Tricia L. Peters

Cell-Specific Gene Expression in Langerhans Cell Histiocytosis Lesions Reveals a Distinct Profile Compared with Epidermal Langerhans Cells

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207+ Langerhans cells (LCs) and lymphocytes that can arise in almost any tissue and cause significant morbidity and mortality. After decades of research, the cause of LCH remains speculative. A prevailing model suggests that LCH arises from malignant transformation and metastasis of epidermal LCs. In this study, CD207+ cells and CD3+ T cells were isolated from LCH lesions to determine cell-specific gene expression. Compared with control epidermal CD207+ cells, the LCH CD207+ cells yielded 2113 differentially expressed genes (false discovery rate < 0.01). Surprisingly, the expression of many genes previously associated with LCH, including cell-cycle regulators, proinflammatory cytokines, and chemokines, were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly overexpressed in LCH CD207+ cells. Furthermore, several genes associated with immature myeloid dendritic cells were overexpressed in LCH CD207+ cells. Compared with the peripheral CD3+ cells from LCH patients, the LCH lesion CD3+ cells yielded only 162 differentially regulated genes (false discovery rate < 0.01), and the expression profile of the LCH lesion CD3+ cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4, and SPP1. Results from this study support a model of LCH pathogenesis in which lesions do not arise from epidermal LCs but from accumulation of bone marrow-derived immature myeloid dendritic cells that recruit activated lymphocytes.

BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups

The Rockefeller University Press

BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children.

30.00 J. Exp. Med. 2014 Vol. 211 No. 4 669-683 www.jem.org/cgi/doi/10.1084/jem.20130977 669 Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions that include pathological langerin+ DCs. LCH has pleotropic clinical presentations ranging from single lesions cured by curettage to potentially fatal multisystem disease. The first descriptions of LCH, including Hand-Schüller-Christian disease and Letter-Siwe disease, were based on anatomical location and extent of the lesions (Arceci, 1999). The diagnosis of high-risk LCH, defined by involvement of “risk organs” which include BM, liver, and spleen, conferred mortality rates >20%, where patients with disease limited to non-risk organs (low-risk LCH) had nearly 100% survival, CORRESPONDENCE Carl Allen: ceallen@txch.org OR Miriam Merad: miriam.merad@mssm.edu

Neither IL-17A mRNA Nor IL-17A Protein Are Detectable in Langerhans Cell Histiocytosis Lesions

The BCOR–CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative ‘Ewing-like’ sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired-end RNA sequencing (RNA-seq) we unexpectedly identified BCOR–CCNB3 fusion transcripts in an undifferentiated spindle-cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR–CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR–CCNB3 sarcoma were males diagnosed in mid childhood (7–13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all six cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR–CCNB3 fusion-positive sarcomas, a newly emerging entity within the undifferentiated unclassified sarcoma category and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors.

An integrated proteomic approach to identifying circulating biomarkers in high-risk neuroblastoma and their potential in relapse monitoring

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions including CD207(+)/CD1a(+) dendritic cells that can result in significant morbidity and mortality. The etiology of LCH remains speculative, and neoplastic and inflammatory origins have been debated for decades. A recent study identified abundant interleukin-17 (IL-17A) protein in dendritic cells in LCH lesions as well as in plasma from patients with active disease. Furthermore, it identified dendritic cells as a novel source of IL-17A expression. However, subsequent studies from our research group failed to identify any IL-17A gene expression from CD207(+) dendritic cells or CD3(+) T cells in LCH lesions. In this study, further investigation once again fails to identify any cells in LCH lesions with IL-17A gene expression. Furthermore, IL-17A antigen is undetectable in LCH lesion lysates with western blotting, immunoprecipitation, spectral analysis, and enzyme-linked immunosorbent assay (ELISA). Western blots, immunoprecipitation, and ELISA experiments also demonstrate that antibodies used in original studies that established the IL-17A hypothesis for pathogenesis of LCH recognize nonspecific proteins. We conclude that evidence for IL-17A as a significant factor in LCH remains inadequate and clinical trials targeting IL-17A remain unjustified.

Activating MAPK1 (ERK2) mutation in an aggressive case of disseminated juvenile xanthogranuloma

Purpose: Despite intensive treatment regimens, overall survival for high‐risk neuroblastoma (HRNB) is still poor. This is in part due to an inability to cure the disease once a patient has reached clinical relapse. Identifying plasma biomarkers of active disease may provide a way of relapse monitoring in HRNB.

CNS Langerhans cell histiocytosis: Common hematopoietic origin for LCH-associated neurodegeneration and mass lesions

Juvenile xanthogranuloma (JXG) is a rare histiocytic disorder that is usually benign and self-limiting. We present a case of atypical, aggressive JXG harboring a novel mitogen-activated protein kinase (MAPK) pathway mutation in the MAPK1 gene, which encodes mitogen-activated protein kinase 1 or extracellular signal-regulated 2 (ERK2). Our analysis revealed that the mutation results in constitutive ERK activation that is resistant to BRAF or MEK inhibitors but susceptible to an ERK inhibitor. These data highlight the importance of identifying specific MAPK pathway alterations as part of the diagnostic workup for patients with histiocytic disorders rather than initiating empiric treatment with MEK inhibitors.

Abstract B79: Detectable BRAF-V600E mutation in circulating peripheral blood of patients with Langerhans cell histiocytosis correlates with risk organ involvement and residual disease

Central nervous system Langerhans cell histiocytosis (CNS‐LCH) brain involvement may include mass lesions and/or a neurodegenerative disease (LCH‐ND) of unknown etiology. The goal of this study was to define the mechanisms of pathogenesis that drive CNS‐LCH.

Frequent BRAF V600E Mutations Are Identified in CD207+ Cells in LCH Lesions, but BRAF Status does not Correlate with Clinical Presentation of Patients or Transcriptional Profiles of CD207+ Cells

Purpose: Langerhans Cell Histiocytosis (LCH) is a clonal disorder characterized by inflammatory lesions with characteristic CD207+ dendritic cells (DCs). LCH has variable clinical presentations ranging from single lesions to potentially fatal multi-system “risk organ” disease. The etiology of LCH remains elusive, with debate of LCH as an inflammatory versus malignant disorder unresolved. The first recurrent somatic genetic mutation in LCH, BRAF-V600E, was recently reported in 57% of LCH lesions (Badalian-Very et al., 2010). Here we investigate the clinical significance of BRAF-V600E as a potential biomarker of risk organ or refractory disease. Methods: Formalin-fixed, paraffin embedded (FFPE) tissue, peripheral blood, and sorted peripheral monocyte/dendritic cell populations were genotyped for BRAF-V600E mutations with allele-specific, real-time PCR assays. The presence of BRAF-V600E mutations was correlated with clinical variables and analyzed with standard statistical methods. A subsequent validation set of 8 patient peripheral blood samples was identified for quantitative analysis of levels of BRAF-V600E positive cells with the BRAF Rotor-Gene Q (RGQ) PCR assay (Qiagen, Valencia, CA), and concordance with results from Qiagen qBiomarker qPCR assay was determined. Quantitation was performed using a delta Ct method of the BRAF-V600E assay, and results were reported as percentage of mutant cells in a background of wild-type cells using standard curves. Results: Lesions from 100 patients with LCH were genotyped, and 64% carried the V600E mutation, which localized to the infiltrating CD207+ DCs. In 16 patients with more than one lesion, BRAF status remained fixed, suggesting somatic mutation of BRAF is an early event. BRAF-V600E did not define specific clinical risk groups or impact overall survival, but it was associated with approximately two-fold higher risk of relapse (p=0.04). Furthermore, the cellular compartment carrying the mutation correlated with disease severity: the ability to detect BRAF-V600E in circulating mononuclear cells defined risk organ LCH with 100% sensitivity/87% specificity. The ability to detect BRAF-V600E in circulating blood cells in patients with risk organ LCH defined clinically detectable disease with 97% sensitivity/100% specificity. For development of a clinically reproducible minimal residual disease assay that would be CLIA-compliant and commercially available, a separate validation sample set was identified. With a limit of detection of 0.02% mutant cells in a background of wild-type cells, the RGQ assay correctly detected BRAF-V600E mutations in all 8 validation specimens and in known BRAF-V600E positive cell lines and did not detect mutations in 10 additional BRAF-V600E mutation negative clinical specimens (analytical specificity = 100%). The RGQ quantitative results correlated with the qBiomarker assay results (R2=0.924) with comparable analytical sensitivity. Conclusions: The molecular foothold of BRAF at the base of LCH pathogenesis will allow therapeutic strategies to move beyond empiric observation to risk-stratified and targeted approaches. Furthermore, effectiveness of therapy may be tested by following BRAF-V600E in peripheral blood cells as a marker of residual disease. Development of validated assays to test for BRAF-V600E in peripheral blood will assist in assigning risk status and assessing therapeutic response. Citation Format: Stephen J. Simko, Marie-Luise Berres, Karen Phaik-Har Lim, Tricia Peters, Jeremy Price, Philip J. Lupo, M. John Hicks, Albert Shih, Kenneth Heym, Kenneth L. McClain, Miriam Merad, Stephen Sarabia, Dolores Lopez-Terrada, Carl E. Allen. Detectable BRAF-V600E mutation in circulating peripheral blood of patients with Langerhans cell histiocytosis correlates with risk organ involvement and residual disease. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B79.