”” 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