MADRID / SPAIN 7–8 Ju Ne 2012 5th FAMILIAL …82_booklet.pdf · 011 SPeAKeRS’ ABSTRAcTS 021...

MADRID / SPAIN7–8 JuNe 2012

5th FAMILIAL cANceR coNFeReNceChair J. Benítez, R. Eeles, H. Vasen

eSo INSIDe TRAcK coNFeReNce

MADRID / SPAIN

7–8 JuNe 20125th FAMILIAL cANceR coNFeReNceEuropEan Schoolof oncology (ESo)SpaniSh national cancEr rESEarch cEntrE (cnio)

5th FAMILIAL cAncer conFerence

chAIrs’ MessAge 3

Dear colleagues,

It is our pleasure to welcome you again to the fifth Madrid Familial Cancer Conference that since 2004 we hold every two years. The present Conference sponsored by the European School of Oncology and hosted at the Spanish National Cancer Research Centre (CNIO), aims to present recent advances in this field from a multidisciplinary point of view.

During this decade, familial cancer has become an important challenge for oncologists, clinicians, geneticists and molecular biologists, due to its implications not only at the individual but also at the familial and social level. Nowadays, bioinformaticians and epidemiologists have joined this multidisciplinary group because of the incorporation of new technologies and the need to extend our knowledge at a population level if we want to give an answer to the new challenges that the familial cancer studies encounter.

This year, the interest for this type of cancer remains and once more we have reached the maxi-mum occupancy of our auditorium. We really hope that the topics, talks, discussions and meetings among professionals and/or students meet everybody's expectations.

Thank you all for coming to Madrid and we hope you enjoy a pleasant and successful Conference.

Madrid, June 7-8th, 2012

Ros Eeles, Hans Vasen and Javier Benitez

INDeX

GeNeRAL INFoRMATIoN007

PRoGRAMMe011

SPeAKeRS’ ABSTRAcTS021

PARTIcIPANTS’ PoSTeRS SeSSIoN 1066

PARTIcIPANTS’ PoSTeRS SeSSIoN 2082

FAcuLTyAND AuThoRS’ INDeX107

GeNeRALINFoRMATIoN5th FAMILIAL cAncer conFerence

generAL InForMAtIon


8


generAL InForMAtIon 9

coNFeReNce VeNueSpanish National Cancer Research Centre Melchor Fernández Almagro, 3E-28029 Madrid, España

oRGANISING SecReTARIAT Mercedes Moro and Virginia de la CruzScientific Events OfficeCentro Nacional de Investigaciones Oncológicas

– CNIO (Spanish National Cancer Research Centre)Melchor Fernández Almagro, 3E-28029 Madrid, EspañaTel.: +34 91 224 69 00Fax: +34 91 224 69 81E-mail: [email protected]

eSo SecReTARIATDaniela MengatoVia del Bollo, 4—20123 Milan, Italia Tel.: +39 02 85 46 45 23Fax: +39 02 85 46 45 45E-mail: [email protected]

oFFIcIAL LANGuAGeThe official language of the course will be English. Simultaneous translation will not be provided.

ceRTIFIcATeS AND AccReDITATIoNA certificate of participation will be issued to all participants. This meeting has received CME accreditation by the Accreditation Council of Oncology in Europe (ACOE). The ACOE has recommended that the event be granted 12 European CME credits.

BADGe The badge is the only official evidence of regis-tration and should be worn at all times during the conference.

LoST AND FouNDFor lost and found objects please go to the registration desk.

INSuRANceThe organisers bear no responsibility for unto-ward events in connection with, before, during and after the conference.

Participants are strongly advised to take out their own personal and travel insurance cov-erage.

MeSSAGe ceNTRePeople can leave messages and other informa-tion for participants at the registration desk.

ReGISTRATIoN PAcKAGeThe full registration package includes:

· Welcome party· Attendance to the scientific sessions· Programme book· Louncheons· Coffee breaks

PoSTeRSThere will be two poster sessions throughout the conference.

Please, consult the poster identification number in this programme book.

We kindly ask participants to display their poster on the morning of the session before the begin-ning of the lectures, and remove them on the evening of each day. The author must be present for discussion during breaks throughout the conference. Coffee breaks will be served in the entrance hall of the CNIO next to the poster exhi-bition area in order to allow sufficient time for viewing and discussing.

SLIDeSAll speakers should take their slides to the technicians in the conference room no less than one hour before the start of their session. All presentations will be uploaded into the auditorium computers (PC or MAC) so we will appreciate it if you could bring it on a CD or pen-drive at the beginning of your corresponding presentation day.

SMoKING PoLIcyThere will be a strict no smoking policy in all areas of the conference.

STAFFStaff members wearing the ESO badge will be pleased to assist you with any further informa-tion you may require.

PRoGRAMMe5th FAMILIAL cAncer conFerence

ProgrAMMe


12


ProgrAMMe 13

WeDNeSDAy, 6 JuNe

19:30 WeLcoMe PArTY For SPeAkerS & PArTIcIPAnTS venue Ac cuzco HoTeL Paseo de la castellana 133, Madrid

ThuRSDAy, 7 JuNe

09:00 WeLcoMe Javier Benítez, CNIO, SP

09:10 HeredITArY cAncer SYndroMeS. WHere

do We go? Javier Benítez, CNIO, SP

09:30 geneTIc vArIAnTS And FAMILIAL cAncer rISk

Roger Milne, CNIO, SP

09:50 geneTIc ModIFIerS oF cAncer rISk For BrcA1 And BrcA2 MuTATIon cArrIerS: IMPLIcATIonS For rISk PredIcTIon

Antonis Antoniou, University of Cambridge, UK

10:10 evALuATIon oF uncLASSIFIed vArIAnTS In FAMILIAL cAncer SYndroMeS

David Goldgar, University of Utah, US

10:30 PIcTure, PoSTerS, coFFee BreAk

11:10 geneTIc counSeLIng In FAMILIAL cAncer

Shirley Hodgson, St. George’s Hospital Medical School, UK

11:30 Short talk A genoMe-WIde

ASSocIATIon STudY on coPY-nuMBer vArIATIon And coLorecTAL

cAncer rISk Clara Ruiz-Ponte, Galician

Public Foundation for Genomic Medicine, SP

11:40 Short talk cLInIcAL And SurgIcAL exPerIence In BrcA PoSITIve PATIenTS AT oncoLogIc InSTITuTe oF veneTo (Iov)

Stefanía Zovato, Veneto Oncology Institute, IT

11:50 round TABLe

GeNeRAL SeSSIoNChair Javier Benítez, CNIO, SP

ProgrAMMe


14


ProgrAMMe 15

12:20 THe FAMILY oF BreAST cAncer geneS

Ana Osorio, CNIO, SP

12:40 PATHoLogIcAL FeATureS oF endoMeTrIAL cAncer In LYncH SYndroMe

José Palacios, Ramón y Cajal Univeristy Hospital, SP

13:00 SeLecTIon crITerIA And cLInIcAL MAnAgeMenT

Diana Eccles, Southampton University Hospital Trust, UK

13:20 LuncH BreAk

15:00 PArP InHIBITorS: ProgreSS In cAncer THerAPY

Françoise Dantzer, University of Strasbourg, FR

15:20 Short talk rAre MuTATIonS In xrcc2 IncreASe THe rISk oF BreAST cAncer

Maroulio Pertesi, International Agency of Research on Cancer, FR

15:30 Short talk mirnA MoLecuLAr cLASSIFIcATIon oF

non-BrcA1/2 FAMILIAL BreAST TuMorS

Miljana Tanic, CNIO, SP

15:40 round TABLe

16:00 coFFee BreAk

16:30 cLInIcAL SeSSIon Hans Vasen, Leiden University

Medical Centre, NL Diana Eccles, Southampton

University Hospital Trust, UK

coMMoN cANceRS IChair Shirley Hodgson, St. George’s Hospital Medical School, UK

FRIDAy, 8 JuNe

08:30 (ePI) geneTIcS And cLInIcAL MAnAgeMenT oF LYncH SYndroMe

Gabriel Capellá, Catalan Institute of Oncology, SP

08:50 THe geneTIcS BASeS oF FAP SYndroMe

Miguel Urioste, CNIO, SP

09:10 MAnAgeMenT oF APC And MUTYH FAMILIeS

Antoni Castells, Clinic Hospital, SP

09:30 FAMILIAL ProSTATe cAncer. geneTIcS And cLInIcAL MAnAgeMenT

Rosalind Eeles, The Royal Marsden Hospital, UK

coMMoN cANceRS IIChair Fred Menko, VU University Medical Center, NL

09:50 Short talk gerMLIne BrcA MuTATIonS Are ASSocIATed WITH nodAL InvoLveMenT, dISTAnT MeTASTASIS And Poor SurvIvAL ouTcoMeS In ProSTATe cAncer

Chee Goh, The Institute of Cancer Research, UK

10:00 round TABLe

10:30 coFFee BreAk

ThuRSDAy, 7 JuNe

mmoro

Text Box

Bárbara Rivera

ProgrAMMe


16


ProgrAMMe 17

11:00 geneTIc HeTerogeneITY

oF FAMILIAL PHeocHroMocYToMA

Mercedes Robledo, CNIO, SP

11:20 geneTIcS And MAnAgeMenT oF HeredITArY PAncreATIc cAncer

Hans Vasen, Leiden University Medical Centre, NL

11:40 BIrT-Hogg-duBÉ SYndroMe. dIAgnoSIS And cLInIcAL MAnAgeMenT

Fred Menko, VU University Medical Center, NL

12:00 cLInIcAL And geneTIc cHArAcTerISTIcS In FAMILIAL MALIgnAnT MeLAnoMA

Nelleke Gruis, Leiden University Medical Center, NL

12:20 Short talk InTegrATIon oF mirnA And mrnA exPreSSIon ProFILeS In PHeocHroMocYToMA And PArAgAngLIoMA IdenTIFIeS genoTYPe-SPecIFIc BIoMArkerS And PoTenTIALLY reguLATed PATHWAYS

Aguirre A. de Cubas, CNIO, SP

12:30 round TABLe

13:00 LuncH BreAk

oTheR heReDITARy cANceR SyNDRoMeSChair Rosalind Eeles, The Royal Marsden Hospital, UK

14:15 THe FAnconI AneMIA SYndroMe

Jordi Surrallés, Autonoma University of Barcelona, SP

14:35 dYSkerAToSIS congenITA: A TuMor Prone dISeASe THAT HArBourS conSITuTIve dnA dAMAge

Rosario Perona, Biomedical Research Institute, SP

14:55 geneTIc SYndroMeS oF THe ras/MAPk PATHWAY

Carmen Guerra, CNIO, SP

15:15 LIFrAuMenI SYndroMe. FroM MoLecuLAr BASeS To cLInIcAL MAnAgeMenT

Thierry Frebourg, Rouen University Hospital, FR

15:35 Short talk SurveILLAnce oF AdoLeScenTS And Young AduLT PATIenTS WITH FAnconI AneMIA (FA): AWAreneSS oF dIAgnoSIng SoLId

TuMorS AT A Young Age Judith Balmaña, Vall d'Hebron

University Hospital, SP

15:45 round TABLe

16:15 coFFee BreAk

RARe TuMoRSChair Mercedes Robledo, CNIO, SP

FRIDAy, 8 JuNe FRIDAy, 8 JuNe

ProgrAMMe


18

16:45 IdenTIFIcATIon oF noveL TArgeTS And regIonS THrougH InTegrATIve genoMIc AnALYSIS

María José García, CNIO, SP

17:05 THe cLInIcAL PoTenTIAL oF SequencIng cAncer genoMeS For PerSonALIzed MedIcIne

Dimitrios H. Roukos, Ionnanina University School of Medicine, GR

17:25 WHoLe exoMe SequencIng In THe SeArcH oF HIgH SuScePTIBILITY-geneS

Alberto Cascón, CNIO, SP

17:45 Short talk MuTATIonAL AnALYSIS oF BrcA1/BrcA2 geneS uSIng nexT-generATIon SequencIng TecHnoLogY: A coMPreHenSIve AnALYSIS ProTocoL

Lucia Pérez-Carbonero, Genomics Systems, SP

17:55 round TABLe

NeW TechNoLoGIeS APPLIeD To FAMILIAL cANceR STuDIeSChair Ana Osorio, CNIO, SP

FRIDAy, 8 JuNe

SPeAKeRS’ ABSTRAcTS5th FAMILIAL cAncer conFerence


speakers’ aBsTraCTs / generAL sessIon 23

GeNeRAL SeSSIoNChair J. Benítez

heReDITARy cANceR SyNDRoMeS.WheRe Do We Go?Javier Benítezhuman genetics group, human genetics Programme, centro nacional de Investigaciones oncológicas (cnIo), Madrid, spain

Hereditary cancer represents around 5-10% of all cancers and it is characterized by an earlier age of onset, bilaterality or multifocality, and the presence of different affected members in the family with the same or other types of cancer. In children it is estimated that this percentage is greater than in adults and that around 1/4 of them are candidates for genetic counseling.

There are three main steps in the study of these cases: 1) the identification and assessment of genetic risk in candidate families. It is one of the most important tasks due to the inherent costs of genetic studies. 2) The genetic study in selected families is the second step and it is based on the possibility to identify individuals at risk and to apply a personalized medicine depending on the mutational status. 3) The surveillance and clinical management of high-risk individuals is the last step and it has the objective to prevent the tumor development whenever possible, to perform and earlier diagnosis and apply a specific treatment or to plan reproductive assistance methods.

There are currently around 45 common hereditary cancers and up to 200 including rare syn-dromes or cancer susceptibility syndromes with a genetic base. However, many of the genes remain unknown. In this way the search for candidate genes is a priority in order to get to know the genetic profile of these cancers. The recent incorporation of the whole exome sequencing technology has opened an important window for gene discovery although until know it has obtained a moderate success. The discovery of low susceptibility alleles contributing to the familial cancer risk has proved to be more successful as demonstrated by different GWAS study-ing several types of cancer. So, during the past years we have increased our knowledge about the number of high susceptibility genes, increased the technical accuracy, lowered the cost of genetic studies and reduced the time of diagnostic response. However, we have learnt that the genetic heterogeneity is greater than we thought and that it is likely that many families with common hereditary cancers such as breast, colorectal, pancreatic or prostate cancer among others, will be explained by a combination of moderate and low-susceptibility genes. On the other hand the problem of unclassified variants not only remains but it is increasing as many genes are available for analysis and more predictive programs (in some cases providing contradictory predictions) have been developed by bioinformatics groups and private companies. The simplification and incorporation of massive sequencing to the clinical practice, the information and education of the families under study and the design of greater number of collaborative studies seem to be some of the objectives we need to address in order to unravel currently unsolved issues in the field of familial cancer genetics.

speakers’ aBsTraCTs / generAL sessIon


24



GeNeTIc VARIANTS AND FAMILIAL cANceR RISKRoger MilnecnIo, Madrid, spain

The genetic determinants of many of the more common familial cancers remain largely unknown, despite large international collaborative efforts to uncover them. Linkage studies have success-fully identified high-risk genes in breast, ovarian and colon cancer, as well as melanoma, among others, but mutations in these genes are not found in most familial cases. Further linkage studies of more specific phenotypes have been largely unsuccessful in uncovering novel major genes. Mas-sive parallel sequencing shows great promise to make furthering genetic discoveries. However, genetic heterogeneity presents a major obstacle for several familial cancers. This may take the form of rare variants that each explain a very small percentage of familial disease aggregation and/or common susceptibility variants with very small effects that act under a polygenic model to explain both familial and sporadic forms of cancer. A review of the most appropriate methods, technologies and study designs is presented, together with the challenges and limitations they present and a discussion of the genetic discoveries they have afforded to date.

GeNeTIc MoDIFIeRS oF cANceR RISK FoR BRCA1 AND BRCA2 MuTATIoN cARRIeRS:IMPLIcATIoNS FoR RISK PReDIcTIoNAntonis C. Antonioucentre for cancer genetic epidemiology, Department of Public health and Primary care, University of cambridge, UK

Pathogenic mutations in BRCA1 and BRCA2 confer increased risks for breast and ovarian cancer and account for approximately 15% of the excess familial risk of breast cancer among first-degree relatives. There is considerable evidence indicating that the BRCA1 and BRCA2 associated cancer risks vary by other genetic and environmental factors clustering in families. In the past few years, based on the availability of genome-wide association data and samples from large collaborative studies, several common alleles have been found to modify breast or ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. These common alleles explain a small proportion of the genetic variability in breast or ovarian cancer risk for mutation carriers, suggesting more modifiers remain to be identified. We review the so far identified genetic modifiers of breast and ovarian cancer risk and how these associations vary with different tumour characteristics in BRCA1 and BRCA2 carriers. We then consider the implications for risk prediction by considering the genetic risk modifiers either in isolation or jointly with other known modifiers of risk. Women carrying BRCA1 and BRCA2 mutations could be among the first to benefit from clinical applications of common variants identified through genome-wide association studies.



26



eVALuATIoN oF uNcLASSIFIeDVARIANTS IN FAMILIAL cANceR SyNDRoMeSDavid GoldgarDepartment of Dermatology, University of Utah school of Medicine, salt Lake city, Us

Clinicians and scientists are accustomed to forming conclusions based on multiple lines of evidence, and to dealing with degrees of uncertainty in these conclusions. Genetic testing for cancer predisposition syndromes often identifies variants whose pathogenicity is uncertain, which poses difficult issues for risk communication in the genetic counseling setting. In the literature these have been designated Variants of Uncertain Significance (VUS), or Unclassified Variants (UV). As next-generation sequencing methods, which allow testing of many (or all) genes simultaneously, make the transition from research to clinical diagnostic testing, this problem will increase dramatically. I will present a number of different approaches that have been employed in order to try and classify such variants. For some variants, case-control, segregation, family history, or other statistical studies can provide strong evidence of direct association with cancer risk. For most variants, other evidence is available that relates to properties of the protein or gene sequence. I will discuss the assessment of prior probability, and how to combine the vari-ous sources of data into a statistically valid integrated assessment with a posterior probability of pathogenicity. Further, I will discuss some of the issues involved in this process and the assump-tions that underpin many of the methods used in the evaluation process. Lastly, I will provide a description and update on an international consortium (ENIGMA) that has been formed to try and classify these variants in a large-scale and systematic fashion.

GeNeTIc couNSeLING IN FAMILIAL cANceRShirley Victoria Hodgsonst. george’s hospital Medical school, London, UK

Genetic counselling in familial cancer presents some unique challenges. Clearly it is important to establish the patient’s agenda at the start, because sometimes the referring physician may not have conveyed their reasons for requesting the appointment for genetic counselling very clearly. People may wish to come and establish their own risk of developing cancer, and ways to reduce these risks, or to find out whether they carry a cancer predisposing mutation inherited from a parent; or they may have cancer themselves and wish to know whether they have a detectable cancer predisposing mutation. This has implications for their own future care, and also opens the possibility of genetic testing for their children and other close relatives. Clearly when a mutation has been identified in an individual it is important to consider who in the family may be at risk and could benefit from genetic testing. This requires arranging for such relatives to be informed of this in an appropriate manner. Sometimes certain individuals prefer not to release the results of their genetic test to their relatives, which leaves the counsellor with the dilemma about whether it is right to breach their confidentiality as a “duty to warn” the at-risk relatives. Genetic tests in at-risk relatives may test an intervening relative inadvertently and this requires careful handling.

New dilemmas are emerging with our increased understanding of genetic testing. Is genetic testing for founder mutations in certain populations appropriate? What counselling should be offered? Should all colorectal cancers in individuals below a certain age be tested for mismatch repair defects, and if so, again, what counselling should be offered?

Other important aspects of genetic counselling in this context are discussions about prenatal and pre-implantation genetic testing. There are also aspects of discovering that there is a strong family history of cancer which may instil great anxiety; confirmation of diagnoses in a family is important, as occasional individuals may invent or be incorrectly informed about the extent of their family history of cancer.

When someone is found to have a cancer susceptibility they will be counselled about prophy-lactic and surveillance options, which must be fully evaluated and explained. Here the remit of the genetic counsellor may broaden into areas of public health; who should be offered what in terms of these options, bearing in mind the costs and risks and benefits?

The discovery of variants in the genome which confer small increases or decreases in risks of cancer is becoming an important area for discussion. Can such tests be used yet in a clinical setting? Would a combination of such tests contribute to determining the level of risk at which certain screening interventions could be made available? This field is evolving fast and we need to keep up with it!



28



ShoRT TALK A GeNoMe-WIDe ASSocIATIoN STuDy oN coPy-NuMBeR VARIATIoNAND coLoRecTAL cANceR RISK

Fernández-Rozadilla Ceres1, Cazier Jean-Baptiste2, Tomlinson Ian2, Brea-Fernández Alejandro1, Bértolo Adriana1, Ferro Marta1, Bujanda Luis3, Bessa Xavier4, Andreu Montserrat4, Jover Rodrigo5, Llor Xavier6, Castells Antoni7, Carracedo Angel1, Castellví-Bel Sergi7,Ruiz-Ponte Clara1 for the EPICOLON consortium1Fundacion Publica galega de Medicina Xenomica (FPgMX)-grupo de Medicina Xenómica, IDIs. santiago de compostela, spain; 2Wellcome trust centre for human genetics, University of oxford, UK; 3Unidad Multidisciplinar de cáncer colorrectal, hospital de Donostia, san sebastian, spain; 4gastroenterología, hospital del Mar, Barcelona, spain; 5gastroenterología, hospital general de Alicante, Alicante, spain; 6Digestive Diseases and nutrition, University of Illinois, chicago, UsA; 7gastroenterología, hospital clinic, cIBerehd, IDIBAPs, U. Barcelona, Barcelona, spain

Colorectal cancer (CRC) is a complex disease, and thus it development is determined by the combination of both genetic as well as environmental factors. Twin studies have estimated CRC heritability to be around 35%. Despite the fact that 5% of the cases may be attributable to hereditary CRC syndromes and an extra 7% is explained by the 18 new moderately penetrant risk loci discovered through genome-wide association studies (GWAS), there is still a considerable proportion of the variation that remains unexplained.

Thus, the analysis of other types of variation, namely copy-number variants, could help us understand part of the remaining heritability fraction. In this study, we have performed a GWAS on CNVs in 881 CRC cases and 667 controls of Spanish origin. By these means, we have been able to identify 4 common CNVs (or copy-number polymorphisms-CNPs), that seem to be related to CRC susceptibility, as well as body mass measures. Despite these results, we recommend future replication efforts in larger cohorts in order to fully ascertain the relationship between these variants and CRC risk.

Keywords GWAS, Colorectal Cancer, CNVs, Risk Variants

This work was supported by grants from Fondo de Investigación Sanitaria/FEDER (08/1276, 08/0024, PS 09/02368) and Fundació Olga Torres.

ShoRT TALK cLINIcAL AND SuRGIcAL eXPeRIeNce IN BRcA PoSITIVe PATIeNTS AT oNcoLoGIc INSTITuTe oF VeNeTo (IoV)

Stefania Zovato, Emma D'Andrea, Marco Montagna, Silvia Tognazzo, Cristina Ghiotto, Enrica Baldan, Fernando Bozza, Giuseppe Opocheroncologic Institute of Veneto (IoV), Padova, Italy

Introduction Constitutive alterations in the genes BRCA1 / 2 can increase the lifetime risk of cancer to 85% for breast cancer and 60% for ovarian cancer in susceptible individuals. Since 2002 at the Oncologic Institute of Veneto (IOV), patients with a family history of breast cancer are studied and followed by a multidisciplinary team consisting of geneticists, surgeons, patholo-gists, oncologists and psychologists.

Materials and methods At the IOV, genetic counseling for patients with breast cancer who have a family history of the disease is carried out. If recommended by the counseling (ie according to the criteria FONCaM), and prior written consent is obtained, the patient is submitted to mutational analysis of BRCA1 / 2 (ie genetic testing). In patients with BRCA pathogenetic mutation, a treatment and medical-surgical follow-up plan is set, according to accredited international guidelines. This present analysis takes into account a subset of 68 BRCA1 / 2 patients, diagnosed with breast cancer currently being cared for both clinically and sugically at the Institute. In the same period, 234 patients with a significantly elevated BRCA-PRO but with an indeterminate genetic test result were identified. The healthy BRCA1 mutation carriers were 29 and those with BRCA2 mutations were 30.

Results Of the 68 patients studied, 30 were BRCA1 positive while 38 were BRCA2 positive. The average age was 47 years (27-75). The tumor was surgically removed in all patients: 34 with quadrantectomy and with radical mastectomy in the remaining half. Twenty-one patients had bilateral cancer, 3 also developed ovarian cancer, and another 5, a second neoplasm (bladder, melanoma, kidney, lung, lymphoma). Four patients were treated with neoadjuvant chemotherapy. Histology showed invasive ductal carcinoma in 41 patients, invasive lobular in 15, DCIS in 9 and other types (tubular / medullary) in 3 patients. The receptor involved 40 positive tumors (20 of the "triple-negative"). About half of the tumors were placed in stage I (35/68) and only 3 patients were already metastatic at diagnosis. Over 50% of patients underwent adjuvant hormonal treatment and 45 patients were also submitted to chemotherapy with anthracyclines. During follow-up, 16 patients were subjected to prophylactic oophorectomy without complications. To date, only 2 patients have died of disease progression; the remaining 66 are disease-free.

Conclusions Patients with hereditary cancer should be followed with particular care in their course of treatment, from surgery to medical follow-up. In our experience, the presence of a multidisciplinary team is a key requirement to implement a correct diagnostic and clinical pathway in individuals at genetic risk of disease.


speakers’ aBsTraCTs / coMMon cAncers I 31

coMMoN cANceRS IChair Shirley Hodgson

The FAMILy oF BReAST cANceR GeNeSAna OsoriocnIo, spain

BRCA1 and BRCA2 are the genes whose implication in hereditary breast and ovarian cancer (HBOC) is best established. Although these two genes were identified more than a 15 years ago [1, 2], their germ-line alteration is still the most powerful predictor of the likelihood of developing breast and ovarian cancer. Individuals carrying mutations in these two genes have a lifetime risk that varies between 40% and 80%, depending on the population and the type of patients studied [3-5]. However, less than 25% of the inherited susceptibility to breast cancer is explained by these muta-tions [6, 7]. Mutations in other genes such as TP53, PTEN, STK11 and CDH1 have been linked to a high risk of breast cancer as part of more broad cancer syndromes but so far no other high-penetrance susceptibility genes, specifically involved in the HBOC syndrome have been identified. The failure to find additional high-risk genes lead to the hypothesis of the polygenic model underlying the excess of familial risk [6, 8] by a combination of low- and moderate-risk alleles. In the last ten years, rare germ-line variants conferring a two- to four-fold increased risk of breast cancer have been found in several genes involved in DNA repair. More recently, the genome wide association studies (GWAS) have identified at least 25 common Single Nucleotide Polymorphisms (SNPs) conferring less than 1.5-fold risk to the disease [9].

Among the moderate-high risk genes identified so far, especially interesting are those involved in the Fanconi Anemia pathway, including BRCA2/FAND1 and others such as BRIP1/FANCJ and PALB2/FANCN [10, 11], that are associated with the Fanconi Anemia phenotype when both alleles are mutated, and confer increased breast cancer risk when mutated in heterozygosis. Investigation of the FA/BRCA pathway, lead to an important discovery in 2010, when the RAD51C/FANCO gene (a RAD51 paralog) was identified as a putative third high-risk gene involved in 1, 3% of hereditary breast and ovarian cancer cases. Although divergent results from subsequent studies have gener-ated controversy about the role of RAD51C, a very recent study from our group suggest that it should be included in genetic testing in the clinical setting, at least for breast and ovarian cancer families [12]. More recently, two other RAD51 paralogs, RAD51D and XRCC2 have also been found to confer increased susceptibility to ovarian and breast cancer, respectively [13, 14]. However, none of these genes account for more that 1% of the hereditary cases each, which leaves around 65% of the excess of familial risk still to be explained.

Taking into account this great genetic heterogeneity, Next Generation Sequencing aroused as a robust approach for the discovery of the remaining breast cancer susceptibility genes. How-ever, the studies published so far using this technology have not yielded conclusive results [15]. Deciphering the genetic basis of the unexplained familial risk and translating those findings to the clinics, has turned out to be more challenging than expected 15 years ago when the BRCA genes were discovered. •

speakers’ aBsTraCTs / coMMon cAncers I


32



references:

1 Miki Y, swensen J, shattuck-eidens D, Futreal pa, Harshman k, Tavtigian s, Liu Q, Cochran C, Bennett LM, Ding W et al: a strong candidate for the breast and ovarian cancer susceptibility gene BrCa1. science 1994, 266(5182):66-71.

2 Wooster r, Bignell G, Lancaster J, swift s, seal s, Mangion J, Collins N, Gregory s, Gumbs C, Micklem G: Identification of the breast cancer susceptibility gene BrCa2. Nature 1995, 378(6559):789-792.

3 antoniou a, pharoah pD, Narod s, risch Ha, eyfjord Je, Hopper JL, Loman N, Olsson H, Johannsson O, Borg a et al: average risks of breast and ovarian cancer associated with BrCa1 or BrCa2 mutations detected in case series unselected for family history: a combined analysis of 22 studies.[erratum appears in am J Hum Genet. 2003 sep;73(3):709]. american Journal of Human Genetics 2003, 72(5):1117-1130.

4 Chen s, parmigiani G: Meta-analysis of BrCa1 and BrCa2 penetrance. J Clin Oncol 2007, 25(11):1329-1333.5 Milne rL, Osorio a, Cajal Tr, Vega a, Llort G, de la Hoya M, Diez O, alonso MC, Lazaro C, Blanco I et al: The average

Cumulative risks of Breast and Ovarian Cancer for Carriers of Mutations in BrCa1 and BrCa2 attending Genetic Counseling Units in spain. Clin Cancer res 2008, 14(9):2861-2869.

6 antoniou aC, easton DF: polygenic inheritance of breast cancer: Implications for design of association studies. Genet epidemiol 2003, 25(3):190-202.

7 Diez O, Osorio a, Duran M, Martinez-Ferrandis JI, de la Hoya M, salazar r, Vega a, Campos B, rodriguez-Lopez r, Velasco e et al: analysis of BrCa1 and BrCa2 genes in spanish breast/ovarian cancer patients: a high proportion of mutations unique to spain and evidence of founder effects. Human Mutation 2003, 22(4):301-312.

8 antoniou aC, pharoah pD, McMullan G, Day Ne, stratton Mr, peto J, ponder BJ, easton DF: a comprehensive model for familial breast cancer incorporating BrCa1, BrCa2 and other genes. Br J Cancer 2002, 86(1):76-83.

9 Ghoussaini M, Fletcher O, Michailidou k, Turnbull C, schmidt Mk, Dicks e, Dennis J, Wang Q, Humphreys Mk, Luc-carini C et al: Genome-wide association analysis identifies three new breast cancer susceptibility loci. Nat Genet 2012, 44(3):312-318.

10 seal s, Thompson D, renwick a, elliott a, kelly p, Barfoot r, Chagtai T, Jayatilake H, ahmed M, spanova k et al: Truncating mutations in the Fanconi anemia J gene BrIp1 are low-penetrance breast cancer susceptibility alleles. Nat Genet 2006, 38(11):1239-1241.

11 rahman N, seal s, Thompson D, kelly p, renwick a, elliott a, reid s, spanova k, Barfoot r, Chagtai T et al: paLB2, which encodes a BrCa2-interacting protein, is a breast cancer susceptibility gene. Nat Genet 2007, 39(2):165-167.

12 Osorio a, endt D, Fernandez F, eirich k, de la Hoya M, schmutzler r, Caldes T, Meindl a, schindler D, Benitez J: pre-dominance of pathogenic missense variants in the raD51C gene occurring in breast and ovarian cancer families. Hum Mol Genet 2012.

13 Loveday C, Turnbull C, ramsay e, Hughes D, ruark e, Frankum Jr, Bowden G, kalmyrzaev B, Warren-perry M, snape k et al: Germline mutations in raD51D confer susceptibility to ovarian cancer. Nat Genet 2011, 43(9):879-882.

14 park DJ, Lesueur F, Nguyen-Dumont T, pertesi M, Odefrey F, Hammet F, Neuhausen sL, John eM, andrulis IL, Terry MB et al: rare Mutations in XrCC2 Increase the risk of Breast Cancer. am J Hum Genet 2012, 90(4):734-739.

15 snape k, ruark e, Tarpey p, renwick a, Turnbull C, seal s, Murray a, Hanks s, Douglas J, stratton Mr et al: predisposi-tion gene identification in common cancers by exome sequencing: insights from familial breast cancer. Breast Cancer res Treat 2012.

PAThoLoGIcAL FeATuReS oF eNDoMeTRIAL cANceR IN LyNch SyNDRoMeJosé PalaciosPathological Anatomy Laboratory service, ramón y cajal University hospital, Madrid, spain

Endometrial cancer (EC) is the second most common type of cancer in patients with Lynch Syndrome (LS). However, the risk of EC in women with LS surpasses their risk of colon cancer. For individuals with documented hMLH1 and hMSH2 germline mutations, the lifetime risk of EC is estimated to be between 40% to 60%. For women with hMSH6 mutations a cumulative risk for EC between 16% to 71% by 70 years of age has been reported. Population-based prevalence stud-ies have identified a 1.8% prevalence of germline mutations in either hMLH1, hMSH2, or hMSH6 among unselected EC patients. However, studies evaluating EC patients less than 50 years of age reported a 9% prevalence of germline mutations in those genes.

In recent years, specific pathologic features associated with colon cancers have been evaluated as to their ability to predict the presence or absence of Lynch syndrome. In colon cancer, pathologic findings of poorly differentiated histology with mucoid and signet cell features, Crohn’s-like reaction, tumor infiltrating lymphocytes and lack of dirty necrosis are all associated with Lynch syndrome-associated colon cancers, and may serve as an impetus for referral to a genetic counselor or for tumor testing. Recently, there has been an attempt to identify certain pathologic factors in Lynch-associated endometrial cancers. It has been reported that the prevalence of LS among women with endometrial cancer of the lower uterine segment (LUS) was up to 29%. This suggests that in cases of pathologically confirmed endometrial cancer originating in the LUS, screening for Lynch syndrome should be considered. Different studies have examined whether routinely assessed morphologic features could reliably predict microsatellite instability status (MSI) (and by extension, Lynch status) in endometrial cancer. As has been shown in CRC, tumor infiltrating lymphocytes (TIL) and peritumoral lymphocytes (PL) in endometrial cancer were shown to be independent predictors of MSI. In addition, a distinctive “undifferentiated” subtype in MSI-H endometrial cancer (both sporadic and associated with LS) has been identified. Although most MSI-positive sporadic EC are of endometrioid histology, an excess of non-endometrioid carci-nomas (mainly clear cell carcinomas) has been reported in women with LS.

Different algorithms for detecting endometrial cancer patients at high risk for Lynch syndrome based on tumor morphology and epidemiologic factors have been proposed. Immunohistochem-istry for mismatch repair proteins and/or molecular analysis of MSI was performed in selected patients. However, given the higher rate of MSH6 mutations in endometrial cancer associated with LS and the lower predictive value of MSI in MSH6-associated LS, the use of IHC, rather than MSI as primary screening for Lynch syndrome in patients with endometrial cancer, should be considered.



34



SeLecTIoN cRITeRIA AND cLINIcAL MANAGeMeNTDiana M Ecclesclinical genetics Department, southampton University hospital trust, UK

Breast cancer is one of the most frequently diagnosed cancers in Europe and the USA. The major-ity of cases are diagnosed in post-menopausal women; about 20-30% have one or more relative somewhere in the family who has also had breast cancer. Genome wide association studies are beginning to map the low penetrance alleles that marginally increase breast cancers risk and often underlie a family history of breast cancer. Currently family history and age used selection criteria for screening. Since low penetrance alleles contribute to breast cancer risk in women with or without a family history, a more complete understanding of these genes may allow tai-loring of screening programmes according to genetic background in the general population in future. A less frequent but more penetrant type of genetic predisposition arises in association with mutations in several genes especially DNA repair genes such as ATM, CHEK2, PALB2. These mutations show only moderate penetrance within families and their contribution to breast cancer risk will be detailed over the coming decade of exome and genome sequencing. A small number of all breast cancer cases (probably < 3%) arise because of a strong genetic predisposi-tion and it is important to recognise these cases. The treating oncologist should understand not only the nature of a family history that might suggest a strong genetic predisposition with high penetrance most often due to BRCA1 or BRCA2 and rarely TP53. The breast cancer pathological features can also point to a specific underlying genetic predisposition. The diagnosis of genetic predisposition has some potential implications for individual cancer management choices in gene carriers but also for the opportunity to offer prevention in the wider family. However the result of genetic testing may not always be straightforward and understanding the benefits and risks and optimum timing for a genetic test is important in discussing management options with the patient.

PARP INhIBIToRS: PRoGReSS IN cANceR TheRAPyFrançoise DantzerPoly(ADP-ribosyl)ation and genome Integrity, UMr7242-IreBs, cnrs, bld. s. Brant, BP10413, 67412 Illkirch. Univerity of strasbourg, France

Poly(ADP-ribosyl) ation is a post-translational modification of proteins catalyzed by Poly(ADP-ribose) polymerases (PARPs, 17 members). In response to genotoxic stress, PARP1, the founding member, senses DNA breaks and through the synthesis of poly(ADP-ribose) restores genome integrity by stimulating a base excision and single-strand break repair process. The recent remarkable and elegant progress in the biological and mechanistic repair functions of PARP1, has placed its inhibition at the forefront of therapeutic strategies following two major schemes: (i) to potential-ize the cytotoxic action of ionizing radiation or clastogenic anti-tumoral drugs; (ii) to sensitize HR-recombination deficient human cancers in a synthetic lethality approach. Although much of the focus has been on PARP1 in DNA repair, recent advances highlight the emergence of other DNA-dependent PARPs (ie PARP2 and PARP3 and possibly Tankyrase) in this process. Given the high degree of conservation of the PARP catalytic domains it seems still unclear how many dif-ferent PARP family members are simultaneously targeted by the currently used PARP inhibitors. A major challenge in the PARP field is to further dissect the biological activities of the emerging DNA-dependent PARPs (ie PARP3, Tankyrase) and to exploid their known structural features for the rationale design of selective and potent PARP inhibitors.



36



ShoRT TALK RARe MuTATIoNS IN XRcc2 INcReASe The RISK oF BReAST cANceR

Park DJ, Lesueur F, Nguyen-Dumont T, Pertesi M, Odefrey F, Hammet F, Neuhausen SL, John EM, Andrulis IL, Terry MB, Daly M, Buys S, Le Calvez-Kelm F, Lonie A, Pope BJ, Tsimiklis H, Voegele C, Hilbers FM, Hoogerbrugge N, Barroso A, Osorio A, BCFR, kConFab, Giles GG, Devilee P, Benitez J, Hopper JL, Tavtigian SV, Goldgar DE, Southey MCgenetic epidemiology Laboratory-University of Melbourne, genetic cancer susceptibility group-International Agency for research on cancer, Beckman research Institute of city of hope, cancer Prevention Institute of california, stanford cancer center Institute, samuel Lunenfeld research Institute, Mailman school of Public health columbia University, Fox chase cancer center, huntsman cancer Institute-University of Utah health sciences center, Victorian Life sciences computation Initiative, Leiden University Medical center, radboud University nijmegen Medical center, spanish national cancer center

Currently, only about 30% of the familial risk of breast cancer has been explained, leaving the substantial majority still unaccounted for. An exome-sequencing study of families with multiple breast-cancer-affected individuals identified two families with mutations in XRCC2 gene, one protein-truncating mutation and one probably deleterious missense substitution. Like most of the known breast cancer genes, XRCC2 is involved in DNA repair by homologous recombination (HR), a fact that prompted us to further screen this gene in a larger series of samples. In particu-lar, DNA samples from affected individuals from 689 families with multiple breast cancer cases were screened in the University of Melbourne. Subsequently, a population-based case-control mutation-screening study was performed at the International Agency for Research on Cancer in which DNA samples from 1, 308 women affected with breast cancer at an early age as well as 1, 120 controls were screened. The latter study identified six probably pathogenic variants in breast cancer cases, whereas no variants were found in the controls. In total, we identified ten breast-cancer cases carrying a protein-truncating or a probably deleterious, according to in silico prediction tools, rare missense variant in XRCC2, implicating this gene in breast cancer susceptibility.

XRCC2 is the first breast cancer susceptibility gene to be identified by exome sequencing technol-ogy and validated in a population-based case-control mutation screening study. This discovery increases the proportion of breast cancers associated with a dysfunction in the DNA repair by HR. Individuals carrying a mutation in this gene could benefit from risk assessment, clinical man-agement as well as specific targeted treatments (PARP inhibitors). This study demonstrates the power of massively parallel sequencing combined with an appropriate study design and analytical strategy for discovering new cancer susceptibility genes, at a faster rate.

Additional centers/organisations Spanish Network on Rare Diseases, Centre for Cancer Epidemiology-The Cancer Council Victoria.

ShoRT TALKmiRNA MoLecuLAR cLASSIFIcATIoNoF NoN-BRcA1/2 FAMILIAL BReAST TuMoRS

Miljana Tanic1, Iván Márquez-Rodas2, Victoria Fernández1, Javier Benítez1, Beatriz Martínez-Delgado1

1human genetics group, spanish national cancer research centre (cnIo); 2Medical oncology service, hospital gregorio Marañon, Madrid. spain

Familial breast cancer comprises 5-10% of all breast cancers. Germline mutations in high risk-breast cancer genes BRCA1 and BRCA2, along with rare moderate-risk genes and common low susceptibility alleles can explain, at best, 40% of the overall excess familial risk, while the remainders of cases are designated as non-BRCA1/2 (BRCAX) tumors. MicroRNA (miRNA) expression deregulation has been extensively implicated in cancer pathogenesis. Specifically, in breast cancer altered miRNA expression profiles have been associated to specific breast cancer subtypes or tumor biological features such as hormone receptor and HER2 status, metastasis or progression. However, there are no publications to date describing miRNA expression profiles in familial breast tumors.

We established miRNA expression profiles associated to different genetic subtypes of familial breast tumors, BRCA1, BRCA2 and BRCAX. Breast tissue series consisted of 80 FFPE breast tissue samples (13 BRCA1, 10 BRCA2 and 43 BRCAX tumors, 10 sporadic and 4 normal breast tissues). Tumor areas were macrodissected and the samples were analyzed using LNA-based miRNA microarray covering more than 800 human miRNA genes. Unsupervised hierarchical clustering revealed clear heterogeneity among BRCAX tumors. Consensus clustering algorithm confirmed the existence of 4 different subgroups (A, B, C and D) with specific miRNA expression signatures. miRNA expression correlation analysis revealed high similarity between BRCAX-C group with BRCA1/2 mutated tumors, while BRCAX-A group highly resembled normal breast tissue (and was significantly enriched for HER2+ tumors. BRCAX-B group stands out as a homogenous group of tumors, while group D lacks any specific miRNA signature.

In summary, miRNA expression profiling contributed to a better definition of BRCAX group of tumors, which can be subclassified into distinct groups related to specific miRNA-expression signature and different histopathological features.


speakers’ aBsTraCTs / coMMon cAncers II 39

coMMoN cANceRS IIChair Fred Menko

(ePI) GeNeTIcS AND cLINIcAL MANAGeMeNT oF LyNch SyNDRoMeGabriel Capelláhereditary cancer Program, catalan Institute of oncology, Barcelona, spain

Lynch syndrome (LS) is characterized by an autosomal dominant inheritance of early-onset colorectal cancer (CRC) and increased risk of other cancers. It is caused by germline mutations in DNA mismatch repair (MMR) genes. The identification of key epigenetic alterations both at somatic and germline levels has opened new perspectives in the management of LS.

Constitutional epimutations are those stable changes in gene expression that do not affect DNA sequence and that are present in normal tissues of a given individual. An epimutation that occurs in the germline or early embryo can affect all or most of the soma, and phenocopy genetic disease. MSH2 epimutations, associated with a strong heritability, have been shown secondary to the pres-ence of deletions in the neighbouring EPCAM gene.The mutations lead to mosaic methylation of MSH2 in EPCAM expressing cells. In our experience, 3 of 118 LS families were carriers of a shared heterozygous deletion in EPCAM exon 9 detected by MLPA (Multiplex Ligation-dependent Probe Amplification). It was a 8,7 Kb deletion encompassing exons 8 and 9 (g.77526_86198del8673), that involves repetitive Alu elements and genotyping suggested a founder origin.

Approximately 40 index cases of constitutional MLH1 methylation have been reported. In a very few cases genetic alterations in cis (gross rearrangements and variants in the promoter region) have been identified as responsible for the methylation. In these cases an autosomal dominant pattern is readily observed. However, in most cases no genetic cause for the epimutation has been identified. So far the inheritance of the epimutation has only been experimentally confirmed in three cases. We have investigated the prevalence of MLH1 epimutations in a series of 34 patients with MLH1-methylated CRC and no detected germline MLH1 mutations. We identified two bona fide MLH1 epimutation carriers (5.9%). In one of them the epimutated allele is maternally transmitted, methylation is present in all embryonic layers, erased in spermatozoa and not transmitted to the next generation. MLH1 methylation screening in lymphocyte DNA from patients with early-onset MLH1-methylated LS-associated tumors allows the identification of epimutation carriers. •

speakers’ aBsTraCTs / coMMon cAncers II


40



In contrast with germline MLH1 methylation screening, the presence of somatic MLH1 promoter hypermethylation has been proposed as a tool for the selection of patients that will not be tested for germline mutation. Methylation of the MLH1 promoter, leading to a loss of MLH1 expression, is strongly associated with sporadic MSI-positive CRCs. However, the identification of hypermeth-ylation in a limited number of LS tumors has made its use controversial. We have assessed the clinical usefulness and cost-effectiveness of the methylation of the MLH1 promoter to improve the yield of the diagnostic algorithm of Lynch Syndrome. A total of 122 colorectal tumors from individuals with family history of colorectal cancer that showed MSI and/or loss of MMR protein expression were studied. MMR germline mutations were detected in 57 cases. Specificity of MLH1 promoter hypermethylation for depiction of sporadic tumors was 66% (31/47) and sensitivity of 96% (23/24). The cost per additional mutation detected when using hypermethylation analysis was lower when compared to BRAF study and germinal MLH1 mutation study. Somatic hyper-methylation of MLH1 is an accurate and cost-effective pre-screening method in the selection of patients that are candidates for MLH1 germline analysis when LS is suspected and MLH1 protein expression is absent.

In conclusion, the identification of epigenetic alterations both at the somatic and the germline level specific in these genes has allowed the identification of novel causes of the disease and is changing its management in a subset of LS patients.

The GeNeTIcS BASeS oF FAP SyNDRoMeMiguel Urioste human genetics cancer Programme. spanish national cancer research centre (cnIo);and centro de Investigaciones Biomédicas en red de enfermedades raras (cIBerer), Madrid, spain

Familial Adenomatous Polyposis (FAP) is a cancer predisposition dominant condition characterized by the development of hundreds of colorectal polyps in early adolescence. It is the second most common inherited form of colorectal cancer with a prevalence of 1 in 10, 000 individuals. Some of the polyps will inevitably evolve into carcinomas if no surgical measures are taken. Approxi-mately 70% of patients also suffer from a variety of extracolonic alterations. In addition to the high risk for colorectal cancer, patients also have increased risk for other cancers like papillary thyroid cancer, hepatobiliary tract tumors, and brain tumors. Classical FAP is diagnosed when more than 100 colorectal adenomas are identified. An attenuated version of the syndrome (AFAP) is diagnosed when patients show from 10-99 colorectal polyps with an onset around 15-20 years later. Mutations in the APC gene are responsible for the great majority of FAP cases and, in a lesser proportion, of AFAP ones. APC is a tumor suppressor with many diverse cellular functions: it is essential for migration, adhesion, differentiation, transcription, cell cycle control, and apoptosis. The germline mutation detection rate in APC reaches 85% in classical FAP, while only in 20-30% of AFAP cases a germline APC mutation is identified. Several studies have tried to establish a relation between the location of the APC mutation and the phenotype. Controversy exists regarding the utility of basing both management strategies and genetic counseling on genotype-phenotype correlation. In 2002, the implications of MUTYH, a BER-pathway gene, were discovered in some recessive, milder course polyposis cases. MUTYH mutations account for 10-20% of classical FAP cases without a mutation in APC, and for 30% of AFAP cases. Two common MUTYH mutations, Y165C and G382D, that account for approximately 80% of the mutations occurring in individuals of Caucasian ancestry. Still, the genetic cause in a considerable number of FAP cases, around 20% in FAP and 50-70% in AFAP, remains unclear. As APC is a tumor suppressor gene that maintains the turnover of b-catenin in the Wnt pathway, other genes implicated in this regulation are also possibly implicated in the syndrome. It is known that AXIN2 is the main negative regulator of the syndrome as well as the scaffold protein that ensembles APC, with the kinase GSK3b, to promote the targeting and degradation of b-catenin. Little is known regarding the real role of AXIN2 in colorectal cancer but three cases of gastrointestinal polyposis and AXIN2 mutations have been previously reported. Recently, germline homozygous mutations in the spindle-assembly checkpoint (SAC) gene BUB1B, have been identified in a patient with adenomatous polyposis and mosaic variegated aneuploidy. This report raises the question about the involvement of BUB1B and other SAC genes in adenomatous polyposis and gastrointestinal neoplasia.

mmoro

Text Box

Barbara Rivera Human Genetics Group, Human Cancer Genetics Programme, CN IO, Madrid, Spain



42



MANAGeMeNT oF APC AND MUTYH FAMILIeSAntoni CastellsDepartment of gastroenterology, clinic hospital, cIBerehd, IDIBAPs, Barcelona, spain

APC-associated polyposisFamilial adenomatous polyposis (FAP) is an autosomal dominant disorder characterized by the presence of hundreds (in its attenuated form) to thousands adenomas (in its classical form) dif-fusely distributed in the colon and rectum. It is responsible for 1% or less of all colorectal cancer cases. In many patients, extracolonic manifestations are present, in particular gastric and duodenal polyps, desmoid tumors, thyroidal and brain tumors, osteomas, congenital hypertrophy of the retinal pigmented epithelium, supernumerary teeth, and epidermoid cysts, among others.

FAP is due to germline mutations in the APC gene. Germline APC mutations are found in more than 70% of patients with classical FAP and about 25% of those with an attenuated form. There is a genotype-phenotype correlation with potential implications in clinical management. In 30-40% of cases, no family history of FAP is present, thus suggesting a de novo origin.

Colorectal cancer screening is justified by the disease’s high penetrance as well as its natural his-tory, since virtually all patients will develop a carcinoma at the age of 40-50 years if colon is left in place. Gene testing allows to perform the most cost-effective screening by driving colorectal examinations to gene carriers.

In families with classic FAP, flexible sigmoidoscopy is an adequate technique because of the almost universal distribution of adenomas. This examination should be performed every 2 years, starting at age 12-14 years. In family members with an identified mutation, endoscopic screening should be continued lifelong because the penetrance of the disease is virtually 100%. In at-risk individuals from families without an identified APC mutation, surveillance should be continued until age 50 years. Once adenomas are detected, colonoscopy should be performed annually.

When an attenuated form is suspected, total colonoscopy is needed. In this setting, examination should be performed every 2 years until polyposis was diagnosed. Screening should start at age 18-20 years and continue long-life because of the more heterogeneous penetrance of the attenuated form. Again, once adenomas are detected, colonoscopy should be performed annually.

Gastroduodenal endoscopy using both front and side-view scopes, with special attention to the papillary area, should be performed every 5 years until adenomas were detected. Since gastroin-testinal adenomas may also develop in the jejunum and ileum, it has been suggested to perform regular screening by barium contrast series or wireless capsule endoscopy.

Surgical resection includes both proctocolectomy with ileal pouch-anal anastomosis and total colectomy with ileorectal anastomosis. The decision on the type of surgery depends on many factors including age, severity of polyposis (i.e. involvement of the rectum), risk of developing desmoids, and site of the mutation. When a diffuse distribution or severe phenotype is present, the former is recommended. On the contrary, when no or scarce adenomas are detected in the rectum and a mild familial phenotype is observed, including the attenuated forms, total colectomy with rectal preservation can be performed.

MUTYH-associated polyposisMUTYH-associated polyposis (MAP) is inherited as an autosomal recessive trait with high pen-etrance. Clinically, MAP resembles the attenuated form of FAP syndrome, with an average age of onset around the mid 50’s with often fewer than 100 adenomas, and, accordingly, patients’ management is very similar.

Biallelic mutations in the MUTYH gene (formerly known as MYH) are responsible for this disorder. Mutations are found in 25-30% of patients with 10-100 adenomas and in 5-30% of patients with more than 100 adenomas. In Caucasian population, more than 80% of mutations correspond to the G382D and Y165C missense variants. Gene testing should start by investigating an affected individual. If the causative mutation is detected, then presimptomatic diagnosis can be offered to at-risk family members (i.e. siblings, because of the recessive nature of the disease).

The high probability of developing colorectal cancer associated with MAP justifies establishing screening strategies in individuals at risk. Because of the similarity with attenuated FAP, individuals should undergo total colonoscopy every 2 years, starting at age 18-20 years and continuing long-life. Although less frequently than in FAP, patients with MAP may develop extracolonic manifestations, i.e. duodenal adenomas. Accordingly, upper endoscopy every 5 years is recommended.

Colorectal management is similar to the one proposed for patients with attenuated FAP. Accordingly, endoscopic polypectomy can be considered in some patients. However, when polyp burden exceed the number that could be safely managed by endoscopy, total colectomy should be offered.

After total colectomy, regular endoscopic surveillance every 6-12 months is recommended for early detection of adenoma recurrence in the rectum. In patients conservatively managed with endoscopic polypectomy, examination of the entire colon and rectum should be performed annually.



44



FAMILIAL PRoSTATe cANceR. GeNeTIcS AND cLINIcAL MANAGeMeNTRosalind EelesProf of oncogenetics, the Institute of cancer research and royal Marsden nhs Foundation trust, UK

There is evidence from twin and epidemiological studies of genetic predisposition to prostate cancer. We now know that there are over 40 SNPs (single nucleotide polymorphisms) which are associated with prostate cancer risk; these are common and will be useful for population risk profiling. There are also rarer, higher risk variants (e.g. those in BRCA2) which are associated with a higher risk of prostate cancer. The prostate cancer occuring in these latter cases has a poorer prognosis and there are now targeted treatments which are starting to be offered to such patients. Risk modelling incorporating family history and genetic profiles will be able to more accurately define risk for targeted screening and treatments.

ShoRT TALKGeRMLINe BRcA MuTATIoNS ARe ASSocIATeD WITh NoDAL INVoLVeMeNT, DISTANT MeTASTASIS AND PooR SuRVIVAL ouTcoMeS IN PRoSTATe cANceR

Elena Castro1, 2, Chee L. Goh1, 2, David Olmos3, 4, Edward J. Saunders1, Daniel A. Leongamornlert1, Malgorzata Tymrakiewicz1, Nadiya Mahmud1, Koveela Govindasami1, Michelle Guy1, Lynne T. O'Brien1, Amanda L. Hall1, Emma J. Sawyer1, Rosemary A. Wilkinson1, Audrey Audern-Jones2, UKGPCS collaborators1, EMBRACE collaborators5, Steve Ellis5, Debra Frost5, Susan Peock5,Antonis C. Antoniou5, Douglas F. Easton5, Zsofia Kote-Jarai1, Rosalind A. Eeles1, 2

1oncogenetics team, the Institute of cancer research, sutton, United Kingdom; 2Academic Urology Unit, the royal Marsden nhs Foundation trust, London, United Kingdom; 3Drug Development Unit, the royal Marsden nhs Foundation trust, sutton, United Kingdom; 4Molecular cytogenetics, the Institute of cancer research, sutton, United Kingdom; 5centre for cancer genetic epidemiology, Department of Public health and Primary care, University of cambridge, UK

Introduction Previous studies have suggested worse prostate cancer (PCa) outcomes in BRCA2 mutation carriers, but were limited by small sample sizes and/or lack of clinical data. Thus, the associated clinical features and the real independent prognostic contribution of germline BRCA1 and BRCA2 mutations remains unclear. This is the largest study to date of clinical features in PCa associated with the presence of mutations in BRCA1 and BRCA2.

Methods PCa patients were identified from two prospective cohort studies (EMBRACE and UKGPCS). Cox regression analysis was used to evaluate the associations between BRCA status and other PCa risk factors with Overall Survival (OS), Cause Specific OS (CSS), and Metastasis Free Survival (MFS).

Results A total of 2019 PCa patients were included (1940 non-carriers, 18 BRCA1 and 61 BRCA2 muta-tion carriers). BRCA1 and BRCA2 associated PCas were more frequently associated with Gleason ≥8 (p=3×10-5), T3/T4-stage (p=0.003), nodal involvement (p=0.0005) and distant metastases (p=0.005) than PCa in non-carriers. OS and CSS were significantly longer in non-carriers than in carriers (median: 12.9 vs 8.1 years, p=1×10-7; and 15.7 vs 8.6 years, p=7×10-8 respectively). For localized PCa, 5-year CSS and MFS were significantly higher in non-carriers (96% vs 82%, p=9×10-8 and 93% vs 77%, p=0.0001, respectively). BRCA1 mutation carriers had similar classical risk factors to BRCA2 carriers at presenta-tion but better survival outcomes. Multivariate analyses showed BRCA2, but not BRCA1 mutations are an independent prognostic factor for PCa outcomes.

Conclusions Our results confirm that BRCA1 and BRCA2 mutations confer a more aggressive PCa phenotype with a higher probability of nodal involvement. BRCA2 mutations are associated with poor survival outcomes and aggressive treatment options should be considered in these patients.

Keywords Prostate cancer, BRCA2, BRCA1, survival outcomes


speakers’ aBsTraCTs / other hereDItArY cAncer sYnDroMes 47

oTheR heReDITARy cANceR SyNDRoMeSChair Rosalind Eeles

GeNeTIc heTeRoGeNeITy oF FAMILIAL PheochRoMocyToMAMercedes RobledocnIo, Madrid, spain

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare tumours of neural crest origin, previously referred to as “the 10 percent” tumor due in part to the frequency of inherited forms associated with three well-known cancer syndromes: von Hippel-Lindau disease, multiple endocrine neoplasia type 2, and neurofibromatosis type 1 due to mutations in VHL, RET, and NF1 respectively. In the early 2000’s the 10 percent rule was challenged after identification of germline mutations in SDHD, SDHB, and SDHC as important causes of familial paraganglioma (PGL) that led to establish that up to a quarter of affected patients carried a PCC/PGL susceptibility gene mutation. Since then four additional susceptibility genes (SDHAF2, SDHA, TMEM127 and MAX) have been identified. Thus, the proportion of hereditary PCC/PGLs may exceed estimates of 40%, which renders PCC/PGL as one of the most inherited tumor entities.

There is no doubt about the importance of detecting a mutation in any of the known genes related to the disease, not only for the affected patient but also for the relatives. Keeping in mind the genetic heterogeneity we face, it is critical the consideration of clinical, biochemical and immunohistochemical features to set up efficient algorithms able to guide a rational genetic screening in each patient.

It is worth highlighting that at least 10% of patients, negative for the genetic study of the so far disease-related genes, are actually good candidates to be carriers of mutations in other suscep-tibility loci not yet identified.

Despite the powerful tools currently available, the identification of new susceptibility genes is not a straightforward issue. Only after a thorough patient’s selection, either due to the pheno-typic features or other OMIC data, the new genes discover will be feasible. The participation of International Consortium is mandatory to this end.

speakers’ aBsTraCTs / other hereDItArY cAncer sYnDroMes


48



GeNeTIcS AND MANAGeMeNT oF heReDITARy PANcReATIc cANceRHans. F. A. Vasen Department of gastroenterology & hepatology, Leiden University Medical centre, Leiden, the netherlands

Pancreatic cancer (PC) has a poor prognosis due to aggressive growth and early dissemination. The overall 5-year survival rate is only 5%. Surveillance programs for PC in high risk groups focus on the detection of small tumors or the known precursor lesions including intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasias (PanINs). Approximately 5-10% of PC cases are associated with an inherited predisposition. Families have been reported with clustering of PC, either site-specific i.e. Familial Pancreatic Cancer (FPC) or as part of a tumor syndrome. Tumor syndromes associated with an increased risk of PC include Peutz-Jeghers syndrome (PJS), Familial Atypical Multiple Mole Melanoma (FAMMM)(P16-gene defect), Hereditary Breast Cancer (c.q. BRCA2 mutation carriers) and possibly Lynch syndrome (1). The lifetime PC risk varies between 5% in BRCA2 mutation carriers to 36% in PJS. There are several imaging techniques available for the detection of small tumors or its noninvasive precursor lesions. EUS (Endoscopic Ultrasound) has been shown to detect small tumors but is an invasive technique and success rate is operator dependent. Magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) is a noninvasive technique that is able to detect small pancreatic lesions. CT scan has also been used as surveillance tool but is less appropriate because of the cumulative radiation dose. A few studies have addressed surveillance for pancreatic cancer. Most studies in FPC showed that the yield of premalignant lesions is relatively high but that of pancreatic cancer low. More studies are needed to assess the value and cost-effectiveness of surveillance programs.

references

1 Canto MI, Hruban rH, Fishman ek et al. Frequent detection of pancreatic lesions in asymptomatic high-risk individuals. Gastroenterology 2012; 142:796-804

2 Vasen HFa, Wasser M, van Mil a et al. MrI surveillance detects early-stage pancreatic cancer in carriers of a p16-Leiden mutation. Gastroenterology 2011; 140: 850-856

3 Canto MI, Goggins M, Hruban rH, petersen GM, Giardiello FM, Yeo C, Fishman ek, Brune k, axilbund J, Griffin C, ali s, richman J, Jagannath s, kantsevoy sV, kalloo aN. screening for early pancreatic neoplasia in high-risk individuals: a prospective controlled study. Clin Gastroenterol Hepatol. 2006 Jun; 4(6):766-81

4 poley JW, kluijt I, Gouma DJ, Harinck F, Wagner a, aalfs C et al. The Yield of first-time endoscopic Ultrasonograhy in screening Individuals at a high risk of developing pancreatic Cancer. The american Journal of Gastroenterology 2009; 104: 2175-81.

5 Langer p, kann, pH, Fendrich V, Habbe N, schneider M, sina M, slater ep, Heverhagen JT, Gress TM, rothmund M, Natrsch Dk. 5 Years of prospective screening of High risk Individuals from Familial pancreatic Cancer Families, Gut 2009; 58: 1410-8.

BIRT-hoGG-DuBé SyNDRoMe. DIAGNoSIS AND cLINIcAL MANAGeMeNTFred H. Menkoclinical geneticist, VU University Medical centre, Amsterdam, the netherlandsMenko, F.h., van steensel, M.A., giraud, s., Friis-hansen, L., richard, s., Ungari, s., nordenskjöld, M., hansen, t.V., solly, J., Maher, e.r.; european BhD consortium. Birt-hogg-Dubé syndrome: diagnosis and management.Lancet oncol. 2009;10:1199-1206

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition, characterized by skin fibrofolliculomas, basally located lung cysts, pneumothorax and renal cancer. The risk of pneu-mothorax and renal cancer until the age of 70 years is about 30% and 15%, respectively.

BHD is due to germline mutations in the FLCN (folliculin). The molecular pathogenesis of BHD is not fully clarified and probably includes several signalling pathways, including mTOR. Thus far no clear genotype-phenotype correlations have been established.

BHD is probably under diagnosed, for several reasons: the skin features are variable and not all patients seek medical advise for skin papules, pneumothorax develops without previous clinical lung disease and mimics spontaneous primary pneumothorax, not all cases of BHD-associated renal cancer show signs of hereditary disease (i.e. early age at diagnosis, bilateral or multifocal tumors and/ or typical histological features).

Management is mainly aimed at early diagnosis and treatment of renal cancer. Probably a yearly or two-yearly renal MRI is the optimal surveillance procedure. Generally the 3 cm threshold rule is applied for intervention which usually consists of nephron-sparing partial nephrectomy.

Further insight in the molecular pathogenesis of BHD is may lead to targeted therapy.

speakers’ aBsTraCTs / other hereDItArY cAncer sYnDroMes


50



cLINIcAL AND GeNeTIc chARAcTeRISTIcSIN FAMILIAL MALIGNANT MeLANoMANelleke GruisDepartment of Dermatology, Leiden University Medical center, Leiden, the netherlands

An increased susceptibility to melanoma may manifest as a family history of melanoma, the development of multiple primary tumours or melanoma in the context of numerous moles and clinically atypical moles. In families, genetic susceptibility to melanoma is related to the inheritance of variation in both high risk genes and lower risk genes. In understanding the impact of these genes, the international melanoma genetics consortium, GenoMEL has established a database in which information on genetic and phenotypic information is included on more than 1500 melanoma families.

The best understood high risk melanoma susceptibility gene is CDKN2A on chromosome 9. Hereditary mutations in this gene underlie susceptibility to melanoma in 40% of families with 3 or more cases of melanoma. The lifetime risk of melanoma in CDKN2A mutation carriers is high, but variable, ranging from 58% in Europe to 91% in Australia by the age of 80 years, which underscores the importance of the environment even in familial melanoma. The probability of identifying a mutation in a familial melanoma case varies by continent but is generally increased if one of the cases has multiple primaries, or if there is onset under the age of 40 years. In most populations, except for Australians, there is also a strong relationship between gene mutation status and pancreatic cancer.

The best understood common low risk melanoma susceptibility gene is the gene coding for the melanocortin 1 receptor (MC1R) which is established as a determinant of susceptible phenotypes such as red hair, sun sensitivity and freckles. Recently, other pigmentary genes (TYR, TYRP1, ASIP) have been identified as low penetrance melanoma susceptibility genes.

Due to the variability in the background incidence of melanoma and penetrance of CDKN2A muta-tions between populations, there is not a single guideline for counselling and genetic testing that would be appropriate to apply worldwide. The current view is that dermatologists, along with other health professionals, must incorporate family history and risk assessment into clinical practice in order to identify patients who might be at increased risk for melanoma. Counselling is important for patients with 3 or more melanoma cases in the family. In countries with lower background popula-tion incidence rates, specialist counselling may be appropriate for 2 case families. The value of gene testing is still debatable but testing should always be preceded by assessment and counselling from a health care professional experienced in genetics. For the majority of families there is no detectable mutation and apparently other hereditary mutations have yet to be discovered. In these families gene testing is therefore unhelpful. Unfortunately, the relative lack of information to predict pancreatic cancer risk in particular families and a lack of proven screening for pancreatic cancer limit the value of testing for CDKN2A mutation positive families. In order to best counsel and inform families on gene testing, it is important to generate pooled data sets from across the world.

ShoRT TALKINTeGRATIoN oF miRNA AND mRNA eXPReSSIoN PRoFILeSIN PheochRoMocyToMA AND PARAGANGLIoMA IDeNTIFIeS GeNoTyPe-SPecIFIc BIoMARKeRS AND PoTeNTIALLy ReGuLATeD PAThWAyS

Aguirre A de Cubas MSc1, L Javier Leandro-García MSc1, Francesca Schiavi PhD2, Iñaki Comino-Méndez MSc1, 3, Lucía Inglada-Pérez MSc1, 3, Agnieszka Maliszewska MSc1, Elena López-Jiménez PhD1, Rocío Letón MSc1, Álvaro Gómez Graña1, Carmen Bernal MD4, Cristina Álvarez-Escolá MD5, Cristina Rodríguez-Antona PhD1, 3, Giuseppe Opocher MD2, 6, Alberto Cascón PhD1, 3, and Mercedes Robledo PhD1, 3

1 hereditary endocrine cancer group, spanish national cancer research centre (cnIo), Madrid, spain.2 Familial cancer clinic, Veneto Institute of oncology, Padova, Italy. 3 centro de Investigación Biomédica en red de enfermedades raras (cIBerer), Madrid, spain. 4 endocrinology and nutrition service, hospital 12 de octubre, Madrid, spain. 5 endocrinology and nutrition service, hospital La Paz, Madrid, spain. 6 Department of Medical and surgical sciences, University of Padova, Italy

Pheochromocytoma (PCC) and paraganglioma (PGL) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. Since microRNAs are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. To identify microRNAs specifically related to the different genetic PCC/PGL classes known so far, we charac-terized microRNA profiles in 70 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX through microarray analysis. To determine the potential roles microRNAs play in tumorigenesis and progression of PCC/PGL, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data.

We identified microRNA signatures specific to, as well as common among, the genetic classes of PCC/PGLs, and the best candidate microRNAs (miR-133b, miR-137, miR-183, miR-210, miR-382, miR-488, miR-885-5p, and miR-96) were validated in an independent series of 30 formalin-fixed paraffin-embedded PCC/PGL samples by qRT-PCR. Pathway analysis of microRNAs commonly down-regulated among all experimental groups showed enrichment in gene networks involved in neuronal and neuroendocrine development.

We demonstrate that PCCs/PGLs have different microRNA profiles according to the underlying primary mutation, suggesting they may be used as specific biomarkers. Our pathway analysis of potential microRNA-regulated gene networks may provide new information on the etiology of these tumors.


speakers’ aBsTraCTs / rAre tUMors 53

RARe TuMoRSChair Mercedes Robledo

The FANcoNI ANeMIA SyNDRoMeJordi Surrallés Autonoma University of Barcelona, genetic and Microbiology, spain

Defects in DNA repair and genome stability genes lead to rare cancer-prone syndromes. Research on these rare diseases is particularly interesting not only to find a cure but also to understand key biological processes required to prevent and understand cancer in the general population. Fanconi anemia (FA) is one of these rare syndromes characterized by progressive bone marrow failure, birth defects, chromosome fragility, hypersensitivity to DNA interstrand cross-linking (ICL) mutagenic drugs and a high predisposition to cancer, including leukemia and solid tumors such as head-and-neck and gynecological squamous cell carcinoma. In this therefore essential to develop screening protocols for early detection of SCC in high cancer risk populations such as FA patients. The only cure of the hematological disease, which is the major cause of early death, is bone marrow transplantation. Those families with unavailable donor rely on the future implementa-tion of gene therapy of hematopoietic progenitors and in a recently reported novel regenerative medicine therapeutic strategy based on the conversion of skin cells to disease free hematopoietic progenitors via induced pluripotent stem (iPS) cells. All these novel therapeutic applications to human health are further complicated by the fact that at least 15 genes (FANC-A, B, C, D1, D2, E, F, G, I, J, L, M, N, O and P) are involved in this disease and their products interact in a complex genome stability and tumor suppression pathway, the so-called FA/BRCA pathway. Notably, 4 out of 15 FA genes (FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1 and FANCO/Rad51C) are breast cancer susceptibility genes in otherwise unaffected mutation carriers. Therefore, a correct diagnosis based on the analysis of chromosome fragility followed by genetic subtyping and mutation detec-tion are essential not only for a proper management and genetic counseling of these families but also for cancer risk follow up in mutation carriers and for the future implementation of gene/cell therapies. Finally, a proper genetic subtyping of FA patients may lead to the identification of novel FA genes potentially involved in familiar breast cancer. In this context we have applied a whole exome sequencing strategy in FA patients where all known FA genes were disregarded to identify a novel FA gene, FANCQ, responsible for a novel FA genetic subtype. Interestingly, this gene turned out to be involved in three genome instability cancer prone diseases.

speakers’ aBsTraCTs / rAre tUMors


54



DySKeRAToSIS coNGeNITA: A TuMoR PRoNe DISeASe ThAT hARBouRS coNSTITuTIVe DNA DAMAGeRosario Perona, Jaime Carrillo, Cristina Manguán-García, Laura Pintado-Berninches, Andrea González de Arce, Rosario Machado-Pinilla Biomedical research Institute csIc/UAM, Madrid, spain cIBer—rare diseases, Valencia, spain

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. DC is caused by defects in telomere maintenance machinery that results in short telomeres in highly proliferating tissues such as bone marrow and skin as a consequence of muta-tions in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). Three different forms of DC have been described (X-linked recessive, MIM 305000; autosomal dominant, MIM 127550; autosomal recessive, MIM 2245230). Mutations in the DKC1 gene appear in 30 % of DC cases (X-linked recessive, XDC) being the most frequent mutated gene and is involved in the more severe variant of DC (Hoyeraal-Hreidarrson syndrome). We have found that three cell lines of dermal fibroblasts derived from XDC patients with different DKC1 mutations also showed a reduction of dyskerin levels when compared with control fibroblasts. We have established a cellular model for XDC, by depleting from DKC1 normal dermal fibroblasts and detected an important decrease in the levels of many ribosomal proteins, which are responsible for the correct folding of rRNA, needed for an accurate cleavage, processing and the assembly of the ribosome in the nucleolus. We also found that fibroblasts of XDC patients and also DKC1 depleted fibroblasts are arrested in G2/M and show deregulation of many cell cycle and DNA damage regulatory genes. Moreover, genes implicated in oxidative stress response are deregulated which could also activate p53-dependent cell cycle arrest or apoptosis.

Telomere shortening prevents the formation of the loop-like structure maintained by shelterin in intact telomeres. This capping structure prevents the otherwise exposed ends of different chromosomes from become fused together by the DNA damage response (DDR). We have found that XDC cells derived from patients, showed increased levels of DNA damage. The DDR response, activated in XDC cells includes ATM, CHK2 and p53 and γ-H2AX foci co-localized with the telomeric probe TRF1. Finally, expression of the GSE24-2 peptide, corresponding to an internal domain of DKC1, rescues DNA damage and telomerase activity in XDC cells representing a novel therapeutic tool for XDC patients.

This work was supported by grants from FIS P11-00949 and Fundación Ramón Areces.

GeNeTIc SyNDRoMeS oF The Ras/MAPK PAThWAyCarmen GuerraMolecular oncology Programme, centro nacional de Investigaciones oncológicas (cnIo), Madrid, spain

The Ras-MAPK pathway is one of the best characterized in human cancer. Recently, it has been described an unexpected new role for mutant proteins of this signaling pathway during human embryo development. Neuro-cardio-facio-cutaneous (NCFC) syndromes, that include Costello (CS), Noonan (NS), cardio-facio-cutaneous (CFC) and LEOPARD (LS) syndromes as well as familial Neurofibromatosis type 1 (NF1), result from de novo germ line mutations that alter the Ras/Raf/Mek signaling pathway. There is significant overlap in their multiple clinical manifestations such as developmental delay, cardiac and musculoskeletal abnormalities, mental retardation and other physiological and neurological defects and, in some syndromes, cancer. Each syndrome is char-acterized by mutations in specific loci. Germline mutations in the HRAS gene are the only genetic cause of Costello syndrome. About half of NS patients carry mutations in PTPN11 and a quarter in SOS1, K-RAS and RAF1. Most CFC patients carry mutations in B-RAF, although other genes of the RAS/RAF/MEK pathways, mainly MEK1 and MEK2 as well as K-RAS, have also been identified. Germ line PTPN11 mutations are the primary cause of LS (90%). Recently, some LS patients harboring a normal PTPN11 locus have been shown to carry RAF1 mutations. Finally, all NF1 patients carry inactivating mutations in NF1, a locus that encodes neurofibromin, a RAS GAP protein. The RAS/RAF/MEK signal-ing pathway plays a central role in the regulation of many cellular responses, including metabolism, cell proliferation and differentiation, and apoptosis. Thus, it is not surprising that malfunction of this pathway during embryogenesis and postnatal development results in the multiple clinical manifestations associated with NCFC syndromes. Moreover, most of these loci are also mutated in human cancers. However, NCFC mutations in K-RAS, B-RAF, and PTPN11 loci are distinct from those in somatic tumors and consistently result in milder activation of their gene products. H-RAS appears to be an exception. Although some mild activating mutations have been recently identified, about 80% of CS patients carry H-RAS alleles harboring mutations previously identified in human tumors. Costello syndrome is a tumor predisposition syndrome with a 15% life time risk for a malignancy, with rhabdomyosarcoma being the most common (60%), followed by neuroblastoma, and bladder cancer. Rhabdomyosarcoma and neuroblastoma are childhood cancers and occur in the typical age range in Costello syndrome. In contrast, bladder cancer is typically a malignancy of older adults, but may occur as early as the second decade of life in individuals affected by Costello syndrome. Understanding the causative role of the RAS/MAPK pathway in NCFC syndromes, requires the development of genetically modified mouse models carrying the same mutations that have been recently identified in these patients. More importantly, these mouse models will be used to study tumor predisposition and more important to assay therapeutic strategies that hopefully will help to treat and or palliate some of the symptoms of these patients. We have generated mouse models for Costello (CS), cardio-facio-cutaneous (CFC) and Noonan (NS) developmental syndromes. Results from the characterization of these mouse models will be presented.

speakers’ aBsTraCTs / rAre tUMors


56



LIFRAuMeNI SyNDRoMe. FRoM MoLecuLAR BASeS To cLINIcAL MANAGeMeNTThierry Frebourgrouen University hospital, France

ShoRT TALK SuRVeILLANce oF ADoLeSceNTS AND youNG ADuLT PATIeNTSWITh FANcoNI ANeMIA (FA): AWAReNeSS oF DIAGNoSING SoLID TuMoRS AT A youNG AGe

Nina Bosch, Neus Gadea, Cristina Heredia, Teresa Olivé, David Valcarcel, Montserrat Munill, Cristina Centeno, Jose Luis Quesada, Jordi Surrallés, Judith BalmañaUnidad de alto riesgo y prevención del cancer, servicio de oncología médica; servicios de hematología de adultosy pediátrica;maxilofacial; ginecología;otorrinolaringología; Vall d’hebron University hospital, spain

A great proportion of FA patients reaches adulthood and become at risk for developing second-ary malignancies. In 2009, the High Risk and Cancer Prevention Unit (HRCPU) at Hospital Vall d’Hebron started a follow-up program for adolescents and adult patients with FA. The goal was to advocate for prevention measures to reduce cancer risk, and coordinate a multidisciplinary surveillance for different neoplasms. During annual HRCPU visits, FA patients undergo a complete anamnesis and receive health education. The surveillance program includes: hematological follow up with hemograms and bone marrow aspiration; examination for head and neck lesions by an otolaryngologist; detailed examination of the oral cavity by a maxillofacial specialist; semestral gynecologic examinations for women with annual cervical cytology testing and breast examina-tion. Finally, HPV vaccination is encouraged. Since 2009, 14 FA patients have been enrolled in our follow-up program. Overall, 50% were women, and median age was 22 years (14-32). Their mean age at diagnosis of FA was 6.5 years, 57% belonged to FANCA group and 57% underwent a bone marrow transplantation (BMT). Follow up has ranged from 1 month to 3 years, 70% of patients have adhered properly with the program and 83% of women have received HPV vaccination. So far, 4 patients (29%) have been diagnosed with a malignant tumor and another two with a premalignant lesion. Premalignant lesions were found in the oral cavity (hyperplasia with mild dysplasia of the tongue) and gynecological (squamous intraepithelial lesion of the cervix CIN1). From those diagnosed with cancer, two of them had head and neck tumors. One was a lingual squamous cell carcinoma pT2N1M0 diagnosed at 24 years which recurred at 4 years and the patient died at age 30. The patient had received a BMT at age 8. The other one was a squamous cell carcinoma of the epiglottis pT4N1M0 diagnosed at 32 years in a patient with mosaicism. The third and fourth patients have developed several basal cell carcinomas in the jaw and back since age 21 and both had undergone BMT at 7 and 8 years. Head and neck tumors are frequent neoplasms in young adults with FA, regardless prior BMT. We need to reinforce surveillance and prevention strategies to reduce the risk of malignancies in these locations.


speakers’ aBsTraCTs / neW technoLogIes APPLIeD to FAMILIAL cAncer stUDIes 59

NeW TechNoLoGIeS APPLIeD To FAMILIAL cANceR STuDIeSChair Ana Osorio

IDeNTIFIcATIoN oF NoVeL TARGeTS AND ReGIoNS ThRouGh INTeGRATIVe GeNoMIc ANALySISMaría José García1human genetics group, spanish national cancer research center (cnIo) and centre for Biomedical network research on rare Diseases (cIBerer), Madrid, spain

In recent years the development of different high-throughput technologies has allowed scien-tists to interrogate multiple aspects of the cell biology in a genome-wide scale. Further integra-tion of individual data sets such as the transcriptome, epigenome, proteome, interactome, or information derived from sequence and copy-number variation analysis is now providing a more comprehensive view of cellular processes and disease mechanisms. In that respect, the recently published integrated genomic analysis of ovarian carcinoma performed by The Cancer Genome Atlas (TCGA) Research Network is a good example of the discovery power delivered by an integrated analysis.

Epithelial Ovarian Cancer (EOC) ranks fifth as the cause of cancer-related death in women in the western world and is the most lethal of gynaecological malignancies. Most patients present with advanced disease at time of diagnosis due to the lack of effective screening methods and warning signs. In addition majority of tumours relapse eventually becoming drug-resistant after initial response. All these factors account for a 5-year survival rate of cases diagnosed at advanced stage of only 35-40%. With this background in mind the efforts of The Cancer Genome Atlas (TCGA) Research Network were focused on defining a catalogue of molecular aberrations and pathways altered in ovarian tumours with the ultimate goal of developing new therapeutic approaches against specific alterations. The data-integration strategy followed by The Cancer Genome Atlas will be commented to illustrate a successful integrative genomic analysis mainly dedicated to characterize sporadic ovarian tumours.

Although the great majority of ovarian carcinomas are sporadic the most important risk factor is a strong family history of breast and ovarian cancer and genetic predisposition is present in about 5-15% of patients. Our group has analyzed the DNA-copy number profile of 53 familial ovarian tumours (21 BRCA1, 6 BRCA2, 26 BRCAX) by using high resolution array-CGH (Agilent 4×180K). The results of this project will be presented as an example of a genome-wide scale project aiming to characterize hereditary ovarian carcinomas. The prospect offered by future integration of the defined LOH regions together with Next Generation Sequencing data from non-BRCA1/BRCA2 mutation carriers will be discussed in the context of discovery of new ovarian cancer susceptibility genes.

speakers’ aBsTraCTs / neW technoLogIes APPLIeD to FAMILIAL cAncer stUDIes


60


speakers’ aBsTraCTs / neW technoLogIes APPLIeD to FAMILIAL cAncer stUDIes 61

The cLINIcAL PoTeNTIAL oF SequeNcING cANceR GeNoMeS FoR PeRSoNALIzeD MeDIcINeDimitrios H. RoukosFounder & chairman; Biosystems & synthetic genomic network Medicine center (BiosyngennetMed), Ioannina University, Ioannina, greece

Currently, single genes (BRCA1/2, mismatch-repair genes, CDH1) testing allows for identification in high-risk families of individuals with ~70 lifetime risk for developing hereditary syndromes such as breast ovarian cancer, colorectal cancer and diffuse gastric cancer. However, approximately 30% of these mutation carriers will never develop cancer. In addition, no robust test exists for the identification of high-risk family members who tested negative for mutation in the reported genes (familial non-hereditary cancer).

The power of next-generation sequencing (NGS) technologies to identify the overall 3 million variants across the human genome which are involved in phenotypes and disease at low cost, accurately and within a short time explain the explosion and popularit of sequencing technolo-gies. Indeed, whole-genome sequencing (WGS), whole-exome sequencing (WES) and RNA-sequencing (RNA-seq) provide the ability to separate somatic from inherited mutations and driver (causal) from passengers (no causative effect) lesions.

In this presentation, I will discuss: first, whether simple identification of driver mutations will be sufficient for overcoming missing heritability; second, how the novel genes and variants identified by NGS can be linked with signaling pathways; and third, how mutations deregulate signaling pathways networks and gene expression patterns. Ultimately, I will describe the challenges in understanding the high heterogeneity, complexity and dynamics in familial cancer development which collectively synthesize multiple hurdles to surpass in order to reach the discovery of robust genome diagnostics and therapeutics with clinical utility in familial cancer.

eXoMe SequeNcING IN The SeARch FoR hIGh SuScePTIBILITy-GeNeSAlberto Cascón cnIo, Madrid, spain

Since the first HapMap exomes were published in 2009, exome sequencing (ES) has been successfully used to identify the highly penetrant causative mutations for several Mendelian diseases. Most alleles (85%) that are known to underlie Mendelian disorders disrupt protein-coding sequences and can be therefore identified using ES. In addition, and despite the obvious limitations that targeted exome capture has relative to whole genome sequencing (WGS), other major considerations such as cost, analysis time, sample size and interpretation of results have made ES the preferred genetic tool to identify the genetic source of a patient’s disease. One of the crucial steps prior to ES is the adequate selection of patients based on a scrupulous phenotypic screening, and/or filtering based on other exclusive “markers”, to avoid undesirable genetic heterogeneity. Taking all these aspects into account, we recently identified for the first time the presence of germline mutations in the MAX (MYC associated factor X) gene in patients with hereditary pheochromocytoma by sequencing the whole exomes of three unrelated patients with a family history of the disease and no mutations in any of the nine known susceptibility genes (Comino-Méndez et al., 20111). The patients were selected as candidates for study because they shared a common clinical phenotype (hereditary adrenal bilateral pheochromocytoma) and because their corresponding tumors showed very similar expression profiles, according to a preliminary analysis performed in our lab (López-Jiménez et al., 20102). The identification of germline mutations affecting the same gene in the three individuals, along with the observed loss of heterozygosity of the wild-type allele and the absence of MAX protein in their tumors, demonstrated that MAX is a new tumor suppressor gene associated with the development of hereditary pheochromocytoma. Further analysis of more than fifty patients, chosen because they had either bilateral pheochromocytoma and/or age of onset less than thirty years, detected 5 additional cases with mutations in MAX. This study was particularly important, from both a diagnostic and a genetic counseling point of view, given the complexity of the disease. In addition, our finding highlights the importance of new massive sequencing technologies, and especially ES, in the identification of the genes responsible for genetic diseases.

1. comino-Méndez I, gracia-Aznárez FJ, schiavi F, Landa I, Leandro-garcía LJ, Letón r, honrado e, ramos-Medina r, caronia D, Pita g, gómez-graña A, de cubas AA, Inglada-Pérez L, Maliszewska A, taschin e, Bobisse s, Pica g, Loli P, hernández-Lavado r, Díaz JA, gómez-Morales M, gonzález-neira A, roncador g, rodríguez-Antona c, Benítez J, Mannelli M, opocher g, robledo M, cascón A. exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. nat genet. 2011; 43:663-7. 2. López-Jiménez e, gómez-López g, Leandro-garcía LJ, Muñoz I, schiavi F, Montero-conde c, de cubas AA, ramires r, Landa I, Leskelä s, Maliszewska A, Inglada-Pérez L, de la Vega L, rodríguez-Antona c, Letón r, Bernal c, de campos JM, Diez-tascón c, Fraga MF, Boullosa c, Pisano Dg, opocher g, robledo M, cascón A. research resource: transcriptional profiling reveals different pseudohypoxic signatures in sDhB and VhL-related pheochromocytomas. Mol endocrinol. 2010; 24:2382-91.

speakers’ aBsTraCTs / neW technoLogIes APPLIeD to FAMILIAL cAncer stUDIes


62

ShoRT TALKMuTATIoNAL ANALySIS oF BRcA1/BRcA2GeNeS uSING NeXT-GeNeRATIoN SequeNcING TechNoLoGy:A coMPReheNSIVe ANALySIS PRoTocoL

Lucía Pérez-Cabornero1, A. Osorio2, A. Barroso2, G. Pita3, F. Ruiz-Espejo4, A. Sarabia Meseguer4, I. Tovar Zapata4, M. Vargas de los Monteros5, M. La Calle Marto5, A. Fontalba6, A. Poyatos7, T. Haro7, B. Sánchez Vega8, N. Chavarria Piudo9, E. Jiménez Orozco9, S. Pedrinaci10, V. Fernández-Pedrosa1, C. Collado1, R. Rodríguez-de Pablos1, S. Zúñiga-Trejos1, JM. Rosa-Rosa1, JC. Triviño1, M. Gil1, J. Benitez2, 3, S. Santillán1

1sistemas genómicos, Paterna (Valencia), spain; 2human genetics group, spanish national cancer center, cnIoand cIBerer, Madrid, spain; 3genotyping Unit-cegen, cnIo, Madrid, spain; 4hospital U. Virgen Arrixaca, Murcia, spain; 5hospital U. Virgen Macarena, sevilla, spain; 6hospital U. Marqués de Valdecilla, santander, spain; 7hospital U.san cecilio, granada, spain; 8geMoLAB, Madrid, spain; 9hospital de Jerez de la Frontera, cádiz, spain;10hospital U.Virgen de las nieves, granada, spain

BRCA1 and BRCA2 are the most important breast cancer susceptibility genes. The conventional BRCA1 and BRCA2 mutation screening, is time consuming and has relatively high costs due to the absence of mutation hot spots and to the high number of exons per gene. Next Generation Sequencing (NSG) is a promising approach to overcome these limitations. The aim of this study was to develop a protocol to reduce the time needed for diagnosis with a high level of sensitivity and specificity. The coding region and adjacent intronic regions of BRCA1 and BRCA2 were ampli-fied and resequenced in 93 amplicons by 5 multiplex PCRs (MASTR v2.1 of Multiplicom) and 454 GS Junior DNA sequencing instrument (Roche Diagnostics), which uses a pyrosequencing system. A pre-sequencing step was performed to assess the presence / absence of indels by fragment analysis and visualization of GeneScan profiles. Bioinformatic analysis was performed by two complementary approaches: Our own pipeline and by the GS Amplicon Variant Analyzer (AVA) software. Significant variants were confirmed by Sanger sequencing and large genomic rear-rangements were detected by Multiplex ligation-dependent probe amplification (MLPA).The successfully validation of this protocol was carried out on a set of 6 cases harboring known vari-ants and a reference HapMap sample. 193 patients with suspected hereditary breast and ovarian cancer, collected by CNIO and Sistemas Genómicos were included in this study. The subsequent application in the cases allowed the identification of 33 pathogenic mutations (7 missense, 5 nonsense, 16 frameshift, 3 splicing variants and 2 large deletions) 27 unclassified variants and a high number of polymorphisms. The mutation spectrum includes both known and novel DNA variants. The comprehensive protocol for the screening of the BRCA1/BRCA2 genes developed in this study is effective as a high throughput, rapid (the whole process can be completed in 30 days) and cost-effective methodology.

Keywords BRCA1, BRCA2, Next Generation Sequencing

PARTIcIPANTS’PoSTeRS5th FAMILIAL cAncer conFerence


parTICIpaNTs’ pOsTers / sessIon 1 67

SeSSIoN 1 1 GeNeTIc ANALySIS oF BReAST cANceR cRITIcAL PReDISPoSING GeNeSMarikkannu Rajeshwari*, Aravidis Christos, Rantala Johanna,Kontham Vinaykumar and Lindblom Annikacenter for Molecular Medicine and surgery (cMM), Karolinska University hospital, stockholm, sweden

Breast cancer is one of the leading causes of death among women in the world, in particular more incidence in Western Europe including Sweden. Though there are a gamut of etiological factors ranging from age and hormonal status to life style attributes, approximately 5-10% breast cancer cases are familial. Family history of breast cancer remains the single most important risk factor for developing the disease. To date, there have been two highly penetrant predisposing genes identified namely BRCA1 and BRCA2 and few other rare high risk genes. They account for less than 25% of the total familial cases, which necessitates intensive studies on identifying the other genetic factors contributing and increasing breast cancer risk in the remaining families. We propose to identify novel cancer critical genes responsible for predisposing breast cancer in the risk families using linkage analysis and association studies. The present genome-wide linkage analysis included 14 non-BRCA1/2 breast cancer families with subjects having other cancer syndromes as well. A total of 540 fluorescently labeled microsatellite markers located on the 23 chromosomes were successully genotyped with approx. 7.25 cM resolution for the whole genome in these families. A multipoint parametric analysis was performed with a dominant mode of mendelian inheritance model. Our study has identified few interesting regions on the chromosomes 5, 6, 8, 11, 18 and 22 with HLOD score >1. These regions were further finemapped, confirming regions on 5q23, 6p21, 18q21 and 22q11 to be linked. Moreover, this analysis also revealed several other linked regions on the other chromosomes viz., 1p31, 2p24, 3p14, 7q33, 9q34, 10q23, 11q13, 13q32, 14q32, 20p12, 21q21, and Xp21 in any one of the individual families. We are further exploring these regions using exome and capture sequencing, followed by sequence analysis, which would help us in identifying the markers associated with disease genes predisposing to breast cancer

Keywords familial breast cancer, genome-wide linkage analysis, microsatellite markers, fin-emapping, HLOD scores

parTICIpaNTs’ pOsTers / sessIon 1


68



2 MuTATIoNAL ANALySIS oF BRcA1 AND BRcA2 IN heReDITARy BReAST AND oVARIAN cANceR FAMILIeS FRoM ASTuRIAS (NoRTh SPAIN). IDeNTIFIcATIoN oF NINe NoVeL PAThoGeNIc MuTATIoNSPilar Blay1, Ana S. Pitiot2, Iñigo Santamaría2, María Luque3, Marta G. Alvarado2,Ana Lastra2, Yolanda Fernández3, Ángeles Paredes1, José M.P. Freije4, Milagros Balbín2

1Unidad de cáncer Familiar, servicio de oncología Médica, Instituto Universitario de oncología del Principado de Asturias (IUoPA), hospital Universitario central de Asturias (hUcA), spain; 2Laboratorio de oncología Molecular, IUoPA, hUcA, spain; 3servicio de oncología Médica, IUoPA, hUcA, spain; 4Dpto. de Bioquímica y Biología Molecular, IUoPA, Universidad de oviedo, spain

The prevalence of BRCA1 and BRCA2 mutations in Spain is heterogeneous and varies accord-ing to the geographical origin of the studied families. The contribution of these mutations to hereditary breast and ovarian cancer has not been previously investigated in Asturian popula-tions (North Spain).

Two hundred and sixteen unrelated high-risk probands with breast and/or ovarian cancer from families living in Asturias were analysed for the presence of a BRCA1 or BRCA2 gene mutation from October 2007 to March 2012. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened by direct sequencing and MLPA.

A total of 54 families (25%) were found to carry a pathogenic germ line mutation, 36 in BRCA1 and 18 in BRCA2. Twenty five additional families carried an unknown significance variant. We detected 24 distinct pathogenic mutations (13 in BRCA1 and 11 in BRCA2). Three mutations in BRCA1 (c.1674delA, c1965C>A and c.2900_2901dupCT) and 6 in BRCA2 (c.262_263delCT, c.2095C>T, c.3263dupC, c.4030_4035delinsC, c.5116_5119del, c.8042_8043delCA) have not been previously described.

The novel mutations c.2900_2901dupCT in BRCA1 and c.4030_4035delinsC in BRCA2 occurred in 7 and 5 families respectively and cluster in two separated small areas suggesting a founder effect. These 2 mutations, together with the Galician BRCA1 mutation c.330A>C (8 families), and the common BRCA1 mutation c.3331_3334del (6 families), account for almost 50% of all mutations. By the contrary, other very frequent mutations in other Spanish series like the BRCA1 Ashkenazi founder mutations c.68_69delAG, was found in only one family.

In this study we report the BRCA1 and BRCA2 spectrum of mutations and their geographical distribution in Asturias, which largely differs from other areas of Spain. Our findings may help to design a panel of recurrent mutations to begin with, when screening high-risk breast and/or ovarian cancer families from this specific area.

3 LARGe GeNoMIc ReARRANGeMeNTS oF BReAST cANceR PReDISPoSING GeNeS IN hIGh-RISK BReAST/oVARIAN cANceR FAMILIeS IN SeRBIAKrivokuća Ana1, Stegel Vida2, Cerkovnik Petra2, Dobričić Jelena1,Novaković Srđan2, Branković-Magić Mirjana*1

Department of experimental oncology, Institute of oncology and radiology of serbia, Pasterova 14, 11000 Belgrade, serbia; 2Institute of oncology Ljubljana, Zaloška 2, 1000 Ljubljana, slovenia

Background Although most of the mutations in high penetrance BRCA1 and BRCA2 genes consist of small deletions, insertions and nonsense mutations, an increasing number of large genomic rearrangements (LGRs) has been identified. Additionally, rare mutations in other genes that interact with BRCA1/2, like CHEK2, increase the risk of hereditary breast/ovarian cancer. The aim of this study was to identify LGRs in BRCA1/2 and CHEK2 genes in Serbian patients with strong family history of breast/ovarian cancer.

Methods Multiplex ligation-dependent probe amplification (MLPA) method which is able to detect LGRs, was used in this study. Selection of high risk breast/ovarian cancer probands was done by BRCAPRO software (CP>10%) and pedigree analysis. All individuals were tested for BRCA1/2 mutations by sequencing whole genes (n=31) and were negative for point mutations and small insertions/deletions. Screening analysis of LGRs within BRCA1/2 and CHEK2 genes by MLPA was carried out in the study group.

Results No LGRs were identified in BRCA genes in the examined group, despite the fact that all of them had specific pre-test probability of BRCA deleterious mutation exceeding 10%. However, in one patient we detected large deletion of exon 9 in CHEK2 gene. This deletion was detected in a female diagnosed with breast cancer at age 32 and with family history of breast cancer (mother and sister were diagnosed with breast cancer at 42. and 36. years of age respectively, BRCA1 CP: 18.1%, BRCA2 CP: 11%).

Conclusions This study has shown that LGRs in BRCA genes are not present in examined group of Serbian high-risk breast/ovarian cancer families. However, we detected large deletion of exon 9 in CHEK2 gene. Considering that deletion which removes both exons 9 and 10 in CHEK2 gene is reported as founder mutation in some populations of Slavic origin, following analysis are needed to reveal the details of detected mutation.

Keywords BRCA1, BRCA2, hereditary breast/ovarian cancer, MLPA

Other info*Corresponding author: Mirjana Branković-Magić, PhDTelephone number: +381112067288



70



4 cLINIcAL ReLeVANce oF GeNoMIc INSTABILITy PATTeRNS IN FAMILIAL AND SPoRADIc ePITheLIAL oVARIAN cANceRKamieniak MM1*, Muñoz I1, 2, Osorio A1, 3, Urioste M1, 3, Rico D4, García-Donas J5,Hernado S5, Robles L6, Ramón y Cajal T7, Cazorla, A8, Sáez R9, Hernández, E10,García-Bueno JM11, Borrego S12, Mendoza E13, Palacios J13, Benítez J1, 3, 14, García MJ1, 3

1human genetics group, spanish national cancer research center, Madrid, spain; 2Pathology Department, hospital Universi-tario de Fuenlabrada, Madrid, spain; 3spanish network on rare Diseases (cIBerer), Madrid, spain; 4structural computational Biology group, spanish national cancer research center, Madrid, spain; 5Medical oncology Unit, hospital Universitario Fundación Alcorcón, Alcorcón, spain; 6Department of oncology, hospital Doce de octubre, Madrid, spain; 7Department of Pathology and oncology, hospital sant Pau, Barcelona, spain; 8Departament of Anatomic Pathology, Fundación Jiménez Díaz, Madrid, spain; 9Laboratory of genetics, hospital Donostia, san sebastián, spain; 10Breast cancer clinical research Unit, span-ish national cancer research center, Madrid, spain; 11Department of oncology, hospital general de Albacete, Albacete, spain; 12 genetics Department, hospital Virgen del rocio, sevilla, spain; 13 Pathology Department, hospital Virgen del rocio, sevilla, spain; 14genotyping Unit, human cancer genetics Programme, spanish national cancer research centre, Madrid, spain

Introduction Epithelial Ovarian Cancer (EOC) is the fifth cause of cancer-related death in women in the Western world and the most lethal of gynaecological malignancies.The most important risk factor is a strong family history of ovarian/breast cancer, however only about 5-12% of the ovarian carcinomas is hereditary.This study was conducted to determine patterns of genomic alterations in familial and sporadic EOC at high resolution level and to define the correlations with clinical data.

Materials and Methods Genomic instability of 53 familial (21 BRCA1, 6 BRCA2, 26 BRCAX) and 15 sporadic EOC was evaluated on high resolution array-CGH (Agilent 4×180K) and correlated with expression of 39 antibodies and survival data. Analyses were performed using R v.2.11, Nexus Copy Number v5.1 and SPSS v.17.

Results Unsupervised hierarchical clustering of all EOC rendered two main clusters, of higher and lower genomic instability level (expressed in number and length of copy number alterations), however it did not differentiate familial and sporadic cases, both types showing intermingled distribution.The tumours were stratified according to their immunohistochemical features; more genomically unstable being mainly high FIGO stage, serous high-grade, of higher proliferative rate and increased expression of p53 and antiapoptotic molecules (Bcl-Xl and survivin).In addition, several copy number changes, significantly differentiating both clusters, were determined.Further integration of survival data revealed their association with patients’ outcome.In particular dele-tion of 6q24-25 was found to be significantly correlated with better overall survival, independent from known factors such as FIGO stage, residual tumour and BRCA status.

Conclusion Familial and sporadic EOC do not differentiate significantly according to the pattern of genomic instability.However pattern of genomic aberrations may be used to stratify patients into clinically relevant groups regardless their BRCA1/2 status.

5 DouBLe heTeRozyGoSITy FoR A PuTATIVe SPLIcING VARIANT BRcA1c.135-5T>c AND The FouNDeR PoRTuGueSe MuTATIoN BRcA2 c.156_157INSALu IN A SPANISh PATIeNTSantamariña M1, Blanco A2, Fachal L2, Vega A2

1cIBerer-grupo de Medicina Xenómica, Universidad de santiago de compostela, spain; 2Fundación Pública galega de Medicina Xenómica, IDIs, cIBerer, santiago de compostela, spain

Introdution Germline mutations in the BRCA1 and BRCA2 genes predispose carriers to breast and ovarian cancer. There are different types of mutations identified in these genes together with missense mutations, silent variations, in-frame deletions and insertions, and intronic variants of uncertain clinical significance. Double heterozygosity is a condition in which both BRCA1 and BRCA2 mutations are present in a family at the same time. It has been rarely described, predominantly involving Ashkenazi Jewish founder mutations.

Case report Our proband is a Galician 44 year-old woman diagnosed with an infiltrating ductal carcinoma grade III at 41 years of age. Her paternal family history included: father diagnosed with breast cancer at age 86, paternal aunt diagnosed with breast cancer at age 56 and her paternal grandmother with gastric cancer at age 70. The maternal family history show a maternal aunt diagnosed with breast cancer at age 75, the mother of the patients was diagnosed with colon cancer at age 43. She had a sister who died from breast cancer at age 39. The BRCA1/2 analyses included bidirectional Sanger sequencing and MLPA. Sequencing results revealed a putative BRCA1 splicing variant c.135-5T>C, was not previously described in the literature. The variant analyses will be presented at the conference. Interestingly, the sequence of exon 3 of the BRCA2 gene presented some background, compatible with the BRCA2 c.156_157insAlu mutation. This BRCA2 mutation has been reported in hereditary breast/ovarian cancer (HBOC) families of Por-tuguese origin and is not detected using the common screening methodologies (Teugels et al., 2005). This Alu insertion leads to the in-frame deletion of exon 3 in the mutated BRCA2 mRNA. To our knowledge this is the first time that this founder BRCA2 Portuguese mutation was identified in a Spanish patient (Peixoto et al., 2011).

Keywords Hereditary breast cancer, BRCA1, BRCA2, minigene



72



6 The SPecTRuM oF BRcA1 & BRcA2 MuTATIoNS IN GReece ReVeALS The PReSeNce oF coMMoN AND uNIque FouNDeR MuTATIoNSTsitlaidou Marianthi1, Stavropoulou Alexandra Vassiliki1, Fostira Florentia1,Pertesi Maroulio1, Glentis Stavros1, Fountzilas George2, Konstantopoulou Irene1,Yannoukakos Drakoulis1

1Molecular Diagnostics Laboratory, national centre of scientific research Demokritos, Athens, greece; 2Aristotle University of thessaloniki, thessaloniki, greece & hellenic cooperative oncology group

The frequency, spectrum and penetrance of mutant BRCA1 and BRCA2 alleles have been esti-mated in several populations. In this study we are reviewing our data since 1998 and presenting new ones from the genetic analysis of approximately 1.000 families with breast and/or ovarian cancer. Selection was based on age of onset of proband and affected relatives, co-occurrence of breast and ovarian cancer, bilateral cancer, presence of male breast cancer, specific histological type etc. The full sequence of both genes was screened while additionally diagnostic PCRs were performed to detect the three Greek founder large genomic rearrangements. In total, 189 families were identified carrying a BRCA1 mutation (78.5%) and 26 families a BRCA2 mutation (21.5%). The mean age of onset for all patients in our cohort was 43.3 years. BRCA1 carriers had a mean age of onset of 42.5 yrs, while BRCA2 carriers 43.8 yrs. The Greek BRCA1 founder mutations include the universally spread c.5266dupC and p.Cys61Gly, and the unique to the Greek population p.Gly1738Arg, p.Arg1751X, deletion of exon 20, deletion of exon 24 and deletions of both exons 23 and 24. The most frequent mutations are c.5266dupC (21%) followed by p.Gly1738Arg (15%), deletion of exons 23-24 (9%) and deletion of exon 24 (7.5%). Correlation with available histopa-thology data shows that 90/107 (84.1%) BRCA1 carriers present a triple negative phenotype while 15/17(88.2%) BRCA2 carriers present estrogen positive tumors.

It’s worth highlighting that about 50% of families with medium-to-strong history of breast-ovarian cancer are negative for mutations in BRCA1 and BRCA2 genes.

Analysis of the BRCA1 and BRCA2 negative families is pursued using next generation sequencing approaches in order to define the contribution of other genes (CHEK2, PALB2, RAD51C, RAD51D etc) in breast and ovarian cancer in Greece.

Keywords BRCA1, BRCA2, Breast-Ovarian cancer, Greece, Germline mutations

Correspoinding author Dr. D. Yannoukakos

7 PReVALeNce oF BRcA1 AND BRcA2 LARGe GeNoMIc ReARRANGeMeNTS IN GALIcIAN (NW SPAIN) BReAST/oVARIAN cANceR FAMILIeSFachal L1, Santamariña M2, Blanco A1, Vega A1

1Fundación Pública galega de Medicina Xenómica, IDIs, cIBerer, santiago de compostela, spain, 2cIBerer-grupo de Medicina Xenómica, Universidad de santiago de compostela, spain

Most of the mutations detected to the date in the breast and/or ovarian cancer susceptibility genes BRCA1 and BRCA2 are point mutations, indels, and small insertions or deletions. Large genomic rearrangements (LGR) account only for a small percentage of all disease causing muta-tions, and its prevalence is highly variable among populations. In Spain, BRCA1 and BRCA2 LGRs explain respectively ~ 2% and ~1% of hereditary breast and/or ovarian cancer (HBOC) families previously tested negative for point mutations. However, the spectrum and prevalence of BRCA1 and BRCA2 mutations in the Galician population show great differences with the rest of the country. Therefore, in order to estimate the prevalence of LGRs among our population, we used multiplex ligation dependent probe amplification (MLPA) to screen for BRCA1 and BRCA2 LGRs in the index case individuals of 485 Galician HBOC families negative for point variations, indels, and small insertions or deletions. Three LGRs were detected in BRCA1 (0.62%), whereas none of the evaluated patients harbour a BRCA2 LGR. Our results confirm the clear population substructure for the BRCA1 and BRCA2 genes in the Iberian population.

Keywords Hereditary breast cancer, BRCA1, BRCA2, large genomic rearrangements



74



8 The uSe oF TeLePhoNe IN GeNeTIc couNSeLINGVeRSuS IN-PeRSoN couNSeLING: A RANDoMIzeD STuDyoN couNSeLeeS’ ouTcoMe.Rantala J, Platten U, Lindblom A, Brandberg Y, Lindgren G, Arver BDepartment of oncology and Pathology, Karolinska University hospital, s-17176 stockholm, sweden; Department of clinical genetics, Karolinska Institutet, s-17176 stockholm, sweden; Department of oncology and Pathology, Karolinska Institutet, s-17176 stockholm, sweden

Increased demand for genetic counseling services necessitates exploring alternatives to in-person counseling. Telephone counseling is a less time-consumingand more cost-effective alternative. So far there is insufficient evidence to support a pre-counseling telephone model. This randomized questionnaire study aims to evaluate the oncogenetic counseling process and to compare the impact of the initial part of the oncogenetic counseling, when conducted via telephone versus inperson. The aspects of evaluations were: patients’ expectations, satisfaction and experiences of genetic counseling, worry for developing hereditary cancer and health related quality of life. A total of 215 participants representing several cancer syndromes were randomized to counseling via telephone or in-person. The questionnaires were completed before and after oncogenetic nurse counseling, and 1 year after the entire counseling process. Overall, a high satisfaction rate with the oncogenetic counseling process was found among the participants regardless of whether the oncogenetic nurse counseling was conducted by telephone or inperson. The results show that a considerable number of participants experienced difficulties with the process of creat-ing a pedigree and dissatisfaction with information on surveillance and prevention. Affected participants reported lower levels in most SF-36 domains compared to non-affected and both groups reported lower levels as compared to a Swedish reference group. The results indicate that telephone pre-counseling works as well as in-person counseling. Emotional support dur-ing genetic counseling and information on recommended cancer prevention and surveillance should be improved.

Keywords Hereditary cancer, Oncogenetic in-person counseling, Oncogenetic telephone counseling, Cancer worry, Oncogenetic nurse

This paper has recently being published in Familial Cancer, 2012 Mar 8

9 FuNcTIoNAL AND STRucTuRAL ANALySIS oF GeRMLINe VARIANTS IN BRcA1Quiles, Francisco1; Fernández-Rodríguez, Juana1; Mosca, Roberto2; Feliubadaló, Lídia1;Brunet, Joan1; Blanco, Ignacio1; Capellá, Gabriel1; Pujana, Miguel Angel1; Aloy, Patrick2, 3; Monteiro, Alvaro4; Lázaro, Conxi1

1hereditary cancer Program, catalan Institute of oncology (Ico-IDIBeLL, Ico-IdIBgi, IcoIgtP), L’hospitalet de Llobregat, gran Via 199-201, 08908, Barcelona, spain; 2Joint IrB-Bsc Program in computational Biology, Institute for research in Biomedicine (IrB) Barcelona, c/ Baldiri reixac 10-12, 08028 Barcelona, spain; 3Institució catalana de recerca i estudis Avançats (IcreA), Passeig Lluís companys 23, 08010 Barcelona, spain; 4Department of risk Assessment, Detection, and Intervention, h. Lee Moffitt cancer center & research Institute, 12902 Magnolia Drive, tampa, FL 33612, Us

Background BRCA1 is a tumor suppressor gene and germ line inactivating mutations in this gene are responsible for the Hereditary Breast and Ovarian Cancer Syndrome (HBOCS). Sequencing analysis of BRCA1 identified about 16% of variants of unknown significance (VUS). Assessment of the pathogenicity or neutrality of these variants is an important challenge for genetic diagnosis and counselling.

Aim The aim of the present study is to apply functional and structural analysis of VUS locatedin the BRCT region of BRCA1 in order to classify them. Material and methods: Structural analysis of VUS was performed by mapping their position on the structures of BRCA1 collected from the Protein Data Bank (PDB, www.pdb.org) and analysing them manually with the help of Pymol (http://www.pymol.org). The structures were collected by querying the PDB with the sequence of the BRCA1 human protein (Uniprot AC P38398) using BLAST. Only four VUS (M1628V, Y1703S, W1718L and p.Ala1823_Val1862del) could be mapped to the structures of the BRCT tandem domains. The transcription activation (TA) assay described by Monteiro’s group has been demonstrated to be functionally informative for the analysis of DNA variants located in the region spanning over 500 amino acids in the Cterminus (aa 1314 to 1863). Here we performed the TA mammalian cell approach that uses a GAL4 DBD:BRCA1 fusion and the firefly luciferase gene under the control of GAL4 binding sites as a reporter, luciferase activity is evaluated in quantitative manner. Seven missense mutations (Q1409L, S1473P, E1586G, R1589H, Y1703S, W1718L, G1770V) and a single exon deletion (p.Ala1823_Val1862del) were analyzed.

Results Computational structure analysis evidenced that Y1703S, W1718L and p.Ala1823_Val1862-del may disrupt the BRCT folding and therefore the formation of the phospho-peptide motif binding site whereas G1770V would likely only disrupt the phosphopeptide binding while not affecting the domain folding. The transcriptional activation assay of the variant Y1703S confirms that this variant causes the loss of function of the BRCT domain while variants S1473P and R1589H showed TA results similar to the wild-type construct. The remaining variants are currently under study and their TA results will be presented in the meeting. •



76



Conclusion Our results suggest that a combined approach of functional and structural analyses could be useful to classify some of the VUS identified in BRCA1. The definition of an algorithm of analysis is crucial to improve genetic diagnosis and subsequent genetic counselling in HBOCS kindred.

Contract grant sponsor Asociación Española Contra el Cáncer; Spanish Health Research Foundation; Ramón Areces Foundation; Carlos III Health Institute; Catalan Health Institute; and Autonomous Government of Catalonia. Contract grant numbers: ISCIIIRETIC RD06/0020/1051, PI09/02483, PI10/01422, PI10/00748, 2009SGR290 and 2009SGR283.

Keywords BRCA1, VUS, FUNCTIONAL STUDIES

10 FIRST DeScRIPTIoN oF AN AcINIc cARcINoMA oF The BReAST IN A BRcA1-MuTATIoN cARRIeRRipamonti Carla B1, Colombo Mara1, Mondini Patrizia1, Manoukian Siranoush2,Peissel Bernard2, Bernard Loris3, 4, Carcangiu MariaLuisa5, Radice Paolo1, 6

1sc Basi molecolari del rischio genetico e test genetici, Dip. Medicina Predittiva e per la Prevenzione, Fondazione Irccs Istituto nazionale dei tumori (Int), Milan; 2 ss genetica Medica, Dip. Medicina Predittiva e per la Prevenzione, Int, Milan; 3Dip. di oncologia sperimentale, Istituto europeo di oncologia (Ieo), Milan; 4consorzio per le tecnologie genomiche (cogentech), Milan; 5Unità di Anatomia Patologica 1, Dip. Patologia e Medicina di Laboratorio, Int, Milan; 6Fondazione Istituto FIrc di oncologia Molecolare (IFoM), Milan, Italy

Acinic cell carcinoma (ACC) is a rare malignant epithelial tumor that resembles salivary gland type tumor for the presence of malignant tubular acinar exocrine gland structures. The lumen of the neoplastic glands contains eosinophilic material. We report on a BRCA1 mutation carrier with a family history of breast/ovarian cancers. At 40 years she developed an invasive ductal carcinoma (IDC). Estrogen receptor (ER) and progesterone receptor (PgR) were positive and HER2 negative. At 44 years a diagnosis of ACC was performed in the contralateral breast. Immu-nohistochemically, the lesion was positive for EMA, S100 and AACT and p53, and negative for Actin 1A4, Maspin, Calponin, GCDFP, ER, PgR, and Her2. The patient is alive and well 19 months after surgery. Loss of heterozygosity (LOH) analysis was carried out by analysing matched peripheral blood leukocytes and tumor DNAs extracted from paraffin-embedded tissues, using the BRCA1 marker D17S855. We detected the BRCA1 allele loss in the ACC as well as in the IDC. By sequencing, we defined that the wild-type allele was lost in both cases. We also detected two somatic TP53 mutations: c.654_655insGTG in the ACC and IVS9-1G>A in the IDC. Four reports describing the presence of ACCs of the pancreas in patients carrying germline mutations in cancer predisposing genes, including BRCA1 and BRCA2, have been published to date. Our case represents the first BRCA1 mutation carrier with a diagnosis of ACC of the breast. The tumor showed immunohistochemical features typical of BRCA1 associated breast cancers, including a triple negative status (i.e. nega-tivity for ER, PgR and Her2) and positivity for p53. Somatic biallelic gene inactivation and TP53 mutations are well documented mechanisms of cancer progression in BRCA1 mutation carriers. The occurrence of phyllodes and metaplastic carcinomas in BRCA1 mutation carriers has been reported. Our findings broaden the spectrum of BRCA1 associated breast malignancies.

Keywords ACC, BRCA1, LOH, TP53



78



11 PReIMPLANTATIoN GeNeTIc DIAGNoSIS FoR INheRITeD cANceR PReDISPoSITIoN AT GeNNeTKrutilkova V., Eliasova I., Brandejska M., Stejskal D. Putzova Mcentre for Medical genetics and reproductive Medicine gennet, Kostelni 9, Praha 7, czech republic

AIM Preimplantation genetic diagnosis (PGD) has been performed in our institute since 2007. Until now we have performed PGD for 53 different monogenic diseases and for other diagnosis PGD is being prepared. About 13% PGD cycles are due risk of hereditary cancer predisposition syndromes. We have been asked for PGD of inherited predisposition to breast and ovarian cancer, Lynch syndrome, MEN2A and neurofibromatosis type I.

Methods and results In our centre we use the genetic haplotyping technique by the multiplex PCR technique on products of MDA (multiple displacement amplification) from 1 blastomere biopsied from the cleavage-stage embryo. All couples interested in PGD undergo a genetic consultation.

PGD was performed in 9 couples at the risk of transmission predisposition to breast and ovar-ian cancer, in 6 couples at the risk of transmission neurofibromatosis type 1 and in 1 couple at the risk of having a child with MEN2A and 1 couple at the risk of transmission Lynch syndrome. Up to this time we have performed 24 PGD cycles in inherited cancer predisposition carriers resulting in 8 pregnancies.

Conclusion PGD allows couples of risk of transmission genetic disease to have healthy children and to avoid the prenatal diagnosis and termination of affected pregnancy. It is the only possibil-ity of that for carriers of hereditary predisposition to cancer with no manifestation in childhood, as the prenatal diagnosis is unacceptable in this country.

Keywords preimplantation genetic diagnosis (PGD), inherited cancer predisposition

12 SeLecTING FoR BRcA TeSTING oN The BASe oF cLINIcAL ReSPoNSe. A NeW cRITeRIuM?Bracci Raffaella MD*, Maccaroni Elena MD*, Bianchi Francesca MD°, Belvederesi Laura* PHD, Alessandra Pagliacci MD*, Stefano Cascinu MD*°

*clinica di oncologia Medica-centro di genetica oncologica, Azienda ospedaliero-Universitaria ospedali riuniti, Ancona °Dipartimento scienze cliniche e Molecolari Università Politecnica delle Marche, Ancona; corresponding Author Adress: clinica di oncologia Medica-centro di genetica oncologica, Azienda ospedaliero-Universitaria ospedali riuniti, Via conca 71, 60126, Ancona, Italy

Background The BRCA1/2 genes mutations account for 5% of breast cancers. Women are usu-ally offered BRCA testing in case of familial clustering or early onset, especially with “basal like” tumor features. Consistent evidences can be found regarding high sensitivity of BRCA1/BRCA2 deficient tumors to platinum compounds. We report a case of a young woman who underwent genetic testing only on the basis of unexpected response to carboplatin. Case description: In 2005, a 37-year-old woman underwent right mastectomy for ductal carcinoma of the breast, stage IIIA (pT3N1M0). The tumor was grade3, estrogen and progesterone receptors negative, HER2 posi-tive (3+). The patient underwent adjuvant chemotherapy with adriamicin/cyclophosphamide for 4 cycles followed by 4 cycles of cyclophosphamide/methotrexate/fluorouracil and 3-weekly trastuzumab for one year. In February 2008, due to dyspnoea, a CT scan was performed, showing bilateral lung nodules. We tried several chemotherapeutic regimens, (trastuzumab/vinorelbine, trastuzumab/docetaxel, capecitabine/lapatinib) with no response. In January 2009 the patients started palliative chemotherapy with carboplatin/gemcitabine, experiencing a rapid disappearance of the dyspnoea. A CT scan after 3 cycles detected a good partial response. Due to this surprising result, genetic testing for BRCA1/2 was offered even in the absence of significant familial history. We detected a BRCA1 point mutation. After 8 cycles with carboplatin/gemcitabine we continued with gemcitabine alone until February 2012, when there was a reappearance of symptoms and progressive lung disease with massive pleural effusion was documented. In March 2012 we rechallenge a platinum compound, and cisplatin/cyclophosphamide chemotherapy was started with a rapid improvement of symptoms. Addendum: The patient’s sister who tested positive for the same mutation developed a small breast cancer.

Conclusion This case suggests that, in breast (and ovarian) cancer patients, response to platinum should be evaluated to expand criteria for BRCA mutation testing. In the next future, mutation carriers not only could have the opportunity to undergo screening, but also to benefit from novel drugs such as poly(ADP-ribose) polymerase inhibitors.

Keywords breast cancer, BRCA1/2, platinum chemotherapy



80



13 ScReeNING oF SLX4/FANcP IN 486 SPANISh BRcAX FAMILIeS.Ruiz de Garibay G1, Tosar A1, Avellaneda M1, Gaviña B1, Romero A1, Garre P1, Vega A2, Blanco A2, Díez O3, Pérez-Segura P4, Díaz-Rubio E1, 4, Caldés T1, de la Hoya M1

1Laboratorio de oncología Molecular, Instituto de Investigación sanitaria san carlos (IdIssc), Madrid, spain; 2Fundación Pública galega de Medicina Xenómica-sergAs, cIBerer, IDIs, santiago de compostela, spain; 3Laboratorio de oncogenética. hospital Universitario Vall d’hebron & Vall d’hebron Instituto oncológico (VhIo). Barcelona; 4servicio de oncología Médica, hospital clínico san carlos. Madrid. spain

Fanconi Anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by development abnormalities, bone marrow failure, and childhood cancers. Compelling evidence indicates a common genetic basis for FA and breast cancer susceptibility. Recently, biallelic germ-line mutations in SLX4 have been demonstrated to cause a previously unknown FA subtype (FA-P). Here we address the role of SLX4 in breast cancer susceptibility by conducting a comprehensive mutation scanning of 486 index cases from non-BRCA1/BRCA2 multiple case breast and/or ovarian cancer (BRCAX) families, ascertained through 3 contributing centers: Hospital Clinico San Carlos, Madrid (N=348), Fundación Publica Galega de Medicina Xenómica, Santiago de Compostela (N=98), and Hospital Vall d’Hebron, Barcelone (N=40). All BRCAX families included a minimum of 3 breast and/or ovarian cancer cases diagnosed at any age in two generations of the same parental branch. Ovarian cancer was present in 76 out of the 486 families. The whole coding sequence and flanking intronic regions of the SLX4 gene were analyzed by High Resolution Melting (HRM) analysis followed by direct sequencing of abnormal melting curve samples.

Fifty-seven different SLX4 sequence variants were identified, including common polymorphisms (f>0.01) and rare changes (12 not previously annotated in dbSNP). We find of particular interest the identification of one truncating mutation (p.Glu1517X), and five missense changes (p.Gly141Trp, p.Arg372Trp, p.Tyr546Cys, p.Arg1445Trp, p.Arg1550Trp) predicted to be deleterious by both Polyphen-2 and SIFT in silico analysis tools.

The c.4549G>T (p.Glu1517X) alteration was identified in a family including multiple cases of breast cancer, but not ovarian cancer. Unfortunately, it was not possible to perform co-segregation analysis in this particular family. The mutated allele, c.4549G>T (p.Glu1517X), codifies for a truncated protein lacking 318 aa at the C-terminal end. The deleted region includes SAP and SBD domains critical for proper SLX4 function.

So far, SLX4 has been analyzed in a minimum of 1158 BRCAX families (including the data here reported). Taking together, these studies suggest that the proportion of BRCAX families explained by SLX4 germ-line mutations (if any) is very low, so that the screening of SLX4 germ-line muta-tions is unlikely to be clinically relevant. Yet, to demonstrate (or discard) further links between FA and breast cancer genetic susceptibility is of outstanding scientific interest, so that further studies in this field are warranted.

14 cuL4A coNTRIBuTeS To BReAST cANceR AGReSSIVeNeSS By MoDuLATING ceLL cycLe PRoGReSSIoN AND PRoLIFeRATIoNSaucedo-Cuevas LP1*, Gracia FJ1, García MJ1, 2, Benítez J1, 2

1human genetics group, spanish national cancer research center (cnIo), Madrid, spain; 2centro de Investigación Biomédica en red de enfermedades raras (cIBerer), Instituto de salud carlos III, Madrid, spain

Comprehensive characterization of a 13q34 amplicon previously defined by our group allowed us to define CUL4A as one of the most likely putative oncogenes. CUL4A is an E3 Ubiquitin Ligase which modulates cellular processes such as cell cycle progression or DNA repair capacity through selective ubiquitylation and degradation of cell cycle mediators and DNA damage sensors. Its deregulation could have implications for oncogenic transformation and treatment response to DNA-damaging agents.

To elucidate the possible implication of CUL4A in the initial steps of the tumorigenic process we conducted the stable up-regulation of CUL4A in human mammary epithelial cells (184B5) by len-tiviral infection. We also stably silenced CUL4A in the human breast cancer cell lines MDA-MB-157 and HCC1937 that present amplification and/or overexpression of CUL4A. With these modified cell lines we performed functional assays in different in vitro systems such as viability experi-ments, colony formation both in plastic and in soft agar. We also conducted cell cycle analysis and determined the half-life of some CUL4A downstream targets. Our preliminary results in CUL4A overexpression in 184B5 model system showed increase in proliferation and colony formation ability. On the other hand, CUL4A silencing in breast cancer cell lines resulted in diminished pro-liferation and decreased colony-forming abilities in both anchorage dependent and independent conditions. By modulating CUL4A expression we were also able to induce cell cycle modifications and protein-level changes in the CUL4A-downstream targets p21 and DDB2.

Our results suggest that CUL4A contributes to breast cancer aggressiveness and progression through modulation of cell proliferation. Additional evaluation of the role played by CUL4A amplification/overexpression in the induction of a transformed phenotype will further clarify the relevance of this ubiquitin ligase in the initial steps of the oncogenic process.

Keywords CUL4A, breast cancer, 13q34 Amplicon, candidate oncogenes, functional charac-terization



SeSSIoN 2 1 coMPouND coNTRIBuTIoN To MISMATch RePAIR DeFIcIeNcy?Jukka Kantelinen, Minttu Kansikas*, Satu Candelin,Heather Hampel, Betsy Smith, Reetta Kariola, Minna Nyström

Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene with a pathogenic mutation causes a dominant susceptibility to a cancer syndrome known as Lynch Syndrome. MMR gene variants of uncertain significance (VUS) have made the interpretation of MMR gene variants challenging. MMR VUS can be pathogenic mutations causing Lynch syndrome or they may cause a moderately increased risk of cancer or even be harmless polymorphisms. Our study suggests that the inherited individually proficient MMR VUS can in a pair with another MMR VUS found in the same colorectal cancer patient have a compound contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in CRC patients were functionally analyzed in an in vitro MMR assay. Of these, a compound defect by a VUS pair which nearly halves the repair capability of the wild type MSH2 protein is presumed to increase the cancer risk significantly. Especially interesting is the role of one of the most frequently reported VUS whose compound defect with another variant may finally explain its frequent occurrence in CRC patients.



84



2 ASSeSSMeNT oF coLoRecTAL cANceR MoLecuLAR MARKeRS FoR LyNch SyNDRoMe IDeNTIFIcATIoN Arias Alonso M., Moreno Laguna S., Montes Díaz M., Gómez Dorronsoro ML.,Janices Zapata MI., García Amigot F., Narvaiza Martínez RC., Moreno Igoa M.,Hernández Charro B. Alonso Sánchez A.

Aim Identification of Lynch Syndrome (LS) patients by using molecular markers on incident colorectal cancers (CRC) in order to evaluate their sensitivity.

Patients and Methods As partial results of a large ongoing research project on Lynch syndrome, we prospectively studied a set of 224 CRC without any selection criteria. Tumours were tested for microsatellite instability (MSI) and mismatch repair MMR proteins inmunohistochemistry (IHC).V600E BRAF mutation and MLH1 promoter methylation were assessed in all MSI-H tumours. BRAF V600E wildtype and non methylated tumours were scanned for MMR genes germline mutations.

Results 13% of all tumours showed MSI-H; with the mono-27 microsatellite being the highest sensitive of those included in the msi-promega panel®. 52% of MSI-H tumours showed MLH1 promoter methylation or V600E BRAF mutation; being the methylation assay more specific than the V600E BRAF mutation. 70% of BRAF wildtype and non-methylated cases, were diagnosed of Lynch Syndrome. Inmunohistochemistry pattern was concordant with the germline mutations in 86%. 57% of the identified germline mutations ocurred in MLH1, 29% in MSH2 and 15% in MSH6. We did not find any mutations in PMS2 gen.

Keywords Microsatellite, Lynch Syndrome, Braf, Methylation

3 The DeTecTIoN oF cIRcuLATING TuMoR ceLLS (cTcs) IN PeRIPheRAL BLooD AS A PoTeNTIAL DIAGNoSTIc, PRoGNoSTIc AND eARLy DeTecTIoN TooL IN PANcReATIc cANceR.Earl J1, Guillén-Ponce C1, Guerrero-Arroyo C1, Mocci E1, Sanjuanbenito A2,Real PX3, Malats N3, Carrato A1

1Medical oncology Department, ramón y cajal University hospital, Madrid, spain; 2gastrointestinal surgery, ramón y cajal University hospital, Madrid, spain; 3spanish national cancer research center (cnIo), Madrid, spain

Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer affecting the exocrine pancreas worldwide an estimated 216, 000 new cases are diagnosed and 213, 000 deaths occur each year. An estimated 5-10% of pancreatic cancers have a familial background either with a high incidence of only pancreatic cancer or in combination with other cancer syndromes such as breast, ovarian, Peutz Jeghers Syndrome (PJS) and Familial Atypical Multiple Mole Melanoma (FAMMM). The prognosis of patients is very poor with an overall 5 year survival of 5%, due to the fact that the majority of patients present with an advanced metastatic disease. The key to improving prognosis lies in early detection and a deeper understanding of the metastatic process. Familial Pancreatic Cancer (FPC) offers an opportunity for early detection in high risk popula-tions. We co-ordinate the multi-centre study of Familial Pancreatic Cancer, PANFAM, the main objectives are the genetic and phenotypic characterization of these families and the evaluation of early detection strategies. Over the last few years the significance of Circulating Tumor Cells (CTCs) in peripheral blood as a prognostic marker in various types of cancer has become clear. The FDA approved CellSearch® system provides a semi automated approach that detects and captures circulating epithelial cells that express markers EpCAM and cytokeratins 8, 18 and 19. This system has proved successful in the study of breast, colon and prostate cancers, although the role of CTCs in pancreatic cancer is poorly studied. Since pancreas cancer is a highly metastatic disease, the study of CTCs in these patients would be of prognostic and diagnostic value and also useful to monitor the efficacy of therapeutic treatments. We used the CellSearch® system for CTC detection in patients diagnosed with pancreatic cancer and also as an early detection screening tool in high risk individuals in FPC. We have detected CTCs in 23% (3/13) of patients diagnosed with metastatic pancreatic cancer before starting treatment, using the cell search system. One CTC was detected in one of 87 high-risk individuals in the screening program. Subsequent follow-up of this individual did not reveal any evidence of cancer, furthermore, the detection of one cell is in concordance with the rate of “false positive” detection of CTCs in healthy individuals. One of the possible drawbacks of the CellSearch® system is that cells that either do not express or express low levels of EpCAM will not be detected. Therefore, we are developing an alternative method of CTC detection using an approach of negative cell selection and CTC identification using both epithelial and mesenchymal cell markers. The identification of pancreatic cancer circulating tumor cells is feasible and may provide a powerful, noninvasive approach for early diagnosis, prognosis and therapeutic response for a specific group of patients in the clinical setting.

Keywords pancreas cancer, familial, CTCs, markers



86



4 PReVALeNce oF heReDITARy cANceR SyNDRoMeS IN AN uNSeLecTeD PoPuLATIoN WITh eNDoMeTRIAL cANceR (ec) IN SWeDeNTzortzatos Gerasimos1, Castro Ofra1, Lindblom Annika2,Gemzell Danielsson Kristina1, Tham Emma2, Mints Miriam1

1Department of obstetrics and gynaecology; 2Department of clinical genetics, Karolinska University hospital, stockholm, sweden

Objective In Sweden, few cases of suspected hereditary cancer are referred to Clinical Genetics from Gynaecological clinics. Previous studies of DNA mismatch repair gene mutation analysis among unselected EC patients found that the frequency of Lynch syndrome (LS) is 1, 8-2, 1% and 9% in EC-patients < 50 years old. Cowden’s syndrome (CS) is characterized by EC, breast and thy-roid cancer with macrocephaly, with a prevalence of 1:200.000. Breast-ovarian cancer syndrome (BR-OVCA) has a prevalence of 1:400 – 1:800. The aim of the present study was to investigate the frequency of cancer syndromes among Swedish EC patients.

Methods Among 830 women with EC undergoing surgery at Karolinska University Hospital 2008-2011, 430 (51, 8%) agreed to be included in the study. Family history was obtained and diag-noses were confirmed via the regional cancer registry. Pedigrees were analyzed and patients who fulfilled the criteria for LS (Amsterdam II or modified Bethesda), CS (National Comprehensive Cancer Network) or BR-OVCA (local screening criteria) were referred for further testing for the relevant syndromes by DNA sequencing and MLPA of DNA isolated from peripheral blood. When the Bethesda criteria were fulfilled, microsatellite instability (MSI) and immunohistochemistry testing on colon cancer samples was performed if available.

Results In 52 (12%) women, hereditary cancer was suspected: 27 (6, 3%) women had both EC and breast cancers, but other criteria for CS were unknown. Of these women, five were screened for PTEN mutations, five women also fulfilled other criteria and were included below; 30 (6,9%) women had a family history that fulfilled: Amsterdam criteria: four (0.9%); Bethesda: 16 (3,7%); BR-OVCA: seven (1,6%); hereditary cancer of other kind: one; Three women have been diagnosed with EC before the age of 40, one woman had a family history that fulfilled two criteria. Of the four Amsterdam criteria families, two had known mutations in MLH1 (c.546-2A>G) and MSH2 (c.1786_17 88del) respectively; for the remaining two families screening analyses are ongoing.

Of the BR-OVCA group, four women had no mutations in BRCA1/2, for two women screening analyses are ongoing and one woman declined screening.

In the Bethesda group, seven women declined further investigation; five MSI analyses and four LS screens are ongoing. One family had three cases of meningioma –final confirmation of diagnosis is pending. In the CS group no PTEN mutations were found, one analysis is ongoing.

Conclusion The proportion of patients with a suspected hereditary cancer syndrome among women with EC is 12%. Of these, up to 3% may have LS, using a criteria-based method. In 6% we may suspect unknown hereditary cancer syndromes. Future studies are needed to identify disease-causing genes in EC-patients.

Keywords Endometrial cancer, Lynch Syndrome, BR-OVCA



88



5 IDeNTIFyING SDhA MuTATIoNS By SDhA IMMuNohISTocheMISTRy IN ADuLT WILD-TyPe AND PeDIATRIc/ADoLeSceNT GASTRo-INTeSTINAL STRoMAL TuMoRSLindsey Oudijk, José Gaal, Esther Korpershoek, Maureen O’Sullivan,Francien H. van Nederveen, Lorna Kelly; Gaia Schiavon, Jaap Verweij, Michael A. den Bakker, Rogier A. Oldenburg, R.L.E. van Loon, Winand N.M. Dinjens, Ronald R. de KrijgerDepartment of Pathology, Josephine nefkens Institute, erasmus Mc, University Medical center rotterdam, the nether-lands; Department of Pathology, our Lady’s children’s hospital, crumlin, Dublin 12, Ireland; Department of Medical oncol-ogy, erasmus Mc, University Medical center rotterdam, the netherlands; Department of clinical genetics, erasmus Mc, University Medical center rotterdam, the netherlands

Most gastrointestinal stromal tumors (GISTs) harbor oncogenic mutations in KIT and platelet-derived growth factor receptor-α (PDGFRA). However, a small subset of GISTs lack KIT or PDGFRA mutations and are termed wild-type GISTs. Germline mutations in one of the subunits of succinate dehydrogenase (SDH) predispose individuals to hereditary paragangliomas and pheochromo-cytomas. However, mutations of the genes encoding succinate dehydrogenase subunits A, B, C or D (SDHA, SDHB, SDHC or SDHD) can also cause GISTs. SDHA and SDHB immunohistochemistry are reliable techniques to identify pheochromocytomas and paragangliomas caused by muta-tions in SDHA, SDHB, SDHC and SDHD. In this study we investigated if SDHA immunohistochem-istry could recognize SDHA-related GISTs as well. Twenty-four adult wild-type GISTs and nine pediatric/adolescent wild-type GISTs were analyzed with SDHB and subsequently with SDHA immunohistochemistry. If SDHA immunohistochemistry was negative, sequencing analysis of the entire SDHA coding sequence was performed. All nine pediatric/adolescent GISTs and seven adult wild-type GISTs were immunohistochemically negative for SDHB. One pediatric GISTs and three SDHB-negative adult wild-type GISTs were immunohistochemically negative for SDHA. In all SDHA-negative GISTs an SDHA c.91C>T transition was found leading to a nonsense p.Arg31X mutation. Our results demonstrate that SDHA immunohistochemistry on GISTs can identify the presence of an SDHA germline mutation. Identifying GISTs with deficient SDH activity in patients warrants additional genetic testing, evaluation and follow-up for inherited disorders and paragangliomas.

Keywords gastrointestinal stromal tumor, pediatric, immunohistochemistry, sequencing analysis, succinate dehydrogenase

6 ANALySIS oF MSh6 MuTATIoNS IN PATIeNTS WITh eNDoMeTRIAL cANceR: PReLIMINARy ReSuLTSMartinez-Bouzas C1, Rubio I1, Ibañez E2, Andrés L3, Maortua H1, Tejada MI1

1Laboratorio de genética Molecular, hospital de cruces; 2sección de oncología ginecológica. servicio de ginecología y obstetricia. hospital de cruces; 3servicio de Anatomía Patológica. hospital de cruces, Barakaldo, spain

The endometrial cancer is the most frequent gynecological tumor, and the most common cancer in patients with Lynch Syndrome. Women with MLH1 or MSH2 mutations have a risk for endometrial cancer of 40-60%, and it has been published that with MSH6 mutations the risk is near 71%.

The purpose of our work was to study MSH6 and MSH2 mutations in women with endometrial cancer to find more Lynch families that could be lost if only Amsterdam criteria are considered.

DNA samples of 56 unrelated index cases were analyzed using PCR-amplification, heteroduplex, CSGE, sequencing and MLPA. Study of microsatellite instability (MSI) was performed in tumor cells. Inmunohistochemical studies (IHQ) were also performed.

For MSI, we used 5 microsatellite markers: two mononucleotide repeats (BAT25 and BAT26) and three dinucleotide repeats (D71S250, D2S125 and D5S346), as recommended. It is necessary to compare normal and tumoral tissue.

We have detected 6 different mutations/variants in 5 patients (6/56= 10.7%): four in MSH6 (7.1%) and two in MSH2 (3.6%). Of these DNA variants, four were pathologic mutations (MSH6; c.1572C>G; c.3514dupA: MSH2 c.1662-?_2664+?del), one of them not described previously (MSH6; c.1173delT). The other two new variants found were benign (MSH6; c.1168G>A: MSH2; c.594A>G).

MSI analysis was done in 47 patients, 35 of them (74.5%) were MSS, 6 (12.8%) were MSI-L and 6 (12.8%) were MSI-H. The remaining pieces of tumor were not available.

Of the 48 tumors tested for immunohistochemistry, 10 (20.8%) showed abnormal staining for at least one MMR protein. Of these 10 abnormal cases, 5 (50%) showed concurrent loss of MLH1 and PMS2, 4 (40%) showed concurrent loss of MSH2 and MSH6 and 1 (10%) showed loss of MSH6 alone.

Our results show a pathological mutation rate of 7.1%, so we can conclude that it is possible to find Lynch Syndrome families if the study begins with women with endometrial cancer.



90



7 heReDITARy cANceR SuB-eXoMe SequeNcING APPRoAch FoR GeNeTIc DIAGNoSTIcSCastellanos E1, Gel B1, Rosas I1, Terribas E1, Sumoy L2, Pluvinet R2,Blanco I3, Capellà G3, Lázaro C3, Serra E1

1 Ico-IMPPc Joint Program on hereditary cancer, Institut de Medicina Predictiva i Personalitzada del càncer (IMMPc) Badalona, Barcelona, spain; 2 genomics Laboratory, Institut de Medicina Predictiva i Personalitzada del càncer (IMMPc) Badalona, Barcelona, spain; 3 hereditary cancer Program, genetic Diagnosis Unit-genetic counseling Unit, catalan Insti-tute of oncology (Ico-IDIBeLL), L’hospitalet de Llobregat, Barcelona, spain

Around 40 inherited diseases caused by mutations in more than 70 genes have been classified as hereditary cancer syndromes. The genetic testing of most of these syndromes is labor intensive, time-consuming and expensive, and is further complicated by the existence of genetic hetero-geneity or the overlapping of certain clinical manifestations.

In order to improve the cost-effectiveness of the genetic testing of hereditary cancer syndromes, we are developing a sub-exome sequencing-based approach to provide a genetic test for all hereditary cancer genes described so far, in a global and unified way. The hereditary cancer sub-exome includes 106 genes, 97 implicated in hereditary cancer syndromes (76 listed in the Cancer Gene Census as Germline mutated cancer genes and 21 additional related ones) and 9 involved in RASopathies (Ras/MAPK pathway deregulated conditions). For these genes, all exons (both constitutive and alternative), intron-exon boundaries and 3’-UTR regions are captured using a custom Agilent SureSelect® target enrichment system and ultra-sequenced in the Ion Torrent PGMTM platform. The huge sequencing capacity allows sample multiplexing, potentially enabling the analysis of all hereditary cancer genes of multiple patients at once, reducing the complexity and cost while maintaining or increasing mutation detection sensitivity.

An automated analysis pipeline is being developed with two different parts. A first general part comprises the alignment of all reads to the genome and the capture and sequencing quality control. A second part, specific for each cancer syndrome, consists in the refinement of the general alignment –taking into account specific pseudo-gene sequences, InDels, SNPs, etc– and the prediction of the functional impact of all variants identified. All results obtained are checked manually and variants and mutations finally selected will be validated using Sanger sequencing. We will present the first results of this pilot study.

8 IDeNTIFIcATIoN oF PATIeNTS WITh LyNch SyNDRoMe IN eSToNIAEgle Rebane, Martin Kask, Olga Kostinacompetence centre for cancer research. tallin, estonia

Hereditary non-polyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an auto-somal dominantly inherited syndrome predisposing to the early development of cancers of colon, rectum, endometrial, small bowel and urinary tract and accounts for ~5% of all colon cancer cases. Molecular testing has emerged as an indispensable strategy for the diagnosis of Lynch syndrome. The diagnostic work-up of at-risk individuals includes a careful family history evaluation and molecular diagnostic algorithm.

The aims of the study were to find optimal molecular diagnostic algorithm for Lynch syndrome and investigate the MMR mutation spectrum in Estonia.

Ninety three unrelated patients were selected for molecular analyses according Amsterdam I, II criteria and our modified criteria of suspected Lynch syndrome. Patient were selected to mutation detection in MMR genes (MLH1, MSH2 and MSH6) based on microsatellite instability (MSI), and immunohistochemical (IHC) analysis of MMR protein expression. MSI occurred in 25 individuals of Lynch suspected family, four of MSI tumors showed normal protein expression in IHC. Fifteen germline MMR gene mutations with clinical significance were found in 25 investigated MSI tumors. Seven mutations were found in MLH1, six in MSH2 and two in MSH6, no PMS2 mutations were detected. Germline MMR gene mutations were found in all four patients with MSI tumors which showed normal protein expression. MLH1 promoter methylation was found in two MSI tumors, patients with the epimutations were considered as non-Lynch syndrome. MMR gene mutations, MLH1 hypermethylation, CIMP phenotype or BRAF mutation were not found in eight MSI tumors, although two of them had germline CHEK2 mutations. MSI and abnormal MMR gene expression of the tumor was left unexplained in six patients, demanding further investigation.

Keywords HNPCC, Lynch syndrome, MMR genes, microsatellite instability



92



9 SeRRATeD PoLyPoSIS WITh FAMILy hISToRy oF coLoRecTAL cANceR AND/oR PoLyPS: A DISTINcT cLINIcAL AND MoLecuLAR eNTITy DIFFeRING BeTWeeN The PRoXIMAL AND DISTAL coLoNSilva, Patrícia1; Albuquerque, Cristina1; Lage, Pedro2, 3; Fontes, Vanessa1; Fonseca, Ricardo4; Vitoriano, Inês1; Fragoso, Sofia1; Rodrigues, Paula3; Moita, Susana1; Ferreira, Sara2, 3;Sousa, Rita2*; Claro, Isabel2, 3; Leitão, Carlos N2#; Chaves, Paula4; Pereira, António D2

1centro de Investigação de Patobiologia Molecular (cIPM), 2serviço de gastrenterologia, 3clínica de risco familiar, 4serviço de Anatomia Patológica—Instituto Português de oncologia de Lisboa Francisco gentil, ePe (IPoLFg, ePe), Lisboa, Portugal; *current address: hospital garcia de orta, Almada, Portugal; #current address: hospital dos Lusíadas, Lisboa, Portugal

Background and aims Serrated polyposis (SPP) is characterized by the development of multiple colorectal serrated polyps and increased predisposition to colorectal cancer (CRC). We aimed to characterize at clinical and molecular level a cohort of SPP patients with or without family history of polyps (multiple or diagnosed at a young age) and/or CRC in first degree relatives (SPP-FHP/CRC).

Methods We analyzed 62 serrated or adenomatous lesions from 11 SPP-FHP/CRC families and 6 sporadic SPP patients for microsatellite instability (MSI), MGMT and MMR gene hypermethylation, and somatic mutations in WNT and RAS/RAF genes.

Results SPP-FHP/CRC patients presented an older mean age at diagnosis (p=0.027), a more het-erogeneous histological pattern of lesions (p=0.032) and a more relevant role of KRAS mutations and MGMT methylation in comparison with sporadic SPP. Two forms of SPP-FHP/CRC appear to exist, according to the preferential location of the lesions, proximal/whole colon and distal, differing mainly in the somatic events found in early lesions. The former presented more fre-quently MGMT methylation, MMR deficiency (mainly through MSH6 and not MLH1 methylation), MSI and WNT mutations [19/29 (65%) vs 7/17 (41%); 17/18 (94%) vs 0/11, p=3×10-7; 15/26 (58%) vs 2/15 (13%), p=0.006; 14/26 (54%) vs 4/20 (20%), p=0.02, respectively]. Distal SPP presented more frequently BRAF mutations [12/20 (60%) vs 7/30 (23%), p=0.009]. Two groups of patients were identified in each form, whose lesions harboured preferentially KRAS or BRAF mutations, respectively. CRC was more frequent in proximal/whole colon SPP following a KRAS (alternate) pathway [4/4 vs 1/8 (12%), p=0.01].

Conclusions SPP-FHP/CRC appear to be a distinct clinical and molecular entity, presenting two different forms, proximal/whole colon and distal, each following either an alternate or a serrated pathway. CRC risk appears to be higher in proximal/whole colon SPP following an alternate rather than a serrated pathway.

10 MIcRoSATeLLITe INSTABILITy DeTecTIoN By hIGh-ReSoLuTIoN MeLTING (hRM) ANALySISRamunas Janavicius, Dovile Matiukaite, Laimonas GriskeviciusVilnius University hospital santariskiu clinics, hematology, oncology and transfusion Medicine center. Vilnius, Lithuania

Background Microsatellite instability (MSI) is an important marker for screening for hereditary nonpolyposis colorectal cancer (Lynch syndrome) as well as a prognostic and predictive marker for sporadic colorectal cancer (CRC). The mononucleotide microsatellite marker panel is a well-established and superior alternative to the traditional Bethesda MSI analysis panel, and does not require testing for corresponding normal DNA. The most common MSI detection techniques-fluorescent capillary electrophoresis and denaturing HPLC (DHPLC)-both have advantages and drawbacks. A new high-resolution melting (HRM) analysis method enables rapid identification of heteroduplexes in amplicons by their lower thermal stability, a technique that overcomes the main shortcomings of capillary electrophoresis and DHPLC. METHODS: We investigated the straightforward application of HRM for the detection of MSI in 70 archival CRC samples. HRM analysis for 2 MSI markers (BAT25 and BAT26) was evaluated, and 2 different HRM-enabled instruments were compared-the LightCycler® 480 (Roche Diagnostics) and the LightScanner(TM) (Idaho Technology). We also determined the analytical sensitivity and specificity of the HRM assay on both instruments using 11 known MSI-positive and 54 microsatellite-stable CRC samples. RESULTS: All MSI-positive samples were detected on both instruments (100% analytical sensitivity). The LightScanner performed better for analytical specificity, giving a combined specificity value of 99.1% compared with 92.3% on the LightCycler 480 [1].

References 1. Janavicius R, Matiukaite D, Jakubauskas A, Griskevicius L. Microsatellite instabil-ity detection by high-resolution melting analysis. Clin Chem. 2010 Nov;56(11):1750-7.



94



11 hNPcc-MSS VeRSuS hNPcc-MSI AND SPoRADIc coLoRRecTAL cANceRGarre P1†, Bando I1, Tosar A1, Llovet P1, Saez C2, de la Hoya M1, Díaz-Rubio E3 and Caldés T1

1Laboratorio de oncología Molecular; 2servicio de Anatomía Patológica, 3servicio de oncología. hospital clínico san carlos. c/ Profesor Martín Lagos s/n, Madrid, spain

Lynch syndrome/Hereditary non-polyposis colorectal cancer is caused by inherited germline mutations in mismatch repair (MMR) genes, and accounts for 2-5% of colorectal cancers (CRC). Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or heredi-tary non-polyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair gene mutations that define this syndrome and result in microsatellite instability (MSI). The carcinogenic pathways and the best diagnostic approaches to detect microsatellite stable (MSS) HNPCC tumors are unclear and Genetic Counseling is difficult in these families.

A few previous studies compare clinical, morphological and molecular features between HNPCC-MSS families and HNPCC-MSI families. From these studies three consistent and repetitive characteristics have been elucidated; these are the slight increase of diagnosis age, increased risk of colorectal cancer and preferential distal localization of tumours. In addition, all authors agree that HNPCC-MSS syndrome is a heterogeneous group of families consisting of different genetic syndromes and some cancer aggregations. Therefore, deep description of such families becomes important for a better classification of this group and search for underlying susceptibility causes.

Our main aim is to compare the clinical, morphological and molecular features (KRAS mutation status) of HNPCC-MSS families, HNPCC-MSI families and a series of sporadic colorectal cancer from the Oncology Unit of the Hospital Clínico San Carlos at Madrid.

Our results are consistent with those described so far. Most of the analyzed data places the HNPCC-MSS families half way between HNPCC-MSI and sporadic CRC. The main features that distinguish HNPCC-MSS families from the other two populations are age at diagnosis, familial cancer risk and polyp burden at the time of cancer detection.

Furthermore, KRAS mutation rates, done by the highly sensitive TheraScreen® kit, were similar in the three study populations (HNPCC-MSS=55.1%; HNPCC-MSI=48.9% and sporadic CRC=49.5%), but they were considerably higher than those described before (HNPCC-MSS=17-40%; HNPCC-MSI=9.5-40%; and sporadic CRC=33-39%).

12 IDeNTIFIcATIoN oF A NoVeL GeRMLINe P53 MuTATIoN IN LI-FRAuMeNI SyNDRoMe: cLINIcAL cASe oF A hIGh RISK BReAST/oVARIAN cANceR FAMILyPatricia Llovet1, Francisco Illana1, Miguel de la Hoya1, Helena Olivera2,Pedro Perez-Segura2, Eduardo Diaz-Rubio2 and Trinidad Caldes1

1Laboratorio de oncología Molecular; 2servicio de oncología Médica. hospital clinico san carlos, IdIssc, c/Martín Lagos s/n. Madrid, spain

Background Hereditary Breast and Ovarian Cancer Syndrome (HBOC) is associated to BRCA1 and BRCA2 mutations. Other high-risk cancer genes for breast or ovarian cancer are P53 (Li-Fraumeni syndrome), PTEN (Cowden syndrome) and STK11 (Peutz-Jeghers syndrome).

Li-Fraumeni syndrome (LFS) has an autosomal dominant inheritance pattern. The most com-mon cancers associated with LFS are breast cancer (30.6%), soft tissue sarcomas (17, 8%), brain tumors (14%), osteosarcome (13.4%) and adrenocortical carcinoma (6.5%). Despite its high penetrance, represents less than 1% of all breast cancer.

Molecular genetic testing for germline BRCA1 and BRCA2 is doing when high risk for HBOC exists. The pedigree and family history of cancer should be evaluated to discard or manage the genetic study to other infrequent possible hereditary syndromes.

Clinical case A sixty year old woman with breast and ovarian cancer arrives at genetic counseling. She has family history of breast, ovarian and laringe cancer; being a case of high risk family to HBOC we offered the study of BRCA1 and BRCA2 genes. The genes were studied by Denaturing Gradient Gel Electrophoresis (DGGE) and Multiplex Ligation-dependent Probe Amplification (MLPA). The whole study of the genes was negative for mutations and deletion/insertion. Few years later we obtained more information about new cancer cases. A family member with osteosarcome (died at 38 years) and a breast Cistosarcoma Phyllodes was diagnosed in the 28 year old daughter. After this new data we decided to study the p53 gene.

The sequencing of the whole coding region and the exon/intron boundaries of P53 detected a dele-tion of 9 nucleotides in exon 5 (c.436del9). This deletion involves the loss of 3 aminoacides but it maintains the open reading frame. This mutation has not be reported in the P53_IARC database.This alteration was studied in the other two members affected with cancer and both were carriers.

The study of the P53 protein by IHC in the Cystosarcoma Phyllodes showed high expression of the protein. P53 is an unstable protein and is not expressed in normal tissue. This data guide the genetic testing for Li-Fraumeni syndrome in patients with breast cancer.

The overexpression of P53 protein and the segregation of the variant c.436del9; p.Trip146_Asp148-del with the disease in this family can indicated that the variant is pathogenic.



96



13 PReVALeNce oF TP53 GeRM LINe MuTATIoNS IN youNG PAKISTANI BReAST cANceR PATIeNTSRashid Muhammad Usman1, 2, Gull S1, Asghar K1, Muhammad N1,Hameed N1, Uddin A1, Amin A3, and Hamann U2

1shaukat Khanum Memorial cancer hospital and research centre (sKMch & rc), Lahore, Pakistan; 2Deutsches Krebsforschungszentrum (DKFZ), Molecular genetics of Breast cancer, heidelberg, germany; 3Levine cancer Institute, carolinas Medical center, charlotte, Us

Women from Pakistan and India are more often diagnosed with early-onset breast cancer than Caucasian women. Given that only 12% of Pakistani women diagnosed with breast cancer at or before 30 years of age have previously been shown to harbor germ line mutations in the breast cancer susceptibility genes BRCA1 and BRCA2, the genetic causes of the majority of early-onset cases are unexplained.

Since germ line mutations in the tumor suppressor gene TP53 predispose women to early-onset breast cancer, we assessed the prevalence of TP53 mutations in 105 early-onset breast cancer patients from Pakistan, who had previously been found to be negative for BRCA1 and BRCA2 germ line mutations. The patient group included 67 women diagnosed with early-onset breast cancer at or before age 30 with no family history of breast or ovarian cancer (EO30NFH group) and 38 women diagnosed with breast cancer at or before age 40 with one or more first- or second-degree relatives with breast or ovarian cancer (EO40FH group). Mutation analysis of the complete TP53 coding region was performed using denaturing high-performance liquid chromatography analysis, followed by DNA sequencing of variant fragments.

One deleterious mutation, c.499-500delCA in exon 5, was identified in the 105 breast cancer patients (1%). This mutation is novel in the germ line and has not been described in other popula-tions. It was detected in a 28-year-old patient with no family history of breast or ovarian cancer. This mutation is rare as it was not detected in additional 157 recently recruited non-BRCA1 and non-BRCA2-associated early-onset breast cancer patients.

Our findings show that TP53 mutations may account for a minimal portion of early-onset breast cancer in Pakistan.

Keywords TP53; germ line mutations; early-onset breast cancer; Pakistan

14 RAPID DeTecTIoN oF APc MuTATIoNS IN SLoVAK FAP FAMILIeS By hIGh ReSoLuTIoN MeLTING ANALySIS AND PRoTeIN TRuNcATIoN TeSTMihok Luboslav, Hlinkova Katarina, Copakova Lucia;national cancer Institute, Bratislava, slovakia

Familial adenomatous polyposis (FAP) is an autosomal dominant predisposition to colorectal cancer and is caused by germline mutations in the adenomatous polyposis coli gene (APC). Clas-sical FAP is characterized by the occurrence of hundreds to thousands of colorectal adenomatous polyps and by several extracolonic manifestations. An attenuated form of polyposis (AFAP) is associated with less than 100 adenomas and later onset of the disease. In this study we analyzed the APC gene for germline mutations in 68 probands from unrelated FAP families of Slovak origin. The mutation analysis of the complete coding region and exon-intron boundaries of the APC gene was performed using a combination of high resolution melting analysis and protein truncation test. All positive findings obtained by both screening methods were verified by sequencing analysis.

We identified 22 different germline APC mutations and 8 of these were novel. Of the identified mutations, 10 were 1 to 5 bp deletions, 2 were 1 bp insertion and 10 were nonsense mutations, 19 leading to the formation of premature stop codon. Three mutations were in the splice-site region of the APC gene. Mutation screening for large genomic alterations in the APC gene was performed by multiplex ligation-dependent probe amplification (MLPA). Large APC deletions were detected in 3 patients, 1 had whole APC gene deletion and 2 had partial deletion involving 4 or more exons.

The total mutation detection rate was 80% in patients with classical FAP and 9% in AFAP patients. The results of the mutation analysis are important for the preparation of individual treatment strategies for FAP patients

Keywords familial adenomatous polyposis, colorectal cancer, germline mutation, APC gene, Slovak



98



15 BeR PAThWAy IN FAMILIAL coLoRRecTAL cANceR TyPe-XMartin L1, Garre P1, Romero A1, de la Hoya M1, Diaz-Rubio E1 and Caldés T1

1Laboratorio de oncología Molecular, servicio de oncología, hospital clínico san carlos IDIssc. c/ Profesor Martín Lagos s/n, Madrid, spain

Introduction Lynch syndrome/Hereditary non-polyposis colorectal cancer is caused by inher-ited germline mutations in mismatch repair (MMR) genes, and accounts for 2-5% of colorectal cancers (CRC)

Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or heredi-tary nonpolyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair gene mutations that define this syndrome and result in microsatellite instability (MSI). These families were designated as familial colorectal cancer type X (HNPCC-MSS). We aimed to characterize a group of HNPCC-MSS families defined by the Amsterdam criteria and MSS tumors at a molecular level. Base excision repair (BER) pathway is the most important cellular protec-tion mechanism responding to oxidative DNA damage. MYH, OGG1, MTH1, PCNA and XRCC1 are members of BER.

Aims To identified genetic variants in MYH, OGG1, MTH1, PCNA and XRCC1 genes present in HNPCC families without MMR deficiency (HNPCC-MSS).

Methods A total of 36 DNAs from HNPCC-MSS index cases, were screened for all coding sequences of MYH, OGG1, MTH1, PCNA and XRCC1 by HRM and/or direct sequencing.

Results We found a total of 27 variants three of which were novel: A95A in OGG1 gene and L87F and E315L in XRCC1.

Seven common variants were detected: S326C in OGG1, D142D in MTH1, Q338H in MYH, P206P, Q399R, c.1481+9 G>A in XRCC1 and c.222-4 C>T in PCNA.

Eight of the remaining variants were missense and they showed an allelic frequency lower than 5% (R46Q, G308E in OGG1, V106M, in MTH1, G396D in MYH and L87F, 315L, N576T in XRCC1). Other two showed frequencies between 5 and 10% (R194W, R280L, in XRCC1).

From the analysis of OGG1, MTH1 and MYH variants, we detected association between MTH1-D142D and HNPCC-MSS families, and also between OGG1-S326C and sporadic CRC. The rare variant OGG1-R46Q segregates with CRC in the family detected and it was shown to affect the splicing leading to an aberrant transcript. Therefore, we suggest OGG1-R46Q could be increasing the cancer risk in this family. Preliminary in silico analysis of XRCC1 variants suggest that L87F and E315L could be pathogenic. Subsequent studies need to be done in order to clarify the pathogenity of these variants and the role as CRC risk factor.

16 ePcAM DeLeTIoNS IN MLh1, MSh2 AND MSh6 NeGATIVe FAMILIeS IN The czech RePuBLIcForetova Lenka FL, Hazova Jana HJ, Svoboda Marek SM,Navratilova Marie NM, Vasickova Petra VP, Machackova Eva MEMasaryk Memorial cancer Institute, Brno, czech republic

Lynch syndrome is caused mostly by germline mutations in MLH1, MSH2, MSH6 and PMS2 mis-match repair genes. Constitutional 3'deletions of EPCAM gene (TACSTD1 – epithelial cell adhesion molecule, tumor associated calcium signal transducer) were shown to be other cause of Lynch syndrome. EPCAM gene has 9 exons and is localized 15kb upstream of MSH2. Deletions of EPCAM 3'end encompassing exons 8-9 and adjacent polyadenyl signal cause disruption of translation termination and create an EPCAM-MSH2 fusion transcript which causes promoter methylation and epigenetic inactivation of MSH2.

227 patients with colon cancer, family history of colon cancer or other Lynch syndrome related cancers, were negatively tested for germline mutations in MLH1, MSH2 and MLH6 genes and for large rearrangements by MLPA. All results of MLPA were checked for possible EPCAM deletions and suspected results were retested by SALSA MLPA KIT P072 MSH6 for classification of deletion.

In one patient only EPCAM deletion without MSH2 deletion was found, in two patients EPCAM deletion together with MSH2 exon 1-8 deletion was detected (1,3%).

Patient with only EPCAM deletion was a male with rectal cancer at 41, his brother had rectal cancer at 43 and his mother had rectal cancer at 70. Other patient, female with EPCAM deletion and MSH2 deletion of exon 1-8 had rectal cancer at 32, both parents had no mutation and this fact was not further investigated. The last patient with EPCAM and MSH2 deletion had cancer at the lienal part of colon, his mother had colon cancer at 37, and there were cancers of stomach and uterus in other mutation carriers.

With the use of more detailed MLPA tests for classification of EPCAM deletions it is possible to increase the detection rate of deleterious mutations (for 1-2%) causing Lynch syndrome in CR.

The testing is supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101).

Keywords Lynch syndrome, EPCAM gene, deletions



100



17 The IMPAcT oF oRIGINAL AND ReVISeD BeTheSDA GuIDeLINeS AND MIcRoSATeLLITe INSTABILITy TeSTING oN The DeTecTIoN oF NeW cASeS oF LyNch SyNDRoMe: The eXPeRIeNce oF A PoRTuGueSe cLINIcAL cANceR ceNTReFilipe Bruno 1, Serrano Miguel2, Lage Pedro2, 3, Francisco Inês1, Belga Sara2, Fonseca Ricardo4, Chaves Paula4, Rodrigues Paula3, Claro Isabel3, Albuquerque Cristina1, Dias Pereira António2

1research Unit of Molecular Pathobiology (UIPM), 2Department of gastroenterology, 3Familial cancer risk clinic, 4Depart-ment of Pathology, Instituto Português de oncologia de Lisboa Francisco gentil, ePe, Lisbon, Portugal

Introduction In 1997 Bethesda Guidelines (BG) were established and in 2004 those criteria were revised (RBG), with the main goal having been to select those colorectal cancers (CRC) that should be subjected to microsatellite instability (MSI) testing. High microsatellite instability (MSI-H) is an intermediate marker for mutational analysis of the mismatch repair (MMR) genes involved in the genesis of Lynch Syndrome (LS).

Aims & methods To evaluate and compare BG/RBG in the detection of MSI-H and subsequent identification of pathogenic mutations associated with new cases of LS. We included 174 patients with CRC and indication for MSI analysis according to BG and/or RBG. MSI testing was performed with the Bethesda markers and mutational analysis of MLH1, MSH2 and MSH6 genes undertaken with DGGE, MLPA and direct sequencing.

Results One hundred fourteen of 174 (65.5%) of the patients fulfilled BG and all of them RBG. Among BG, MSI-H was detected in 37/114 (32.5%) of the CRCs and mutational analysis was posi-tive in 14/37 (37.8%), corresponding to 14/18 (77.8%) of the cases in which it was concluded. The RBC led to the detection of MSI-H in 49/174 (28.2%) of the CRCs, having the mutational analysis been positive in 16/49 (32.7%), which represents 16/21 (76.2%) of the patients with completed analysis. We could identify 14/114 (12.3%) new cases of LS, through BG and 16/174 (9.2%) via RBG. Eleven pathogenic mutations were found in MLH1, 5 in MSH2 and none in MSH6.

Conclusion BG, although more restrictive in case selection, showed a similar overall percentage for the detection of MSI-H and mutations, when compared with RBG. Although RBG implicated the analysis of a superior number of patients, they allowed the detection of two additional cases of LS. This difference has a significant impact in all the mutation-carriers belonging to these families, mainly for CRC prevention.

Keywords Revised Bethesda Guidelines, Lynch Syndrome, Microsatellite instability

18 A NoVeL DeeP INTRoNIc MuTATIoN IN APc uNDeRLyING AN ATTeNuATeD FAP PheNoTyPe

Rivera B1*, Sánchez-Tomé E1, Mercadillo F1, Muñoz I2, Pedrinaci S3, Robledo M4, 5,Benítez J1, 5, Urioste M1, 5

1human genetics, spanish national cancer research centre (cnIo), Madrid; 2servicio de Anatomía Patológica, hospital Universitario de Fuenlabrada, Madrid; 3genética Molecular, hospital Universitario Virgen de las nieves, granada; 4hereditary endocrine cancer, spanish national cancer research centre (cnIo), Madrid; 5centro de Investigaciones Biomédicas en red de enfermedades raras (cIBerer); Madrid, spain

Familial Adenomatous Polyposis (FAP) is a colorectal cancer syndrome caused by mutations in the APC gene or in MUTYH gene in some cases. However, the genetic cause of 20% of classic forms and 50% of attenuated forms of the disease remain unclear.

We have studied in deep APC and MUTYH genes in a series of 18 FAP families with a negative result in the conventional study of both genes. The study of APC mRNA identified an aberrant transcript containing a 39 bp insert between exon3 and 4 in an attenuated FAP family. In order to depict the causal germline mutation that should be affecting to a cryptic splice site, we sequenced the intron 3 in 3 carriers of the insert and 2 noncarriers family members. We found a heterozygous variant c.423-3958c>T in all three carriers. Bioinformatics tools predict the creation of a new cryptic and a new enhancer binding site for the SPf-55 protein. Moreover we discarded the presence of the variant in a 855 control population.

The 39bp insert will give rise to the creation a new in frame pseudoexon and therefore to a longer transcript that might be still partially functional. This is in agreement with the attenuated version of the syndrome in the family. We discard LOH in two adenomas from one patient which matches with the first hit-second hit correlation theory. Moreover, inmunohystochemical b-catenin expression pattern showed overexpression in the adenomatous polyps compared with normal colorectal epithelium from the probandus thus confirming an over activation of Wnt signaling.

This new description of a deep intronic mutation pinpoints the importance of cDNA screening in APC/MUTYH negative FAP patients.



102



19 MoLecuLAR chARAcTeRIzATIoN oF coLoRecTAL cANceR TyPe X FAMILIeS IDeNTIFIeD AMoNG eARLy-oNSeT coLoRecTAL cANceR INDeX cASeSDaniel Rueda1, Alicia Canal1, Joaquín Martinez-López1, Yolanda Rodríguez1,Edurne Álvaro1, Irene Osorio1, Cristina Alegre1, Jose C. Trapero1, José C. Marín1, José Díaz-Tasende1,Javier Benítez2, Miguel Urioste & 1Jose Perea2

1 hospital Universitario 12 de octubre, Madrid, spain; 2 human genetics group, human cancer genetics Program, spanish national cancer centre (cnIo), and centro de Investigaciones Biomédicas en red de enfermedades raras (cIBerer), Madrid, spain

Introduction Familial colorectal cancer type X fulfills Amsterdam criteria used for Lynch syn-drome identification but from a molecular point of view there is no evidence of DNA mismatch repair deficiency. The aim of this study was the molecular characterization of familial colorectal cancer type X tumors identified in a series of patients selected by an early onset correctal cancer diagnosis.

Material and Methods We analyzed 88 individuals with CRC diagnosed at an age of 45 years-old or younger collected from our institution from 2003 to 2008. We collected anatomoclinical features and familial cancer history of all cases. We analyzed tumor samples for microsatellite instability (MSI), BRAF mutation status and the CpG Islands Methylator Phenotype (CIMP). CIMP panel were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of the following genes: CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1. In those cases showing MSI, MMR genes mutations analysis was carried out. We specifically selected those cases defined as Microsatellite Stables (MSS) Amsterdam type II positive cases and described their anatomoclinical, familial and molecular characteristics.

Results Within early-onset CRC we observed that a high percentage of patients showing familial aggregation, with half of the patients with aggregation for Lynch neoplasms. Six patients showed Familial Adenomatous Polyposis, while ten patients (11, 4%) were identified as Lynch syndrome cases as they showed mutations on MMR genes. However, five other patients fulfilled Amsterdam criteria, but with microsatellite stable phenotype (5, 7%). Those cases, compared with the oth-ers, showed a predisposition for right colon location (60% vs 23%), and when they associated polyps, those were purely adenomatous or hiperplastic. Molecular characterization showed no mutations on BRAF gene and CIMP negative or low phenotype. Familial cancer history showed only aggregation for Lynch syndrome neoplasm

Conclusions We identified the absent of CIMP-High phenotype as new molecular signature for early-onset CRC with Familial Colorectal Cancer type X, and also the absent of Lynch unrelated neoplasias familial history.

20 heReDITARy LeIoMyoMAToSIS AND ReNAL ceLL cANceR IN chILDReNCastillejo A1, Sánchez Heras, AB2, Esquembre C3, Castillejo MI1,Perea R2, Hernández Illán E1, Barbera VM1, Trigueros M4, Soto JL1

1 Laboratorio de genética Molecular, hgU elche; 2 Unidad de consejo genético en cáncer, servicio oncología Médica, hgU de elche; 3 servicio oncología Pediátrica, servicio Pediatría, hgU Alicante; 4 servicio Anatomía Patológica, hgU Alicante, spain

Introduction and objectives The hereditary leiomyomatosis and renal cell cancer (HLRCC) is a cancer predisposition syndrome caused by mutations in the fumarate hydratase gene (FH). HLRCC is an autosomal dominant syndrome with variable expressivity and high penetrance. The typical clinical manifestations are cutaneous leiomyomas (85%), uterine (25-77%) and renal cancer (20%). Kidney tumors are typically unilateral and have type 2 papillary histology or ductal. To date, about 200 families have been reported. We describe a family diagnosed with HLRCC from diagnosis of kidney cancer in a 10 years.

Materials and methods Clinical, pathological and genetic criteria for a family with HLRCC. Genetic analysis by PCR and sequencing the entire coding region and intron-exon junctions of the FH gene. Predictive tests for at risk relatives. Follow-up recommendations for early diagnosis in mutated individuals.

Results Index case: 10 years old male with a diagnosis of unilateral and multifocal renal oncocytic tumor. Family history: father with cutaneous leiomyomas and colon cancer (dx 50a). Paternal grandfather with pancreatic cancer (dx 58a). Paternal great-uncle by renal angiomyolipoma (dx 20a). Genetic studies of the index case revealed the presence of heterozygous mutation c.1118A> G; p.Asn373Ser in exon 8 of the FH gene. This alteration has been previously described in this syndrome. Predictive genetic tests have been performed in 14 relatives. Seven of them were positive. Follow-up recommendations for early diagnosis were provided: abdominal CT or MRI yearly, annual dermatological examination and ultrasound uterine gynecological examination in women.

Conclusions HLRCC genetic diagnosis and predictive studies on individuals at risk, allows to offer a program for early diagnosis of the clinical manifestations.



104

21 The coMPARATIVe MoLecuLAR cLASSIFIcATIoN SuGGeSTS The eXISTeNce oF TWo DIFFeReNT GRouPS WIThIN MIcRoSATeLLITe STABLe eARLy-oNSeT coLoRecTAL cANceR WITh AN IMPoRTANT FAMILIAL coMPoNeNTPerea J1, Rueda D1, Canal A1, Rodríguez Y1, Álvaro E1, Osorio I1, Alegre C1, Trapero JC1, Martinez-López J1, Benítez J2, Urioste M2

1hospital Universitario 12 de octubre, Madrid, spain; 2human genetics group, human cancer genetics Program, spanish nation-al cancer centre (cnIo), and centro de Investigaciones Biomédicas en red de enfermedades raras (cIBerer), Madrid, spain

Introduction Early-onset colorectal cancer (CRC) is an indicator of a hereditary component, but recently this is changing. The aim of our study was to compare the CRC molecular classification from the three main carcinogenetic pathways within early-onset CRC, in order to identify any particular group in this population.

Material and Methods We analyzed a total of 67 early-onset CRC patients from 2002 to 2008. We collected clinico-pathological data and familial pedigree from each proband. We analyzed the microsatellite instability (MSI) and CpG Islands Methylator Phenotype (CIMP), classifying them into 4 main subtypes: MSI with CIMP-high (group A), MSI with CIMP low or 0 (group B); Microsatellite Stability (MSS) with CIMP-high (group C), and MSS with CIMP low or 0 (group D). For MSI cases Mismatch Repair genes analysis was carried out. We analyzed the clinic-pathological and familial differences between all the groups.

Results We observed that in group A and B were 100% and 50% of Lynch syndrome cases, being group A (MSI and CIMP-High) correspondently the one in which Lynch syndrome features are more frequent, except mucinous tumours (younger age of onset, right colon, earlier stages, Multiple Primary Neoplasm and Amsterdam type II positive cases). More remarkable are the characteristics of the MSS groups, according with CIMP status. Early-onset MSS CRC with CIMP-high were: more frequently right colon cancers, with an important component of poor differentiated tumours, as well as mucinous, with mainly hiperplastic associated polyps (83, 3%). This group of tumours had an important familial component regarding mainly Lynch related neoplasias. The last group, early-onset MSS CRCs showing CIMP-low or null, were male, rarely right colon cancers, homogeneously distributed stages, with mainly adenomatous associated polyps, and the least proportion of Primary Multiple Neoplasm. This group showed an important familial cancer component: 24, 5% and 20, 4% of early-onset CRC had mainly familial history for Lynch syndrome-related and unrelated cancers, respectively. In this subset of tumours were also a 10, 2% of Familial Colorectal Cancer Type X.

Conclusions We identified two differential groups within early-onset CRC, with MSS, depending on the CIMP subtype, showing group C anatomo-clinical and familial differences (Lynch related neoplasias), and group D familiar cancer history differences.

Keywords Early-onset Colorectal Cancer. Familial Colorectal Cancer. Microsatellite stability. Lynch Syndrome

FAcuLTyAND AuThoRS’INDeX5th FAMILIAL cAncer conFerence

FAcULtY


108


FAcULtY 109

antoniou, antoniSUniversity of Cambridge, UK

BEnitEz, javiEr ChairCNIO, Spain

capEllá munar, gaBriElCatalan Institute of Oncology, Spain

caScón, alBErtoCNIO, Spain

caStEllS, antoniClinical Hospital of Barcelona, Spain

DantzEr, françoiSEUniversity of Strasbourg, France

EcclES, DianaSouthampton University Hospital Trust, UK

EElES, roSalinD ChairThe Royal Marsden Hospital, UK

frEBourg, thiErryRouen University Hospital, France

garcia, maría joSéCNIO, Spain

golDgar, DaviDUniversity of Utah, US

gruiS, nEllEkELeiden University Medical Center, The Netherlands

guErra, carmEnCNIO, Spain

FAcuLTy

hoDgSon, ShirlEy victoria ChairSt. George's Hospital Medical School, UK

mEnko, frED h.VU University Medical Center, The Netherlands

milnE, rogErCNIO, Spain

oSorio, ana ChairCNIO, Spain

palacioS, joSéRamón y Cajal University Hospital, Spain

pErona, roSarioBiomedical Research Institute, Spain

roBlEDo, mErcEDES ChairCNIO, Spain

roukoS DimitrioS h.Ioannina University School of Medicine,Greece

SurralléS, jorDiAutonoma University of Barcelona, Spain

urioStE, miguElCNIO, Spain

vaSEn, hanS f.Leiden University Medical Center, The Netherlands

AUthors


110


AUthors 111

ariaS, m.mVirgen del Camino Hospital, Spain

Balmaña, jVall d’Hebron University Hospital, Spain

Blay, pUniversity Central Hospital of Asturias (HUCA), Spain

Bracci, rMedical Oncology Clinic-University Hospital Company-United Hospitals, Italy

canal, aDoce de Ocubre University Hospital, Spain

caStEllanoS, EInstitute of Predictive and Personalized Medicine for Cancer (IMPPC), Spain

caStillEjo, aUniversity General Hospital of Elche, Spain

AuThoRS

DE cuBaS, a. a.CNIO, Spain

DE la hoya, mSan Carlos Clinical Hospital (IdISSC), Spain

Earl, jRamón y Cajal University Hospital, Spain

fachal, lGalician Public Foundation for Genomic Medicine (FPGMX), Spain

filipE, BPortuguese Institute of Oncology of Lisbon (IPOLFG), Portugal

forEtova, lMasaryk Memorial Cancer Institute, Czech Republic

garrE, pSan Carlos Clinical Hospital (IdISSC), Spain

goh, cThe Institute of Cancer Research, United Kingdom

janaviciuS, rVilnius University Hospital Santariskiu Clinics, Lithuania

kamiEniak, mCNIO, Spain

kanSikaS, mUniversity of Helsinki, Finland

korpErShoEk, ECNIO, Spain

krivokuca, aInstitute for Oncology and Radiology, Serbia

krutílková, vCentre for Medical Genetics and Reproductive Medicine (GENNET), Czech Republic

llovEt, pSan Carlos Clinical Hospital, Spain

marikkannu, rCenter for Molecular Medicine and Surgery, Sweden

martin, lSan Carlos Clinical Hospital (IdISSC), Spain

martinEz, cCruces Hospital, Spain

mihók, l'National Cancer Institute, Slovakia

pErEa, jDoce de Octubre University Hospital, Spain

pérEz, lGenomics Systems, Spain

pErtESi, mInternational Agency for Research on Cancer, France

AUthors


112

QuilES, f Bellvitge Biomedical Research Institute (IDIBELL), Spain

rantala, jKarolinska Institute, Sweden

raShiD, mShaukat Khanum Memorial Cancer Hospital & Research Centre, Pakistan

rEBanE, ECompetence Centre for Cancer Research, Estonia

ripamonti, cNational Cancer Institute (IRCCS), Italy

rivEra, BCNIO, Spain

ruiz-pontE, cGalician Public Foundation for Genomic Medicine (FPGMX), Spain

Santamariña, mBiomedical Network Research on Rare Diseases (CIBERER), Spain

SaucEDo, l.pCNIO, Spain

Silva, pPortuguese Institute of Oncology of Lisbon (IPOLFG), Portugal

tanic, mCNIO, Spain

tSitlaiDou, mThe National Centre of Scientific Research Demokritos, Greece

tzortzatoS, gInstitute of Women's and Child's Health, Sweden

zovato, SVeneto Oncology Institute, Italy

5th FAMILIAL cAncer conFerence 5th FAMILIAL cAncer conFerence


european school of oncologyVia del Bollo, 420123 Milan, Italytel.: +39 02 8546451Fax: +39 02 85464545www.eso.net

spanish national cancer research centre (cnIo)Melchor Fernández Almagro, 328029 Madrid, spainwww.cnio.es

coordination and editionMercedes Moro and Virginia de la CruzDesign by underbauProduction by Gráficas anya

this work is subject to copyright. All rights are reserved, wether the whole or part of the material is concerned, specifically the rights to translation, reproduction on microfilms or in any other way and storage in data banks.

© Fundación cnIo carlos III, 2012

Printed in spain

european school of oncologyVia del Bollo, 420123 Milan, Italytel.: +39 02 8546451Fax: +39 02 85464545www.eso.net

spanish nationalcancer research centre (cnIo)calle Melchor Fernández Almagro, 328029 Madrid, spainwww.cnio.es

ORGANISERS

As a non-profit organisation, we would like to thank all those who sponsored this conference.such contribution helps us to ensure that our conferences will continue to establish the cnIo as a pointof reference for the international cancer research community.

MADRID / SPAIN 7–8 Ju Ne 2012 5th FAMILIAL …82_booklet.pdf · 011 SPeAKeRS’ ABSTRAcTS 021...

Documents

Transcript of MADRID / SPAIN 7–8 Ju Ne 2012 5th FAMILIAL …82_booklet.pdf · 011 SPeAKeRS’ ABSTRAcTS 021...