”” DiferentialDiferential Gene Expression in Gene Expression in

BreastBreast CancerCancer””Dirce Maria CarraroDirce Maria Carraro

Head, Genomic and Molecular Biology LaboratoryHead, Genomic and Molecular Biology Laboratory

CIPECIPE--International Center of Teaching and Research International Center of Teaching and Research

AC Camargo HospitalAC Camargo Hospital

Inovações no diagnóstico e tratamento do câncer de mama - 02 e 03 de Março de 2012OUTUBRO ROSA INCA Rio de Janeiro, 03-04 Outubro, 20 12

Gene expression for assessing Gene expression for assessing tumor biology and biomarkertumor biology and biomarker

• Progression of in situ Ductal Carcinoma• Sens-Abuázar C, et al. Transl Oncol. 2012

• Castro NP, Breast Cancer Res. 2008

• Hereditary Breast Cancer: Influence of BRCA1 mutation in

Gene Expression of Triple Negative Tumors• Carraro et al., Journal of Human Genetic – submitted

• Lisboa, B; Silva F, Pena M et al – submitted

• Ferreira et al., not published

– Ductal carcinoma (80%)

– In situ DCIS - 20-30% of all Ductal carcinoma (DC) detected by mammography

screenening

– Incert evolution (Rapid progression or slow evolution)

– Histologic Classification

• Comedo or Non-comedo

• Grade: Low, Medium or High

• Estrogen/Progesteron Receptor

– progride to invasive disease

� Definition of molecular events necessary for the epithelial cells acquire the ability to

invade the surround tissue

� Due to the technological advances in detecting very small or non-palpable lesions, the number of

women diagnosed with DCIS and early breast cancer lesions is continuously increasing.

Progression of Progression of in situin situ Ductal Carcinoma Ductal Carcinoma (DCIS)(DCIS)

In situ invasive

Gene Expression Profile

Normal PureDCIS in situ component DCIS/IDC ~ IDC

Morphological Features

Normal PureDCIS ~ in situ component DCIS/IDC IDC

IDC (10)IDC (10)Normal (4)Normal (4) Pure DCIS (5)Pure DCIS (5)

In situ In situ component ofcomponent of

DCISDCIS--IDC (10)IDC (10)

Laser Microdissection for capturing epithelial cells from tumor lesion

Molecular Divergence: number of differentially expressed genes (DEG) as distance measure

(the higher the number of DEG – the more distant the group is allocated)

Castro et al ., Breast Cancer Res. 2008

Molecular Divergence of Tumor Epithelial cellsMolecular Divergence of Tumor Epithelial cells

Two important pointsTwo important points

• 1) From pure DCIS to in situ component of

DCIS-IDC happen most transcriptional

alterations

• 2) Alterations in gene expression occur before

the cells manifest their morphological aspects

of invasion

Different malignant potential

147 Differentially Expressed Genes

Molecular Difference between cells from intraductal componentsPure DCIS (5)

At least 5 years follow-up

In situ component

DCIS-IDC (10)

The earliest molecular alterations of The earliest molecular alterations of epithelial cellsepithelial cells

Castro et al ., Breast Cancer Res. 2008

pureDCISpureDCIS

In situ In situ componentcomponent

DCIS/IDCDCIS/IDC

Non Non neoplasicneoplasic

100% of in situ component of DCIS-IDC was discrimin ated from 60% of Pure DCIS

Putative genes involved in the progression Putative genes involved in the progression of of in situ in situ DCISDCIS

The progression of in situ DCIS seems to be markedly

characterized by gene downregulation

ValidationValidation in in independentindependent SampleSample setset((epithelialepithelial cellscells capturedcaptured byby laser laser –– Ferreira et al., Ferreira et al., DiagnDiagn Mol Mol PatholPathol , 2010, 2010))

• 61 genes: 32 (52,4%) in concordance with microarray results (Fold change 2; p<0.01)

ANAPC13 ANAPC13 mRNAmRNA andand proteinprotein

MCF-7 SKBR-3MFC-7 SK-BR-3

20 kDa

15 kDa

Re

lati

ve

exp

ress

ion

(LO

G2

)

Protein: cytoplasmic and nuclear

staining – 74 amino acids

Pure DCIS in situ component of DCIS-IDC

Positive Negative P

Pure DCIS 25 (69,50) 11 (30,50) 0,02

in situ componentof DCIS-IDC 11 (40,80) 16 (59,20)

• Chromosome 3q 22.2.• Encodes a component of Anaphase complex (subunit 13 ) (APC/C) – cell cycle.• Highly conserved among the species

Pure DCIS: 41 cases In situ component DCIS-IDC: 36 cases

ANAPC13ANAPC13 expression along tumor progression expression along tumor progression

weak

moderate

strong

ANAPC13

absent

ne

ga

tiv

ep

osi

tiv

e

qRT-PCR

ANAPC13

Re

lati

ve

mR

NA

ex

pre

ssio

n

In situ

DCIS-IDC

pure

DCIS

IDC

mRNA level – epithelial cells

negative

positive

In situ

DCIS-IDC

pure

DCIS

IDC

* *

***Protein level

DCIS: 41 specimensin situ component of DCIS-IDC: 36 specimens IDC: 187 specimens

Sens-Abuázar et al ., Translational Oncology. 2012

Frequency and Intensity

Negative Positive P

w/o progression 7 (35,00) 13 (65,00) 0,18

with progression 5 (71,40) 2 (28,60)

ANAPC13 as BiomarkerANAPC13 as BiomarkerFor progression of pure DCISFor progression of pure DCIS

For prognosis in Invasive Ductal carcinoma (IDC)For prognosis in Invasive Ductal carcinoma (IDC)Survival curves based on ANAPC13Kaplan Meier curve

• 187 Invasive Ductal Carcinoma

0 30 60 90 120 150 1800

20

40

60

80

100

Dis

ease

free

sur

viva

ll p-value=0.04

Ove

rall

surv

ival

p-value=0.004

0 30 60 90 120 150 1800

20

40

60

80

100

ANAPC13ANAPC13is an independent prognostic factor in is an independent prognostic factor in

invasive breast cancerinvasive breast cancer

• women diagnosed with ANAPC13 negative tumor has twice the risk of dying from the disease

than patients with positive tumor

Sens-Abuázar et al ., Translational Oncology. 2012

copy number alterations (CNAs)

ANAPC13ANAPC13 expression expression versusversus genomic genomic instabilityinstability

qRT-PCR in 42 IDC cases

High expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 2122 X Y20

Chr 1 Chr 8 Chr 17

Chr 1(q25.2-q25.3)

Chr 8 Chr 17(q24.2)

Low expression

1 2 3 4 5 6 7 8 10 11 12 13 14 16 21 X Y15 2017 229 1819

Gains and losses according to ANAPC13 expression level

lossesgains

*

• Participation in cromatides separation in cell division

Sens-Abuázar et al ., Translational Oncology. 2012

•cDNA microarray platform G4851A 8X60K (Agilent®) (ANAPC13 expression: low, medium and high level)•Short Time-series Expression Miner (STEM)

Gene Gene expressionexpression modulatedmodulated byby ANAPC13ANAPC13expressionexpression levellevel

• Enrichment of Cell Cycle-related Biological ProcessesProfile A

Profile B

Profile B (79 genes)Profile A (52 genes)

P<0,001 P<0,001

ANAPC13

GAPDH

ANAPC13

GAPDH-3

-2

-1

0

1

2

3

Fol

d ch

ange

(LO

G2)

-3

-2

-1

0

1

2

3

Fol

d ch

ange

(LO

G2)

Low Medium High Low Medium High

• MCF7: Tumorigenic human breast cell lines

•• ANAPC13 ANAPC13 sensesense andand antisenseantisense ORF ORF werewere insertedinserted intointo pCDNA3.1/pCDNA3.1/mycmyc--His vectorHis vector

*

Nakahata, A, Ricca T; not published

Cell number

xCELLigence System monitors cellular events

in real time

**

*

**

**

***

**

ANAPC13 overexpressing cellsANAPC13 downregulating cellsMOCK2MCF7

ANAPC13 expression level interferes in Cell ANAPC13 expression level interferes in Cell Proliferation RateProliferation Rate

Glucose Uptake assay

SummarySummary1) The most dramatic changes in gene expression profile of

epithelial cells occur in the transition from pure DCIS to in situ

component of DCIS-IDC during the course of breast tumor

progression.

2) Gene expression program for invasion is established in

epithelial cells before morphological manifestation

3) ANAPC13 is potencial biomarker for pure DCIS and IDC of

the breast

4) ANAPC13 expression level modulated the cell proliferation

rate

5) Loss of ANAPC13 is associated with genomic instability

60 s

hots

0sho

ts

refe

renc

e

LCM capH&E staining

myoepithelial-enriched cell population

LCM capH&E staining

fibroblast cell population Transcriptional analysis • Microarray

• RNAseq (NGS approaches)

PerspectivesPerspectives

• Mutation Profile – Pre-invasive lesions (distinct malignant potential – Exome sequencing)

Hereditary Breast Cancer: Hereditary Breast Cancer: Influence ofInfluence of BRCA1 BRCA1 mutation in Gene mutation in Gene

Expression of Triple Negative (TN) Expression of Triple Negative (TN) TumorsTumors

• Hereditary BC (HBC) is an autosomal dominant disease

• germ line mutations in BRCA1 and BRCA2 genes

• higher risk of developing breast and ovarian cancer

• (HBOC - Hereditary Breast and Ovarian Cancer syndrome)

• 240 women screened for BRCA1 and BRCA2 genes

• Point mutations and indels – Gene sequencing

• Chromosomal rearrangements- MLPA and CGH

• (~ 25% mutation rate)

Identification of a gene signature of Identification of a gene signature of BRCA1/BRCA2BRCA1/BRCA2 associated tumorsassociated tumors

• Fifty-four patients under 35 years old (median age of 31 years old - range 22-35),

• Agilent platform

• 9 BRCA1/2 associated and 23 BRCA1/2 negative tumors

• 48 differentially expressed genes

100% of BRCA1/BRCA2 associated

tumors was discriminated from

91% BRCA1/BRCA2 negative

tumors (21 out of 23)

• Up-regulated genes in BRCA1/2

associated tumors - DNA repairs and

mitotic cell cycle-related processes

• Up-regulated genes in BRCA1/2 negative

tumors - cell signaling and metabolic

pathway-related processes

Carraro et al., submitted

Distinct mechanisms is

involved in triggering

tumorigenesis in BRCA1

associated and negative

tumors

Distinct mechanisms is

involved in triggering

tumorigenesis in BRCA1

associated and negative

tumors

BRCA1 BRCA1 mutation status and its relation mutation status and its relation with tumor subtype and familial historywith tumor subtype and familial history

Different biological pathways

• Young patients diagnosed with TN tumors – 46% mutation rate in BRCA1

gene (6 out of 13)

• Young patients diagnosed with TN tumors and with positive familial history

– 72% mutation rate in BRCA1 gene (5 out of 7)

Brazilian young patients with TN tumor is fair mandatory for the

BRCA1 mutation screening

BRCA1 mutation triggers a significant proportion of TN tumors

Carraro et al., submitted

Triple negative breast cancer (TNBC)Triple negative breast cancer (TNBC)- TNBC- ER/PR, HER2 negative

- Very aggressive Breast Tumor subtype

- BRCA1 mutation in TN – sensitivity in PARP inhibitor – Plummer et al, 2011

- BRCA1 associated tumor [non-sense mutation - R1751X (e20)] – deleterious

- BRCA1 unclassified variant (UV) associated tumor [missense mutation - Q356R (e11)] – no deleterious

- BRCA1/BRCA2 negative tumor (wild type patient)

Ferreira et al., not published

RNA-seq (whole transcriptome from tumor and normal adjacent tissues) in SOLID platform

6 out of 8 (75%) genes showed difference in

expression level between BRCA1-associated and

negative TN tumors

T x N

SummarySummary

• Distinct mechanisms might be involved in triggering tumorigenesis in

BRCA1 associated and negative tumors

• Germ line mutation in BRCA1 gene can have high prevalence in negative

TN tumors in Brazilian young patients

PerspectivesPerspectives• Definition of BRCA1 mutation prevalence in TNBC

– High-throughput screening of BRCA1 gene in HR(-) tumors to establish

the prevalence of somatic and germline BRCA1-mutations

• Barcode approach based on Carraro et al., PLoS One, 2011

– (ROCHE-454 Junior)

• Association with clinical characteristic and drug response

Laboratory of Genomics and Molecular Biology Laboratory of Genomics and Molecular Biology –– CIPE/AC Camargo HospitalCIPE/AC Camargo Hospital

•• Elisa N Ferreira, PhD Elisa N Ferreira, PhD

•• Bianca Bianca LisboaLisboa. . MsCMsC

•• Marcia Pena, Marcia Pena, MscMsc

•• Felipe Silva, PhDFelipe Silva, PhD

•• Tatiana Tatiana RiccaRicca, PhD, PhD

•• Alex Alex FioriniFiorini, PhD, PhD

•• Adriana Adriana MitiMiti NakahataNakahata, PhD, PhD

FacilitiesFacilities

•• Hugo Froes, MD, PHD Hugo Froes, MD, PHD -- BiobankBiobank AC Camargo Hospital AC Camargo Hospital

•• Eloisa Eloisa OlivieriOlivieri, , MsCMsC / Louise Motta, Biologist, Ana / Louise Motta, Biologist, Ana -- Macromolecules laboratoryMacromolecules laboratory

Collaborators of CIPE/ AC Camargo Hospital Collaborators of CIPE/ AC Camargo Hospital

•• Ana Cristina Ana Cristina KrepischiKrepischi, PhD , PhD –– Laboratory of Structural GenomicsLaboratory of Structural Genomics

•• Fernando Fernando SoaresSoares, MD, PhD; Cynthia Osorio, MD, , MD, PhD; Cynthia Osorio, MD, MsCMsC –– Pathology DepartmentPathology Department

•• Emmanuel Dias Emmanuel Dias NetoNeto, PhD / Diana , PhD / Diana NunesNunes, PhD , PhD -- Laboratory of Medical GenomicsLaboratory of Medical Genomics

•• Maria Isabel Maria Isabel AchatsAchats, MD, PhD. , MD, PhD. DepartamentDepartament of of OncogeneticsOncogenetics

•• Maria Socorro Maria Socorro MacielMaciel, MD, PhD, , MD, PhD, MastologyMastology DepartmentDepartment

CollaboratorsCollaborators

•• Maria Mitzi Maria Mitzi BrentaniBrentani, MD, PhD , MD, PhD –– FaculdadeFaculdade MedicinaMedicina –– USPUSP

•• Helena Helena BrentaniBrentani, MD, PhD , MD, PhD –– FaculdadeFaculdade MedicinaMedicina –– USPUSP

•• Anamaria Camargo, PhD / Anamaria Camargo, PhD / EricoErico Costa Costa –– SirioSirio LibanesLibanes Hospital / Ludwig InstituteHospital / Ludwig Institute

AcknowledgmentsAcknowledgments

Ricardo Renzo Ricardo Renzo BrentaniBrentaniin memoriamin memoriam

Financial Support:

Laboratory of Genomics and Molecular Biology - CIPE

-- Elisa Napolitano e Ferreira, PhD, Biologist, Junior ResearcherElisa Napolitano e Ferreira, PhD, Biologist, Junior Researcher

-- Alex Alex FioriniFiorini CarvalhoCarvalho, Biologist, PhD, Senior Researcher, Biologist, PhD, Senior Researcher

-- Felipe Felipe CarneiroCarneiro Silva, Biologist, PhD, Senior TechnicianSilva, Biologist, PhD, Senior Technician

-- Bianca Bianca LisboaLisboa, Biologist, PhD, Senior Technician, Biologist, PhD, Senior Technician

-- Tatiana Tatiana IervolinoIervolino RiccaRicca, Biologist, PhD, Postdoc, Biologist, PhD, Postdoc

-- VaninaVanina ElianeEliane Elias, PhD, Elias, PhD, PosdocPosdoc

-- Andrea Andrea YaguiuYaguiu, PhD, , PhD, PosdocPosdoc

-- BrunaBruna DurDurããeses de de FigueiredoFigueiredo Barros, Biologist, PhDBarros, Biologist, PhD--studentstudent

-- Carolina Carolina SensSens AbuazarAbuazar, Biologist, MSc, PhD, Biologist, MSc, PhD--studentstudent

-- GiovanaGiovana TardinTardin TorrezanTorrezan, Biologist, MSc, PhD, Biologist, MSc, PhD--studentstudent

-- JosJoséé Roberto de Oliveira Ferreira, Pharmacist, MSc, PhDRoberto de Oliveira Ferreira, Pharmacist, MSc, PhD--studentstudent

-- Mabel Mabel GiglioliaGigliolia PinillaPinilla FernFernáándezndez, Medical Technologist, MSc, Medical Technologist, MSc--studentstudent

--MMáárciarcia FigueiredoFigueiredo Pena, Biologist, MSc, PhDPena, Biologist, MSc, PhD--studentstudent

--Mayra Toledo de Castro, Biologist, MScMayra Toledo de Castro, Biologist, MSc--studentstudent