GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Stefanía Bahamonde OlayaDiana Agudelo Jaramillo

Teacher Lina Martínez Medicine Faculty

III Semester

GENERALIDADES

LEUKEMIA is a neoplastic disease that occurs as a result of unregulated proliferation and incomplete modulation of cellular precursors of hematopoietic and lymphoid tissues.

In BONE MARROW tumor cells displace the remaining normal series and later spreads to other sides of the body:

Lymph-nodes Liver

Spleen

GRENERALIDADES

It is a type of cancer that begins in the white blood cells in the bone marrow.

Over time these cells tend to accumulate and many people with the disease do not show symptoms for at least a few years.

We have discovered two types: -one fast progress.- one slower progress.

GRENERALIDADES

B lymphocytes B lymphocytes (cells that protect the body against invading germs by maturing into plasma cells that produce antibodies) are the cells that are affected more frequently in CLL.

SYMPTOMS SYMPTOMS -Weakness -Sensation of fatigue -Weight-Loss -Fever -Enlarged lymph-nodes

INTRODUCTION

GALNT11 GALNT11 is a variety of the 14 GALNTs this overexpressed in patients with CLL, in B cells.

TN ANTIGEN TN ANTIGEN is the first product GalNAc transferase.

MOLECULAR MARKER MOLECULAR MARKER is a character or a gene may be used to indicate the presence of another gene.

INTRODUCTION

THIS ANALYSIS IS COSTLY AND DIFFICULT

THERE ARE TWO PROFILES AND A WIDE RANGE OF DIFFERENT MANIFESTATIONS. •(IGHV) MUTATED (M) ---> BETTER PROGNOSIS •(IGHV) UNMUTATED (UM) --> WORSE PROGNOSIS

THE ARTICLE IS BASED ON RECENT DEVELOPMENTS. SUCH US

INTRODUCTION

ALTERNATIVES WERE SEARCHED FOR AS MARKERS ZAP-70 CD38 LPL

ASSOCIATED WITH UM

BUT THE CORRELATION IS NOT ABSOLUTE.

INTRODUCTION

THEREFORE THE EXPRESSION OF THE ENZYMES OF THIS PROCESS IN CANCER CELLS (GALNTS) WAS INVESTIGATED.

SPECIFICALLY GALNT11

EXPRESION OF RESULTING OF AN INCOMPLETE O-GLYCOSYLATION EXPRESION OF RESULTING OF AN INCOMPLETE O-GLYCOSYLATION

ONE OF THE MOST IMPORTANT ALTERATIONS IN CANCER CELLS

TN ANTIGENTN ANTIGEN

GENERAL OBJETIVE

THE GOAL IS TO STUDY GALNT11 AS POSSIBLE NEW MOLECULAR

MARKER IN CHRONIC LYMPHOCYTIC LEUKEMIA.

TO USE IT AS A DIAGNOSTIC TEST FOR THIS SPECIFIC TYPE OF LEUKEMIA AND BUILD MORE APPROPRIATE AND SPECIFIC

MATERIALES Y MÉTODOSMATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

SANGRE PERIFÉRICACONTROLES

SANOS PACIENTES

CLL

CELULAS BCELULAS T

CENTRIFUGARONPURIFICARON

MATERIALES Y MÉTODOS

EXTRACCIÓN DE RNA

SE TRATÓ CON AMBION(TRATAMIENTO LIBRE DE DNA)

PARA IDENTIFICAR LA EXPRESIÓN DE DIFERENTES TEJIDOS

VARIOS CICLOS DE PCR CONVENCIONAL

MMLV(TRANSCRIPTASA REVERSA)

RT-PCR

LPL GALNT

MATERIALES Y MÉTODOS

QUANTITATIVE REAL TIME PCR (q-PCR)

SYBR GREEN (SUSTITUTO DE BROMURO DE ETIDIO) TINCIÓN

CONTROL DE CONTAMINACION( 6 DONANTES SANOS)PCR POR DUPLICADO(TODO) FUSION DE CURVAS

PRIMERS

AMPLIFICACIÓN GAPDH (GEN DE REFERENCIA)

CD3 (MARCA DE LINFOCITOS T)

GALNT11

AMBION Y RETROTRASNCRIPCIÓN

FLUORESCENCIA EN RELACIÓN A LA CANTIDAD DE ÁCIDOS NUCLEICOS

MATERIALES Y MÉTODOS

ANTICUERPOS Y LECTINAS

SE FORMO

DETECTAN PRESENCIA DE TN ANTIGENO

ANTIGALNAC-ANTIBODY DE

CONEJO

MAB B72.3 (AC)

MAB 83D4 (AC) IGM

DETECTA PROTEÍNA RECOMBINADA DE GALNAC-T11

PROTEINAS DE UNION ESPECIFICA Y REVERSIBLE A CARBOHIDRATOS

VVB4 LECTIN (SIGMA)

MATERIALES Y MÉTODOS

EXTRACCION DE PROTEINAS,SDS PAGE, WESTERN BLOT

SEPARACION DE PROTEINAS SEGUN TAMAÑO POR EF EN PAGE

FILTRO

EN ESTA EF SON DISUELTAS EN SDS(DETERGENTE DUODENIL SULFATO ) CARGA

NEGATIVA

GAPDH AC FUE USADA COMO CONTROL

INCUBACION CON AC

CUANTIFICACION DE PROTEINAS POR BCA-ENSAYO

CUANTIFICACION RELATIVA POR GENTOOLS

QUIMIOLUMINISCENCIA

MATERIALES Y MÉTODOS

CITOMETRÍA DE FLUJO

IDENTIFICA INCLUSO PEQUEÑAS POBLACIONES CELULARES

UNION AC CON MOLECULA FLUORESCENTE ( ABSORBE LUZ UV)

CON ALTA ESPECIFICIDAD

A PARTIR DEL CD Y MIDE VARIOS PARAMETROS

INMUNOFLUORESCENCIA

REACCION ANTIGENO-AC

TRANSCRIPTASA INVERSA

PBS( BUFFER FOSFATO SALINO)

DETECCIÓN DE HORMONAS Y PROTEÍNAS

RESULTS

Control

VVB4VVB4

Daudi

Anticuerpo secundario

RESULTS

Jurkat Tn (VVB4) 20 Tn (VVB4)

5/6 Muestra de pacientes con CLL, se encuentran en el rango de 22-51% con marcador de VVB4.

RESULTS

Jurkat Tn (83D4) 20 Tn (83D4)

Muestra de pacientes con CLL, se encuentran en el rango de 0.1–0.9% con marcador de mAB 83 D4.

RESULT

A549 sialyl-Tn 41 sialyl-Tn

6 CLL PACIENTS

Control Positivo

ANTI SIALYL-TN ANTICUERPO B72.3

ANTI SIALYL-TN ANTICUERPO B72.3

RESULTS

CONTROL NEGATIVO

Intensidad depende de la concentración

de la PCR

Intensidad depende de la concentración

de la PCR

POSITIVO- CLL- CÉLULAS T- JURKAT

NEGATIVO- CÉLULAS B- DAUDI

RESULTS

?

CD3MARCADOR CÉLULAS T

CD3MARCADOR CÉLULAS T

Buena expresiónde GalNAc-Tn

RESULTS

Intensidad según expresión de proteína.

Proteínas GalNAc-Tn se expresan bien en muestras

de CLL

RESULTS

ANTI-T11

ANTI-CD19CD19

MARCADOR CÉLULAS B

CD19MARCADOR CÉLULAS B

RESULTS

PBMC from healthy donors

T11 CD19 T11 + CD19

ANTI-T11ANTI-CD19

RESULTS

ANTI-T11

ANTI RABBIT TOTAL Ig

ANTI-T11 + PEPTIDO

MUY ESPECÍFICOMUY ESPECÍFICO

RESULTS

MUTADOS = MEJOR PRONÓSTICO

NO MUTADOS = MAL PRONÓSTICO

RESULTS

DISCUSSION

DISCUSSION

CONCLUSION

CONCLUSION

MAPA

Stefanía Bahamonde Olaya

MAPA

Diana Agudelo Jaramillo

BIBLIOGRAPHY

- MARTINEZ SÁNCHEZ, Lina María. (2012) . Class book , Molecular Biology , 7 ed. Colombia, Medellín: UPB Medical Faculty, Editorial Universidad Pontificia Bolivariana pp.7-8.

- M.G. Libisch, M. Casás, ML. Chiribao, P. Moreno, A. Cayota. (2013 September 17). GALNT11 as a new molecular marker in chronic lymphocytic leukemia. Elsevier B.V. Retieved from: http://www.sciencedirect.com/science/article/pii/S0378111913012572