Riesgo genético y familiar. Javier Benitez Centro Nacional Investigaciones Oncológicas Madrid

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1 Riesgo genético y familiar Javier Benitez Centro Nacional Investigaciones Oncológicas Madrid

2 Cancer: An example of complex disease 80-85% of cancers Polygenic model (combination of X genes) Low penetrance genes ( > 1000 genes). OR: <1.3 Interaction between genetic and environmental factors

3 Family with breast/ovarian cancer breast cancer breast cancer 62 yrs breast cancer 32 yrs ovarian cancer 54 yrs breast cancer 45 yrs breast cancer 47 y breast cancer 37 yrs -High penetrance genes BRCA1/2 -Dominant model -Early age of onset -Bilaterality Familial Breast Cancer represents about 5% of Breast Cancer

4 Breast/ovarian cancer syndrome BRCA1: Mapped in q Cloned in 1994 Associated to breast/ovarian cancer BRCA2: Mapped in q Cloned in 1995 Associated to breast cancer in males Spain: 1.5/1.000 women are carriers >25.000

5 1: Selection of candidate families for genetic studies. Criteria (flexible) of HRF At least three or more women with breast/ovarian cancer (One under 50 years old) Families with breast cancer and at least one male with breast cancer Young women (under 35) Jewish antecedents

6 1: Selection of candidate families (400 Spanish high risk families ) 26% with mutation in BRCA1/2-3 women with breast cancer 15% - 5 or more women with breast cancer: 25% - Breast cancer plus ovarian cancer: 52% - Breast cancer in males: 60% -Young women (<35) 6% -Young women (<35), triple negative, 10-12% -It is necessary the search for new diagnostic markers -Identification of BRCAX genes O.Díez et al. Hum Mutat 2003

7 Penetrance and expressivity 80 dx 45 BRCA1+ 79 dx 67 BRCA1+ 81 dx 69 BRCA1-61 BRCA dx59 BRCA Dx55, 60 BRCA1 + BRCA1 - BRCA1 + BRCA1 - BRCA1-38 Dx35 BRCA dx30 BRCA dx33 BRCA BRCA1 - Unilateral breast cancer Prostate cancer Bilateral breast cancer Pancreatic cancer Ovarian cancer

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9 MODIFICADORES DE RIESGO Localización y tipo de mutación Genes modificadores Factores exógenos

10 Location and type of mutation

11 Modification of Risk in BRCA1/2 Carriers. COGS project Min, Max 5%, 95% Average 82% Min, Max 5%, 95% Average 90% 48% 38% Breast Cancer Loci: FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, 1p11.2, ZNF365, CDKN2A, 9q31.2, 12q24

12 Nickels S et al. PLoS Genet Mar;9(3):e doi: /journal.pgen Epub 2013 Mar 27. Evidence of gene-environment interactions between common breast cancer susceptibility loci and established environmental risk factors. COGS project: breast cancer patients and controls Analysis of ten environmental risk factors Overall, the per-allele odds ratio (95% confidence interval) for LSP1-rs was 1.08 ( ) in nulliparous women and ranged from 1.03 ( ) in parous women with one birth to 1.26 ( ) in women with at least four births. For CASP8-rs , the per-allele OR was 0.91 ( ) in those with an alcohol intake of <20 g/day and 1.45 ( ) in those who drank 20 g/day. These data provide first strong evidence that the risk of breast cancer associated with some common genetic variants may vary with environmental risk factors.

13 Types of supressor genes: Gatekeepers : Regulan DIRECTAMENTE el crecimiento del tumor APC, VHL, RB Caretakers: Regulan INDIRECTAMENTE el crecimiento del tumor HNPCC, XP, BRCA1/2...

14 Methylation!! Osorio et al, Int J Cancer 2002

15 BRCA 1/2 son genes supresores Explica la Penetrancia Variable Explica el riesgo de : Segunda Neoplasia Modelo de Herencia Dominante a nivel de susceptibilidad Modelo de Herencia Recesiva a nivel de desarrollo del tumor

16 BRCA1 and BRCA2* Functions Integrity of the genome & Chromosomal stability DNA repair DSBs repair & NER* Checkpoint control Block G2/M & G1/S phases* Chromatin remodelling BRCA1 Replication Daughter strand gap repair Histone deacetylasa function Transcriptional activator Transcription Narod & Foulkes., Nature Reviews, 2004 & Boulton, Biochemical Society, 2006 Ubiquitylation Protein degradation Meiotic sex chromosome inactivation Sterility Telomeres Oxidative stress

17 Análisis de mutaciones recurrentes BRCA1 185delAG Int5>G 1294del del4 Arg1443Ter 5385insC BRCA2 3034delAAAC 6174delT 9254del5 9326insA

18 Recurrent mutations E.10 E.11 CNIO, Febrero 2010 Revisión > 2000 familias

19 Técnicas alternativas de prescreening SSCP Single Strand Conformation Polymorphism CSGE Conformation Sensitive Gel Electrophoresis DHPLC High Performance Denaturing Liquid Chromatography HRM High Resolution Melting

20 Secuenciación directa por el método de Sanger: gold standard para el análisis de BRCA1/2 Secuencia normal NGS Next Generation Sequencing Secuencia mutada Sensibilidad cercana al 100% Alto coste en tiempo y dinero debido al gran tamaño de los genes

21 APLICATIONS OF MASSIVE SEQUENCING IN FBOC -1)The study of BRCA1/2 genes is faster, cheaper, more accurate -2)The study of all high and moderate susceptibility genes -3)The search of new high susceptibility genes (Whole Exome Sequencing)

22 1) The study of BRCA1 and BRCA2 is expansive and time comsuming CNIO: DHPLC+Sanger until September 2011 Results 3-4 months 1-2 semanas Samples/year around Cost/sample % % 3.5% 1% Similar results 22,5% BRCA1/2 + 11% USV 6% 66% BRCAX 17,5% BRCA1/2+ 16% USV 5% 66,5% BRCAX

23 2)The study of all high and moderate susceptibility genes h

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25 Searching for breast cancer susceptibility genes Candidate pathways Linkage studies New technologies

26 Searching for high susceptibility genes BRCA1 and BRCA2 explain up to 25% of familial breast cancer The identification of new high susceptibility genes in BRCAX families International study 7 Families with 8-10 breast cancer women Sequencing of 2 affected women Solexa platform

27 Plos One, February, NO FAMILIES WITH HIGH SUSCEPTIBILITY GENES DESPITE THE SIZE OF FAMILIES (only rare variants with unknown significance). Similar results in other studies with different approaches - Rosa-Rosa JM et al, PLOs One 2011 ( 3 regions with LODs >2) - Snapek et al BCRT 2012 (50 BRCAX probands) - Thompsom et al PLOs Genet 2012 (15 small families) - Hilbers FS et al. PLOs One 2013 (6 regions with LOH by acgh)

28 Monogenic vs polygenic model High risk mutation in BRCA1/2 Low and moderate susceptibility variants

29 The landscape of breast cancer genes (2012) -RARE VARIANTS 20% HIGH-RISK BRCA1/2 -NON CODING REGIONS -INFINITESIMAL MODEL -OTHER UNEXPLAI NED 28% 5% 5% Other HIGH- RISK MOD-RISK LOW-RISK

30 Conclusiones Only 30% of cases can be currently explained by genetic mutations. The genetic architecture of familial breast cancer is more complex than we thought Great Genetic Heterogeneity Several genes, few families explained per gene Polygenic model explains part of the FRR - To translate these results to the clinical practice will be complicated

31 Bioinformatic pipeline BWA+BFAST, SAMTOOLS GATK, SOAP Modification of Rosa-Rosa pipeline, Plos One 2011

32 POLYGENIC MODEL

33 Polygenic effect of several risk alleles 25% Eight loci in population 20% 15% 10% Population Cases 5% 0% Relative risk

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35 BRCA1 protein BRCA1