Genetics of Langerhance Cell Histiocytosis Dr Mohammad Keramatipour MD, PhD Tehran University of Medical Sciences

Overview Langerhance Cell Histiocytosis (LCH) ?? Genetics of LCH Genetic testing Role in targeted therapy and prognosis Role of new sequencing technologies in LCH management

Langerhans Cell Histiocytosis Langerhans Cell Histiocytosis (LCH): a systemic disease associated with the proliferation and accumulation (granulomas) of Langerhans cells in various tissues Langerhans cells play an immunologic role in protecting against environmental antigens

Langerhans Cell Histiocytosis Epidemiology 1-2/100,000 Age of diagnosis: ~ 1-3 years Etiology Inflammatory, immunologic, or even neoplastic ?? Inflammatory myeloid neoplasia Genetics?? Familial clustering Concordance rate in monzygotic twins

LCH Pathogenesis RAS/RAF/ERK signaling pathway: Key role in cell growth and proliferation

RAS/RAF/ERK Pathway

BRAF gene BRAF gene A proto-oncogene located on 7q34 21 exons Encodes B-RAF protein 766-amino acid protein Member of the RAF kinase family Substantial role in regulating the RAS/RAF/ERK signaling pathway B-RAF: three conserved domains: conserved regions 1-3

B-Raf protein CR1 CR2 CR3 B-Raf activation RAS interaction A flexible linker that connects CR1 and CR3, acts as a hinge CR3 B-Raf enzymatic kinase domain 457- 530: N-lobe, responsible for ATP-binding 535- 717: C-lobe, binds substrate proteins B-Raf activation

BRAF Mutations BRAF germline mutations / AD conditions Cardio-facio-cutaneous syndrome, type 1 Leopard syndrome, type 3 Noonan syndrome, type 7 BRAF somatic mutations Melanoma Lung cancers Colorectal cancers ……

BRAF mutations BRAF somatic missense mutations A majority of mutations within the kinase domain p.V600E The most common mutation (90%) of the BRAF gene The most common mutation tested for in clinical laboratories 66% of malignant melanomas and at lower frequency in a wide range of human cancers non-Hodgkin lymphoma colorectal cancer thyroid carcinoma non-small cell lung carcinoma hairy cell leukemia adenocarcinoma of lung

BRAF mutations Other more common pathogenic mutations: R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, A727V. Most of these mutations are clustered to two regions: Glycine-rich P loop of the N lobe Activation segment and flanking regions These mutations change the activation segment from inactive state to active state.

BRAF mutations Mutated BRAF proteins elevated kinase activity cancer cell lines with the V600E mutation have a RAS-independent growth Vemurafenib (PLX4032) A highly selective and potent inhibitor of BRAF V600E. It has marked antitumor effects against melanoma cell lines with the BRAF V600E mutation but not against cells with wild-type (non-mutated) BRAF. Vemurafenib improves overall and progression-free survival in patients with advanced melanoma with the V600E mutation.

Redefining LCH 1982: RAS mutations causing tumor ! Targeted therapy prevents tumor growth Mutations in others genes has the same effect RAF genes: BRAF, ARAF MEK genes: MAP2K1, MAP3K1 Genetic testing in cancer Personalized Cancer Management

LCH Pathogenesis

RAS/RAF/MEK Pathway

Genetic Testing in Cancer Genetic testing in cancer management Screening Hereditary cancers Assisted diagnosis Targeted therapy Molecular classification of tumors Monitoring treatment / recurrence

Genetic Testing in LCH First / Second lines: NGS for all related genes Tumor sample / blood sample ??? First / Second lines: Testing for V600E (c.1799T>A), and T599A Analysis of exons 2 and 3 of MAP2K1 NGS for all related genes Panel: BRAF, ARAF, MAP2K1, MAP3K1, ERBB3 Whole Exome Sequencing

Challenges in Genetic Testing Detecting somatic mutations Solutions: Sampling Analysis: NGS depth / Sanger sequencing

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