aplicaciones de la biología molecular en diagnóstico clínico€¦ · molecular diagnostics ~10%...

Aplicaciones de la Biología Molecular

en Diagnóstico Clínico

Jacobo Zuñiga Castillo PhD

~48% Molecular

Diagnostics

~10% Applied Testing

~19% Pharma

~23% Academic Research

SALES QIAGEN’s adjusted net sales grew 6% CER in FY 2017

$1.42 bn

EMPLOYEES QIAGEN has been repeatedly recognized as an employer of choice with leading HR programs.

4,800

EXPERIENCE QIAGEN was founded in 1984 by scientists from the Heinrich Heine University in Düsseldorf, Germany.

1984

LOCATIONS QIAGEN maintains subsidiaries in 20 countries and has more than 70 sales offices worldwide. 35

CUSTOMERS QIAGEN serves a truly global customer base across various markets in life science and healthcare.

500,000

PATENTS QIAGEN invests ~10% of its sales into R&D to continuously drive innovation and growth.

>2,200

PRODUCTS QIAGEN markets a broad range of consumables and instruments for various customer needs.

500

Facts and figures 2018

3

QIAGEN‘s Global Reach

QIAGEN subsidiary

Distributor

Venlo

Shanghai Germantown

Hilden

Over 35 subsidiaries in more than 20 countries

Distributor network in 70 countries

Dynamic growth in top 7 emerging markets

47%

20%

32%

4

We help customers gain insights to improve healthcare and advance science

• Complex Sample

•Sample Preparation •Pure Genes •Results

•Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, Immunoglobulins, DNA, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Carrier proteins

Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, Immunoglobulins, DNA, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Carrier proteins

DNA Information/

Insights

Virus detected

5

Multi-channel approach based on modern technologies

SAMPLE TO INSIGHT

Sample Tech

Assay Tech

Bioinfor- matics

Insights Samples

Our Core Our Customers Our Channels

Field Sales /Service

Inside Sales

Telemarketing

eCampaigns

QIA-Webshop

QIAcabinet, B2B digital

Academia

Pharma

HID/Forensic/ Food/Vet

Customer

Customer

Customer

… and with a very strong focus on Customer Intimacy

Diagnostics Customer

Clinical Sales Force

6

Cost of Sequencing is falling fast

9

QIAGEN: el primero en revolucionar la extracción de DNA…

…mediante el concepto de mini columnas de alta calidad!!!

1993 1953 …...la época oscura……

Estructura de DNA descrita Y publicada

Purificación de DNA con ultracentrifugación

QIAGEN introduce la mini columna

Extracción con fenol cloroformo

Cell cycle Angiogenesis

P53

MDM2

Cyclins

CDKs

VEGF

bFGF

ANGPT

PDGF

IL8

TLR

IFNγ

TNFβ Ind

ivid

ual

p

arti

cip

ants

Inflammation

Biological Markers Define Biological Processes

Path

way

Cell cycle Angiogenesis Inflammation

Complete Biological Story Built on Pathway / Network Analysis

• Assessing Changes (often in diseased/treated samples)

VEGF Expression Angiogenesis

Non-Cancer Cancer

0 10 YES

0 0 ?

If VEGF does not change in expression, traditionally you might interpret as no Angiogenesis.

But what if FLT3 (the VEGF receptor) or a downstream signaling partner is up-regulated…and makes the signaling pathway active?

Angiogenic signals will be activated in the nucleus.

\ A complete biological story is based on assessing all participants

Differential (∂) Gene Expression Analysis

10,000

89

1

# genes

Focused Pathway

Single Gene

Transcriptome

384

30,000+

5

Discovery

Focused Biological Discovery / Validation

Validation

12

Where Does The RT2 Profiler PCR Array fit into my Research?

•84 Pathway-Specific Genes of Interest

•5 Housekeeping Genes

•Genomic DNA Contamination Control

•Reverse Transcription Controls (RTC)

•Positive PCR Controls (PPC)

•Any Gene (up to 4)

Anatomy of “Modified” RT2 Profiler PCR Array

96 well version (1 sample/PCR Array) (384 well version available (4 samples/PCR Array)

Algunas definiciones….

Definicion de PCR

PCR = Reacción en cadena de la Polimerasa

Método in vitro rápido, simple, sensible y versatil para

amplificar selectivamente secuencias/regiones de DNA/RNA

a partir de una muestra compleja de ácidos nucleicos.

aguja

Definición PCR

Técnica de Biología Molecular que permite la producción

de grandes cantidades de una secuencia específica de

DNA a partir de un templado de DNA (mezcla compleja de

DNA) empleando una reacción enzimática sin la

participación de un organismo vivo (in vitro)

PCR: Fundamento

En 1983 Kary Mullis desarrolló el procedimiento conocido como PCR (reacción en cadena de la polimerasa)

Se desarrolla en tres etapas:

• Desnaturalización del templado (95°C)

• Alineamiento de Primers (50-60°C)

• Elongación de la cadena (72°C)

F:/QIAGEN/Cursos y Presentacions de Valaner/PCR.swf

Ventajas de un PCR •Rapidez

•Alta sensibilidad

•Método robusto

•Alta Especificidad

•Alta Fidelidad

•Flexibilidad de la metodología

•Standarización de los métodos

•Facilidad de uso

Limitaciones de la PCR

•Se necesita conocer la secuencia a amplificar

•El tamaño de los fragmentos amplificados es

relativamente pequeño

•La PCR estándar no es una técnica adecuada para

análisis cuantitativos

¿De dónde obtenemos el DNA para trabajar?

Las muestras que utilizamos son muy variadas y PEQUEÑAS: sangre, biopsias, cultivo celular, tejidos varios etc.,

En nuestro genoma hay pocas copias de cada gen, entonces NO SON SUFICIENTES para realizar estudios posteriores.

El DNA es una muestra compleja, ¿cómo podemos aislar específicamente las secuencias que nos interesan?

PROBLEMA OBVIO: ¿Cómo obtener MUCHAS copias IDENTICAS de los genes o secuencias que nos interesan?

Diagrama de flujo para una PCR

Obtención de la muestra y

estabilización del DNA

Lisis de la muestra

y purificación del DNA

PCR Análisis

de la PCR

Factores que afectan una PCR

•Diseño de los primers

•Calidad del templado de DNA: presencia de contaminantes e

inhibidores.

•Buffer en donde se conserva el templado de DNA

•Concentración de dNTPs

•Concentración de primers

•Concentración de magnesio

•Concentración de templado

•Temperatura de alineamiento

Materiales utilizados para la PCR

•Reactivos – Polimerasas – Primers – dNTPs – Buffer – Templado de DNA

•Equipo: Termocicladores

– Estandar – Gradiente – In situ – Tiempo Real

Duplex DNA

94oC Desnaturalizacipón y alineamiento de primers

Primers forward y Reverse

60oC

72oC Extension

Ciclo

Desnaturalización -- alineamiento -- Extensión

El ciclo de la PCR

Gen - Específicos

Random hexamers

Degenerados

Tipos de Primers

¿Cómo definimos a un primer? Fragmentos cortos de DNA que son complementarios a la región blanco, es decir, aquella que se desea amplificar

Primers

Herramientas para el diseño de primers

•MethPrimer

•Oligo Analizer

•Oligo calc

•Webprimer

Polimerasa

Sus características determinan el tipo de amplificación:

Procesividad: La “distancia” que la enzima permanece unida al DNA, determinan el tamaño del fragmento

Termoestabilidad: La estabilidad a altas temperaturas, determinan tamaño y rendimiento

Fidelidad: (actividad PROOFREADING): Corrección de errores al sintetizar fragmentos de DNA, procedimientos como clonación

Tipo de extremos: Dos tipos de extremos blunt o romos y overhang o cohesivos, determinan posibilidad de clonación

Templado de DNA

Algunas recomendaciones generales son las siguientes:

•El DNA o RNA debe de encontrarse íntegro

•Se debe evitar la contaminación de las muestras de RNA con DNA

•Las muestras deberán ser purificadas cuidadosamente para eliminar los

inhibidores de la PCR (iones, solventes orgánicos etc)

•Preferentemente las muestras de DNA se almacenan en Tris 10 nM pH 7.5

Fuente • plásmido

• genómico

• viral

• cDNA Cantidad

• 0.1-10ng plasmido

• 0.1-1ug gDNA

Calidad

Sin inhibidores

Características del DNA molde

SDS >0.005% (W/V)

Fenol >0.2% (V/V)

Etanol >1%

Isopropanol >1%

Acetato de Sodio > 5 mM

Cloruro de Sodio > 25 mM

EDTA > 0.5 mM

Hemoglobina >1 mg/ml

Heparina > 0.15 I.U./ml

PCR Inhibidores

SDS Phenol EtOH NaAc NaCl EDTA

- - - - - -

Efecto de Inhibidores

Hay que optimizar

– [Mg2+]

– Temperatura de alineamiento

– Concentración de aditivos

– Número de ciclos

– Secuencia de los primers

– Concentración de los templados

– Tiempos de alineamiento y extensión

[Mg2+] mM Temperatura

[Mg2+] y Temperatura

Factores que influyen en la especificidad de la PCR

•Cantidad inicial de templado

•Diseño de las primers

•Cationes contenidos en los buffers de reacción

•Generación inicial de productos no especificos por la acción de la taq

polimerasa.

Composición exclusiva del buffer de reacción

Destabilization

Templado Primer

Stabilization Stabilization

Mayor

especificidad

+

NH4+ Buffers

Desestabiliza la unión no especifica de primers

Comparación de dos tipos de buffer en relación a la concentración de magnesio

PCR: 150 ng DNA genómico,.

NH4+ buffer

M M 1.5 1.5 4 4 mM Mg2+

750 bp

Sin buffer NH4+

NH4+ Buffers

Desestabiliza la uniòn no especifica de primers

Dímeros de primers en NTC muestran reacciones no especìficas

NH4+ Buffer Buffer sin NH4

+

Gene: Human BAX cDNA: 100 pg – 10 ng Primers: Hs_Bax_Primer Assay Cycler: Rotor-Gene 3000

Señal de dìmeros de primers en

NTC

No hay señal de dimeros en NTC

Señal de dimeros de primers en NTC

Tecnicas de PCR • RAPD

• AFLP

• PCR de colonias

• SSCP PCR (análisis conformacional de polimorfismos de cadena sencilla)

• PCR Hot Start

• PCR Inverso

• PCR In situ

• PCR para fragmentos largos

• RT – PCR

• PCR en Tiempo Real

• PCR de una célula

• PCR Multiplex

RT-PCR

Transcriptasa Reversa PCR

RT-PCR

Las principales aplicaciones de esta técnica son:

• Análisis de expresión:

– Cuáles genes están encendidos

– Estimado de cuánto están encendidos

• Construcción de bibliotecas de expresión (cDNA)

El templado inicial es RNA total o RNA mensajero

¿Entonces que necesitamos para amplificar RNA?

Una enzima que transforme el RNA en DNA para poder hacer una PCR:

La Transcriptasa Reversa (Transcripción reversa de la reacción en cadena de la polimerasa)

Cuáles son los reactivos necesarios para una RT-PCR?:

•Transcriptasa Reversa •Oligos •RNAsa •Reactivos para PCR

Aplicaciones en genómica y expresión

Genómica Expresión

• Identificación de genes • Cambios en la secuencia: • Polimorfismos • Mutaciones • Deleciones • Inserciones • Clonación • Identificación de

promotores

• Función de los genes • Mecanismos celulares • Regulación génica • Patrones de expresión

• Diseño y evaluación molecular de tratamientos

• Detección/diagnóstico de enfermedades

PCR en TIEMPO REAL

DNA RNA Proteína Genes y Promotores

Genotipificación

Clonación de Genes y promotores

Expresión

Manifestaciones Físicas o Fenotipo Actividad Función

Genotipificación: Secuencia de Genoma Identificar genes Encontrar diferencias, mutaciones

Puntos de regulación: Ensayos in vivo e in vitro como tansfecciones, CHIPs etc,

Interacciones DNA-Proteína (cromatina), RNAi, miRNA, epigenética

¿Cuáles genes se están transcribiendo

? ¿Qué tanto se están

transcribiendo?

¿Son activas/funcionales? ¿Qué tan activas? ¿Se unen a otras

proteínas?

Dogma Central de la Biología Molecular: Aplicaciones

Cuantificación

DNA DNA

GEN A GEN B

TRANSCRIPCIÓN = EXPRESIÓN

4 COPIAS

9 COPIAS

DNA DNA

GEN A GEN B


7 COPIAS

2 COPIAS

Células Epiteliales Normales: Perfil de Expresión

Células Epíteliales Cancerosas: Perfil de Expresión

RNA RNA

Perfiles de expresión

Gen de Referencia

– Se utiliza un gen de referencia para normalizar él ensayo:

• El gen de referencia es un gen cuya expresión debe ser constante en cualquier

condición analizada (tratamientos, enfermedades etc)

– La normalización con un gen de referencia permite:

• Corregir variaciones por la cantiodad de muestra (sangre, tejido, etc)

• Corregir las variaciones de RNA agregado en cada reacción

• Corregir variaciones debido al pipeteo

• Corregir la eficiencia de la RT

• Recobro de RNA en el proceso de extracción etc.

DNA DNA

GEN A GEN B


4 COPIAS

9 COPIAS

DNA

GEN HK

DNA DNA

GEN A GEN B


7 COPIAS

2 COPIAS

DNA

GEN HK

2 COPIAS 2 COPIAS

Perfiles de expresión: Normalización



Perfiles de expresión: Análisis

DNA DNA

GEN A GEN B


4 COPIAS 9 COPIAS

DNA

GEN HK

DNA DNA

GEN A GEN B


7 COPIAS 2 COPIAS

DNA

GEN HK

2 COPIAS 2 COPIAS



Normalización = GOI/GHK

VN = 2 VN = 4.5 VN = 3.5 VN = 1

Normalización = GOI/GHK

Gru

po

Co

ntr

ol

Gru

po

Mu

est

ra

Calibradores

Nivel de expresión = Muestra

Calibrador

GEN A

GEN B

1.75

0.29

=

=

Un poco de historia… ¿Cómo se hacía antes?

PCR Punto Final

DOR 2.6 =

Muestra Calibrador

Extracción RNA RNA RT - PCR Gel Agarosa

http://www.jbc.org/content/277/15/12854/F1.large.jpg

http://www.jbc.org/content/277/15/12854/F1.large.jpg

¿Cuáles son las limitaciones de la PCR en punto final?

Limitaciones del PCR punto final utilizando sistemas de detección Basados en análisis electroforético

Bromuro de etidio no es cuantitativo,

Pobre precisión

Baja sensibilidad

Baja Resolución

Bajo rango dinámico (2 logs)

Resultados subjetivos

Procesamiento Post PCR (gel agarosa)

5 10 15 20 30 40 50 0

¿Es posible cuantificar?

¿Es cuantitativa la PCR punto final?

Detección con Bromuro de Etidio

PCR Punto Final

Can

tid

ad d

e D

NA

Número de Ciclos

PCR Cuantitativo: PCR en tiempo real

La PCR en Tiempo Real permite monitorear los productos de PCR en el instante en el que se generan durante

cada ciclo.

Principio de la PCR en Tiempo Real

En la PCR en Tiempo Real la detección de los productos de PCR se realiza midiendo la fluorescencia que emiten:

Fluorescencia =

Absorbe luz de baja longitud de onda y emite luz de alta longitud de onda

Producto ds PCR

Producto ds PCR

Principio de la PCR en Tiempo Real

En cada ciclo se incrementa la intensidad de la fluorescencia

Producto ds PCR

...... ...... ...... ......

1

2

4

8

Detección y monitoreo en Tiempo Real

PCR convencional (punto final)

Detección con Bromuro de

Etidio

NTC

Fase Plateau

Fase Log-linear

baseline region

Curva de amplificación de un PCR en Tiempor Real

Fase Exponential

PCR Punto Final

Bajo número de copias (e.g. ~103 copies)

Señ

al

(F

luo

resc

enci

a n

orm

aliz

ada)

Tiempo (ciclos de amplificación)

threshold = umbral de deteccción

Curva de Amplificación QPCR

Alto número de copias (e.g. ~108 copies)

Fase inical lag No detecta señal

Fase log Señal ~ el doble cada ciclo

Fase plateau Reacción es lenta & se detiene cuando se tiene un reactivo limitante.

Punto Final (no cuantitativo)

Bajo

= mas copias, amplifica

antes

Alto CT

= menos copias,

amplifica despues

CT

CT = threshold cycle (Numero de ciclo en donde la señal de amplificación cruza el umbral de detección.

Curva de amplificación QPCR

Dos tipos de formatos de Detección

Sondas secuencia específica: Hibridación e hidrólisis

HybProbes Dual-labeled probes (sondas TaqMan)

Detección inespecifica

utilizando colorantes que se intercalan en el DNA de doble cadena.

Solo hay un colorante

Es más económico

SYBR® Green

Formato SYBR Green

SYBR Green

ss DNA

Taq polymerase

ds DNA

Sondas de Hibridación e Hidrólisis: Fundamento

FRET: Fluorescence-Resonance-Energy-Transfer Definition

Transferencia de energia a un fluoroforo excitado (donador) a otro fluoróforo (aceptor) o bien a una molecula no fluorescente

Dark Quencher Fluorophore

heat

Fluorophore 2 Fluorophore 1

input radiation

output radiation

input radiation

PCR en Tiempo Real

1. Sondas de Hibridación

Incrementa la fluorescencia en cada ciclo

La señal depende unicamente de la hibridación

1. Sondas de Hidrólisis

Incrementa la fluorescencia en

cada ciclo La señal depende unicamente de la

hibridación

Hybridization Probes Molecular Beacons

Chemistry Considerations

There are many chemistry types TaqMan, Eclipse, Beacons, Adjacent dual (FRET) Probes, LUX primers, Scorpion probes, etc

There are many chemistry variants MGBs, LNAs, “superbases”, inosine substitutions, etc

Trend: the chemistry provider designs the assay

Providers understand idiosyncrasies of their chemistry They usually have proprietary design software They usually design for free if you purchase

Recommended you always consult chemistry provider about assay design!

Ventajas de la PCR en Tiempo Real

Riesgo de contaminación Mínimo

Quantificación

Reduce los procesos de estandarización y variaciones de operarios

Ideal para diagnóstico clínico

Rapidez

Mayor sensibilidad

Mayor especificidad

Mayor precisión

Ethidium-Gel

detection

Cuantifiación absoluta

– Se realiza cuando se desea conocer los niveles de expresión de un

gen indicados como un número absoluto (copias, concentración, pg

etc), en NUMEROS.

– Para la cuantifiación se requiere realizar una curva estándar con al

menos cinco estándares por triplicado

– Los estándares deben representar preferentemente diluciones

1:10, para esperar una diferencia de Ct de 3.162 entre cada dilución

– Importante: La calidad de la cuantifciación absoluta esta dada por:

• Pipeteo correcto

• Preparación de los estándares

• Buena conservación de los estándaes

amount

5,000 50,000 500,000 5,000,000 50,000,000 500,000,000

CT

amount

Muestra

desconocida

determinada

por la curva

estándar

5,000 50,000 500,000

5,000,000

50,000,000 500,000,000

CT

Cantidad

1. Graficar CT vs log concentración 2. Utilizar la gráfica que mejor se ajuste

3. Calcular la eficiencia a partir de la gráfica La ecuación es la siguiente, donde m es la penciente E= 10(-1/m) – 1 E=1=100%.

4. Eficiencia de correlación (R2) correlación entre el valor teórico y e valor real. La correlación debe ser lo más cercana a 1

Cuantificación absoluta y eficiencia de reacción

Panel Transplante

•Infecciones Diversas después del Transplante

– infecciones Virales

– infecciones Fúngicas

.Herpes virus

CMV

EBV

HSV 1/2

VZV

Aspergillus

Panel Encefalitis

.Panel Encefalitis

Patógenos virales más comunes

HSV-1/2

Otros virus

Influenza

CMV , EBV (Herpes)

Patógenos Bacteriales

Mycobacterium tuberculosis

Panel Infecciones Intra-Hospitalarias

MRSA S. aureus C. difficile

Panel Infecciones Salud de la Mujer

C. trachomatis N. gonorrhoeae GBS

Panel Sangre

HIV

Virus Hepatitis

HBV

HCV

PHC therascreen - Fast results for timely treatment decisions

Personalized Health Care

Companion diagnostics

Patients

Payers

Providers Pharma companies

Physicians

All stakeholders benefit

Industry-leading portfolio of co-development projects

Project Partner Indication Biomarker Status

Eribitux (cetuximab)

Colorectal cancer

KRAS U.S. regulatory approval in July 2012

Vectibix (panitumumab)

Colorectal cancer

KRAS U.S. regulatory approval in June 2014

PF-00299804 (dacomitinib)

Lung cancer (NSCLC)

KRAS In development

Iressa (gefitnib)

AstraZeneca Lung cancer (NSCLC)

EGFR CE-IVD kit in Europe and other non-U.S. markets

Gilotrif (afatinib)

Lung cancer (NSCLC)

EGFR U.S. regulatory approval in July 2013

Early-stage compound Blood cancer JAK2 In development

Various projects

Not disclosed Various In development

Not disclosed Confidential oncology Confidential In development

Not disclosed Confidential Non-oncology Confidential In development

Therascreen portfolio to address cancer biomarkers

•Disease Areas

Brain

MGMT

IDH1 & IDH2

(BRAF)

Colorectal

KRAS

NRAS

BRAF

PIK3CA

UGT1A1

Melanoma

BRAF

NRAS

* Under development

NSCLC

EGFR

ALK

KRAS*

(NRAS)

(BRAF)

GIST

KIT & PDGFRA

(BRAF)

www.mycancergenome.org

Leukemia (ipsogen)

JAK2 V617F

BCR-ABL1

PML-RARA

WT1

NPM1

….

Non-small cell lung carcinoma (NSCLC)

Drug efficacy correlates with EGFR mutation status

IPASS (IRESSA Pan-ASia Study): randomised, parallel-group study to assess the efficacy, safety and

tolerability of gefitinib (novel drug) versus carboplatin/paclitaxel (standard therapy) as first line treatment in a

clinically selected patient population from Asia.

Gefitinib or Carboplatin–Paclitaxel in Pulmonary Adenocarcinoma. TS. Mok, Y-L. Wu, S. Thongprasert, C-H. Yang, D-T. Chu, N, Saijo, et al., NEJM, Sep 2009; 361: 947-957.

IPASS Study

Afatinib versus pemetrexed/cisplatin as a first-line treatment in advanced lung cancer harbouring EGFR-activating mutations. Yang JC-H, et al. ASCO 2012: Abstract LBA7500, LUX-Lung 3:

Lux Lung 3 trial

Non-small cell lung carcinoma (NSCLC)

Drug efficacy correlates with EGFR mutation status

Afatinib

Pemetrexed/cisplatin

Only patients EGFR mutation positive included in the study

tested with Qiagen kit Patients benefit from Afatinib (Gilotrif) Kit and drug FDA approved

Technology Landscape: Advantages therascreen

PCR:

Real-time

Detection

PCR

Clean-up

Pyro-

Sequencing

Reaction

PCR

Heat

Destroy

Polym

Invader

Reaction

Detection

via

Plate Read

therascreen RGQ & Pyro PCR/Invader

PCR

Clean-up

Sequencing

Reaction

Clean-up

PCR/Sequencing

Capillary

Sequencing

= Transfer product/ add reagent/ open tube

PCR

Clean-up

Sequencing

Reaction

Clean-up

SNaPshotTM

Capillary

Sequencing

PCR

PCR

Clean-up

Sequencing

Reaction

Nested PCR/ Seq

Clean-up

Capillary

Sequencing

1

Steps

2

3

4

5

6

PCR:

Real-time

Detection

PCR

Clean-up

Pyro-

Sequencing

Reaction

PCR

Clean-up

Pyro-

Sequencing

Reaction

PCR

Heat

Destroy

Polym

Invader

Reaction

Detection

via

Plate Read

therascreen RGQ & Pyro PCR/Invader

PCR

Clean-up

Sequencing

Reaction

Clean-up

PCR/Sequencing

Capillary

Sequencing

= Transfer product/ add reagent/ open tube= Transfer product/ add reagent/ open tube

PCR

Clean-up

Sequencing

Reaction

Clean-up

SNaPshotTM

Capillary

Sequencing

PCR

PCR

Clean-up

Sequencing

Reaction

Nested PCR/ Seq

Clean-up

Capillary

Sequencing

1

Steps

2

3

4

5

6

We offer the fastest, easiest to use, most sensitive and CE-IVD approved methods and kits

ARMS Scorpions qPCR

Highest sensitivity on the

market

Selective mutation detection

Ease of use

One-step procedure

Pyrosequencing

Sequence information

Complex mutation testing

with simple data interpretation

Small amount of starting material

QIAGEN solutions for molecular diagnostic

KRAS

EGFR

BRAF

And more…

Mutation testing

Criteria for choosing PCR or Pyro kits

Parameter PCR Pyro

What type of information is needed Restricted to mutations that are

clinically relevant (closed system) Sequencing data (open

system)

Technology preference of the customer PCR Sequencing

Most important criteria of the assay Faster result and minimum handling Detailed report

Report format Results YES/NO Sequencing data

General Lab profile Diagnostic or routine service lab Diagnostic lab with R&D

activities

Experience in interpreting results

clinical/technical background of user Basic skills Experience required

Experience in molecular biology No Yes

therascreen RGQ Kit Portfolio

Sample assessment run

Result interpretation

~ 2h ~ 15 min

RGQ Kit workflow overview

PCR Setup

~ 20min

PCR Setup

82

Mutation detection run

~ 2h ~ 20min

Myeloproliferative neoplasms (MPN) spectrum

Philadelphia

(Ph) -

Philadelphia

(Ph) +

MPN

Polycythemia

vera (PV)

Essential

thrombocythemia (ET)

Primary

myelofibrosis (PMF)

Chronic myelogenous

leukemia (CML)

~ 65%

JAK2 V617F

positive

~ 55%

JAK2 V617F

positive

Sources: Tefferi, A. (2008) Leukemia; Patnaik, M.M. (2009) Leukemia; Tefferi, A. (2010) Leukemia..

~ 96%

JAK2 V617F

positive

ipsogen JAK2 RGQ PCR Kit CE Content

4 dilutions for JAK2 Wild-Type quantification:

• WT-1: 50 copies standard • WT-2: 500 copies standard • WT-3: 5 000 copies standard • WT-4: 50 000 copies standard

4 dilutions for JAK2 V617F quantification:

• MT-1: 50 copies standard • MT-2: 500 copies standard • MT-3: 5 000 copies standard • MT-4: 50 000 copies standard

JAK2 Mutant Control (MTC): genomic DNA harboring 100% JAK2 V617F allele

JAK2 WT Control (WTC): genomic DNA harboring 100% JAK2 wild type allele

JAK2 WT Rx (Primers & Probes reaction mix) - FAM specific for JAK2 Wild-

Type allele - HEX specific for IC

JAK2 MT Rx (Primers & Probes reaction mix) - FAM specific for JAK2 V617F

mutant allele - HEX specific for IC

QIAGEN qPCR master-mix including Internal control (IC)

IVD

Catalog number: 673623

LOD at 0.042% JAK2 V617F

Water for NTC TE buffer for sample dilution

100%

10%

1%

0.1%

0.01%

0.001%

0.0032%

LoD of previous kit : ipsogen BCR-ABL1 Mbcr Kit

LoD of previous kit : ipsogen BCR-ABL1 Mbcr IS-MMR Kit

Limit of detection (LOD) and molecular response

•Improved sensitivity

0.0069%

0.08%

LoD of new ipsogen BCR-ABL1 Mbcr RGQ RT-PCR kit

0.0030% MR4.5

MR5

MR4

MMR

Myeloproliferative Neoplasm (MPN) Spectrum

Philadelphia

(Ph) -

Philadelphia

(Ph) +

MPN

Polycythemia vera (PV)

22% in EU

Essential thrombocythemia

(ET) 33% in EU

Primary myelofibrosis

(PMF) 10% in EU

~ 55-65%

JAK2 V617F

positive

~ 50-60%

JAK2 V617F

positive

~ 97%

JAK2 V617F

positive

Simplified from Tefferi, A., Vardiman, J.W. (2008) Leukemia. 22, 14. Patnaik, M.M., Tefferi, A. (2009) Leukemia. 23, 834. Tefferi, A., et al. (2010) Leukemia. 24, 105. Barbui et al. (2015) Blood Cancer Journal. 5, e337. Incidence and prevalence in the American Journal of Hematology, Vol. 00, No. 00, Month 2014.

>90%

BCR-ABL1 Mbcr

Chronic myelogenous leukemia

(CML) 35% in EU

~ 20-25%

CALR

positive

~ 20-25%

CALR

positive

~ 7%

MPL

positive

~ 3%

MPL

positive

Myeloproliferative Neoplasm (MPN) Spectrum

Philadelphia

(Ph) -

Philadelphia

(Ph) +

MPN

Polycythemia vera (PV)

22% in EU

Essential thrombocythemia

(ET) 33% in EU

Primary myelofibrosis

(PMF) 10% in EU

~ 55-65%

JAK2 V617F

positive

~ 50-60%

JAK2 V617F

positive

~ 97%

JAK2 V617F

positive

>90%

BCR-ABL1 Mbcr

Chronic myelogenous leukemia

(CML) 35% in EU

~ 20-25%

CALR

positive

~ 20-25%

CALR

positive

Epigenetics Definition and Mechanisms

DNA Methylation * Adding of a methyl group on C residues of CpG dinucleotides

* CpG islands are often found in promoter regions * Genes in methylated DNA are silenced

Histone Modification * Acetylation, methylation, and phosphorylation of histones * Histones are associated with DNA as chromatin * Acetylation of histones results in chromatin that is generally transcriptionally active

miRNA * Post-transcriptional control of gene expression * microRNA can bind to 3’-UTR of mRNA and affect translation of mRNA * microRNA downregulate gene expression

Epigenetics Mechanisms

The study of reversible inheritable influence on gene activity that is

not accompanied by a change in the DNA sequence

What Is Micro RNA?

.Endogenously expressed small functional RNAs (~ 21-23 nt)

.Regulate mRNA expression post-transcriptionally • Primarily translation inhibition, but also mRNA

degradation

.Only partial sequence complementarity required • One miRNA can regulate multiple mRNA • One mRNA can be regulated by multiple miRNA

.Another layer of complexity for regulating gene expression

A look back 10 years Unique miRNA signatures are

found in human cancer

– miRNAs located in genomic regions amplified in cancers (e.g. miR-17-92 cluster) can function as oncogenes, whereas miRNAs located in portions of chromosomes deleted in cancers (e.g. miR-15a-miR-16-1 cluster) can function as tumor suppressors.

– Abnormal expression of miRNAs has been found in both solid and hematopoietic tumors.

– miRNA expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggression and response to therapy.

In the last 10 years, a substantial number of studies and reviews have associated the

presence of various miRNAs with cell proliferation, resistance to apoptosis, invasiveness, and differentiation in cancer cells.

Potential events leading to disruption of ‘normal’ miRNA : target interaction in disease

microRNA Gene

mature miRNA

Pre-miRNA

Drosha-DGCR8

Exportin

DICER-TRBP

Pri-miRNA

Ago

Target Transcript

miRNP

Altered Transcription

Methylation

Histone Modification

Transcription Factor

Drosha Processing

Genomic Instability

Amplification/Deletion

Translocation

Insertional Mutagenesis

Loss of miRNA Binding Site in target

SNP or Mutation

Alternative Splicing

Loss/Change of 3’-UTR

Dicer Processing

miRNA in Blood RBC, WBC, platelets, CTC, “other cells”, extracellular?

What is blood? RBC, WBC, platelets, other cells (e.g. circulating tumor cells) Serum (post clotting) Plasma (no clotting)

High levels of nucleases present in plasma

Freely circulating RNA should be rapidly degraded

Surprisingly, stabile miRNA can be detected in serum and plasma

1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9:654-659

2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e3694 3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-17452 4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma,

Proc. Natl. Acad. Sci 108: 5003-5008 5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol

13:423 6) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic

Acids Res. 2010 Nov 1;38(20):7248-59.

Exosomes & micro vesicles1,2,3

miRNA

Ago

Ago-2-miRNA complexes4

HDL mediated miRNA transport5

Other “protective” protein6

Stable miRNA in circulation ~2007 Contained in exosomes, microvesicles, other: An evolving story

Extracellular miRNAs are in all body fluids

Weber et al., (2010) Clinical Chem. 56: 1733-1741 miRNeasy, miScript PCR System

Microbiome: From identification to characterization

Humans or Superorganisms? •Cellular composition of the organism

97

Human

Microbiota

Estimations of the number of microbial cells that live in and on the human body, human cells are outnumbered by a factor of 10. Nomenclature: Microbiota are the microbes that live in a specific location, e.g. the human body, the gut, soil, etc. Metagenomics is the study of the collection of genomes derived from a specific sample or community. Microbes are microscopic organisms that can be either single or multicellular. For the purposes of this webinar, we will use microbiome to describe the collective genomes of the microbiota that inhabit a specific location.

Microbiota composition •Microorganisms cluster by body site

98

•Cataloguing efforts by the NIH Human microbiome project suggest:

•~10,000 organisms live with us

•~ 8 ×106 genes in this “second genome”

•Identifying microbiota in healthy individuals revealed:

•Different body sites have unique communities

•Race, Age, Gender, Weight or Ethnicity have no effect

Complexity and function of genomic content

•Function of microbiome enables individual survival

99

•Each organism has developed genetic content for its own survival in a specific environment.

•Metabolism tuned to local nutrient sources

•Virulence factors for stable colonization

•Antibiotic resistance genes to metabolize toxins

Current Methods for Microbial Analysis

•Identifying microbes and their genes

• Culture – Nutrients – Antibiotics

• 16S rRNA gene clone library construction – Pan 16S rRNA PCR amplification cloned sequenced

• Microarray

• Next generation sequencing – 16S rRNA sequencing – Whole genome sequencing

• MALDI

• qPCR – Target dependent (16S rRNA or gene)

100

16S rRNA gene as a phylogenetic marker for

bacterial ID

– Classification from the variable sequences

16s rRNA sequence similarity

95% genus level, 97% species level, 99% strain level

101

16S rRNA gene

- Conserved region - Variable region

Association of the human microbiota and disease

– Gut

• Intestinal infections

• Obesity

• Inflammatory Bowel Disease

– Airway

• Pneumonia and other respiratory infections

• Chronic Obstructive Pulmonary Disease

• Cystic Fibrosis

– Urogenital

• Bacterial Vaginosis

• Urinary Tract Infections

• Sexually Transmitted Disease

– Blood

• Sepsis/Blood-stream infections

– Cancer

– Heart disease

– Neurological disorders

– Oral

• Periodontitis

• Gingivitis

102

Correlate qPCR assay performance with NGS results

•Profiles of vaginal flora by qPCR and whole genome sequencing

103

Microbial DNA qPCR Arrays Protocol

•Identification or Profiling

104

Antibiotic resistance gene reservoirs in the body

•Screening of the gut for presence of antibiotic resistance genes

105

500 ng of genomic DNA from stool samples originating from five healthy adults were analyzed for presence of antibiotic resistance genes. 87 unique antibiotic resistance genes were tested for by qPCR. Positive (+) / negative (blank) result for each antibiotic resistance gene was determined using a threshold cycle cutoff.

Cervical flora: Gardnerella vaginalis positive vs. BV negative

106

Cervical flora: Gardnerella vaginalis positive vs. BV negative

In samples with high Gardnerella vaginalis abundance, there was an increase in co-occurrence of BV-associated microorganisms and decrease in abundance of the normal flora, Lactobacillus crispatus.

107

Antibiotic resistance genes in our food supply?

108

Mejorando vidas mediante el impacto clinico de NGS Presentando el sistema de NGS GeneReader

• QIAGEN NGS Basics

110

NGS 101: The Basics NGS Basics Content Responsibility Additional Materials Time

Introduction to NGS Background of NGS GPM NOW 1 training Slides

NGS Market Sequencers/ Companies / Opportunities

GPM

NGS Applications and Experiments at QIAGEN

WGS, WES, Targeted Panel(DNA), transcriptome, Targeted RNA, miRNA, liquid biopsy,

GPM

Basic Overview of Portfolio

DNA library prep GPM Portfolio flyer/ training deck

Selling NGS: Questions /Selection Guide/Key Terms

GPM+ specialist or regional marketing

Selection Guide

Feedback

What does NGS do? •Decipher the nucleotide composition in a DNA sample

CONFIDENTIAL, DO NOT DISTRIBUTE.

DNA sample

ACCTAGTCATGAGTCATGCAT

Nucleotides

Transform the DNA sample into a collection of nucleotides

What makes a sequencing method “Next Generation”?

1

1

3

1 Cluster (DNA spot) = 1 read

What are we talking about?

NOW training-NGS Basics 114

1st Generation Sequencing Sequence many identical

molecules

Using any old sequencing chemistry

In a tube

2nd Generation Sequencing (NGS) Sequence many identical

molecules (usually)

Using a reversible, step-wise sequencing chemistry

Immobilized on a surface

General NGS Workflow

NOW training-NGS Basics

Data Analysis &

Interpretation

2nd generation Sequencing

Sample Extraction

Library Preparation

What does NGS data look like?

PowerPoint Style Guide 116

Short answer: Unintelligible times a billion

(seriously, these files are huge and will make your computer cry if you try to open them)

How do you sequence? •You use NGS platforms


gDNA


Nucleotides

Ion Torrent NGS platforms

Illumina NGS platforms

Why NGS?

What NGS library prep does

1. Create DNA fragments

2. Add platform-specific sequences to every fragment

3. Amplify library molecules for QC and sequencing (if necessary)

“Stuff”

Library Preparation

Data Analysis &

Interpretation

2nd generation Sequencing

Sample Extraction

NGS Library? •NGS platforms sequence libraries


DNA


Nucleotides

Library

DNA needs to be turned into libraries before it can be sequenced Library = millions of small DNA fragments

What is a library? •Pieces of DNA to be sequenced flanked by adapters


DNA


Nucleotides

DNA to be sequenced

Adapters

How do I generate the small pieces of DNA?


DNA


Nucleotides Library

Small pieces of DNA

Shearing – physically breaking DNA into small pieces

Fragmentation – enzymatically digesting DNA into small pieces

Multiplex PCR – use primers to generate small amplicons

Enables targeted sequencing using panels

What do adapters do?


•Multiplexed Sequencing: Multiple samples sharing one sequencing reaction to reduce per-sample costs and control the amount of data generated per sample.


124

What the adapters do? : Add barcodes for multiplexing

Barcode 1

Each 96-plex adapter includes two 8-base barcodes 8 Barcode 1’s x 12 Barcode 2’s = 96 combinations

Pool barcoded libraries

single multiplexed

NGS run

Demultiplexing

fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq

Separate fastq data files for each sample

Adapter Ligation Point

How do you sequence? •NGS platforms are not enough. You need to use a flow cell or chip on NGS platforms


gDNA


Nucleotides

GeneReader workflow uses flow cells

Ion Torrent NGS platforms use

beads on chips

Illumina NGS platforms use flow cells

Flow cells and chips are important because the entire sequencing run takes place on their surfaces

You generated reads. Now what? •Reads have to be analyzed through data analysis (bioinformatics) pipelines


Variants (mutations)

Read 1000000

Read 1000

Read 1

What is the principle of data analysis? •Compare DNA sequences in your sample to the DNA sequence of the reference genome


ACAGTTAAGCCTGAACTAGACTAGGATCGTCCTAGATAGTCTCGATAGCTCGATATC

AACTAGACTAGGATCGTCCTAGATAGTCTCG

Reference sequence

Aligned reads

AACTAGACTAGGATCGTCCTACATAGTCTCG

AACTAGACTAGGATCGTCCTACATAGTCTCG

GATCGTCCTAGATAGTCTCGATAGCTCGAT



There is a nucleotide change from G to C at this position in my sample compared to the

reference genome (which is considered a normal genome)

One can take an additional step to interpret what those genetic changes

mean (data interpretation)

Putting it all together


DNA purification

Data Analysis &

Interpretation

Library preparation

Sequencing Sample

Collection and Stabilization

Tradeoff Between Depth and Coverage


All Bases

1. Whole Genome

2. Hybrid Capture

(Exome)

mRNA-Seq (Transcriptome)

3. DNA Targeted

Panels

Affordable Sequence Depth

30X WGS

100X Exome

1000X+ Amplicon Seq RNA

Targeted Panels

4. RNA

1X Low-pass (SC)

Selected NGS Applications

•Large fraction of genome = greater costs, but greater ability to compare samples or patients across studies •Small targeted panels = easier and less expensive to complete the project, but limited ability to follow up on new hypotheses or discover new associations


Whole Genome Exome Sequencing DNA Amplicon Panels RNA-Seq (Transcriptome)

Free circulating nucleic acids

RNA and DNA from dead cells shed into the bloodstream, can contain cancer-related mutations.

What is Liquid Biopsy? •A minimal invasive technology for detecting signs of cancer and other diseases

Tissue samples

FFPE tissue samples of tumor extracted from the patient’s body through an invasive procedure.

Comprehensive portfolio offering proven ways to access nucleic acids

Exosomes

Tiny microvesicles found in body fluids that transport RNA between cells.

Circulating Tumor Cells

Tumor cells shed from a tumor into the bloodstream carrying genetic information.

Oncología: Líder en Medicina Personalizada

Launch Event GeneReader Mexico City 132

GeneReader NGS First complete Sample to Insight NGS solution, expansion to companion diagnostics

Lung Breast Colon Hematology

ipsogen Widest range of molecular tests for blood cancer

therascreen No. 1 portfolio of solid tumor companion diagnostics

Cervical

digene The proven gold standard for cervical cancer detection

Providing critical diagnostic solutions across clinical disease progression

insights along cancer care continuum Industry-leading pharma collaborations

Disease unknown

Hybrid Capture (HC2) Population screening

Disease suspected

Next-generation sequencing Metastatic cancers

Initial diagnosis

Translational research

Disease diagnosed

Targeted PCR / NGS Resistance detection

Treatment modification

Companion diagnostics

Disease monitoring

Targeted PCR / NGS Refraction

Recurrence

La promesa en NGS Consolida las pruebas para incrementar la eficiencia en su laboratorio

Limitaciones de los ensayos moleculares tradicionales:

• Múltiples pruebas para detectar diferentes variantes y tipos de las mismas

• Riesgo de no tener suficiente material

• Incrementar el tiempo de respuesta y hacer eficientes los rescursos

NGS resuelve estos problemas

¿Qué evita a los laboratorios de convertir en realidad la promesa de NGS?

Altos costos

Flujos de trabajo fragmentados

Análisis e Interpretación de datos

¿Qué evita a los laboratorios de convertir en realidad la promesa de NGS?

Seamless Bioinformatics

Simple workflow Cost-effective

¿Qué hace diferente al sistema de NGS GeneReader?

La primera solución completa a nivel mundial que cubre de la

muestra al detalla en el campo de NGS

Diseñada para entregar información con impacto clínico

“The QIAGEN GeneReader NGS System is

the first truly integrated solution that is

focused on delivering critical insights with

maximal efficiency and minimal adoption

hurdles.”

—Scott Steelman, PhD, Associate Director of Technology Labs

Launch Event GeneReader Mexico City

“If you combine different

products and technologies from different

companies, you never

know how well they will fit and function

together.”

“People tend to largely underestimate the

true cost of running NGS, not considering

the cost of an often lengthy and pricy

optimization process. With the

GeneReader NGS System, I expect to see

a major advantage from a fully integrated

workflow.”

CAP Today, March 2017

1. Evitar el dolor de sincronizar equipos accesorios de otros proveedodres

2. Ahorrar el tiempo de recursos especializados para desarrollar y optimizar el análisis bioinformático

3. Proveer una solución verdadera con un secuenciador previamente validado en su flujo de trabajo

Beneficios de la integración de los flujos de trabajo

1

3

9 Integrado

Actionable

Flexible

Accesible

Cofiable

Not a Sequencer---but a System

L

a

u

n

c

h

E

v

e

n

t

G

e

n

e

R

e

a

d

e

r

M

e

x

i

c

o

C

i

t

y

1

4

0

Nucleic Acid Extraction

Target Enrichment

Library Preparation

Next-Generation Sequencing

Data Analysis

Interpretation

QIAxcel QIAxcel RGQ

QC

GeneRead Assistant (iPad and App included)

Support

Data Management

GeneRead Link and LIMS GeneRead Databank

QIAcube QIAcube RGQ GeneRead QIAcube

GeneReader QCI-Analyze QCI-Interpret

Automation

El panel que aporta más información de impacto clínico

• KRAS

• NRAS

• KIT

• BRAF

• PDGFRA

• ALK

• EGFR

• ERBB2

• PIK3CA

• ERBB3

• ESR1

• RAF1

Enfocado en tumores sólidos con:

• La prevalencia más alta

• Necesidad más grande de detección

• Variantes asociadas a información clínica

QIAGEN Knowledge Base

Lung

Breast

Ovarian Colorectal

Melanoma

12 genes

Integrado

Actionable

Flexible

Accesible

Confiable

QIAact BRCA 1/2 Panel: no solo para el cancer de mama hereditarios

•

Tipo de Cancer Tipos de mutaciones Guías NCCN/SGO

recomiendan Clinical Actionability

• Mama • Ovario

• Germinales • Somaticas

• Toda mujer en edad reproductiva

• Pacientes con alto riesgo de cancer mamario

• Inhibuidor PARP

• Protocolos Activos para mutaciones somaticas

Detección de mutaciones en Sangre, FFPE y múltiples tipos de canceres

QIAact Lung MB Panel – need for test consolidation

•

Cambios geneticos complejos , por ejemplo:

Mutación Puntual: EGFR T790M

Deleción grande MET: exon 14

CNV: ALK

Fusiones: RET, ROS1

Pruebas actualmente en uso: • PCR • IHC • FISH • Arreglos

Detección en un sólo ensayo que proporciones todos estos resultados

QCIA – Intuitive and flexible variant calling and visualization

•Fully automated alignment, quality control, variant calling

El QIAact panel surge de la base de conocimiento generada por QIAGEN

Base de conocimientos de QIAGEN

• Comprende la infromación generada por la empresa líder del segmento

• Compila multiples fuentes publicas y privadas

• Está depurada y continuamente actualizada

El diseño del panel está basado en :

• Medicamentos disponibles

• Protocolos Clinicos

• Guías Profesionales

Integrado

Actionable Flexible

Accesible

Confiable

Un panel diseñado para aportar información con impacto clínico relevante

QCII – Intuitive and transparent variant interpretation

•Designed, built and validated in collaboration with clinical labs

Customizable Reporting

ability to capture /persist internal annotations, integration into LIMS and EMR systems

Comprehensive Curation of Clinical Evidence

reported cases, drug labels, professional guidelines

Decision Support Software classify variants, identify treatment options, and

geographical clinical-trial matching

Treatment and Trial Identification

based upon levels of evidence, expected drug response, prognosis and lab defined rules

QIAact Panel Portfolio

Actionable Insights Tumor Panel Lung DNA & Fusions

Panel

Myeloid Leukemia Panel

BRCA 1/2 Panel

Custom Panel

Based on QIAGEN knowledge base, enabling maximum actionability Verified from sample to insight

No Invasive Pre-Nata Test (NIPT)-Natera

Now Now Now

November with UMIs

Q4

Now

Q3

QIAGEN es el líder global en calidad y servicio

1

4

8

>35 offices

worldwide

>550 service

specialists

>15,000 installations

>4,000 employees

>500,000 customers

Integrado

Actionable

Flexible

Accesible

Confiable

Flexibilidad que se ajusta a sus necesidades

•Un batch se compone de 1 a 10 pacientes; GeneReader puede procesar hasta 4 Flow Cells

new new

GeneGlobe inside

1-10 pacientes 1 Amplificación

clonal 1 Flow Cell 1-10 Reportes

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data

Patient/ clinical

data Patient/ clinical

data

40 pacientes 4 Amp Clonal 4 Flow Cell 40 Reportes

120 pacientes 12 Amp Clonal 12 Flow Cell 120 Reportes

Por corrida

Por semana

Por Flow Cell

Molecular Oncohemathology Workflows: From Sample to Insight

Jacobo Zuñiga Castillo Ph.D. Regional Marketing Manager

QIAGEN Mexico, Central America and Caribbean

aplicaciones de la biología molecular en diagnóstico clínico€¦ · molecular diagnostics ~10%...

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