archontis Zampogiannis1, Maria Adamaki1, Natalia Tourkantoni1, John Nikas2 1 Hematology/Oncology Unit, 1st Department of Pediatrics, University of Athens, Pediatric Oncology Unit “MARIANNAV VARDINOYANNIS-ELPIDA;” 2Radiology Department, “Agia Sophia” Children’s Hospital, Athens, Greece

Purpose: Patients under the age of 24 months usually present with multisystem LCH. The outcome is poor in this type of LCH. The spectrum of clinical presentations and outcome of LCH in children younger than 24 months of age was the aim of our study. Methods: Eleven children, who were diagnosed with LCH in the first two years of age and treated in our Unit, were enrolled in this study. The age ranged from 26 days to 24 months (mean: 10 months). All patients were treated according to the LCH protocol and completed the chemotherapy regiment at least 2–7 years ago. All patients remain in follow-up. Results: Five cases presented with multisystem disease in the first 6 months of life, including intestine involvement, 3 cases had multiple bone lytic lesions in rare sites, one case had periorbital mass and 2 cases and juvenile xanthogranuloma in the first month of life. All cases received chemotherapy according to the LCH protocol. Four cases (36%) with multisystem disease presented reactivation within 8–12 months from the end of the chemotherapy. They achieved second remission. All the cases still remain in remission. Conclusion: Although children with LCH under the age of 24 months have a poor outcome, the new protocols of LCH achieve improvement of the survival for this group. Reactivation of the disease is still high but reactivated cases had a second chance for stable remission.

HAEMATOPOETIC STEM CELL TRANSPLANTATION FOR LANGERHANS CELL HISTIOCYTOSIS (LCH): OUTCOME BY INTENSITY OF CONDITIONING Vasanta Nanduri1, K. Scott Baker2, Andrea Biondi3, Jeffery Davis4, Gretchen Eames5, Maarten Egeler6, Robert Krance7, Edoardo Lanino8, Wing Leung9, Susanne Matthes10, Christina Peters10, Ollie Ringden11, Angie Smith12, Paul Veys1 1 Great Ormond Street Hospital, London, United Kingdom; 2Fred Hutchinson Cancer Research Center, Seattle, WA USA; 3Ospedale San Gerardo, Monza, Italy; 4British Columbia Children’s Hospital, Vancouver, BC Canada; 5Cook Children’s Medical Center, Fort Worth, TX USA; 6Hospital for Sick Children, Toronto, ON Canada; 7Baylor College of Medicine, Houston, TX USA; 8Institute G. Gaslini, Genova, Italy; 9St Jude Children’s Hospital, Memphis, TN USA; 10St Anna Children’s Hospital, Vienna, Austria; 11Karolinska Institute, Stockholm, Sweden; 12University of Minnesota, Minneapolis, MN USA

Purpose: Treatment strategies for refractory LCH with “risk organ” involvement include the combination of 2-chlorodeoxyadenosine and cyatarbine and/or allogeneic haematopoietic stem cell transplantation (HSCT) with myeloablative (MAC) or reduced intensity conditioning (RIC) regimens. The current analyses describe outcomes after HSCT. Methods: Data were obtained from CIBMTR and EBMT registries for transplants between 1993 and 2011. Eighty-seven patients received HSCT (MAC, N ¼ 59; RIC, N ¼ 28). Results: The median age at HSCT was 2 years and median time to HSCT, 10 months for MAC and 14 months for RIC. In the MAC group, busulfan with cyclophosphamide was the predominant regimen and in RIC group, melphalan with fludarabine and Alemtuzumab or ATG. A third of patients in both groups received grafts from HLA-matched siblings, 50% from unrelated donors and the remainder, from mismatched family donors. The median follow-up of MAC and RIC recipients was 7 and 5 years, respectively. The day-100 incidence of grade 2– 4 acute GVHD was higher after MAC compared to RIC (35% vs. 12%, p ¼ 0.01). The 5-year probability of overall survival was 57% and 70% after MAC and RIC HSCT (p ¼ 0.25). Disease progression/relapse occurred in 11/59 and 4/28 patients after MAC and RIC HSCT. Six of 27 evaluable patients with full donor chimerism and 2/10 patients with mixed chimerism exhibited recurrent disease. Conversely, 3 of 5 patients with autologous reconstitution remain disease-free. Conclusion: The data suggest MAC or RIC HSCT salvage over half of refractory LCH patients. With this sample size we were unable to detect a statistically significant survival advantage with RIC; however, with equivalent disease control, RIC may offer the advantage of

HISTIOCYTE SOCIETY 30th ANNUAL MEETING ABSTRACTS reducing the risk of late effects. Recurrent disease despite achievement of donor chimerism is reminiscent of neoplastic rather than non-malignant disease, a finding supported by the finding of BRAF mutations in LCH samples.


A REGIONAL STUDY OF ADULTS WITH HISTIOCYTIC DISORDERS IN NORTHEAST ENGLAND Jane Salotti1, Venetia Bigley2, Katrina Wood3, Matthew Collin2, Kevin Windebank4


Department of Pediatric Hematology-Oncology, Agia Sofia Children’s Hospital, Athens, Greece; 2Department of Endocrinology, P&A Kyriakou Children’s Hospital, Athens, Greece; 3 Neurosurgery Clinic, HYGEIA Hospital, Athens, Greece Purpose: For isolated suprasellar lesions, secure diagnosis to guide treatment is difficult without detectable peripheral lesions to be biopsied, like bone lesions in Langerhans cell histocytosis (LCH) or measurable alpha-fetoprotein (a-FP)/ beta-human chorionic gonadotropin (b-HCG) in germ cell tumors (GCT). Slow growing lesions explain the reluctance of families/physicians to undertake biopsy. We report two cases where prompt biopsy led to definitive diagnosis and appropriate treatment. Methods: Case 1: A prepubertal 8 y/o girl was evaluated for short stature (height percentile drop from 15 to 5% over 3 years). Detailed history revealed long-standing diabetes insipitus (DI), not appreciated previously. Suprasellar mass clinical diagnosis lead to MRI imaging that revealed a centrally located mass in the sella turcica and suprasellar region, with low signalintensity on T2-weigthed images, intermediate signal-intensity on T1 and heterogeneous, intense contrast enhancement. The pituitary stalk and gland could not be delineated from the tumor. The optic chiasm appeared deformed and dislocated. Case 2: An adolescent 15.5 y/o girl was followed for 2.5 years for DI and menses irregularities with a known slowgrowing pituitary stalk thickening (initially 3.8 mm, pre-surgery 5.5 mm). Seven months later, central hypopituitarism was added. MRI scan indicated an endo- and suprasellar enhancing lesion. Results: Both patients in excellent clinical condition had no other lesions upon total body imaging. Repeated serum and cerebrospinal fluid (CSF) measurements for a-FP and b-HCG were within normal limits. Their families were under the impression that they had LCH, a “benign condition”. Following repeated discussions, they were persuaded to proceed to transsphenoidal partial excision of the masses. Pathology proved that they suffered from malignant GCT and they were treated accordingly, both being in remission. Conclusion: Suprasellar lesions, without positive serum/CSF markers and accompanying imaging findings should be biopsied promptly. Biopsy proven diagnosis benefits patients’ long-term health by establishing diagnosis and guiding appropriate treatment.


Institute of Health & Society, Newcastle University, Newcastle upon Tyne, United Kingdom; Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom; Pathology Department, Newcastle Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom; 4Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom 2 3

Purpose: There is currently no UK registry of patients with histiocytic disorders and the scale of the problem is unknown. Adults in Northeast England (adult population 2.1 million) may attend a dedicated clinic in Newcastle with haematological, dermatological and paediatric histiocytosis expertise. However, it is not known which other specialists are treating patients. With a view to establishing a national register, this study will assess: the frequency of biopsyproven histiocytic diseases by age/sex; presentation, treatment and outcome; services received for disease or sequelae. Methods: Cases diagnosed from 2000–2012 aged 16 years have been identified by searching 8 hospital pathology department databases covering Northeast England for appropriate Snomed codes: Langerhans cell histiocytosis (LCH), haemophagocytic lymphohistiocytosis (HLH), Rosai-Dorfman (R-D), Erdheim Chester (ECD), juvenile xanthogranuloma (JXG). Questionnaires are being mailed to referring clinicians for further information. Results: Pathology data were received from all 8 hospitals. Snomed codes included reactive histiocytoses as well as primary histiocytic disorders and subsequently, pathology reports were screened for eligibility. 169 eligible patients were identified aged 16–81 years, M:F ratio 1.25:1. The most common disorders were JXG (42%) and LCH (29%). One case had ECD (bone marrow/spleen/liver involvement) and one case of HLH also had Hodgkin’s Disease. The most frequent LCH sites were lung, bone and lymph. Additionally, 40 cases had fibrous histiocytoma and 6 had reticulohistiocytosis. In the majority of JXG cases, local excision was complete and these cases are not being followed up. Although some cases may be diagnosed radiologically or remain undiagnosed, the minimum IR for these disorders is approximately 6.2 per million per year and 1.7 for LCH cases. Conclusions: The study has highlighted a higher incidence of histiocytic disorders than expected from numbers of attendees at specialist clinics. A UK registry would increase the number of reported cases and raise awareness of these disorders.

CENTRAL NERVOUS SYSTEM IMAGING IN CHILDHOOD LCH Stefan Scho¨ning1, Elke Hattingen2, Jan So¨rensen1, Luciana Porto2, Thomas Lehrnbecher1 Hospital for Children and Adolescents: 2Neuroradiology Department of the Johann Wolfgang Goethe University, Frankfurt/Main, Germany





Jennifer Picarsic , R. Maarten Egeler , Kudakwashe Chikwava , Kathleen Patterson4, Ronald Jaffe1 1 Department of Pathology, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA USA; 2Department of Hematology/Oncology, The Hospital for Sick Children, University of Toronto, Toronto, ON Canada; 3Department of Pathology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA USA; 4Department of Pathology, Seattle Children’s Hospital, Seattle, WA USA

Purpose: Thymic involvement by Langerhans cell histiocytosis (LCH) has been described mainly in isolated case reports. The recent literature suggests thymic involvement with systemic LCH follows an aggressive clinical course. However, a detailed description of the histopathologic patterns of LCH in the thymus together with their therapeutic implications has not been previously addressed. Methods: The pathology consultation files at Children’s Hospital of Pittsburgh of UPMC were reviewed for cases of thymic involvement by LCH together with a review of the literature. The histopathology and clinical course of these thirty patients were collected. Results: Seven consultation cases of thymic involvement were reviewed together with 23 cases in the literature that provided histopathologic description (n ¼ 30), revealing three distinct clinico-pathologic conditions that also appear to have unique therapeutic implications. Seven cases showed microscopic collection of LCH-like cells (so called LCHlike “hyperplasias”) in incidental thymectomies of patients without LCH disease. This pattern appears reactive in nature and requires no further treatment. Ten cases of solitary and/or cystic LCH presented as a unifocal thymic mass lesion, with at least three cases resolving without systemic therapy. Thirteen cases showed thymic involvement as part of multisystem LCH disease and required adjuvant therapy with a higher rate of death in this group, including a congenital LCH case dying of disease. Conclusions: Three histopathologic patterns of thymic LCH-like/LCH involvement associated with divergent clinical presentations are presented. Incidental hyperplasia is not associated with LCH disease. The solitary (single organ, single site) type may also benefit from observation alone, after exclusion of systemic LCH. The multisystem type may be associated with younger age and more aggressive clinical course with future studies addressing the benefit of mediastinal ultrasound imaging as part of routine LCH staging.

Pediatr Blood Cancer DOI 10.1002/pbc

Purpose: Langerhans cell histiocytosis (LCH) is a rare disease, and the clinical presentation ranges from a single bone lesion to widespread multiorgan involvement. The incidence of manifestations of LCH on the central nervous system (CNS) is often underestimated, and the diversity of CNS lesions leads to difficulties in comparing treatment and outcomes. The aim of this study was to evaluate and to categorize the cerebral MR imaging abnormalities in children with LCH. Methods: As the reference center for German childhood LCH, we retrospectively reviewed cranial magnetic resonance imaging (MRI) examinations in children and adolescents with LCH. Only patients in whom 1) sagittal T1-weighted (w) images after contrast, permitting evaluation of the infundibulum, 2) T2-w images, fluid attenuated inversion recovery (FLAIR) and 3) T1w images before and after contrast in at least two different section planes were available were included in the analysis. The incidence of typical cerebral pathologies of LCH was rated regarding their signal intensity on T2-w images and on contrast-enhanced T1-w images. Results: Unifocal and multifocal bone disease was present in 9 and 7 children, respectively; localized LCH of the skull was seen in 8, multisystem LCH in 7 children. Ten children suffered from diabetes insipidus. The time from diagnosis to cranial imaging ranged from 0 days to 12.9 years. The most common MR changes were osseous (55%), followed by pineal enlargement (45%), pituitary stalk mass (32%) and signal changes of dentate nucleus (32%), respectively. Occasionally, hyperintensity in hippocampus, parenchymal and meningeal enhancement as well as white matter hyperintensity were observed. Conclusion: Excluding bone lesions, the most common sites of CNS manifestations of LCH were pineal gland and the hypothalamic-pituitary system. Signs of neurodegeneration may begin at early stage of disease. Standardized imaging may allow better comparison of treatment regimens and outcome.

CLINICAL PRESENTATION AND OUTCOME OF SECONDARY HAEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS (HLH) Santanu Sen1, Vasanta Nanduri2 1 Kokilaben Dhirubhai Ambani Hospital, Mumbai, India; 2Watford General Hospital, Watford, United Kingdom

Purpose: Secondary HLH can be difficult to recognize and diagnosis is often delayed. We describe our experience of diagnosing and treating secondary HLH in a tertiary hospital in Mumbai, India.



Methods: All cases diagnosed with secondary HLH between 2010 and 2014 were studied. Clinical and laboratory parameters and outcome were retrospectively reviewed. Genetic testing ruled out primary HLH in all cases. Results: Thirteen patients were diagnosed in this period, age range 2 to 65 years. Results at presentation showed a mean value (range) of: haemoglobin 7.5gm/dl (6.2–9.6); white cell count 2.06  109/L (0.8–7.3); platelets 29.3  109/L (10–46), ferritin 43738 (3000 to 122000), triglyceride 350 (252–496). Haemophagocytosis was found in the marrow in 5 of 13 cases. Investigations for the underlying cause revealed 2 patients each with Epstein Barr Virus (EBV) infection, malaria, dengue fever and SLE and 1 each of typhoid, tuberculosis and staphylococcal sepsis. Two patients were diagnosed with secondary HLH following chemotherapy for leukemia and lymphoma. Eight of the 13 patients survived. Three (EBV, malaria and typhoid) had spontaneous recovery with treatment for the underlying infection. The 2 patients who had HLH following chemotherapy recovered from their acute HLH with supportive care, but unfortunately died later due to complications of the malignancy. Eight patients were treated according to HLH2004 protocol. Therapy could be stopped after 8 weeks of initial therapy for 3 patients, but was continued until week 40 in the rest due to persistent active disease. Three of the 8 patients on treatment died: 1 HLH reactivation and 2 gramnegative sepsis. Conclusion: HLH can go unrecognized in acute infectious diseases seen in tropical countries and may contribute to increased mortality. Our small series shows that awareness and thorough investigation for infectious diseases can lead to early diagnosis and treatment with improved outcome.

SPINAL LANGERHANS CELL HISTIOCYTOSIS (LCH) IN SINGLE INSTITUTION Jong Jin Seo, Seong Wook Lee, Jin Kyung Suh, Kyung-Nam Koh, Ho Joon Im Division of Pediatric Hematology/Oncology, Department of Pediatrics, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea Purpose: To delineate the clinical characteristics of spinal involvement of LCH in children focusing on the reactivation, spinal reconstitution and neurologic and orthopedic sequelae. Methods: We retrospectively analyzed the data of 20 patients with spinal LCH who were diagnosed at Asan Medical Center from 1990 to 2010. Total 27 vertebrae were involved including 24 vertebral bodies, 2 spinal processes and 1 pedicle portion. Results: The median age at diagnosis of spinal LCH was 4.2 years (range, 0.6–12.3). Fourteen were male and 6 female. The distribution of spinal lesions revealed predominance in the thoracic spine (n ¼ 13), followed by lumbar (n ¼ 9) and cervical (n ¼ 5). Radiologic finding of spinal LCH mostly revealed symmetric vertebral plana (VP) in 19 cases, and paraspinal mass was detected in 8. Among 24 VP lesions, 16 were severe lesions (50%–100% collapse) and 6 mild (0–50%). Seventeen patients were treated with chemotherapy, 2 with local curettage and 1 with radiotherapy. LCH recurrence occurred in 8 patients (4 spine, 2 other bones, 1 scalp, 1 lymph node), and 6 reactivated patients received salvage chemotherapy. Eighteen assessable patients excluding 2 cases of follow-up loss are alive in complete remission of LCH. In followup imaging assessment at a median follow up period of 5.7 years (0.5–12.0), 16 (66.7%) of 24 VP lesions showed improvement in the height of vertebral bodies, especially in mild cases. At a median follow-up period of 9.4 years (3.5–14.4), no patient had significant neurological deficit. Four cases showed orthopedic sequelae including 2 scolioses and 2 kyphoses, but none of them received orthopedic intervention. Conclusion: Higher prevalence of VP lesions in the thoracic spine was noted in children with spinal LCH and these symmetric collapses did not contributed to the development of orthopedic complications requiring surgical intervention. Most patients with VP showed improvement in the height of vertebral bodies without neurologic sequelae, therefore early surgical management of spine LCH requires circumspection.

HEREDITARY SPHEROCYTOSIS AND HEMOPHAGOCYTIC SYNDROME(HPS). CASE REPORT AND LITERATURE REVIEW Bibi Shahin Shamsian1,2, Fatima Malek2, Hossein Esfahani3, Samin Alavi1,2, Nahid Arabi2, Mohammad Taghi Arzanian1,2 1 Pediatric Congenital Hematologic Disorders Research Center, Mofid Children’s Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 2Department of Pediatric Hematology-Oncology, Mofid Children Hospital, Shariati Avenue, Tehran, Iran; 3Department of Pediatric Hematology-Oncology, Besat Hospital, Hamedan University of Medical Science, Hamedan, Iran

Introduction: Hemophagocytic syndrome (HPS) is clinically defined as a combination of fever, liver dysfunction, coagulation abnormalities, pancytopenia, progressive macrophage proliferation throughout the reticuloendothelial system and cytokine overproduction, which may be primary or secondary to other disease including infectious. The association between HPS and infections has been widely documented and both familial or sporadic cases are often precipitated by acute infections. A better understanding of the pathophysiology of HPS may clarify the interactions between the immune system and the variously implicated potential infectious agents. A number of viral infections associated with HPS are including: EBV, CMV, adenovirus, paramyxovirus (leading to measles and mumps), rubella, virus, human parainfluenza viruses, Inf (rare), Hepatitis, Enterovirus infection and parvovirus B19. EpsteinBarr virus (EBV) infection has been prominently associated with HPS, with clonal proliferation and the hyperactivation of EBV-infected T cells. Although associated with

Pediatr Blood Cancer DOI 10.1002/pbc

substantial morbidity and mortality, early recognition and prompt therapy may result in successful treatment of EBV-induced HPS. Also bacterial, fungal and parasitic infections have been reported in association with HPS. Case report: An 8-month-old-girl, 4th child after normal vaginal delivery, term, body weight 3400 gr, from non cansanguinous parents was reffered in our hospital due to persistant fever since 2 months ago, pancytopenia and hempatosplenomegaly. She had history of jaundice, severe hemolytic anemia (Hb; 5Gr/dl), high reticulocyte count; (12%), blood transfusion and 1 time IVIG treatment in age of 40 days of life. Blood group of child was O negative. The specific diagnosis for hemolytic anemia was unegative and other children were healthy. Before starting of fever she had history of 2–3 times of blood transfusion duo to hemolytic anemia. During the follow up for anemia at age of 6 months she progressed to fever, hepatosplenomegaly and pancytopenia. She was referred in our hospital for more evaluation. Laboratory tests revealed: WBC:1300/mm3, RBC:2.300.000/mm3, Hb:7Gr/dl, MCV:84 Fl Platelet:45000/mm3, Reticulocyte: 0.2%. Repetition of reticulocyte count in next laboratory test also showed low reticulocytes (0.4%). Direct and indirect coombs tests: Negative, SGOT:110IU/l, SGPT:30IU/l, LDH:951 IU/mL, Bilirubin Total: 2.1 Mg/dl, Bilirubin indirect 0.9 Mg/dl, Albumin: 2.2 g/l, ESR:40, Uric acid:3.5mg/dl, PT:19.5 second (control; 13), INR: 1.2, PTT:50 second (control 43),TG:534Mg/dl( 2000 Ng/ ml, BUN:2Mg/dl, Cr:0.4 Mg/dl, IgA:124 Mg/dl(19–220), IgE:5Mg/dl (up to 10), IgG:2732Mg/dl(800–1000), IgM:854Mg/dl(20–100).TORCH study: negative, CMV-PCR: neg, EBV IgG: positive, EBV IgM: positive but EBV-DNA peripheral whole blood (PCR): negative, Parvovirus B19 PCR:negative, HbsAg: negative: HbsAb: positive, HCV AB: negative and HIVAb: negative. Wright, Coombs wright and Widal tests: negative. Blood culture: negative. CXR:Normal, Abdominal Sonography and color Doppler sonography showed: Large spleen, Liver; upper limit Normal and mild increase echogenicity, No collateral vein, portal vein normal. CSF analysis: normal, Bone survey: normal. Eye examination: normal, First bone marrow aspiration (BMA) and bone marrow biopsy (BMB) showed normocellular marrow with erythroid hyperplasia and maturation arrest in myeloid series. Report of CD-Flow cytometry: normal. However, in second BMA some hemophagocytic cells were seen. So due to clinical findings and laboratory tests sampling for evaluation of hereditary HLH were sent and treatment with IVIG 1gr/ kg/ day  2 days and prednisone 2mg/kg/ day oral were stated. Her response to treatment was very good. After 2 weeks of treatment she improved clinically and the result of CBC revealed: WBC:13400/ mm3, Neut 38%, lymph:62% RBC:2,730.000/mm3, Hb: 8.2Gr/dl, MCV:86Fl, pltatelet:464000/mm3, BilirubinTotal:4.3Mg/dl, Bilirubin direct: 0.6Mg/dl, Reticulocyte:18%. She followed and in next CBC; Hb: 9gr/dl and Reticulocyte; 25%. In peripheral blood smear; 2þ spherocytic cells was reported. The results of other laboratory tests 2 months after the last blood transfusion revealed increased osmotic fragility tests (OFT) before and after incubation. Autohemolysis tests without incubation: 25% and after adding glucose 10%.G6PD, pyruvate kinase tests were normal. The result of gene mutation for hereditary HLH including perforinand syntaxin11 were negative. Other types of gene mutation were not possible to do. Now our patient has just splenomegaly 3 cm below costal margin and she is on follow up for anemia dueto underlying disease, hereditary spherocytosis. Conclusion: In patients with persistentfever, pancytopenia and hepatosplenomegaly, it is necessary to think about HPS, but we should be careful about underlying disease. So it might not be necessaryto start aggressive treatment and based on etiology, patients with HPS may have good response to mild treatments such as prednisone and IV IG.

UNUSUAL PRESENTATION AND DIFFERENTIAL DIAGNOSIS OF PEDIATRIC SKELETAL LANGERHANS CELL HISTIOCYTOSIS Yoko Shioda1, Osamu Miyazaki2, Tomoo Osumi1, Keita Terashima1, Chikako Kiyotani1, Nobuhito Morota3, Noriko Morimoto4, Atsuko Nakazawa5, Kimikazu Matsumoto1 Division of Children’s Cancer Center, 1Radiology; 2Neurosurgery; 3Otolaryngology; 4 Pathology; 5National Center for Child Health and Development, Tokyo, Japan Purpose: Radiologic studies are critical in evaluating the lesions associated with Langerhans cell histiocytosis (LCH). In pediatric LCH patients, the bone is usually affected, especially the axial skeleton (skull, spine, and pelvis) and long bones. The characteristic features of LCH include sharply-defined lytic bone defect and lack of marginal sclerosis associated with adjacent soft tissue mass. Such features reflect a rapid progression of the disease. The clinical and radiologic presentations of LCH may vary and, in some cases, resemble other conditions. Characterization of LCH by radiological findings potentially eliminates the need for unnecessary invasive examination in typical benign cases. Methods: To clarify the characteristics of unusual skeletal LCH, 60 children with bone lesions of biopsy-proven LCH were retrospectively reviewed. The clinical and radiological reports of pediatric patients whose findings resemble LCH were also reviewed to establish a clear distinction between LCH and other benign conditions. Results: Unusual skeletal manifestations of LCH involving carpal bone, sternum, coccyx, pedicle of transverse or posterior process and skull base were detected along with pain and adjacent soft tissue swelling. Some lesions were asymptomatic. Between 2007 and 2014, benign conditions listed for differential diagnosis of LCH in our institution include lytic bone lesion of the skull (dermoid and epidermoid cyst, hemangioma, sinus pericranii, Pacchionian depression/arachnoid granulations), mastoid (myofibroma), mandible (odontogenic keratocyst, necrotizing periodontitis) and destruction of humerus and soft tissue mass (osteomyelitis). Identifying the location of lesions coupled with radiological findings helped in the differential diagnosis. Thin sclerotic margins, cortical thickening and solid periosteal reaction were noticed in the benign bone lesions, suggesting a slow-growing process.

HISTIOCYTE SOCIETY 30th ANNUAL MEETING ABSTRACTS Conclusion: In LCH, any type of bone can be involved. Hence, radiological findings should be interpreted with caution. In this review, several benign conditions revealed an oval-shaped lytic bone lesion similar to that of typical LCH. By classifying clinical and radiological findings such as location, age, growing process, pain, marginal sclerosis, periosteal reaction, enhancement and signal pattern, differential diagnosis can be achieved. The involvement of a radiologist with sufficient knowledge of normal and characteristic imaging features is imperative.

INTERFERON (IFN)–g RECEPTOR DEFICIENCY PRESENTING AS FATAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS (HLH): REPORT OF TWO CASES Elena Sieni1, Bianca Tesi2,3, Francesca Romano4, Benedetta Ciambotti1, Conceic¸a˜o Neves5, Samuel Chiang6, Luisa Galli7, Stefano Avenali8, Annalisa Tondo1, Ana Isabel Cordeiro5, Heinrich Schlums6, Jan-Inge-Henter2, Clementina Canessa4, Francesca Lippi4, Magnus Nordenskjo¨ld3, Amy Hsu9, Steven M. Holland9, Joao Farelas Neves5, Chiara Azzari4, Yenan Bryceson6 1 Department Pediatric Hematology Oncology, Anna Meyer Children’s University Hospital, Florence, Italy; 2Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden; 3 Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; 4Division of Pediatric Immunology, Department of Health Sciences, University of Florence and Anna Meyer Children’s Hospital, Florence, Italy; 5Primary Immunodeficiencies Unit, Hospital Dona Estefania, Pediatric University Hospital, Lisbon, Portugal; 6Centre for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; 7Division of Pediatric Infectious Diseases, Department of Health Sciences, University of Florence and Anna Meyer Children’s University Hospital, Florence, Italy; 8Department of Pediatric Anesthesia and Intensive Care, Anna Meyer Children’s University Hospital, Florence, Italy; 9National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA

Purpose: HLH is a life-threatening hyper-inflammatory syndrome typically associated with mutations in genes required for lymphocyte cytotoxicity. We report two cases first diagnosed with HLH and subsequently with disseminated tuberculosis. Case report: Case 1. A 2-month-old Caucasian girl was admitted for impetiginized atopic eczema. She later developed a systemic syndrome with eosinophilia resembling a drug reaction and subsequently HLH. The patient was treated with dexamethasone, cyclosporine and anti-thymocyte globulin (ATG). Later, the patient was found positive for CMV infection and disseminated Mycobacterium bovis infection. Specific treatment was started, but the child developed acute respiratory distress syndrome and died at the age of 4.5 months. Case 2. A 4-year-old Chinese girl was admitted for fever and mediastinal mass. She suddenly developed full-blown HLH with elevated EBV-DNA plasma levels. Chemo-immunotherapy (including dexamethasone, anti-CD20, cyclophosphamide and etoposide) was administrated, achieving partial remission of HLH, clearance EBV-DNA plasma levels and mediastinal mass necrosis. Five weeks later she reactivated and received ATG. At this point, disseminated Mycobacterium tuberculosis was diagnosed and a 5-drug anti-tubercular therapy was started with transient control followed by death. Results: Genetic analyses identified homozygous IFNGR2 deletions and homozygous missense IFNGR1 mutations in the respective patients, whereas no mutations were identified in genes required for lymphocyte cytotoxicity. Plasma levels of IFN-g were markedly elevated in both patients. Cellular analyses demonstrated impaired IFN-g-mediated STAT1 phosphorylation in the one patient tested, whereas cytotoxic lymphocyte degranulation and perforin production was normal in both cases. A paucity of peripheral blood NK cells was noted in both patients and NK cell activity was defective in the one tested. Conclusions: Our findings of fatal HLH in IFN-g receptor deficient patients argue for the use of refined functional assays in clinical diagnosis of HLH and indicate that mechanisms beside lymphocyte cytotoxicity may contribute to the pathophysiology of the disease.

MUTATIONS OF FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS (FHL) RELATED GENES AND ABNORMALITIES OF CYTOTOXICITY FUNCTION TESTS IN PATIENTS WITH MACROPHAGE ACTIVATION SYNDROME (MAS) OCCURRING IN SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS (SJIA) Elena Sieni1, Claudia Bracaglia2, Martina Da Ros1, Carmela De Fusco3, Concetta Micalizzi4, Valentina Cetica1, Coniglio ML1, Ciambotti B1, Antonella Insalaco2, Fabrizio De Benedetti2, Maurizio Arico`5 1 Department of Pediatric Hematology-Oncology, Anna Meyer Children University Hospital, Florence, Italy; 2Division of Rheumatology, Department of Pediatric Medicine, IRCCS Ospedale Pediatrico Bambino Gesu`, Rome, Italy; 3Department of Pediatric HematologyOncology, Pausillipon Children Hospital, Naple, Italy; 4Department of Pediatric Hematology-Oncology, G. Gaslini Children Hospital, Genoa, Italy; 5Istituto Toscano Tumori (I.T.T.), Florence, Italy

Purpose: MAS is a severe complication of rheumatic diseases, mostly sJIA. Clinical and laboratory features are similar to those of FHL resulting from mutations in selected genes involved in the cytotoxicity pathway. We investigated the presence of mutations of FHL-

Pediatr Blood Cancer DOI 10.1002/pbc


related genes and of abnormalities in degranulation and perforin expression in patients with MAS occurring in the context of sJIA. Methods: From the HLH Italian National Registry, we selected patients with MAS defined according to the HLH 2004 criteria and with confirmed diagnosis of sJIA based on ILAR criteria. Mutation analysis was performed by Sanger sequencing of FHL-related genes. Perforin expression and degranulation were analyzed using flow-cytometry. Results: We identified 31 patients (17 females; 25 Southern European, 6 Indian) with MAS and sJIA. Eleven patients (35%) had 14 monoallelic mutations in PRF1(n ¼ 7), UNC13D (n ¼ 1), STX11(n ¼ 1), STXBP2 (n ¼ 4), and Rab27a(n ¼ 1). Three patients had mutations in 2 genes. Both degranulation and perforin expression were evaluated in 18 patients. At least one test was defective in 11 patients (61%). The clinical and laboratory features of patients with monoallelic mutation and/or with abnormalities in at least one functional test were not different from those of the remaining patients. However, re-occurrence of MAS tended to be more frequent in patients carrying mutations (mutated 27% versus non-mutated 10%) and in patients showing abnormalities in at least 1 functional test (abnormal 18% versus 0%). One patient died of MAS: she carried the N252S PRF1 variant and showed reduced perforin expression. Conclusion: Monoallelic mutations in FHL-related genes and partial defect in either perforin expression or degranulation capacity are frequently observed in patients with sJIA who develop MAS. Additional genetic studies are warranted to identify additional genes potentially linked to MAS development.

ROSAI-DORFMAN DISEASE (RDD): REPORT OF 16 PATIENTS Elena Sieni1, Alessandra Lombardi2, Carmen De Fusco3, Monica Cellini4, Stefania Varotto5, Elena Mastrodicasa6, Antonella Sau7, Camilla Rosa1, Maurizio Arico`8 1 Department Pediatric Hematology Oncology, Anna Meyer Children University Hospital, Firenze, Italy; 2Department of Pediatric Hematology-Oncology, IRCCS Ospedale Bambino Gesu`, Roma, Italy; 3Department Pediatric Hematology Oncology, Pausillipon Hospital, Napoli, Italy; 4Department Pediatric Hematology Oncology, Policlinico Modena, Modena, Italy; 5Department Pediatric Hematology Oncology, University of Padua, Padova, Italy; 6 Unit of Pediatric Oncology and Hematology, S. Maria della Misericordia Hospital, Perugia; 7 Department Pediatric Hematology Oncology, Civile Hospital, Pescara, Italy; 8Istituto Toscano Tumori (I.T.T.), Florence, Italy

Purpose: RDD is a non-Langerhans cell histiocytic disorder characterized by histiocytic cell proliferation within lymphnode sinuses and lymphatics in extranodal sites. The rarity of the disease limits our knowledge. We describe the Italian experience. Methods: Data on demographics, presenting features, treatment and outcome of patients with histologically proven RDD were collected. Results: Sixteen patients (10 males) were diagnosed between 1998 and 2013, at a median age of 3.3 years (range: 7 months-38 years; quartiles: 2, 3.3, 7.7 years). Nodal involvement was present in 12, and was the presenting manifestation in 10. Head and neck lymph nodes were affected in all cases; inguinal, axillary, mediastinal, abdominal nodes were also reported. Extranodal involvement was present in 8/16 (50%) patients: nasal cavity/paranasal sinuses (n ¼ 2), central nervous system (CNS) (n ¼ 2), retro-orbital tissue (n ¼ 2), thigh soft tissue (n ¼ 1) and parotid gland (n ¼ 1). Both nodal and exatranodal sites were involved in 4/16 (25%). Two patients with CNS/orbital lesions had systemic symptoms including fever, sweating and weight loss. Associated diseases reported in 2 patients were: Autoimmune Lymphoproliferative Syndrome and Kawasaki disease. Spontaneous resolution was reported in 7/16 (44%) patients with nodal involvement; the remaining patients underwent surgery (n ¼ 3), surgery þ chemo/immunotherapy (n ¼ 3) or chemo/immunotherapy alone (n ¼ 3). Chemo/immunotherapy regimens included: prednisone alone, prednisone þ vinblastine, cladribine, immunoglubulin, anti-CD20. Two patients experienced reactivation, one was refractory to initial therapy; at last follow-up all are alive, two of them never achieved complete remission. Conclusions: The clinical manifestations of RDD are highly variable. None of the patients developed life-threatening conditions, despite extranodal involvement in one half of cases. Although steroid monotherapy or LCH-directed chemotherapy are often applied, the real need for specific therapies remains controversial. A prospective diagnostic and therapeutic study, also allowing biologic studies, appears warranted.

CYTARABINE MONOTHERAPY FOR DE NOVO AND RECURRENT LANGERHANS CELL HISTIOCYTOSIS Stephen Simko, Harshal Abhyankar, Philip Lupo, John Hicks, Jeremy Jones, Huy Tran, Kenneth McClain, Carl Allen Baylor College of Medicine and Texas Children’s Cancer Center, Houston, TX USA Purpose: While vinblastine/prednisone remains the standard initial therapy for Langerhans cell histiocytosis (LCH), fewer than 50% of patients are cured without prolonged or additional therapy. Although cytarabine has known activity against LCH, its role as a first-line, single agent therapy in de novo or progressive disease has not been evaluated. Methods: Medical records of all patients treated at Texas Children’s Hospital for LCH from 2005–2013 were reviewed. Patients treated with regimens utilizing standard-dose cytarabine (100–170 mg/m2 per day over 3–5 days) were evaluated for outcome and toxicity.



Results: Thirty-one patients were treated with cytarabine, including 13 patients with de novo disease and 18 patients with progressive disease. For patients with de novo disease, 12/13 had non-active disease within one year after cytarabine monotherapy, and no patients developed disease reactivation during therapy. Three patients had recurrent disease after completing one year of cytarabine, all within six months of therapy cessation. Presence or absence of BRAFV600E mutation was known in 8/13 de novo cases; the three patients with recurrence all had wild-type BRAF. Of the 18 patients treated with cytarabine for disease reactivation, 12 (67%) had disease improvement within three months, including 5/6 patients with risk organ involvement. Estimated 3-year progression-free survival (PFS) for patients receiving cytarabine for progressive disease was 41%; 3-year overall survival was 100%. Patients with progressive disease who received cytarabine monotherapy had higher 3-year PFS than those who received cytarabine plus other chemotherapy agents (83.3% versus 25%, P ¼ 0.0433). Excluding expected neutropenia and chemotherapy-associated fever, toxicity requiring hospitalization occurred in 3/31 patients. Conclusion: Standard-dose cytarabine monotherapy may be effective therapy for patients with de novo or recurrent disease. Prospective studies are warranted to determine whether cytarabine monotherapy is superior to vinblastine/prednisone in treating de novo disease, particularly in disease involving risk organs or harboring BRAFV600E mutation.

TREATMENT OF RECURRENT LANGERHANS CELL HISTIOCYTOSIS WITH CLADRIBINE IN CHILDREN Karel Svojgr1, Josef Malis1, Martin Kyncl2, Roman Kodet3, Vratislav Smelhaus1, Jan Stary1, Hubert Mottl1 University Hospital Motol, 1Department of Pediatric Hematology and Oncology, 2 Department of Radiology, 3Department of Pathology and Molecular Medicine, Prague, Czech Republic Purpose: Langerhans cell histiocytosis (LCH) is a rare malignant disease with frequent relapses. 2-chlorodeoxyadenosine (cladribine) is a potent agent in treatment of LCH. Methods: From 1997 to 2007, 17 patients with recurrent LCH were treated with cladribine at our institution. We analyzed response and survival of patients treated with cladribine. Results: Median age at diagnosis of patients with LCH was 1.24 years (0.25–17.2 years). All patients were pretreated with systemic therapy, 15 patients were pretreated with LCH II or LCH III protocol, one was pretreated with 2 courses and one with 3 courses of systemic therapy. Median time from diagnosis to start cladribine treatment was 14.4 months (1.9–79.7); median age of patients starting cladribine was 3.35 years (0.4–17.9). Cladribinetherapy was initiated in 11 cases for SS-LCH, in 6 cases for MS-LCH. In 3 cases, risk organs (lungs or liver) were affected (lungs now not considered as high-risk organ). Thirteen patients were treated with 6 cycles of cladribinemonotherapy, 1 received only 4 cycles; 2 were treated with cladribineand cytosine arabinoside and 1 received cladribineand maintenance therapy with vinblastine, mercaptopurine and prednisone. All patients responded to cladribine treatment, only 3 patients relapsed after cladribine therapy, but relapses were local only and were easily managed with subsequent therapy. From 3 relapsed patients, 2 were rescued with radiotherapy, 1 received radiotherapy and maintenance vinblastine. Event free survival is 82.4%, overall survival is 100%. Conclusions: Cladribine is highly active in treatment of LCH. Supported by MHCZ – DRO, University Hospital Motol, Prague, Czech Republic 00064203.

Conclusion: We report this case to increase awareness of familial HLH in the differential diagnosis where neonatal hemochromatosis is a consideration.

TERTIARY LYMPHOID NEOGENESIS IS COMMON IN LANGERHANS CELL HISTIOCYTOSIS-AFFECTED TISSUES Astrid G.S. van Halteren1, Willemijn T. Quispel1, Susy J. Santos1, Lianne Koens2, Cor van den Bos3, Reina E. Mebius4, R. Maarten Egeler5 1 Department of Pediatrics/Willem Alexander Childerens Hospital/Leiden University Medical Center, Leiden, the Netherlands; 2Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands; 3Emma Childrens Hospital/Academic Medical Center, Amsterdam, the Netherlands; 4Department of Molecular Cell Biology & Immunology, VU Medical Center, Amsterdam, the Netherlands; 5Division of Hematology/Oncology, Hospital for Sick Children, Toronto, Canada

Purpose: Chronically inflamed non-lymphoid tissues often contain structured lymphocyte aggregates, so-called Tertiary Lymphoid Structures (TLS). TLS closely resemble classical lymph nodes with regard to cellular organization, chemokine expression and specialized vasculature called High Endothelial Venules (HEV). TLS likely act as local sites of antigenpresentation and lymphocyte activation. Their presence in tumors is associated with a survival benefit. Methods: Neoplastic Langerhans cell histiocytosis (LCH)-cells and lymphocytes are prototypic components of LCH-affected tissues. We studied by immunofluorescent and conventional immunohistochemical staining lymphocyte organization and the expression pattern of some of the key components for TLS formation in LCH specimens collected at diagnosis. Results: TLS-like lymphocytic aggregates were detected in 31/52 (60%) hematoxylin-eosin stained tissue sections. In 9 out of 18 biopsies, lymphoid aggregates contained segregated CD3þ T cells and CD79þB cells as well as CD35þ Follicular Dendritic Cells (FDC) and Bcl-6þ centrocytes. The B cell follicle-organizing chemokine CXCL13, predominantly produced by macrophages but not by LCH-cells, and its receptor CXCR5 were also abundantly present. In addition, TLS-containing biopsies displayed podoplaninþ cells which most likely represent FDC or fibroblastic reticular cells, two other key cell types in TLS formation. The HEV-specific marker MECA-79 was detected in 9/18 biopsies which co-expressed all other components necessary for TLS formation. The latter biopsies contained the highest absolute numbers of T cells, the highest number of CD4þ T-cells as well as the highest percentage of regulatory T-cells when compared to n ¼ 22 LCH lesions wherein MECA-79 was absent (p ¼ 0,0005, p ¼ 0,01 and p ¼ 0,04 respectively). Conclusions: Various stages of lymphocyte aggregation are commonly seen in LCH-affected tissues. The presence of fully structured TLS, which are readily detectable in hematoxylineosin stained tissue sections, is associated with the highest influx of T cells. We are currently addressing the activation status of these T cells in relation to LCH outcome.


FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS MIMICKING NEONATAL HEMOCHROMATOSIS Julie Talano, Monica Thakar, James Casper, Sara Szabo, Regan Veith, David Basel, Bernadette Vitola Children’s Hospital of Wisconsin, Medical College of Wisconsin, Milwaukee, WI Purpose: We describe a case of neonatal hemochromatosis diagnosed in a stillborn infant of an affected sibling who subsequently presented with hemophagocytic lymphohistiocytosis (HLH). Methods/Results: A prima gravida mother delivered a 37-week stillborn male with hydrops fetalis. An autopsy determined the cause of death to be presumptive neonatal hemochromatosis. With the subsequent pregnancy, this mother received weekly IVIG on protocol for 20 weeks prior to this child’s birth for prevention of alloimmune neonatal hemochromatosis. He was born at 36 weeks via planned C-section and remained in the NICU briefly for mild respiratory distress and jaundice requiring phototherapy. Evaluation at birth revealed an INR of 1.26 and ferritin 200; therefore, it was elected to follow him. He was well until he presented at 2 months with a 3 day history of fever of 102.6˚F, sleepiness and poor feeding. A septic workup was initiated. Laboratory evaluation was significant for an AST 280, ALT 136, albumin 2.5, total bilirubin 1.3, PT 17.4, INR 1.49, PTT 44, D-dimer 8.96, fibrinogen 242, ferritin 5210 ng/dl, WBC 3, Hg 8.3, platelets 74,000, ANC 1500. He had prominent hepatosplenomegaly and was intubated for respiratory failure. An MRI of the liver/ pancreas revealed low signal intensity throughout the liver consistent with suspected primary hemochromatosis. However, due to the atypical presentation, the patient underwent a lower lip biopsy to obtain minor salivary gland tissue to document iron deposition. The specimen obtained was inadequate for evaluation. Further testing revealed sIL2r 38,872 units/ml. NK cell and B cell counts were zero, granzyme B was present but no perforin was detected. Genetic testing for familial HLH revealed a mutation in Munc 13-4. Genetic studies on the stillborn brother confirmed Munc 13-4.

Pediatr Blood Cancer DOI 10.1002/pbc

Department of Hematology, Beijing Friendship Hospital, Capital Medical University, Beijing, China Purpose: To observe the clinical value of splenectomy for treatment of refractory hemophagocytic lymphohistiocytosis (HLH) in adult cases with unknown origin. Methods: We report here 19 adult cases of refractory HLH with unknown origin treated with splenectomy at Department of Hematology, Beijing Friendship Hospital from June 2007 to March 2014. Their clinical data were analyzed. Results: To rule out a possible underlying disease, e.g. virus or bacterial infection, autoimmune disease, neoplasms and primary HLH, we selectively and systematically performed a series of laboratory tests, including positron emissiontomography (PET)/CT, HLH-associated gene defects and in some cases lymph-node biopsy. There were more than 2 times relapse in all cases who were received more than 2 kinds of therapies. 12 cases achieved partial response (PR), 7 cases achieved no response (NR) at the time of splenectomy. There was an increase of radioactivity of PET-CT in 8 cases, 7 cases of them were diagnosed as lymphoma according to spleen pathology. Infection and hemorrhage were main complication of splenectomy in HLH cases. 18 cases were evaluated, 7 cases who were diagnosed as lymphoma according to pathology received chemotherapy, 4 cases achieved complete response (CR),1 case achieved PR, 2 cases achieved NR. Treatment was discontinued 2 or 3 months after splenectomy in 11 remaining cases, 5 cases achieved CR, 4 cases achieved PR, 2 cases achieved NR. 11 of 18 cases were alive with a median follow up of 25 months (range 3– 79), 5 cases died due to HLH, 1 case died due to pulmonary infection after splenectomy and 1 case died due to complication of allogeneic hematopoietic stem cell transplant after HLH relapse. 12 and 36-month PFS rates were 48% (95% CI, 38%–58) and 24% (95% CI, 14%– 34%), respectively; 12- and 36-monthOS rates were 57% (95%,47%–67%) and 25% (95% CI, 15%–35%), respectively. Conclusion: These results demonstrated splenectomy may be a new effective method of diagnosis and treatment in adult refractory cases with HLH of unknown origin. Further study is needed to establish the value of splenectomy in this disease.


3 4 5

Yini Wang, Zhao Wang , Wenqiu Huang, Na Wei, Jia Zhang, Jingshi Wang, Lin Wu Beijing Friendship Hospital, Capital Medical University, Beijing, China Purpose: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome. Current standard therapy increased the complete remission (CR) rate to more than 50%. Even so, about 30% patients fail the standard treatment. This study aimed to investigate the efficacy of liposomal doxorubicin together with etoposide and high dose methylprednisolone (DEP) as a salvage therapy for adult refractory HLH. Methods: Refractory HLH was defined as patient who did not achieve at least partial remission (PR) 2 weeks after initial standard HLH therapy. Effective evaluation referred to Marsh RA [Pediatr Blood Cancer. 2013, 60(1):101–109]. The DEP regimen included liposomal doxorubicin 25mg/m2 d1, etoposide 100mg/m2 Qweek and methylprednisolone 15mg/kg d13, 2mg/kg d4-6, 1mg/kg d7-10, 0.75mg/kg d11-14. We estimated the curative effect after 2 weeks therapy and 4 weeks therapy. Results: We observed 41 refractory HLH patients from Mar 2013 to Dec 2013, including 28 males and 13 females. The median age was 31 years old (Range 18–62). Twelve patients (29.3%) achieved CR and 20 patients (48.8%) achieved PR. The overall response reached 78.1% (32/41). The underlying disease of HLH were diagnosed within 2 to 8 weeks after HLH diagnosis, including FHL-2 (one case, CR), lymphoma associated HLH (20 cases, 3 CR and 12 PR), and EBV associated HLH (12 cases, 3 CR and 6 PR). There were still 8 cases that had unknown underlying disease (5 CR and 2 PR). The patients who had no response to DEP died within 2 to 4 weeks after salvage therapy. Sixty-three percent of patients who achieved PR or CR survived to undergo subsequent chemotherapy, allogenic hematopoietic stem cell transplantation (allo-HCT) or splenectomy. Conclusion: DEP regimen appears to be an effective salvage protocol for refractory HLH. Prospective randomized controlled trials of DEP regimen will further estimated the curative effect and define optimal dosing levels and schedules.



Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan; Department of Pediatrics, Jichi Medical University of Medicine, Shimotsuke, Japan; Department of Pediatrics, Ehime University Graduate School of Medicine, Toon, Japan

Purpose: Epstein-Barr virus (EBV) infection is the most common trigger of secondary hemophagocytic lymphohistiocytosis (HLH) in children. Immunochemotherapy such as HLH-94/2004 protocol reduced recurrence and improved survival in EBV-HLH patients. However, little is known about molecular markers capable of predicting therapeutic response, thus minimizing the treatment intensity. We have recently reported that BIOMED2 multiplex PCR method in combination with GeneScan analysis was useful for monitoring of rearrangement of T-cell receptor (TCR)b and/or TCRg genes in children with EBV-HLH (Matsuda et al., 2011), although the clinical significance is still obscure. Thus we conducted a prospective study to identify the association of TCR clonality and clinical response to treatment of EBV-HLH. Methods: Children with EBV-HLH who were registered in the Japan Pediatric Leukemia/ Lymphoma Study Group (JPLSG) for the treatment of HLH-2004 protocol were eligible for this study. Rearrangements of TCRb/g genes and viral load of EBV in peripheral blood were serially analyzed by BIOMED-2/GeneScan methods and quantitative PCR at onset and 2, 4 and 8 weeks after the start of treatment. Results: A total of 40 patients with EBV-HLH had sex ratio of 1:1 and a median age of 2 years (range: 0–14 years). EBV load at onset ranged from 3.3  10^2 to 2.2  10^6 (median, 1.8  10^5) copies/mgDNA in whole blood cells and 2.2  10^2 to 1.2  10^7 (median, 9.5  10^5) copies/mL in serum. TCR clonality was detected in 27 of 40 patients (67.5%) at onset; rearrangement of TCRb in 2, TCRg in 10, and TCRb/g genes in 15. Clinical course of patients who had no TCR clonality was favorable, while response to treatment was heterogeneous in the TCR clonality positive group. Conclusion: Detection at onset and serial monitoring of T-cell clonality may be useful to predict therapeutic response and could be incorporated in the next study for the treatment of EBV-HLH.


Zhao Wang, Yini Wang, Wenqiu Huang, Xiangzong Zeng, Jia Zhang, Jingshi Wang, Lin Wu, Na Wei

Department of Hematology, Beijing Friendship Hospital, Capital Medical University, Beijing, China

Beijing Friendship Hospital, Capital Medical University, Beijing, China Purpose: This study aimed to analyze the clinical manifestations of adult hemophagocytic lymphohistiocytosis (HLH), the important laboratory tests, treatments and prognosis. Methods: A multicenter retrospective study was carried out to analyze the causes, clinical features, laboratory tests, diagnosis, treatments and clinical outcomes of 423 adult patients with HLH from 46 institutions in China, from 2006 to 2013. Results: Among the 423 cases, there are 144 cancer-related patients and followed by the infection-related patients, with the number of 138. Referring to HLH-2004 diagnostic criteria, the incidence rate of criteriawere: fever (99.53%), Ferritin (94.09%), soluble interleukin-2 receptor (sCD25) increased (91.96%), the activity of NK-cells decreased or absent (91.73%), cytopenias (78.01%), splenomegaly (69.50%), hemophagocytosis in bone marrow or spleen or lymph nodes (61.70%), hypofibrinogenemia (52.72%), hypertriglyceridemia (40.43%). In addition, 92.43% of patients with liver disorders, 73.05% of patients with coagulopathy. Comparison among tumor, infection and rheumatic group shows statistically significant difference of some diagnostic criteria, which are white blood cells, platelet count, alanine aminotransferase, aspartate aminotransferase, direct bilirubin and albumin. Besides, the difference of white blood cells, bilirubin, albumin, lactate dehydrogenase, creatinine, urea nitrogen between survival group and death group is still statistically significant. Conclusion: This clinical analysis of HLH cases show that adult primary HLH is not rare anymore, and it should be taken seriously. The secondary HLH is related to various potential diseases. Haematological malignancies, especially non-Hodgkin lymphomas, become the main reason of adult HLH, followed by the EB virus infection. The diagnostic sensitive indicators are persistent fever, ferritin, low or absent NK-cell activity, sCD25 increased. But the blood cells of rheumatology associated HLH may not reduce in the early phase. Patients with low serum albumin levels and white blood cell may have a markedly worse survival. Renal insufficiency and coagulopathy play an important role in prognosis.

Purpose: NK cell activity is an important indicator to determine anti-tumor, anti-viral infection and immune regulation function. NK cell activity detection methods are mainly traditional 51Cr release assay, LDH release assay and MTT colorimetric assay and the new generation of flow cytometry method, but each of them still haveflaws and shortcomings. Methods: We have established a novel method of flow cytometry in detecting human peripheral blood NK cell activity by lentiviral transfection technology to build a stable K562 target cell strain expressing GFP and flow cytometry detectionwith Annexin V-PE/7AAD double staining mark. After NK cell cytotoxicity, apoptosis of target cells reflects the proportion of NK cell killing activity. This paper selected 32 adult normal healthy volunteers which were located in different effector cell/target cell concentration gradient ratio 5:1,10:1,20:1 and incubation time 2h, 4h, 6h, 8h, from which to select the optimal effect/target (E/T) ratio and incubation time. In addition, randomly selected 37 adult patients with diagnosis of HLH, using the optimal detection method, and compared the differences of NK cell activity between HLH patients and normal people. Results: NK cell activity in a certain incubation time increases with the E/T ratio increasing, 4  6h basically reached the peak, 6h after a steady decline. In the conditions of E/ T ratio of 10:1 and the incubation time of 4h, HLH patients showedNK cell activity (11.79  2.73)% significantly lower than that of normal healthy volunteers (20.98  4.61)%, P 3,000 mg/L in 46% of the patients (median 2,099 mg/L), whereas 1,000 U/L in 49.1% (median 984 U/L). 87.3% had hypoalbuminemia (median 27.8 g/L). 32.5% had either renal dysfunction or pathological imaging of kidneys. Pathological MRI of the brain was seen in 9.5% of the patients. 17.1% of patients gave up treatment on parental demand after HLH was diagnosed, mostly due to financial difficulties. 116 (82.9%) of the patients underwent further treatment and analysis. 56.9% of the patients were treated with etopside (VP16), corticosteroids and cyclosporine (CSA), and 10.3% only with corticosteroids. 11.2% of the patients were only treated for their underlying diseases. Among patients that died, 89.8% died within 3 months and 51% died before initiation of HLH-specific treatment. Delayed chemotherapy by more than 7 days after diagnosis had a significant negative impact on survival (P ¼ 0.028). Conclusions: EBV was the most common trigger of HLH in Chinese children. Timely diagnosis and prompt treatment were key points to further reduce early mortality.

Pediatr Blood Cancer DOI 10.1002/pbc

LANGERHANS CELL SARCOMA: CASE REPORT AND REVIEW OF WORLD LITERATURE Ted Zwerdling1, Eric Won2, Lisa Shane3, Ramin Javahara4, Ronald Jaffe5 1 Department of Pediatrics, Miller Children’s Hospital, Long Beach, CA USA; 2Department of Pediatrics, University of California, Irvine, Irvine, CA USA; 3Department of Pathology, Miller Children’s Hospital, Long Beach, CA USA; 4Department of Neurosurgery, Miller Children’s Hospital, Long Beach, CA USA; 5Department of Pathology, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA USA

Langerhans cell sarcoma (LCS) is a rare malignancy with only 1 pediatric case (< 15 years of age) reported. Here we report the second case of LCS in a child who presented with cord compression. This patient was treated with extensive surgical resection, post-operative chemotherapy and involved-field radiation therapy. She completed therapy and remains in remission for 27 months. A review and analysis of all 53 cases published in the world literature is provided to help guide physicians treating this disease. Recently discovered genetic mutation involving BRAF is also discussed.

Abstracts from the 30(th) Annual Meeting of the Histiocyte Society Toronto, Ontario, Canada October 28-30, 2014.

Abstracts from the 30(th) Annual Meeting of the Histiocyte Society Toronto, Ontario, Canada October 28-30, 2014. - PDF Download Free
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