ALBERT

Description: Langerhans cell histiocytosis (LCH) is a clinically and histologically heterogeneous disorder. Its classification as either reactive inflammatory or neoplastic has been a matter of debate. However, the recent finding of frequent BRAFV600E mutations in LCH argues for the latter. The exact cell type that harbors the mutation and is responsible for proliferation remains to be identified. We here apply a BRAFV600E mutation-specific antibody to detect the BRAF mutant cells in lesions from 89 patients with LCH. We found BRAFV600E mutations in 34 of 89 (38%) lesions. In lesions with the BRAFV600E mutation, the majority of cells coexpressing S-100 and CD1a harbored mutant BRAFV600E protein. These cells also expressed CD14 and CD36, whereas various fractions exhibited CD207. On the other hand, CD80 and CD86 expression was also present on BRAFV600E-positive cells. Thus, cells of variable maturation, exhibiting an immunohistochemical profile compatible either with myeloid cell or with dedifferentiated Langerhans cell antigens, carry the BRAFV600E mutation. In conclusion, we identify and characterize the neoplastic cells in LCH with BRAFV600E mutations by applying a mutation-specific marker and demonstrate feasibility for routine screening.

Description: Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are frequently found in patients with acute myeloid leukemia (AML) and several other tumors. Mutant IDH1 produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, and leads to a block in differentiation to promote tumorigenesis. Methods: We developed a novel, highly active oral pan-IDH1 inhibitor, BAY-1436032, for clinical evaluation. Its inhibitory potency was evaluated in primary human AML cells in vitro for the five major IDH1R132 mutation types and in two patient derived AML xenograft (PDX) models in vivo, in which BAY-1436032 cleared leukemic blasts in peripheral blood and prolonged survival by induction of differentiation and inhibition of leukemia stem cell self-renewal. Results: R-2HG production by mutant IDH1 was effectively inhibited in patient derived AML cells with all reported IDH1R132 mutations ex vivo by BAY-1436032 with an IC50 between 3 to 16 nM. AML cells cultured ex vivo showed morphologic differentiation and marked upregulation of the myeloid differentiation markers CD14 and CD15. For in vivo experiments, human AML cells from two patients were transplanted into sublethally irradiated NSG mice. After stable engraftment at 17 (PDX1) or 90 (PDX2) days post transplantation, mice were treated with BAY-1436032 orally every day at a dose of 150 mg/kg or vehicle for 100-150 days (n=10 per group). The R/S-2HG ratio in serum was reduced to near normal levels by BAY-1436032. Leukemic cell counts in peripheral blood constantly increased in control mice, while leukemic cells declined from day 30 of BAY-1436032 treatment onwards with morphologic and immunophenotypic evidence of differentiation (Figure). Importantly, all BAY-1436032 treated PDX1 mice survived until the end of treatment at 150 days. In contrast, vehicle-treated mice died with a median latency of 91 days (range 70-95, P

Description: Histiocytoses are rare disorders of unknown origin with highly heterogeneous prognosis. BRAF mutations have been observed in Langerhans cell histiocytosis (LCH). We investigated the frequency of BRAF mutations in several types of histiocytoses. Histology from 127 patients with histiocytoses were reviewed. Detection of BRAFV600 mutations was performed by pyrosequencing of DNA extracted from paraffin embedded samples. Diagnoses of Erdheim-Chester disease (ECD), LCH, Rosai-Dorfman disease, juvenile xanthogranuloma, histiocytic sarcoma, xanthoma disseminatum, interdigitating dendritic cell sarcoma, and necrobiotic xanthogranuloma were performed in 46, 39, 23, 12, 3, 2, 1, and 1 patients, respectively. BRAF status was obtained in 93 cases. BRAFV600E mutations were detected in 13 of 24 (54%) ECD, 11 of 29 (38%) LCH, and none of the other histiocytoses. Four patients with ECD died of disease. The high frequency of BRAFV600E in LCH and ECD suggests a common origin of these diseases. Treatment with vemurafenib should be investigated in patients with malignant BRAFV600E histiocytosis.

Description: Abstract 4040 Background: Activating mutations of the serine/threonine kinase BRAF are observed in various solid and hematologic malignancies with the point mutation V600E being the most common. However, both frequency and relevance of the mutation differ greatly between entities. This year, a small molecule inhibitor specifically targeting V600E-mutated BRAF has been approved for metastatic melanoma. Recently, BRAF mutations in multiple myeloma (MM) have been reported by several groups in the context of next generation sequencing projects. However, the clinical relevance of these mutations remains unclear. Objective: We report on frequency and clinical characteristics of MM patients harboring the BRAF V600E mutation. Furthermore, first clinical data on treatment with vemurafenib in MM with BRAF V600E mutation is presented. Methods: A V600E-mutation specific monoclonal antibody was recently developed at our institution. We screened 338 tissue samples by immunohistochemistry (315 bone marrow, 23 soft tissue plasmocytoma) from 310 patients with monoclonal gammopathy collected at out center between April 1999 and May 2011. Positive results were verified by direct sequencing. Of 310 analyzed patients, 205 had symptomatic MM, 12 symptomatic light chain amyloidosis, 45 smoldering MM and 48 MGUS. MM subtypes comprised 174 IgG, 60 IgA, 55 Bence Jones, 14 IgM, 4 IgD, 1 biclonal, 4 asecretory. From 25 patients serial bone marrow samples or tissue from different compartments were available. Median age at biopsy was 61.4 years (range 29–87) and 244 samples were obtained at from newly diagnosed patients. Results: Six patients (2%) were positive for BRAF V600E mutation both by immunohistochemistry and by confirmatory sequencing. A brief overview of patient characteristics is shown in Table 1. Five patients received bortezomib-based first line treatment whereas one received conventional chemotherapy. Four patients underwent autologous transplantation. Interestingly, PFS after start of first-line treatment was relatively short with a median PFS of 10.5 months. Remarkably, four of these six patients developed extramedullary myeloma in their disease course (soft tissues in 2, CNS in 1, both soft tissues and CNS in 1). In two of these patients a re-biopsy after two and three lines of therapy, respectively, was available. In both cases the mutation remained present in all cells without signs of clonal evolution in regard to BRAF V600E. One of the BRAF V600E positive patients had relapsed with multiple soft tissue plasmocytomas after autologous transplantation followed by multiple treatment lines containing bortezomib, lenalidomide and bendamustine. After informed consent we started the patient on vemurafenib 480mg BID and increased the dosage to 720mg BID after one week. Vemurafenib was well tolerated and no grade III/IV adverse events were noted. Already after the first cycle (four weeks), a partial response was achieved according to IMWG and RECIST criteria for serological and radiological assessment, respectively. As assessed by immunohistochemistry, a dramatic decrease in proliferative activity (MIB-1) accompanied by a sharp increase in apoptosis, as well as loss of MAP kinase signaling (p-ERK) could be observed in tumor samples under treatment with vemurafenib. Conclusions: Immunhistochemistry is a rapid and reliable method for the detection of BRAF V600E and provides a useful tool especially if applied to entities with low mutation frequency, such as MM. In our cohort, however small, patients harboring the mutation show a conspicuous course of disease with a comparably short PFS and an unusually high frequency of extramedullary myeloma. Individualized treatment with vemurafenib seems feasible and rapid response was observed. A detailed follow-up of the clinical course will be presented at the meeting. Disclosures: Off Label Use: Vemurafenib is a BRAF inhibitor FDA approved for treatment of metastatic melanoma.