Molecular and clinicopathologic characterization of pediatric histiocytoses.

The spectrum of somatic mutations in pediatric histiocytoses and their clinical implications are not fully characterized, especially for non-Langerhans cell histiocytosis (-LCH) subtypes. A cohort of 415 children with histiocytosis from the French histiocytosis registry was reviewed and analyzed for BRAFV600E . Most BRAFWT samples were analyzed by next-generation sequencing (NGS) with a custom panel of genes for histiocytosis and myeloid neoplasia. Of 415 case samples, there were 366 LCH, 1 Erdheim-Chester disease, 21 Rosai-Dorfman disease (RDD), 21 juvenile xanthogranuloma (JXG, mostly with severe presentation), and 6 malignant histiocytosis (MH). BRAFV600E was the most common mutation found in LCH (50.3%, n = 184). Among 105 non-BRAFV600E -mutated LCH case samples, NGS revealed mutations as follows: MAP2K1 (n = 44), BRAF exon 12 deletions (n = 26), and duplications (n = 8), other BRAF V600 codon mutation (n = 4), and non-MAP-kinase pathway genes (n = 5). Wild-type sequences were identified in 17.1% of samples. BRAFV600E was the only variant significantly correlated with critical presentations: organ-risk involvement and neurodegeneration. MAP-kinase pathway mutations were identified in seven RDD (mostly MAP2K1) and three JXG samples, but most samples were wild-type on NGS. Finally, two MH samples had KRAS mutations, and one had a novel BRAFG469R mutation. Rarely, we identified mutations unrelated to MAP-kinase pathway genes. In conclusion, we characterized the mutational spectrum of childhood LCH and clinical correlations of variants and subtypes. Variants responsible for JXG and RDD were not elucidated in more than half of the cases, calling for other sequencing approaches.

Functional evidence for derivation of systemic histiocytic neoplasms from hematopoietic stem/progenitor cells.

Langerhans cell histiocytosis (LCH) and the non-LCH neoplasm Erdheim-Chester disease (ECD) are heterogeneous neoplastic disorders marked by infiltration of pathologic macrophage-, dendritic cell-, or monocyte-derived cells in tissues driven by recurrent mutations activating MAPK signaling. Although recent data indicate that at least a proportion of LCH and ECD patients have detectable activating kinase mutations in circulating hematopoietic cells and bone marrow-based hematopoietic progenitors, functional evidence of the cell of origin of histiocytosis from actual patient materials has long been elusive. Here, we provide evidence for mutations in MAPK signaling intermediates in CD34+ cells from patients with ECD and LCH/ECD, including detection of shared origin of LCH and acute myelomonocytic leukemia driven by TET2-mutant CD34+ cell progenitors in one patient. We also demonstrate functional self-renewal capacity for CD34+ cells to drive the development of histiocytosis in xenotransplantation assays in vivo. These data indicate that the cell of origin of at least a proportion of patients with systemic histiocytoses resides in hematopoietic progenitor cells prior to committed monocyte/macrophage or dendritic cell differentiation and provide the first example of a patient-derived xenotransplantation model for a human histiocytic neoplasm.

Circulating cell-free BRAFV600E as a biomarker in children with Langerhans cell histiocytosis.

The BRAFV600E mutation is reported in half of patients with Langerhans cell histiocytosis (LCH). This study investigated the detection of the BRAFV600E allele in circulating cell-free (ccf) DNA in a paediatric LCH cohort. Children with BRAFV600E -mutated LCH were investigated to detect ccf BRAFV600E at diagnosis (n = 48) and during follow-up (n = 17) using a picolitre-droplet digital PCR assay. At diagnosis, ccf BRAFV600E was positive in 15/15 (100%) patients with risk-organ positive multisystem (RO+ MS) LCH, 5/12 (42%) of patients with RO- MS LCH and 3/21 (14%) patients with single-system (SS) LCH (P < 0·001, Fisher's exact test). The positive BRAFV600E load was higher for RO+ patients (mean, 2·90%; range, 0·04-11·4%) than for RO- patients (mean, 0·16%; range, 0·01-0·39) (P = 0·003, Mann-Whitney U test). After first-line vinblastine-steroid induction therapy, 7/7 (100%) of the non-responders remained positive for ccf BRAFV600E compared to 2/4 (50%) of the partial-responders and 0/4 of the complete responders (P = 0·002, Fisher's exact test). Six children treated with vemurafenib showed a clinical response that was associated with a decrease in the ccf BRAFV600E load at day 15. Thus, ccf BRAFV600E is a promising biomarker for monitoring the response to therapy for children with RO+ MS LCH or RO- LCH resistant to first-line chemotherapy.

Variation of mutant allele frequency in NRAS Q61 mutated melanomas.

BACKGROUND: Somatic mutations of BRAF or NRAS activating the MAP kinase cell signaling pathway are present in 70% of cutaneous melanomas. The mutant allele frequency of BRAF V600E (M%BRAF) was recently shown to be highly heterogeneous in melanomas. The present study focuses on the NRAS Q61 mutant allele frequency (M%NRAS). METHODS: Retrospective quantitative analyze of 104 NRAS mutated melanomas was performed using pyrosequencing. Mechanisms of M%NRAS imbalance were studied by fluorescence in situ hybridization (FISH) and microsatellite analysis. RESULTS: M%NRAS was increased in 27.9% of cases. FISH revealed that chromosome 1 instability was the predominant mechanism of M%NRAS increase, with chromosome 1 polysomy observed in 28.6% of cases and intra-tumor cellular heterogeneity with copy number variations of chromosome 1/NRAS in 23.8%. Acquired copy-neutral loss of heterozygosity (LOH) was less frequent (19%). However, most samples with high M%NRAS had only one copy of NRAS locus surrounding regions suggesting a WT allele loss. Clinical characteristics and survival of patients with either <60% or ≥60% of M%NRAS were not different. CONCLUSION: As recently shown for M%BRAF, M%NRAS is highly heterogeneous. The clinical impacts of high M%NRAS should be investigated in a larger series of patients.

Detection of BRAF p.V600E mutations in melanomas: comparison of four methods argues for sequential use of immunohistochemistry and pyrosequencing.

BRAF p.V600 mutation detection recently became necessary to treat metastatic melanoma patients with vemurafenib. This study compares different methods of detection of BRAF mutations. Melanoma samples from 111 patients were analyzed for BRAF mutations, and for 89 of them, results were obtained with the four following methods: Sanger sequencing, real-time PCR, immunohistochemistry, and pyrosequencing. All samples contained at least 60% of tumor cells. Directional Sanger sequencing of PCR products failed to detect 3 of 40 p.V600E-mutated cases (7.5%) (sensitivity, 92.5%; 95% CI, 78.5% to 98.0%). BRAF p.V600E-specific real-time PCR identified 39 of 40 p.V600E-mutated cases (97.6%) (sensitivity, 97.5%; 95% CI, 87.1% to 99.6%) and all 39 wild-type (WT) cases and surprisingly was also positive for 6/6 p.V600K (specificity, 87.8%; 95% CI, 75.8% to 94.3%). However, other mutations, p.V600R (n = 1), p.K601E (n = 2), and p.600_601delinsE (n = 1), were not detected. Immunohistochemistry with VE1, specific for p.V600E, identified all p.V600E and WT cases (sensitivity, 100%; 95% CI, 91.2% to 100%) but was negative for all other BRAF mutations. Pyrosequencing successfully identified all WT and mutated cases. Immunohistochemistry is highly specific for p.V600E, and could be used as a first-line method, as is currently performed for HER2 amplification detection. Pyrosequencing proved to be the most efficient method to detect BRAF mutations in melanomas and could be performed on VE1-negative or uninterpretable cases.