Kenneth Matthew Heym

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

The Rockefeller University Press

Contact-activated monocytes: efficient antigen presenting cells for the stimulation of antigen-specific T cells.

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

Outcome of children with multiply relapsed B-cell acute lymphoblastic leukemia: a therapeutic advances in childhood leukemia & lymphoma study

Mature dendritic cells (DCs) are potent antigen presenting cells (APCs) that have been used in vaccine studies and adoptive immunotherapy protocols. For many clinical studies DCs are derived from monocytes in the presence of cytokines, which are expensive and often unavailable for clinical use. Here we describe a cytokine independent method for the differentiation of monocytes into APCs for the reactivation of antigen-specific memory T cells from both healthy donors and cancer patients. Contact activation of monocytes resulted in secretion of proinflammatory cytokines, such as IL-8, and increased cell surface expression of costimulatory molecules. To determine if activated monocytes (actMo) like DC can reactivate antigen-specific CTL, they were transduced with adenoviral vectors encoding the subdominant Epstein Barr virus antigens, latent membrane proteins (LMP) 1 and 2, which are expressed in Epstein Barr virus-positive malignancies. Stimulation of peripheral blood mononuclear cells with LMP1- and LMP2-expressing actMo activated LMP1- and LMP2-specific T cells, which could be further expanded with LMP1 or LMP2 expressing lymphoblastoid cell lines. The use of actMo as APCs simplifies the production/manufacture of antigen-specific T cells for clinical trials.

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

The survival of pediatric patients with multiply relapsed and/or refractory (R/R) B-cell acute lymphoblastic leukemia has historically been very poor; however, data are limited in the current era. We conducted a retrospective study to determine the outcome of multiply R/R childhood B-ALL treated at 24 TACL institutions between 2005 and 2013. Patient information, treatment, and response were collected. Prognostic factors influencing the complete remission (CR) rate and event-free survival (EFS) were analyzed. The analytic set included 578 salvage treatment attempts among 325 patients. CR rates (mean ± SE) were 51 ± 4% for patients with bone marrow R/R B-ALL who underwent a second salvage attempt, 37 ± 6% for a third attempt, and 31 ± 6% for the fourth through eighth attempts combined. For patients achieving a CR after their second, third, and fourth through eighth attempts, the 2 year EFS was 41 ± 6%, 13 ± 7%, and 27 ± 13% respectively. Our results showed slight improvement when compared to previous studies. This is the largest and most recent study to date that evaluates the outcome of this patient population. Our data will provide detailed reference for the evaluation of new agents being developed for childhood B-ALL.

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.

Invasive Candida Infections in Pediatric Patients Treated on the Pilot Study of Decitabine and Vorinostat with Chemotherapy for Relapsed ALL: A Report from the Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Consortium

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.