BRAF MUTATIONS IN HAIRY CELL LEUKEMIA

Enrico Tiacci, Vladmir Trinof, Gianluca Schiavoni, Antony Holmes, Wolfgang Kern, Maria Paola Martelli, et al.

Jenniffer Correa GutierrezDaniela Castaño Correa

Molecular Biology Universidad Pontificia Bolivariana

2011

Leukemia

• Acute or chronic neoplastic disease of the bone marrow.

• Unrestrained proliferation of white blood cells.

• Anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen.

Introduction

• Leukemia cells crowd out

the normal blood cells.

• Spreads to the lymph nodes.

• Causes swelling or pain.

Leukemia

Leukemia

• Cell proliferation• Cell differentiation• Cell survival

RAF Family

BRAF Gene

BRAF protein kinase

MEK phosphorilation

RAS- RAF signaling

Mutated BRAF

Increased MEK and ERK

activation

Excessive cell proliferation,

survival to apoptosis

nucleus

RAF

V600E mutation

Oncogenic BRAF:

• BRAF mutations at position

600 (BRAFV600) can lead to

overactive BRAF signaling.

• Amino Acid substitution of

glutamic acid for valine at

position 600 of BRAF protein.

RAF

Hairy Cell Leukemia

Hairy Cell Leukemia

Hematological malignancy

Accumulation of abnormal B

lymphocytes

Sequestration, marginalization, and destruction of blood cells in the spleen

Abundant cytoplasm: Hairy like projections

Hairy Cell Leukemia

Bone marrow, spleen and liver

This pathology is caused by a mutation on the BRAF

protein kinase which normally travels to the nucleus to

control the cell functions. In HCL, the BRAF protein

prevents helps cancer development.

Relationship of RAF with HCL

Oncogenic BRAF signaling:

• Resistance to apoptosis.

• Progression and

maintenance of cancer.

Relationship of RAF with HCL

Scarce tumor cells for analysis.

Low proliferative index of leukemic cells.

Inability to grow HCL in immunodeficient mice.

Main obstacles in HCL characterization:

Relationship of RAF with HCL

Main Objective

• To identify genetic

mutations on hairy

cells through massive

parallel sequencing of

patients with hairy cell

leukemia.

Muestras:• 47 pacientes con presentacion

clinica y morfologica de HCL.

• Positivos para annexin A 1:

analisis immunohistolgico.

• Coexistencia de CD11c, CD25 y

CD103 en citometria de flujo, o

ambos.

Métodos

• Muestras de sangre periferica de paciente índice.

• Secuencial de exomas en celulas leucemicas

purificadas CD19- positivas: tiempo de latencia de

la enfermedad (>90%).

• Secuencial de exomas en celulas leucemicas

purificadas CD19- negativas mononucleadas:

quimioterapia.

Métodos

• Consenso informado: analisis genético.

• Protocolo aprovado por el comité de la Universidad

de Perugia.

• Otros pacientes: consenso informado oral.

Métodos

Secuencial de exoma:

• Seleccion de regiones que codifican en un genoma

humana para identificar genes asociados con

desordenes poco comunes.• Exomas secuenciales: Se encontraron mutaciones somaticas en los candidatos por medio de Genome Analyzer IIx (Illumina).

Métodos

PCR: polymerase-chain-reaction

• Amplificacion enzimatica in vitro directa de un gen o

un fragmento de DNA o indirecta de RNA.

• Desnaturalizacion del DNA para dar hebras sencillas.

• Hibridacion especifica: primer.

• Replicacion: DNA polimerasa.

• Rastreo de mutaciones.

Métodos

Inmunofluorescencia:

• Determina proteinas y

hormonas.

• El fluorocromo es una

sustancia que absorbe luz

(200-400nm).

• Emite luz (400-700nm).

Métodos

Western Blot

• Variación de transferencia southern.

• SDS- PAGE poliacrilámida gel

electroforesis.

• SDS : detergente con carga (-)

• Incubación con anticuerpos.

• Determina proteína.

Métodos

Resultados

Resultados

Pruebas inmunohistologicas y Western Blot:

Celulas HCL expresaron fosforilacion de MEK y ERK.

Activacion constitutiva de RAF-MEK-ERK, mediante la via protein-kinasa en HCL

activada por mitogeno.

El PLX-4720 es un inhibidor especifico del BRAF activo que marca una disminucion en

la fosforilacion de ERK y MEK.

Resultados

Resultados

DiscussionAuthor’s Name What the author said Do you agree with the

author?

Keshet Y and Seger R.

The BRAF protein ( RAS-RAF-MAPK ) signaling pathway.It plays a role in regulating cell survival, proliferation and differentiation.

Yes

Wan PT, Garnett MJ, Roe SM, et

al.

The patients who carried BRAF mutations had the V600E phosphomimetic substitution, which markedly enhances its kinase activity in a constitutive manner.

Yes

Secuencial

Author’s Name What the author said Do you agree with the author?

Slupsky JR, Kamiguti AS, Harris RJ,

Cawley JC, Zuzel M.

ERK provided a survival signal for the leukemic

hairy cellsYes

Lee JW, Yoo NJ, Soung YH, et al.

BRAF mutations have been reported in peripheral B

cell lymphomaYes

Conclusions

• The study of molecular biology

laboratory techniques has helped

identify the characteristics of

cells that are specific to a

disease.

• The BRAF V600E mutation was

present in all patients with HCL,

which can lead in the future to a

greater understanding of this

pathology.

Conclusions

• This study can incite

scientists to research an

effective treatment for

patients with HCL.

• Knowledge of this gene can

lead to future early

prevention of HCL and other

kinds of leukemia.

Jenniffer Correa

Daniela Castaño

Bibliography

• WebMD. Leukemia - Topic Overview. [ August 3, 2011]. [Internet];

Available on: http://www.webmd.com/cancer/tc/leukemia-topic-

overview

• Bio Oncology. What is BRAF? [ August 3, 2011]. [Internet]; Available

on: http://www.biooncology.com/research-education/braf/braf/in

dex.ht ml

• Martinez S LM. Biología Molecular. Ed 6. Medellin. 2011. Pg 113, 131,

143.

THANK YOU!