Prostate And Breast Cancer Surveillance In Unaffected Male BRCA2 And BRCA1 Mutation Carriers

August 2012 Mantra Resort, NSW

PROGRAMME

A combined meeting of kConFab, Australian Breast Cancer Family Study, Australasian Colorectal Cancer Family Study, Australian Ovarian Cancer Study, Family Cancer Clinics of Australia and New Zealand

“Familial Aspects of Cancer 2012 Research and Practice”

Tuesday 21st August FCC Day –Plantation Room

Sponsored by:

9.00 – 9.10 Welcome: Gillian Mitchell

FCC Session 1. Massively parallel sequencing Chairperson: Julie McGaughran 9.15 – 10.30 Next generation sequencing: the promises and requirements of a transformative genomic technology. Tony Roscioli

Massively parallel sequencing: issues for clinical practice. Graeme Suthers

Followed by panel discussion: Graeme Suthers, Tony Roscioli, Sean Grimmond, Cliff Meldrum, Andrew Biankin, Alison Trainer, Ian Campbell

10.30 – 11.00 Morning tea

FCC Session 2 A smorgasbord for clinical practice Chairperson Nicola Poplawski 11.00 – 12.30 Risk-reducing behaviour of female BRCA1 and BRCA2 mutation carriers; long-term follow-up of participants in the Kathleen

“Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, NSW

Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). Ian Collins

Germline PTEN gene mutations and Cowden Syndrome: a plastic condition with implications for clinical practice? Marion Harris

A proposed screening study in Li Fraumeni Syndrome. Gillian Mitchell

Non-cancer outcomes of risk-reducing bilateral salpingo- oophorectomy: a proposed clinical trial. Martha Hickey

12.30 – 1.30 Lunch

12.30 – 12.45 COSA FCC Group - Plantation Room

FCC Session 3 1.30 – 1.50 eviQ update – current activity and future plans in familial canc er Robyn Ward

1.50 - 3.15 Counselling adolescents at risk of genetic syndromes Chairperson: Margaret Gleeson

Developing a youth friendly model of genetic counselling Mary-Anne Young

Adolescent Clients in the Clinical Genetics Setting: Using predictive testing for FAP as a case-study for exploring developmentally appropriate care. Rony Duncan

Support and counselling of adolescents with cancer, contrasts and similarities with hereditary cancer syndromes. Kate Thompson

3.15 – 3.45 Afternoon Tea

FCC Session 4

3.45 – 5.30 Pancreatic/GI cancer Chairperson: Lara Lipton

Screening for pancreatic cancer in high risk individuals. Alina Stoita

A new way to screen for pancreatic cancer?: KRAS serum screening. Lara Lipton

A fresh look a SMAD 4. Noralane Lindor

CAPP3 trial in Lynch syndrome – an update Finlay Macrae

Follow on meetings in the Plantation room:

IMPACT meeting 5.30 – 6.15 PM

CONFIRM meeting 6.15 – 6.45 PM

Wednesday 22nd August ABCFS, ACCFS, kConFab and AOCS – Plantation Room

8.30 – 8.40 Welcome: Stephen Fox

Session 1 – Plantation Room Chairperson: Georgia Trench

8.40 – 9.20 Epigenetic Epidemiology: Exploring the epigenome to find biomarkers of cancer risk and prognosis. James Flanagan

9.20 – 9.40 Lobular breast cancer GWAS Ian Tomlinson

9.40 – 10.00 A GWAS-based cross-disease approach to find genes predisposing to both endometriosis and endometrial cancer. Jodie Painter

10.00 – 10.20 Genotypic and phenotypic analysis of familial male breast cancer shows under representation of the HER2 and basal subtypes in BRCA-associated carcinomas. Stephen Fox

10.20 – 10.40 Genome- wide scan of methylation of DNA extracted from archival blood biospecimens. Chol-hee Jung

10.40 – 11.10 Morning Tea

Plantation Room 11.10 – 11.50 The Jeremy Jass Memorial Lecture Introduction & Chair: John Hopper

Presented by: Looking Forward in Lynch Syndrome Noralane Lindor

Session 2 Chair: Daniel Buchanan

11.50 - 12.10 Relative risk of colorectal cancer risk in Lynch syndrome is inversely associated with age: a review. Mark Jenkins

12.10 – 12.30 Is breast cancer a Lynch syndrome cancer? Aung Ko Win

12.30 – 12.45 Prostate cancers arising in Lynch syndrome mutation carriers frequently show DNA mismatch repair deficiency Mike Walsh

12.45 – 1.05 Methylation of DNA Repetitive Elements from Peripheral Blood DNA is Associated with Early-Onset Colorectal Cancer Daniel Buchanan

1.05 - 2.00 Lunch

Session 3 – CONCURRENT SESSION: Psychosocial and Cohort Research Moss Room, Peppers Chair: Rachel Williams

2:00 – 2:20 Disclosure obligations for participants donating a DNA sample for genetic research to lifepool. What does the woman in the street need to know and what does she need to tell insurance companies? Jeff Gleeson

2:20 – 2:40 Evaluation of the efficacy of two models of delivering information about treatment-focused genetic testing among your women newly diagnosed with breast cancer Bettina Meiser

2:40– 3:00 The attitudes of people with sarcoma, their family and health professionals towards genomics and ancillary information arising from genetic research. Mary Anne Young

3:00 – 3:25 What happens when uncertainty remains? A systematic review and quantitative exploration of distress in unaffected high-risk individuals who decline, delay or remain ineligible for predictive genetic testing Louise Heiniger

3.25 – 3.45 Improving mutation notification when new genetic information is identified in research: A trial of two strategies in familial breast cancer employed by kConFab. Heather Thorne

3.45 – 4.05 What matters to women diagnosed with breast cancer during their reproductive years? Preliminary results from a qualitative investigation using a population-based sample. Maggie Kirkman

Session 4 – CONCURRENT SESSION Plantation Room Chairperson: Melissa Southey

2.55 – 3.15 Population-based estimate of prostate cancer risk for carriers of the HOXB13 missense mutation G84E Robert MacInnis

3.15 – 3.35 Colorectal cancer risk for MET T992I missense variant carriers from the Australasian Colorectal Cancer Family Registry Daniel D. Buchanan

3.35 – 3.50 Gene regulatory elements as breast cancer susceptibility loci Melissa Brown

3.50 – 4.10 Gap in the Middle of Familial Colorectal Cancer – Moderately Penetrant Genetic Variants Deborah Neklason

5.00 – 7.00 Poster Session + Wine and Cheese in the main foyer

6.00 Plantation Room: The following selected posters will give a 3 minute oral presentation. Chair Judy Kirk.

Order of presentation:

Poster # 5. Kirsty Mann. Interdisciplinary patient file reviews and follow-up of Peutz-Jeghers syndrome management

Poster # 10. Michelle Bowman. SDHB immunohistochemistry: four cases from clinical practice.

Poster # 18. Emma Steel. Risk factors for breast cancer: how do women with a family history obtain and interpret this information?

Posters # 19 & 20. Skye Simpson. Analysis of germline variants in DNA damage repair genes in pancreatic cancer identified by next- generation sequencing, and, The Australian familial pancreatic cancer cohort: screening for pancreatic cancer in high risk Australians

Poster # 31. Bryony Thompson. Mismatch Repair Gene Isoforms: Issues in the Interpretation of Aberrant Splice Transcripts

Poster #27. Mike Walsh. BRAF V600E immunohistochemistry – A new approach to screen colorectal cancers for Lynch Syndrome

Poster # 22. Sarah Sawyer. The association of identified breast cancer common genomic variants and pathological features in individuals with Hereditary Breast and Ovarian cancer.

Delegates Organise their own Dinner

Thursday 23rd, August ABCFS, ACCFS, kConFab and AOCS - Plantation Room

Session 5 - Plantation Room Chairperson: Gillian Mitchell

9.00 – 9.20 Cancer 2015” (Vic Cancer Genome Cohort): translating discoveries into cures John Parisot

9.30 – 9.50 CASCADE: A CAncer tiSsue Collection After DEath programme to improve our understanding of the progression from primary stage cancer to metastatic, treatment-resistant disease. Kathryn Alsop

9.50 – 10.00 Introducing an online bowel cancer risk calculator to promote colorectal cancer awareness in the community. Jody Simmons

10.00 – 10.20 A case of two mutations in trans in a women diagnosed with breast cancer at the age of 30 years. Margaret Gleeson

10.20 – 10.40 Evaluation of BRCA1/2 variants of uncertain clinical significance using a combination of multifactorial likelihood analysis, bioinformatic and in vitro approaches Phil Whiley

10.40 - 11.10 Morning tea

Session 6 – Plantation Room Chairperson: Prue Cowin

11.10 – 11.50 Cloning of the gene for mixed polyposis Ian Tomlinson

11.50 – 12.10 FAVR (Filtering and Annotation of Variants that are Rare): methods to facilitate the analysis of rare germline genetic variants from SOLiD and Illumina datasets Tú Nguyen-Dumont

12.10 – 12.30 Identification of rare DNA copy number variants overlapping mismatch repair pathway genes in endometrial cancer patients and their potential contribution to disease risk Logan Walker

12.30 – 12.50 Scope Vs Hope: when should studies of “missing heritability” go whole-genome? Khalid Mahmood

12.50 – 1.10 Mapping the genetic basis of sarcoma in a high risk kindred David Thomas

1.10 - 2.10 Lunch

Session 7- Plantation Room Chairperson: Anna de Fazio

2.10 – 2.30 Invasive epithelial ovarian cancer and BRCA mutations: the South Australian perspective Nicola K Poplawski

2.30 – 2.50 LRP1B deletion in high-grade serous ovarian cancers is associated with acquired chemotherapy resistance to liposomal doxorubicin Prue Cowin

3.50 – 3.10 Clinically, molecularly and functionally annotated models of HG-SOC in which to perform pre-clinical analysis of targeted therapies. Monique Topp

3.10 – 3.30 Molecular profiling of pre-invasive serous ovarian tumours Sally Hunter

3.30 – 3.50 Genomic profiling of benign, borderline and invasive mucinous ovarian tumours identifies key and novel genetic events. Kylie Gorringe

4.30 – 5.30 Pavillion Room John Hopper – updates on the Monday and Tuesday meeting

4.30 – 5.00 Plantation Room FFPE Based Molecular Analysis - The Illumina Story Brett Kennedy, Product Marketing Manager, Australia and New Zealand

6.00 – 8.00 Conference Cocktail drinks pool side @ Peppers. All delegates welcome.

Delegates organise their own dinner

Friday, 264h August ABCFS, ACCFS, kConFab and AOCS - Plantation Room

Session 8 – Plantation Room Chairperson: Michael Bogwitz

8.30-8.50 Improving early diagnosis of MEN2b: raising awareness and stimulating debate Hilda High

8.50 – 9.10 Variability of Clinical Features in MEN2 Charlotte Slade

9.10 – 9.30 Review of mutation detection rate for women diagnosed with breast cancer aged 30 years and younger Annabelle Ng

9.30 – 9.50 When do we test for Cowden Syndrome? Leonie Noon

9.50 – 10.10 The Evolution of Li-Fraumeni Syndrome and Challenges for Counselling Linda Cicciarelli & Alexandra Lewis

10.10-10.30 Clinical genetic testing for hereditary cancer using next generation sequencing Lesley Andrews

10.30 - 11.10 Morning Tea

Session 9 – Plantation Room Chairperson: Mandy Spurdle

11.10 – 11.50 Modelling genetic susceptibility to breast and ovarian cancer: updates to the BOADICEA risk prediction model, and results on genetic modifiers of cancer risk for BRCA1 and BRCA2 mutation carriers from the iCOGS custom genotyping array. Antonis C. Antoniou

11.50 – 12.10 The breast cancer risk prediction model BOADICEA: calibrated for Australian women and a new easy-to-use batch processor Robert MacInnis

12.10 – 12.30 Multiple independent TERT variants associated with telomere length and risks of breast and ovarian cancer

Georgia Chenevix-Trench

12.30 – 12.50 Risk prediction models for mismatch repair gene mutations: a systematic review and a meta-analysis. Aung Ko Win

12.50 – 1.10 Tumour morphology of early-onset breast cancers predicts breast cancer risk for first-degree relatives John Hopper

End of Meeting: Lunch will be served

PROGRAMME

Tuesday 21st

A combined meeting of kConFab, Australian Breast Cancer Family Study, Australasian Colorectal Cancer Family Study, Australian Ovarian Cancer Study, Family Cancer Clinics of Australia and New Zealand.

“Familial Cancer 2012: Research and Practice”

FCC Session 1:

Plantation Room

Chairperson: Julie McGaughran

MASSIVELY PARALLEL SEQUENCING: ISSUES FOR CLINICAL PRACTICE

Graeme Suthers, Chair, Genetics Advisory Committee, RCPA; SA Clinical Genetics Service, SA Pathology, Women’s & Children’s Hospital, North Adelaide SA 5006.

Quality in genetic testing encompasses issues that will be familiar to everyone involved in the delivery of diagnostic testing: choosing the right test, collecting the right sample, doing the correct analysis, making the right interpretation, providing a clear and timely report to the requestor, and trusting that the information will be used appropriately for decision-making.

However, the scope of genetic testing is huge, both in terms of the variety of tests available and the potential to impact the health of the patient’s relatives. The introduction of massively parallel sequencing compounds these challenges. This introduces some additional challenges for each of these steps:  Should test selection be restricted to loci known to be associated with disease – or to potential loci that may be relevant?  Sampling errors occur and are likely to be missed unless there is corroboration of key test results.  The required quality control for a single analyte is stringent. QC for multiple analytes is challenging. Quality measures in sequencing represent the average quality over multiple nucleotides – but clinical decision-making is based on the quality of the specific variants identified.  The clinical significance of many variants is unknown, and processes to systematise their consistent interpretation are rudimentary.  Clinicians already struggle to read reports of older familiar tests accurately. Most are unskilled in the newer field of genetics.  A test result may have both immediate and latent significance for the patient or relatives. How should these be reported?  The comparative novelty of genetic tests can give them inappropriate influence in decision-making.

The concept of a genetic test should be re-cast as a consultation with a laboratory professional if massively parallel sequencing is to be effectively translated into quality information for patients and their clinicians.

Programme

Tuesday 21st

FCC Session 2:

Plantation Room

Chairperson: Nicola Poplawski

RISK-REDUCING BEHAVIOUR OF FEMALE BRCA1 AND BRCA2 MUTATION CARRIERS; LONG TERM FOLLOW-UP OF PARTICIPANTS IN THE KATHLEEN CUNINGHAM FOUNDATION CONSORTIUM FOR RESEARCH INTO FAMILIAL BREAST CANCER (KCONFAB)

Ian Collins 1, Prue Weideman1, John Hopper2, Sue-Anne McLachlan3, Roger Milne4 , Michael Friedlander5, KConFab Investigators, Kelly-Anne Phillips1,2, Division of Cancer Medicine, Peter MacCallum Cancer Centre, Melbourne, Melbourne School of Population Health, University of Melbourne, Oncology Department, St. Vincent's Hospital, Melbourne, Spanish National Cancer Research Centre, Madrid, Spain, Department of Medical Oncology, Prince of Wales Hospital, Sydney

Background Several interventions can prevent breast and ‘ovarian’ cancer in women with a pathogenic mutation in BRCA1 or BRCA2. Knowledge of the contemporary uptake of these interventions in Australian women may help guide future policy and clinical practice.

Methods Study subjects were female BRCA1 or BRCA2 mutation carriers enrolled in kConFab between 3/11/1997 and 4/9/2008, unaffected with any cancer at the time of enrolment, who had completed at least one three-yearly self-reported follow-up questionnaire (collecting demographic information, cancer events and uptake of risk reducing measures, including bilateral mastectomy (BRRM) or salpingo-oophorectomy (RRSO)) since enrolment. Follow-up was censored at a cancer diagnosis, death or date of last follow-up. Descriptive statistics were calculated using MS Excel.

Results Of 1317 women from families segregating a mutation in BRCA1 or BRCA2, 447 had a mutation (237 BRCA1 and 210 BRCA2). Of these, 325(73%) reported knowing their mutation status (175 BRCA1 and 150 BRCA2), 122(27%) reported being unaware of their mutation status. During 2447 person-years of follow-up (median 9 years, range 2- 14), 68 incident cancers were reported, including 52 breast cancers, 9 “ovarian” cancers, 3 melanomas and 1 of each of bowel, gastric, pancreas and unknown primary.

Event N (%) Age at event (years)

Median Range

Cohort entry 325(100) 37 18-78 Disclosure of genetic results 325(100) 39 18-78 Incident cancer diagnosis 68(21) 45 26-80 BRRM1 69(21) 40 26-67 RRSO at any age2 110(34) 44 30-77 RRSO by age 40 26(12)3 n/a n/a RRSO by age 50 79(65)4 n/a n/a Both BRRM and RRSO 34(10) n/a n/a Enrolment in medical prevention 9(3) 36 35-56 trial Risk reducing tamoxifen 4(1) 37 35-49 Tubal ligation 71(22) 32 20-54 1 7 before cohort entry. 28 Before cohort entry. 3224 women aged 40+ at last follow- up, 4122 aged 50+ at last follow-up

Of the 71 who reported tubal ligation, 29 subsequently underwent RRSO, including 3 with occult serous cancers. Median time between genetic result disclosure and subsequent risk reducing behaviour was 1 year [range 0-15].

Conclusion Most Australian BRCA1 and BRCA2 mutation carriers do not undergo BRRM, few undergo RRSO before age 40 and 35% had not had RRSO by age 50. Medical prevention is rarely used. Interestingly 22% undergo tubal ligation (lowering “ovarian” cancer risk) presumably for contraception. The reasons for the low uptake of risk-reducing interventions need investigation and intervention is required if genetic knowledge is to optimally translate into real reductions in cancer incidence in these women.

COWDEN SYNDROME – THE RELEVANCE OF THE CONDITION FOR FAMILIAL CANCER PRACTICE IN 2012

Marion Harris, Southern Health Familial Cancer Centre, Victoria

Cowden syndrome ( CS) is a rare tumour- hamartoma syndrome caused by a germline mutation in the PTEN gene. There is a concern that cases of CS are missed in the BRCA negative high risk breast cancer population seen by familial cancer services. The clinical features of the condition will be reviewed and the relevance of the condition for familial cancer practice in 2012 will be discussed.

A PROPOSED SCREENING STUDY IN LI FRAUMENI SYNDROME

Gillian Mitchell 1, David Thomas1, Mandy Ballinger1, Kate Mahendran2, Nick Ferris3, Martin Delatycki4, Janet Hiller5, Mary-Anne Young1, Australian Familial Cancer Centres. 1 Peter MacCallum Cancer Centre VIC, 2 Westmead Hospital NSW, 3 Southern Health VIC, 4Austin Hospital VIC, 5 Australian Catholic University VIC.

The Li Fraumeni syndrome is associated with significant risk of cancers arising at multiple body sites. There is no accepted screening program for adults with this syndrome other than breast MRI screening for adult women. A recent eviQ guideline committee meeting reviewed the evidence for screening in this syndrome and only advised annual breast MRI screening for women with additional annual faecal occult blood testing and 5-yearly colonoscopy for both men and women. Whole-body MRI screening is now possible and is attractive for Li Fraumeni families as it avoids exposure to ionising radiation and can access multiple body sites in a single screening test. This presentation will outline the proposed whole-body MRI screening study for Li Fraumeni families in Australia which will form the pilot for a proposed international screening study in this syndrome.

Programme

Tuesday 21st

FCC Session 3:

Plantation Room

Chairperson: Margaret Gleeson

DEVELOPING A YOUTH FRIENDLY MODEL OF GENETIC COUNSELLING

Mary-Anne Young 1, Ann-Maree Duncan1, Lucy Holland2, Kate Thompson2

1 Familial Cancer Centre, Peter MacCallum Cancer Centre, Victoria, Australia, 2 ONTrac at Peter Mac, Peter MacCallum Cancer Centre, Victoria, Australia,

Evidence suggests that healthcare professionals trained in either adult or paediatric models of care find it difficult to provide adolescents and young adults (AYAs) services which meet their developmental and healthcare needs. Genetic counselling is no exception and anecdotal evidence suggests that it is particularly difficult for these professionals to provide adolescent care which meets the expectations of both clients and the counsellor. AYAs present for genetic counselling at a time when they are already grappling with the developmental challenges of adolescence. Additional anxiety due to the potential risk of a genetic disease during this lifestage may overwhelm the coping resources of young people. A lack of specific models of genetic counselling and counselling strategies for working with young people who are psychosocially vulnerable compromises the quality of genetic counselling and care received by AYA clients accessing such services.

To address this problem a reference group was formed to develop a model of AYA specific genetic counselling. This group comprised experts in adolescent health and genetic counselling practice and research. A literature review was undertaken following which a youth friendly model of genetic counselling was developed. This model will be presented with particular emphasis on genetic counselling practice when working with AYAs.

ADOLESCENT CLIENTS IN THE CLINICAL GENETICS SETTING; USING PREDICTIVE TESTING FOR FAP AS A CASE STUDY FOR EXPLORING DEVELOPMENTALLY APPROPRIATE CARE

Rony Duncan, PhD, Centre for Adolescent Health Royal Children’s Hospital, Murdoch Children’s Research Institute

Predictive genetic tests are routinely offered to young people during adolescence as long as medical benefit is conferred by the test. Nonetheless, it can be highly challenging to engage, work with and support young people through this process due to (i) their unique developmental stage of life, (ii) the frequent and simultaneous involvement of multiple family members, and (iii) the lack of training provided to genetic health professionals in adolescent health and development. Young people sit between childhood and adulthood; old enough to have their developing autonomy respected yet too young to be treated exactly as adults. For these reasons, young people require a different clinical approach from that provided to adults and this is now gaining strong recognition internationally.

This presentation will draw on findings from in-depth interviews with young people who underwent predictive testing for familial adenomatous polyposis (FAP) aged 10-17 years at the time of their test. Using these case-studies and first-hand accounts from young people, the key challenges associated with seeing adolescent clients, and their families, in a clinical genetics setting will be highlighted. These findings will then be placed in the context of existing clinical recommendations for working with adolescents in the medical setting more broadly. Questions will be raised about what aspects of this broader guidance are transferable to the clinical genetics setting, with the aim of working towards a model for best practice with adolescents in clinical genetics internationally.

Dr Rony Duncan is a Senior Research Fellow at the Centre for Adolescent Health, Murdoch Childrens Research Institute. She has a background in bioethics, adolescent health and clinical psychology. Much of Rony’s past research has focused on the implications of predictive genetic testing in young people. Central to this work were a series of in-depth interviews with adolescents and young adults who had undergone genetic tests for Huntington Disease and FAP. Rony’s current program of research focuses on the ethical issues that arise in adolescent health care, with a particular focus on confidentiality. In addition to her research, Rony supervises a number of postgraduate research students, sits on two clinical ethics committees, lectures in ethics and adolescent health at the University of Melbourne and appears fortnightly on Triple R Radio as an ethics commentator.

SUPPORT AND COUNSELLING OF ADOLESCENTS WITH CANCER, CONTRASTS AND SIMILARITIES WITH HEREDITARY CANCER SYNDROME

Kate Thompson 1, Mary-Anne Young2, Lucy Holland1 & Ann-Maree2 1ONTrac at Peter Mac Victorian Adolescent & Young Adult Cancer Service, 2Victorian Familial Cancer Centre, Peter MacCallum Cancer Centre

Background: A number of developmental changes occur during the adolescent and young adult (AYA) years that distinguish this life stage from both childhood and adulthood. Within the healthcare sector, AYA patients present with unique needs related to biological, psychological and social factors pertinent to this period of development. Currently these patients receive care within either paediatric or adult healthcare sectors. However, evidence suggests that healthcare professionals trained in either model of care find it difficult to provide young people with services which meet their needs. The specific challenges faced by healthcare professionals include understanding the AYA life stage and how illness impacts on development, adjustment and quality of life.

ONTrac at Peter Mac Victorian Adolescent and Young Adult Cancer Service was developed in 2004 to meet the needs of young people with cancer and their families. ONTrac at Peter Mac is a Multidisciplinary team which works from the foundational principles of adolescent development including importance of AYA communication, promoting autonomy and flexibility working in family models of care. In 2011, in conjunction with the Familial Cancer Centre at Peter Mac, it was recognised that there was no specific model of AYA genetic counselling. This was thought to both compromise the quality of care for young people and present complexities for the professionals working in this field as work with this patient group is typically accompanied by unique ethical and practice challenges.

Method: To address this gap, a reference group was formed to develop a model of AYA specific genetic counselling. This group comprised experts in adolescent health and genetic counselling practice and research.

Discussion: Principles of practice in work with young people with cancer will be discussed. The application of these principles to the model of care for working with young people who present for genetic testing will be delineated. Considerations for the implementation of this model will be reviewed.

Programme

Tuesday 21st

FCC Session 4:

Plantation Room

Chairperson: Lara Lipton

SCREENING FOR PANCREATIC CANCER IN HIGH RISK INDIVIDUALS

Alina Stoita, St Vincent’s Hospital, Sydney

Ten percent of pancreatic cancers are due to a genetic predisposition (familiar pancreatic cancer and inherited pancreatic cancer syndromes). The lifetime risk of pancreatic cancer of an individual with 3 first degree relatives with pancreatic cancer is 40% and with 2 first degrees is 8-12%. Inherited cancer syndromes at high risk of pancreatic cancer are hereditary pancreatitis, Peutz-Jeghers sydrome, FAMMM, breast ovarian cancer syndrome and Lynch syndrome. In familial pancreatic cancer, smoking increases the risk of pancreatic cancer by 2-3 folds and decreased the age of onset by 10 years.

Screening for pancreatic cancer targets individuals with a 5% of greater lifetime risk of pancreatic cancer. Multiple international screening programs are using endoscopic ultrasound (EUS) as the main screening modality. Results have shown that EUS can identify precursor or early lesions and the diagnostic yield is 10% (5-23%). Genetic counseling in individuals screened was perceived as very useful and these patients would seek genetic testing if available.

Australian pancreatic cancer screening program, a collaboration of St Vincent’s Hospital Sydney, NSW Family Cancer Clinics and Garvan Institute targets these high risk groups. Screening started in 2011 and involves genetic counseling, research blood tests and EUS.

A NEW WAY TO SCREEN FOR PANCREATIC CANCER? KRAS SERUM SCREENING

Lara Lipton, Royal Melbourne Hospital, Melbourne

Pancreatic cancer has one of the poorest outcomes of all epithelial cancers. At present no effective screening tests exist for detecting pancreatic cancer at an early, presymptomatic and potentially curable stage. Although not a common cancer in the general population, certain individuals and families are at substantial risk due to inherited causes and chronic pancreatic disease. Over 95% of pancreatic cancers harbour mutations in KRAS. Current technology is available to detect tiny amounts of mutant DNA in plasma. This study will collect circulating DNA pre and post-surgery for operable adenocarcinoma of the pancreas and KRAS mutation testing will be performed on these samples. If sensitivity appears high, further studies will be performed in populations of high risk patients.

A FRESH LOOK AT SMAD 4

Noralane Lindor, Research Program, Mayo Clinic, Arizona, USA

As long ago as 1980, clinicians have described rare families with the combination of juvenile polyps of the colon associated with digital clubbing and cutaneous and pulmonary arteriovenous malformations. In 1998, a mutation in SMAD4 was reported in a large Juvenile Polyposis Syndrome (JPS) family and subsequently it was learned that SMAD4 mutations cause about 20-25% of autosomal dominant JPS with another 20-25% being explained by mutations in BMPR1A (and the remainder not yet mapped). On a different front, mutations in ENG and ALK1(ACVRL1) cause about 90% of cases of Hereditary Hemorrhagic Telangiectasia syndrome (HHT; Osler-Weber-Rendu syndrome), a vascular malformation disorder. It is notable that all 4 of these genes are involved with the transforming growth factor-beta signaling pathway. In 2004, 6 families that co-segregated the JPS and HHT phenotypes were all found to have mutations in SMAD4 and in 2010, 15/19 families with JPS/HHT were confirmed to have SMAD4 mutations. The phenotypic spectrum of disorders produced by SMAD4 has expanded now to also include aortopathy, mitral valve dysfunction, and a disorder of short stature and thick skin known as Myhre syndrome. This talk will review new information on SMAD4-related disorders with a special emphasis on the JPS/HHT syndrome that leads easily to the conclusion that any JPS patient with a SMAD4 mutation merits evaluation for HHT and any HHT patient with a SMAD4 mutation merits evaluation for JPS.

Programme

Wednesday 22nd

Session 1:

Plantation Room

Chairperson: Georgia Chenevix-Trench

EPIGENETIC EPIDEMIOLOGY: EXPLORING THE EPIGENOME TO FIND BIOMARKERS OF CANCER RISK AND PROGNOSIS

James M. Flanagan, Breast Cancer Campaign Fellow, Epigenetics Unit, Department of Surgery and Cancer, Imperial College London

Epigenetic epidemiology aims to use the natural variation present in the epigenome, in DNA methylation as well as histone modifications, to look for associations between particular epigenotypes and disease risk or prognosis. We use a quantitative bisulphite pyrosequencing as well as methylation microarray based approaches, including custom tiling microarrays and the Illumina 450K methylation beadchip, to analyse methylation in peripheral blood DNA. We have analysed blood samples collected from prospective cohorts including European Prospective Investigation into Cancer (EPIC) and the Breakthrough Generations Study (BGS) as well as pre-diagnostic blood samples collected by KConFab. We have recently shown that an intragenic DNA methylation marker within the ATM gene showed an increased risk of breast cancer limited to women in the highest quintile (OR=1.89 (1.36-2.64); P = 1.64×10-4) indicating that blood DNA methylation levels at ATM could be a marker of breast cancer risk. We have also analysed blood samples from an ovarian cancer clinical trial, SCOTROC1, to identify prognostic biomarkers of progression free survival, overall survival, response and toxicity. This study has identified decreased methylation within the SFN gene associated with better progression free survival as a continuous measure in test (p=0.048, n=430) and validation sample sets (p=0.026, n=283, Interquartile Hazard Ratio=1.3 (1.1-1.6), (p=0.033). Finally, in a recent KConFab pilot project we have begun to investigate the possibility of familial aggregation of tumour methylation profiles from non-BRCA1/2 breast cancer families. From these translational studies, it is now apparent that the epigenetic variation between individuals is yielding markers of both cancer risk and prognosis.

LOBULAR BREAST CANCER GWAS

Ian Tomlinson, The Wellcome Trust, Centre for Human Genetics, Nuffield Department of Clinical medicine, University of Oxford, UK

A GWAS-BASED CROSS-DISEASE APPROACH TO FIND GENES PREDISPOSING TO BOTH ENDOMETRICAL CANCER

Jodie N Painter 1, Stuart Macgregor2, Ian Tomlinson5, Dale R Nyholt3, Krina T Zondervan5, Deborah Thompson6, Alison Dunning6, Douglas Easton6, Grant W Montgomery4, Amanda B Spurdle1

Molecular Cancer Epidemiology1, Genetics and Computational Biology2, Neurogenetics3 and Molecular Epidemiology4 Laboratories Queensland Institute of Medical Research, Brisbane Australia Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK5 Centre for Cancer Genetic Epidemiology, University of Cambridge, Strangeways Research Laboratory, Cambridge, UK6

The gynaecological diseases endometriosis and endometrial cancer have serious consequences on quality of life for affected women and impose significant costs to the healthcare system in Australia, together accounting for >28,000 hospital bed days in 2009-10. A convincing link between these diseases has been difficult to establish epidemiologically, although both are hormonally regulated diseases of abnormal endometrium growth and share a number of risk factors and pathological features.

Utilising previously published and unpublished (I Tomlinson) GWAS data we recently applied a cross-disease approach to explore the genetic relationship between the two diseases. Genetic prediction analyses revealed significant (P=7.8x10-10) shared genetic architecture underlying endometriosis and endometrial cancer, indicating that there are genetic loci that influence the risk of both diseases. We then performed a cross-disease meta-analysis which revealed two loci exceeding a genome-wide level of significance (Chr11 P=2.6x10-8, OR=1.16; Chr17 P=3.6x10-8, OR=1.15) and another two loci just under this significance threshold (Chr17 P=1.8x10-7, OR=1.16; Chr6 P=2.9x10-7, OR=1.19).

Three of these regions harbour genes previously linked to other hormonal (breast, ovarian and prostate) cancers, suggesting that multiple disease susceptibility variants may be present at these loci. Imputation of the published GWAS datasets to the latest 1000Genomes release has suggested additional SNPs for follow-up in the future, and all four regions are being investigated for potential functional SNPs/sequences using a bioinformatics approach. While our results require validation to confirm risk loci, they indicate that a number of loci contribute to both endometriosis and endometrial cancer susceptibility, suggest plausible candidate genes, and provide evidence of the value of cross-disease meta-analyses for finding new genes contributing to complex genetic diseases.

GENOTYPID AND PHENOTYPIC ANALYSIS OF FAMILIAL MALE BREAST CANCER SHOWS UNDER REPRESENTATION OF THE HER2 AND BASAL SUBTYPES IN BRCA- ASSOCIATED CARCINOMAS

Siddhartha Deb1,2,3, Jia-Min Pang1,3, Hongdo Do4, Elena Takano4, Nicholas Jene1, kConFab investigators5, Alexander Dobrovic4, Stephen B Fox 1,3,4, Department of Anatomical Pathology, Peter MacCallum Cancer Centre, East Melbourne,, Victorian, Cancer Biobank, Victorian Cancer Council, Carlton, Department of Pathology, University of Melbourne, Parkville 3052, Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne 3002, Kathleen Cuningham Foundation Consortium for research into Familial Breast Cancer, Peter MacCallum Cancer Centre, East Melbourne

Background: Male breast cancer (MBC) is an uncommon and relatively uncharacterised disease accounting for less than 1% of all breast cancers. A significant proportion occurs in families with history of breast cancer. Here, we characterise clinicopathological and genomic features of BRCA1 and BRCA2 mutation carriers and males from BRCAX families. Methods: Cases (n=60) ascertained through kConFab included 3 BRCA1, 25 BRCA2 mutation carries, and 32 non-BRCA1/2(BRCAX) cases with strong family histories of breast cancer. Clinicopathological factors, mutation status of PIK3CA, AKT, KRAS and BRAF and methylation of multiple tumour suppressor genes were examined. Results: Phenotypic correlation: Compared with the general population, MBC incidence was higher in BRCA1 and BRCA2 carriers and in BRCAX families. There was no correlation between mutation status and age of onset, disease specific survival or other clinicopathological factors (all > 0.05). Comparison of our familial cohort with sporadic MBC studies shows similar clinicopathological features and prognostic variables including primary tumour size (p=0.003), age (p=0.002), lymphovascular (p=0.019) and perineural invasion (p=0.027). Fifty-four (90.0%) were ductal carcinoma of no special type and 2 were lobular (3.3%). Four (6.7%) tumours were invasive papillary carcinoma and a further 8 (13.3%) had foci of micropapillary differentiation, showing a trend for occurrence in BRCA2 carriers (p=0.058). Notably, there was no association between BRCA1 carrier status and basal cell phenotype. Five BRCA2 carriers and 5 BRCAX males but no BRCA1 carries developed a second non-breast primary cancer, most commonly prostate cancer (n=5). Mutation Analysis: Six of 57 cases (10.5%) of familial male breast cancers had a PIK3CA mutation. Presence of the mutation was associated with carrier status, with 5 BRCAX patients and 1 BRCA1 mutation carrier having a PIK3CA mutation, but no mutations seen in BRCA2 mutation carriers (p=0.0317). Mutation of AKT, BRAF or KRAS was not observed. Methylation: Of the 5 genes examined (BRCA1, TWIST, RASSF1A, APC and RARB), moderate to high levels of methylation were most frequently seen in BRCA2 carriers (average of 2.05 genes, p=0.0070), and least frequently in BRCAX patients (average 1.29 genes, p=0.0081). RARB methylation was more commonly seen in BRCA2 mutation carriers (47.6% vs 20.0%, p=0.04). Compared to sporadic male breast cancer, less methylation of RASSF1A was seen in familial male breast cancer (94.1% vs 42%, p=0.0003). BRCA1 methylation was not seen. Conclusion: MBC in BRCA1/2 carriers and BRCAX families is different to females. While a clear BRCA1 phenotype is not seen, a possible BRCA2 phenotype with micropapillary histology. Some genomic differences are also seen between the subgroups. Comparison with sporadic MBCs and female BRCA-associated cancers shows some differences making further recruitment and investigation of familial MBCs of value in further understanding these rare and possibly unique set of tumours.

GENOME-WIDE SCAN OF METHYLATION OF DNA EXTRACTED FROM ARCHIVAL BLOOD BIOSPECIMENS

Chol-hee Jung 1, Melissa Southey 2, Dallas English 3,4, Andrew Lonie 1, Alicia Oshlack 5, Helen Tsimiklis 2, John Hopper 4, Gianluca Severi 3,4, Graham Giles 3,4, Laura Baglietto 3,4 1 Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, Carlton, Victoria, 3010, Australia 2 Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne 3 Cancer Epidemiology Centre, Cancer Council of Victoria, Melbourne 4 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne 5 Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville

Within the framework of a large case-control study on methylation and breast cancer risk in the Melbourne Collaborative Cohort Study, we measured genome-wide methylation in DNA from 1,311 women. DNA was extracted from biospecimens collected approximately 20 years ago, including frozen lymphocytes and buffy coats and dried Guthrie card blood spots. Methylation was measured using the Illumina 450k Infinium array. Samples were arranged in 15 plates each containing 8 batches of 12 samples. In order to measure the repeatability of the methylation measures, we included 22 within plate/within batch duplicates and 45 between plates/between batches duplicates. We evaluated the performance of the 450k Infinium array on our samples in terms of number of probes detected at a probability lower than 0.01 and in terms of correlation in methylation between duplicates. The results of this study provide us with insights into the feasibility of measuring genome-wide methylation in DNA extracted from different archival samples with implications in the design of epidemiological studies and future potential relevance in the clinical context.

The Jeremy Jass Memorial Lecture Plantation Room

Introduction & Chair: John Hopper

Presented by: Noralane Lindor

THE JEREMY JASS LECTURE

LOOKING FORWARD IN LYNCH SYNDROME

Noralane Lindor, Research Program, Mayo Clinic, Arizona, USA

The basics of Lynch Syndrome were pieced together about 20 years ago now and these understandings have formed the basis for clinical care. There have, however, been a number of significant advances in both the laboratory and clinical understanding of hereditary DNA mismatch repair. Is universal screening for Lynch syndrome coming and is that a good thing? How often is Lynch syndrome inherited? How much difference does the underlying gene make in disease penetrance? Do tumors arising in the context of Lynch syndrome respond to 5-FU-based chemotherapy like other tumors with microsatellite instability? How much colon should be removed when a Lynch- related colon tumor is diagnoses? Are breast and prostate cancers part of the Lynch syndrome spectrum? Are there new serologic methods to diagnose Lynch syndrome or to track the tumor course? Is aspirin use warranted in Lynch syndrome? An overview of these and other notable advances in Lynch syndrome will be presented.

RELATIVE RISK OF COLORECTAL CANCER RISK IN LYNCH SYNDROME IS INVERSELY ASSOCIATED WITH AGE: A REVIEW

Mark A. Jenkins, Aung Ko Win, James G. Dowty, John L. Hopper, Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Parkville

Introduction: Carriers of a germline mutation in a mismatch repair gene (MLH1, MSH2, MSH6, PMS2) are at increased risk of colorectal and other cancers compared with non- carriers (using the population incidences as a comparison). Once ascertainment of mutation carriers have been adjusted for, the average relative risk of colorectal cancer is 20-30 fold. However, several studies have assessed the relative risk by age to answer the question: Does the degree of increased risk vary with age?

Methods: PubMed was used to identify all studies estimating relative risk of colorectal cancer for carriers of a mismatch repair gene compared to the general population, by age. We also added unpublished data of our own. Studies were excluded if they did not adjust for ascertainment of mutation carriers in their analysis. A meta-analysis (random effects) was conducted for each sex-gene combination available.

Results: Five published studies were identified [1-5] plus one unpublished study of the Colon Cancer Family Registry (Dowty, personal communication). All of these studies observed that the relative risk for colorectal cancer was highest at young ages and decreased with age. This was observed for both sexes and for MLH1, MSH2 and MSH6. Meta-analysis showed the following approximate increased risks of colorectal cancer for MMR mutation carriers relative to the general population (relative risks): 85-fold (30-39 years), 65-fold (40-49 years), 30-fold (50-59 years), 10-fold (60-69 years) and 7- fold (70-79 years).

Conclusion: The relative risk of colorectal cancer for carriers of mutations in mismatch repair genes appears to be inversely related to age. At young ages, the magnitude of the increased risk compared to the general population is substantially greater than at older ages. This has also been observed previously for BRCA1 and breast cancer risk [6]. The reason for this is not known but it adds to the other complexities of MMR gene mutation penetrance including parent-of-origin effect, polygenic effects, and modifying effects of gender, personal characteristics, and environmental factors. There is still much to learn about the effect of MMR gene mutations on colorectal and other cancers that have both clinical and aetiological research implications.

Baglietto, L., et al., J Natl Cancer Inst, 2010. 102(3): p. 193-201. Choi, Y.-H., et al., Hereditary Cancer in Clinical Practice, 2009. 7. Jenkins, M.A., et al., Clin Gastroenterol Hepatol, 2006. 4(4): p. 489-98. Quehenberger, et al., J Med Genet, 2005. 42(6): p. 491-6. Stoffel, E., et al., Gastroenterology, 2009. 137(5): p. 1621-7. Antoniou, A., et al., Am J Hum Genet, 2003. 72(5): p. 1117-30.

IS BREAST CANCER A LYNCH SYNDROME CANCER?

Aung Ko Win 1, Mark A. Jenkins1 1 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Parkville, Victoria, Australia.

Lynch syndrome is an autosomal dominantly inherited disorder of cancer susceptibility caused by germline mutations in the DNA mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. It is generally accepted, based on numerous studies, that these mutation carriers have a substantial burden of increased risks of cancers of the colon, rectum, endometrium and several other organs which generally occur at younger ages than for the general population. Initially raised by Henry Lynch and colleagues several decades ago, the issue of whether breast cancer risk is increased in Lynch syndrome has been debated with evidence for and against this association. Generally, this research falls into either molecular/pathological studies of breast tumour in mutation carriers or analysis of family data to determine whether breast cancer occurs more often than expected in mutation carrying families. Molecular/pathological studies have observed genotype-phenotype correlations, i.e. that breast tumours in women who carry a mutation demonstrate mismatch repair deficiency. However a limitation of these studies is that they were not able to address whether the mismatch repair deficiency caused breast cancer or was a phenotype arising in breast cancer caused by another factor(s). In previous family studies, the evidence for increased risk of breast cancer has been inconsistent. Of the studies that investigated breast cancer risk in Lynch syndrome, six studies did not observe evidence for an association while seven studies found an increased risk of breast cancer ranging from 2 to 13-fold increase. I will discuss the current evidence of an elevated risk of breast cancer in Lynch syndrome including the quality and design of the studies and discuss what further studies are needed to conclusively answer this question.

PROSTATE CANCERS ARISING IN LYNCH SYNDROME MUTATION CARRIERS FREQUENTLY SHOW DNA MISMATCH REPAIR DEFICIENCY

Michael D. Walsh 1,2, Christophe Rosty1,3,4, Sally Pearson1, Mark Clendenning1, Rhiannon Walters1, Belinda Nagler1, David Packenas1, ACCFR Investigators, Joanne P. Young1, John L. Hopper5, Mark A. Jenkins5, Daniel D. Buchanan1, 1Cancer and Population Studies Group, Queensland Institute of Medical Research, Bancroft Centre, Herston QLD, 2Department of Histopathology, Sullivan Nicolaides Pathology, Taringa QLD , 3University of Queensland, School of Medicine, Herston, QLD , 4Envoi Specialist Pathologists, Herston QLD , 5Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne, Carlton

Background: Lynch syndrome is an inherited predisposition to developing cancers of the colorectum, endometrium, small intestine and upper urinary tract. Mutations in the DNA mismatch repair enzymes MLH1, MSH2, MSH6, and PMS2 underpin Lynch syndrome (LS). While prostate adenocarcinoma is not considered one of the spectrum tumours for LS, several reports have documented loss of expression of MMR proteins associated with germline mutations in the corresponding MMR gene, and some studies have suggested that there is an increased risk of developing prostate cancer in mutation carriers. We sought to investigate the incidence of MMR loss of expression and to identify clinicopathological tumour features associated with MMR deficiency in a series of LS carriers enrolled in the AACFR. Methods: DNA mismatch repair (MMR) immunohistochemistry was performed on tissue samples of prostate carcinoma. Germline MMR-gene mutations were identified via sequencing and MLPA. Results: Prostate cancers (n = 12) were examined from proven or obligate MMR-gene mutation carriers recruited to the Australasian Colorectal Cancer Family Registry. Participants ranged in age from 45.5 to 73.7 yr (mean age = 62.8 yr). Pedigrees were available for ten families (two individuals were from the same family). All families met modified Amsterdam criteria. Six individuals had been diagnosed with at least one colorectal carcinoma preceding their diagnosis of prostate carcinoma by 5 to 34 yr. One individual was diagnosed with pancreatic carcinoma two years following the diagnosis of prostate carcinoma at the age of 73 yr, and prostate carcinoma was the only malignancy diagnosed in four individuals. MMR-deficiency was detected in 10/12 (83%) cases: two tumours showed loss of MLH1 and PMS2 expression, while eight tumours showed loss of MSH2 and MSH6 proteins. The pattern of loss of MMR expression was consistent with the individual’s germline mutation in all cases. All twelve cancers were of acinar type. One tumour had a Gleason score (GS) of 6 (this tumour had normal MMR expression and arose in an MSH6 mutation carrier), five tumours were GS 7, and six tumours were poorly differentiated with a GS ≥8. Tumour infiltrating lymphocytes were noted in low numbers in six of the tumours, and perineural invasion was noted in four of the five assessable cases. While many of the tumours were typified by high GS, other markers of aggressive tumour behaviour were commonly absent including p53 expression (1/8 tumours positive) and ERG expression (0/6 tumours positive).Conclusions: These findings indicate MMR-deficiency is commonly observed in prostate adenocarcinomas arising in Lynch syndrome mutation carriers. The prostate tumours with MMR- deficiency from these carriers demonstrated features in common with MMR-deficient cancers from other sites (e.g. poorly differentiated). Prostate carcinoma may thus be considered a suitable option for tissue testing in the absence of other tumours for triaging patients suspected of having Lynch syndrome, particularly in the absence of other cancers.

METHYLATION OF DNA REPETITIVE ELEMENTS FROM PERIPHERAL BLOOD DNA IS ASSOCIATED WITH EARLY-ONSET COLORECTAL CANCER

Rhiannon Walters1, Elizabeth Williamson2, Dallas English2, Joanne P Young1, Christophe Rosty1,3,4, Mark Clendenning1, Michael D. Walsh1, Sally Pearson1, Belinda Nagler1, David Packenas1, ACCFR Investigators, Susan Parry5,6, Aung Ko Win2, John L. Hopper2, Mark A. Jenkins2, Daniel D. Buchanan 1.

1Cancer and Population Studies Group, Queensland Institute of Medical Research, Herston, QLD, Australia, 2Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne, Carlton VIC 3053, Australia 3University of Queensland, School of Medicine, Herston, QLD 4006, Australia 4Envoi Specialist Pathologists, Herston Q4006 5New Zealand Familial Gastrointestinal Cancer Registry, Auckland City Hospital, 6Department of Gastroenterology and Hepatology, Middlemore Hospital, Auckland, New Zealand Background: Methylation levels of DNA from peripheral blood have been shown to be associated with an elevated risk of cancer. The aim of this study was to investigate the association between methylation of DNA repetitive elements LINE-1, Alu and Sat2 in DNA from peripheral blood and: 1) early-onset colorectal cancer (CRC), and 2) subgroups of CRC including inherited and familial CRC. Methods: White blood cell (WBC) DNA from 539 probands with CRC diagnosed before 60 years of age and 242 sex and age frequency-matched healthy controls from the population-based resources of the Australasian Colorectal Cancer Family Registry (ACCFR) were assessed for methylation levels across three DNA repetitive elements (Alu, LINE-1 and Sat2) using MethyLight. The percentage of methylation compared to a reference (PMR) was calculated for each marker. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were generated using multivariable logistic regressions. Results: Probands with early-onset CRC demonstrated significantly higher median PMRs for LINE-1 (p=<0.001), Sat2 (p=<0.001) and Alu repeats (p=0.02) compared with controls. Probands with PMR values in the highest quartile were at a significantly elevated risk of CRC compared with those in the lowest quartile for each of the repetitive elements (LINE-1 OR= 2.38; 95% CI: 1.50-3.78; p = <0.001, Alu OR= 1.82; 95% CI: 1.16-2.84; p = 0.01, and Sat2 OR= 1.78; 95% CI: 1.13-2.82; p = 0.01), after adjusting for all measured confounders: age at blood draw, sex, BMI, smoking, drinking, ethnicity, folate, aspirin and ibuprofen two years ago, and family history (1 st or 2nd degree relative) of CRC or polypectomy. These associations were not significantly altered by chemotherapy treatment with or without 5FU (LRT LINE-1 p= 0.91, Alu p=0.06, Sat2 p=0.79). We found no statistical evidence for differential effects when stratified by family history of CRC or polypectomy, germline MMR or MutYH mutation status, age at diagnosis (<40, 40-<50, and 50+), or tumour characteristics (MMR deficiency, BRAF V600E status, site, or T-stage). Conclusions: We have shown an association between increasing methylation levels of DNA repetitive elements in WBC DNA and the risk of early-onset CRC. This increased risk was not related to a family history of CRC or polyps or inherited CRC-predisposing germline mutations nor was it related to BRAF V600E status, a surrogate marker for a hypermethylated CRC tumour phenotype.

Programme

Wednesday 23rd

Session 3:

Moss Room, Peppers

Chairperson: Rachel Williams

DISCLOSURES OBLIGATIONS FOR PARTICIPANTS DONATING A DNA SAMPLE FOR GENETIC RESEARCH TO LIFEPOOL. WHAT DOES THE WOMAN IN THE STREET NEED TO KNOW AND WHAT DOES SHE NEED TO TELL INSURANCE COMPANIES?

Jeff Gleeson 1, Gabi Crafti2, Sue Viney3, Sue Timbs4, Ian Campbell5, Stephen Fox5, John Hopper6, Anne Kavanagh7, Bruce Mann8, Vicki Pridmore3, Lisa Devereux5, Gillian Mitchell5

Affiliations: Senior Counsel, Isaacs Chambers, Lonsdale Street Melbourne, Barrister at Victorian Bar, Joan Rosanove Chambers, Lonsdale Street, Melbourne, BreastScreen Victoria, Breast Cancer Network Australia, Peter MacCallum Cancer Centre, Centre for MEGA Epidemiology, University of Melbourne, Key Centre for Womens’ Health, Gender and Society, University of Melbourne, The Royal Melbourne and Royal Women’s Hospital

Background Lifepool is a prospective cohort of women recruited from the Victorian population. Funded by the National Breast Cancer Foundation (NBCF), lifepool is a collaboration between BreastScreen Victoria, Peter MacCallum Cancer Centre, University of Melbourne and The Royal Melbourne Hospital. Through BreastScreen Victoria, women are invited to join lifepool at the same time as they are invited to participate in routine mammographic screening. The vast majority of women have not had breast cancer at the time they join lifepool. Consent to participate in lifepool is a two-stage process. The Stage I Consent Form requests baseline health and lifestyle data via completion of a questionnaire, and permission for lifepool to access, over time, mammogram data and to track participants’ health via linkage to health databases. Participants are also asked for permission to access any remnant tumour tissue and associated clinical information, if it becomes relevant. Participants agree to use of this data in future, although currently unspecified, research into breast cancer and other important women’s health issues. Access to lifepool data and biological samples is open to national and international researchers, subject to stringent scientific and ethical review. Stage II operates only if the participant has indicated that she would be happy to be contacted in the future regarding other specific research. Some of those women will be contacted and asked to donate a blood or saliva sample from which DNA will be extracted for genetic research. The lifepool project maintains data in a re-identifiable format to facilitate feedback of significant health information to an individual woman. The genetic research results may be of relevance to the woman’s blood relatives. Aim To review the Stage II Participant Information and Consent Form (PICF) in respect of a woman’s obligation to disclose to her insurers and any future insurers her participation in long-term cohort studies that include unspecified genetic research. A key consideration for this review is the recommendation by the Financial Services Council to its insurer members to include questions in their application forms about applicants’ participation in genetic research. The challenge, in a setting of population cohorts, is to provide information that is accurate, clear and succinct whilst not dissuading participation. Discussion We will present a review of the duty of disclosure to insurers in the context of genetic research and discuss our development of the lifepool Stage II PICF. We will also discuss the potential for the duty of disclosure to apply in the context of close blood relatives.

EVALUATION OF THE EFFICACY OF TWO MODELS OF DELIVERING INFORMATION ABOUT TREATMENT-FOCUSED GENETIC TESTING AMOUNG YOUNG WOMAN NEWLY DIAGNOSED WITH BREAST CANCER

Meiser B, 1,2, Rahman B,1,2 Watts KJ,1,2 Tucker K,1,2Kirk, 3 Saunders C,4 Barlow-Stewart5 Gleeson M,6 Mitchell G7

1) Department of Medical Oncology, Prince of Wales Hospital, NSW, Australia; 2) Prince of Wales Clinical School, University of New South Wales, NSW, Australia; 3) Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, NSW, Australia; 4) Department of Surgery, University of Western Australia, WA, Australia; 5) Centre for Genetics Education, NSW, Australia; 6) Hunter Family Cancer Service, NSW, Australia; 7) Peter MacCallum Cancer Centre, Vic, Australia; WA, Australia;

Background: Increasingly, women newly diagnosed with breast cancer with a relevant cancer family history or other high risk features, in particular young age and triple negative pathology, are being offered BRCA genetic testing to guide their treatment (Treatment-Focused Genetic Testing ‘TFGT’). However, there are concerns that urgently referring many women newly diagnosed with breast cancer to genetic clinics for detailed counselling may overwhelm both the women and these services and so a simpler strategy is needed. In this non-inferiority randomised controlled trial, we evaluate two ways of offering information about TFGT to young women at diagnosis, testing the hypothesis that patient outcomes will be no worse if only a simple information leaflet is given rather than a detailed interview at a family cancer clinic (FCC).

Methods: A total of 128 women (<50 years) at diagnosis (before definitive breast cancer surgery) with either a suggestive cancer family history or other high risk features, are invited to participate by their surgeon in this ongoing study. After completion of a baseline questionnaire, participants are randomised to receive information about TFGT either: a) in the form of brief educational materials (Intervention) or, b) at a FCC (Control). Free BRCA genetic testing is offered; results are disclosed at a FCC. Self-report questionnaires assess demographic information, decisional uncertainty about TFGT, and psychosocial and surgical outcomes. The second questionnaire is administered immediately after the intervention or at an equivalent time point; the third and fourth questionnaires are completed 2 weeks and 12 months after results disclosure, respectively.

Results: As of June 2012, results for change in decisional conflict (the primary outcome variable) are reported for 89 women who completed the first and second questionnaires, all of whom opted for TFGT. Decisional conflict decreased following receipt of information about TFGT, with no statistically significant difference in mean change between the two groups (Intervention M = -13.3, SD = 21.1; Control M = -16.1, SD = 27.3).

Conclusions: These early data suggest that decisional conflict about genetic testing is no worse in the intervention group compared to the control group, and that an abbreviated counselling protocol may be a safe and efficient way of offering TFGT to women.

THE ATTITUDES OF PEOPLE WITH SARCOMA, THEIR FAMILY AND HEALH PROFESSIONALS TOWARDS GENOMICS AND ANCILLARY INFORMATION ARISING FROM GENETIC RESEARCH

MA Young 2, J.L Halliday1, A. Herlihy1, D Thomas2, M.Ballinger2, Gillian Mitchell2 1Murdoch Childrens Research Institute, Parkville, Victoria, Australia 2Peter MacCallum Cancer Centre, Victoria, Australia

The International Sarcoma Kindred Study is a population based study recruiting individuals with sarcoma (probands), their spouses and genetic relatives. Biospecimens, clinical and epidemiological data have been collected. Within the study questionnaire is a section asking about their 1. Beliefs about the genetic component of aspects of health and well-being 2. Feelings towards new genetic discoveries 3. Attitudes towards genetic testing for inherited conditions 4. Attitudes towards predictive testing for sarcoma 5. Attitudes towards the possibility of “incidental findings” as a result of genomic investigations

Results were available on 524 probands, 514 genetically related family members and 162 spouses. For further comparison, the question related to point 1 above was also given to 80 health professionals/ researchers in the field of sarcoma. The beliefs amongst probands, spouses and genetic relatives about the genetic contribution to a number of conditions and characteristics was similar, although was markedly different from health professionals. Older participants, females and those who were more highly educated had higher odds of feeling more positive about genetic discoveries. The questionnaire used four hypothetical scenarios to determine what conditions people would like to be informed about when ancillary information arises as a result of participating in research. These scenarios were 1) risk for a disease caused by a single gene for which there is no prevention e.g. inherited blindness, 2) risk for a disease caused by single gene for which there is screening/treatment which can modify risk e.g. breast or bowel cancer, 3) risk for a disease caused by many genes which has a major impact in health for which there is treatment or lifestyle changes which can modify risk e.g. diet, stopping smoking, exercise 4) risk for a disease caused by many genes which have a lower impact on health for which there is treatment and lifestyle changes which can modify risk e.g. asthma. A higher proportion of spouses as compared to probands and family members thought research participants would like to be informed about ancillary information indicative of a single gene disorder for which there is no prevention (p=0.05). For the remaining three scenarios, almost all participants thought people would like to be given the ancillary information. These results add to the body of emerging evidence that show that research participants wish to be informed about ancillary information that arises as a result of participating in genomic research for reasons including clinical and personal utility.

WHAT HAPPENS WHEN UNCERTAINTY REMAINS? A SYSTEMATIC REVIEW AND QUANTITATIVE EXPLORATION OF DISTRESS IN UNAFFECTED HIGH-RISK INDIVIDUALS WHO DECLINE, DELAY OR REMAIN INELIGIBLE FOR PREDICTIVE GENETIC TESTING

Louise E Heiniger 1, Phyllis Butow1, Melanie Price1, Margaret Charles1, kConFab Psychosocial Group on behalf of the kConFab Investigators 1 Centre for Medical Psychology and Evidence-based Decision Making (CeMPED), School of Psychology, University of Sydney

Background: Research on psychosocial aspects of genetic testing typically focuses on carriers and non-carriers of genetic mutations. However, the majority of unaffected high-risk individuals remain untested. Emerging evidence suggests that remaining ineligible for genetic testing may be more distressing than receiving an unfavourable genetic result, and that declining or delaying testing, when it is available, is associated with less distress. The aims of this project were to: 1) review studies of unaffected individuals who decline, delay or remain ineligible for genetic testing, and 2) compare distress in women who opt for, delay, decline or remain ineligible for predictive testing for familial breast/ovarian cancer

Systematic Review

Method: Medline, CINAHL, PsychINFO and PubMed were searched, along with reference lists of eligible articles and relevant reviews to identify articles for review and quality assessment.

Results and conclusions: Of 1898 potentially eligible articles, 23 (19 different studies) were included. Findings suggest definitions of delaying and declining are not always clear cut, there is almost no research on delayers and few studies on those who remain ineligible for testing. Distress in decliners tended towards lower initial distress with deterioration over time in some cases. Prospective studies suggest those who remain ineligible for testing may also be at risk for poorer psychological outcomes. A number of psychological, personality and family history vulnerability factors were identified for decliners and individuals who are ineligible for testing. Further research is needed to improve our understanding of the psychological correlates of being denied the option of testing, declining and delaying testing.

Quantitative study

Method: This study compared women who opt for (n=120), decline (n=56), delay (n=9 0) or remain ineligible (ineligibles; n=865) for predictive testing with regard to cancer-r elated distress (CRD; Impact of Events Scale), anxiety and depression (Hospital Anxiety and Depression Scale) in 1131 unaffected women enrolled in the kConFab psychosocial study. Controlling for potential confounders (age, education, perceived risk and cumulat ive life event stress), statistical and clinical significance of differences between groups in distress was investigated.

Results: Delayers and testers reported significantly higher CRD than decliners (ps≤.00 1) and ineligibles (ps<.05). Moderate-high CRD was observed in 14% of decliners, 24% of ineligibles, 34% of delayers and 42% of testers (χ2=24.73, p<.001). Decliners reported lower anxiety than delayers (p=.005) and testers (p=.011), with possible/definite anxiet y disorder in 25% of decliners, 34% of ineligibles, 53% of delayers and 44% of testers (χ 2=19.21, p<.001). Depression was significantly higher in delayers (p=.036) and testers (p=.009) compared with decliners, and in testers compared with ineligibles (p=.006). H owever these differences were not clinically significant and prevalence of depression di d not differ between groups (χ2=3.49, p=.322).

Conclusion: Decliners and delayers have distinctly different distress profiles and shoul d be considered as separate groups. Delayers report significantly higher distress, similar to those who opt for testing, indicating that these two groups have the greatest need for psychological support and would benefit most from supportive interventions.

IMPROVING MUTATION NOTIFICATION WHEN NEW GENETIC INFORMATION IS IDENTIFIED IN RESEARCH: A TRIAL OF TWO STRATEGIES IN FAMILIAL BREAST CANCER EMPLOYED BY KCONFAB

Claire E. Wakefield1,2, Heather Thorne,3 Judy Kirk,4,5 Eveline Niedermayr,3 Emma L. Doolan,1,2 the Kathleen Cuningham National Consortium for Research into Familial Breast Cancer (kConFab) and Kathy Tucker.6,7

1School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW, 2 Centre for Children’s Cancer and Blood Disorders, Sydney Children’s Hospital, Randwick, NSW, 3 Peter MacCallum Cancer Centre, Research Department, Kathleen Cuningham National Consortium for Research into Familial Breast Cancer (kConFab), Melbourne, 4 Familial Cancer Service, Westmead Hospital, Westmead, NSW, 5 Faculty of Medicine, Westmead Clinical School, Westmead Millennium Institute for Medical Research, University of Sydney, NSW, 6 Hereditary Cancer Clinic, Department of Medical Oncology, Prince of Wales Hospital, Randwick, NSW, 7 Prince of Wales Clinical School, University of New South Wales, Sydney, NSW.

Aim: The Kathleen Cuningham Foundation Consortium for Research into Familial Aspects of Breast Cancer (kConFab) is a large-scale research study which notifies participants when new, personally relevant, information is discovered. In 2009, kConFab instituted an intensive notification process to ensure at-risk individuals were effectively notified. This study: i) evaluated the impact of intensive notification on genetic testing uptake; ii) identified those most likely to undergo testing post-notification, and; iii) identified those most likely to acknowledge that they had been notified.

Methods: Clinical/demographic data were retrieved from the kConFab database. Logistic regression analyses were conducted to identify potential predictors of testing uptake and notification acknowledgment using SPSSTM.

Results: 155/1812 individuals underwent testing after standard notification (8.6%). In comparison, 23/291 individuals (7.9%) notified using the ‘intensive’ approach underwent testing (χ2=0.14; p=0.71). After controlling for notification process, females, and participants with a previous cancer, were most likely to have undergone testing (p<0.006). Older individuals (50+ years) were most likely to acknowledge they had been notified (p=.038).

Conclusion: Increasing the intensity of participant follow-up did not increase genetic testing uptake. The challenge to effectively notify participants, and increase the proportion whose risk is managed clinically, remains, particularly for males and individuals unaffected by cancer.

WHAT MATTERS TO WOMEN DIAGNOSED WITH BREAST CANCER DURING THEIR REPRODUCTIVE YEARS? PRELIMINARY RESULTS FROM A QUALITATIVE INVESTIGATION USING A POPULATION-BASED SAMPLE

M Kirkman 1,2, C Apicella2, J Graham2, M Hickey3, J Hopper2, L Keogh2, I Winship3, JRW Fisher1,2 1. The Jean Hailes Research Unit, Monash University, Victoria 2. School of Population Health, The University of Melbourne, Victoria 3. School of Medicine, The University of Melbourne, Victoria

Background: Women diagnosed with breast cancer during their reproductive years confront a threat not only to their survival but also to their reproductive health and future motherhood: Breast cancer treatments may result in loss of fertility. Little is known about the impact on women’s emotional wellbeing of reduced fertility and impaired reproductive health associated with cancer.

Aim: To investigate psychosocial aspects of sexual and reproductive health and fertility in women diagnosed with breast cancer when of reproductive age, with the goals of informing and enhancing patient care and addressing women’s short- and long-term psychological needs.

Method: Participants were drawn from a population-based sample of women diagnosed with breast cancer between the ages of 18 and 40, forming part of the Australian Breast Cancer Family Study. Using the ABCFS protocol, all eligible women diagnosed either in 1996-2000 or in 2009 were offered the opportunity to be invited to participate in the research. Participants were interviewed in depth about their fertility- related experiences, expectations, and reflections. Written transcripts of interviews were qualitatively analysed using narrative and thematic techniques.

Results: About 50% of eligible women (51% older, n=27/53; 48% younger, n=23/48) accepted the invitation and gave informed consent. There are few differences between participants and non-participants in age at diagnosis, partnership status, and number of children. Six women in the older group had given birth since diagnosis, in contrast with non-participants, none of whom had given birth since diagnosis. The participating women described diverse motherhood desires and experiences including being pregnant at diagnosis, having completed or not having begun their childbearing, and hoping for children at the conclusion of treatment, with hopes fulfilled and unfulfilled. Whilst all emphasised staying alive, often for the sake of existing children, many women talked of a parallel need to maintain reproductive options but reported varied consideration of fertility management in their professional care. Some (mostly older) women did not recall ever having had the topic of fertility raised by their healthcare team. At the other extreme, some women (most of whom were in the more recently diagnosed group) assessed their fertility-related care as appropriately suited to their needs.

Programme

Wednesday 22nd

Session 4:

Plantation Room

Chairperson: Melissa Southey

POPULATION-BASED ESTIMATE OF PROSTATE CANCER RISK FOR CARRIERS OF THE HOXBI3 MISSENSE MUTATION G84E

Robert J. MacInnis 1,2, Gianluca Severi1,2, Laura Baglietto1,2, James G. Dowty2, Mark A. Jenkins2, Melissa C. Southey3, John L. Hopper1, Graham G. Giles1,2

1 Cancer Epidemiology Centre, Cancer Council Victoria, Victoria, Australia, 2 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, The University of Melbourne, Victoria, Australia, 3 Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Victoria, Australia

The HOXB13 missense mutation G84E (rs138213197) has been reported to be associated with increased risk of prostate cancer, but the current estimate of increased risk has wide confidence intervals and may be misleading. Using a population-based series of early-onset prostate cancer cases (probands) and their families, we found that 15 of 1,091 (1.4%) probands carried the missense mutation and of these only three probands had a family history of prostate cancer.

We estimated the age-specific cumulative risk of prostate cancer (penetrance) for carriers to be 8% (95% CI 2-31%) at age 60 years, 22% (95% CI 7-53%) at age 70 years and 47% (95% CI 15-82%) at age 80 years. The age-specific incidence for carriers was estimated to be, on average, 3.6 (95% CI 1.2-10.3) times that for the Australian population. Our study has provided a more accurate estimate of the average risk of prostate cancer for HOXB13 missense mutation G84E carriers, and its precision, that can be used to guide clinical practice and research.

This study has also shown that the majority of hereditary prostate cancers due to the HOXB13 missense mutation are ‘sporadic’ in the sense that unselected cases with the missense mutation do not typically have a family history of prostate cancer.

COLORECTAL CANCER RISK FOR METT9921 MISSENSE VARIANT CARRIERS FROM THE AUSTRALASIAN COLORECTAL CANCER FAMILY REGISTRY

Daniel D. Buchanan 1, Aung Ko Win2, Mark Clendenning1, Michael D. Walsh1, Rhiannon Walters1, Sally Pearson1, Belinda Nagler1, David Packenas1, ACCFR Investigators, Joanne P. Young1, Christophe Rosty1,3,4, John L. Hopper2, Mark A. Jenkins2., 1Cancer and Population Studies Group, Queensland Institute of Medical Research, Bancroft Centre, Herston QLD, 2Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne, Carlton VIC 3053, 3University of Queensland, School of Medicine, Herston,, 4Envoi Specialist Pathologists, Herston

Background: Colorectal cancer (CRC) has a strong familial component with 15-30% of cases attributed to having a family history of the disease (depending on the age at diagnosis). Only 2-5% of all CRCs are caused by the known highly penetrant inherited syndromes, and therefore, additional CRC-predisposing genes remain to be identified. Recently, the T992I missense variant within the MET oncogene was shown to be associated with familial CRC. The aim of this study was to estimate the risk of CRC for MET T992I variant carriers.

Methods: Case-probands were population-based, recent CRC diagnosed before age of 60 years unselected for family history of colorectal cancer (n=600). Control-probands were identified from electoral rolls and sex and age-frequency matched to case- probands and had no previous diagnosis of CRC (n=243). All cases and controls were from the Australasian Colorectal Cancer Family Registry (ACCFR) and were genotyped for the T992I variant of MET using HRM assay followed by sequencing to confirm aberrant melting profiles. Relatives of carrier probands with DNA available were also genotyped. We excluded one family with a co-occurring MLH1 mutation and one family with de novo MET T992I variant. MET T992I carrier status, sex, age and history of cancer and polypectomy were sought from 19 probands and 717 relatives (359 females and 377 males). We estimated the hazard ratios (HR) of CRC incidence for T992I variant carriers relative to the general population (based on age-, sex- and country-specific cancer incidences), and hence the age-specific cumulative risks (penetrance) using a modified segregation analysis that incorporated both genotyped and ungenotyped relatives and conditioned on ascertainment to produce unbiased estimates.

Results: We identified 21 probands that were heterozygous for the MET T992I variant (14 case probands and 7 control probands); median age at diagnosis for case-probands was 54 years (range 31-60) and the CRCs were predominantly in the left colon (64%) and were of low grade (73%). For the 717 relatives of carriers, 20 were diagnosed with CRC (median age 73 years (range 34 to 89). The HR for CRC for the MET T992I variant carriers was 2.56 (95% confidence interval, 1.06-6.19; p=0.04) with no evidence for difference between males and females (p=0.77). The cumulative risks to age 70 years were estimated to be 9% (4-20%) for males and 6% (3-14%) for females.

Conclusions: The MET T992I missense variant carriers were at elevated risk of CRC, consistent with the T992I variant being a low- to moderately-penetrant CRC- predisposing mutation. Further investigation of this variant in larger studies is needed in order to refine the risk estimates and determine the clinical and biological significance of the MET T992I variant with CRC.

GENE REGULATORY ELEMENTS AS BREAST CANCER SUSCEPTIBILITY LOCI

Brooke L Brewster1, Philip Whiley2, Peter Bailey1, Ania Wronski1, Ming Wong3, Juliet D French1, Stacey L Edwards1, kConFab4, ENIGMA5, Etienne Rouleau6, Melissa C Southey3, Paolo Peterlongo7, Amanda B Spurdle2, Melissa A Brown 1 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. 2 Queensland Institute of Medical Research, Brisbane, Australia. 3Department of Pathology, The University of Melbourne 4Kathleen Cuningham Consortium for Research into Familial Breast Cancer. 5Evidence-based Network for the Interpretation of Germline Mutant Alleles. 6Institut Curie, Hoˆpital Rene ́ Huguenin, Saint-Cloud, France. 7Fondazione Istituto FIRC di Oncologia Molecolare and Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.

Many mutations that confer a predisposition to breast cancer have been described; however these mostly affect protein coding regions and only account for a small proportion of the genetic risk of this disease. It is becoming increasingly clear that disease associated variants that affect gene expression also play an important role in breast cancer susceptibility, however, the precise nature and contribution of such variants remains to be determined. Variants affecting gene expression have been identified in transcriptional control elements, including promoters and proximal and distal enhancers, and in post-transcriptional control elements, including those found in the 3’UTR. Our group has a long-standing interest in elucidating the transcriptional and posttranscriptional regulation of breast cancer associated genes, including BRCA1. Our studies have led to the identification of regulatory sequences mapping to BRCA1 promoter, intronic regions, 3’UTR and extragenic sequences. We have also identified proteins and miRNAs that target these sequences. We have shown that genetic and epigenetic changes in these sequences affect gene expression and may be associated with altered targeting by proteins or miRNAs. For example, BRCA1 c.*1340_1342delTGT, identified in a breast cancer case and located in the 3’UTR, introduces a functional miR-103 binding site, whilst c.*291C>T disrupts the effect of the RNA-binding protein, HuR on wild-type BRCA1 3’UTR regulatory activity. Current studies involve identifying new regulatory variants through the ENIGMA consortium, elucidating the role of these variants in mammary tumourigenesis, and determining the clinical utility of these variants as biomarkers of cancer susceptibility. The results of this project will enable more comprehensive genetic testing and more accurate genetic counselling, will facilitate the development of accurate risk prediction models for carriers of all non-coding variants and will potentially form the basis of new RNA-based therapeutics for minimizing breast cancer risk and improving breast cancer outcomes.

GAP IN THE MIDDLE OF FAMILIAL COLORECTAL CANCER – MODERATELY PENETRANT GENETIC VARIANTS

Deborah Neklason 1,2, Michelle Done1, Nykole Sargent1, Matthew Bailey3, Craig Teerlink3, Richard Kerber4, Randall Burt1,3 , 1Huntsman Cancer Institute, 2Department of Oncological Sciences, 3Department of Medicine, University of Utah, Salt Lake City, UT, USA; 4Department of Epidemiology, University of Louisville, Louisville, KY, USA. [email protected] (801) 587-9882

Background The presence of family history is a well-established risk factor for colorectal cancer (CRC) with 25-35% of CRCs attributable to inherited and/or familial factors. The highly penetrant inherited colon cancer syndromes account for approximately 5% and genome wide association studies have identified low penetrance common variants in the population (relative risk <2) that account for an additional 5% of colorectal cancers. This leaves greater than 15% of inherited CRCs without clear genetic definition. This undefined population is composed of both moderately penetrant genetic variants and rare “private mutations”. Identification of this next tier of unknown genetic risk factors is important because increased cancer surveillance would be justified in this population. In addition, by defining the molecular pathways involved we will gain a better understanding in the development of CRC and thus suggest targets for cancer prevention in this at-risk population. We report on the successes and the challenges in identification of the moderate risk inherited factors in cancer, a complex disease involving interplay of genes and environment.

Methods Our population consists of CRC affected relative pairs and large multigenerational families with excess clustering of cancers. The major source of our research population is an extraordinary resource of over 15 million records that link cancer and medical records to extensive genealogies dating back to the 1800s and housed in the Utah Population Database (UPDB). In affected relative pairs and large families, known syndromes are excluded, phenotypes confirmed with medical records, and genetic analysis performed by either 1) linkage analysis of whole genome STR or SNP panels or 2) exome sequencing with variant analysis and sharing in affected individuals.

Results We have recently reported on a germline non-synonymous change in the MET proto- oncogene (p.T992I also reported as p.T1010I) in ~ 5% of two cohorts of first degree relative pairs affected with CRC. This mutation is present in ≤ 1% of the general population. MET T992I was found to segregate with CRC, melanoma, and a large sessile serrated adenoma in a UPDB CRC family. An additional UPDB cohort of “cancer clusters” was tested for germline MET T992I and found it in 1.8% of all cases, but a noteworthy 3/44 melanomas (7%). We present a mechanistic hypothesis on how this specific variant is driving cancer progression. More recently, we have applied exome sequencing in four CRC cases from three branches in a large UPDB family. We are working on the model that any one of the cancers could be sporadic and, like MET T992I, the genetic change may be present in dbSNP and may cause a subtle change to the protein. Each individual has ~30,000 exon variants, and pairs of individuals share

~10,000 coding changes (amino acid change, stop codon, indel). Our current challenges, approaches, results will be presented.

Conclusions In the field of familial CRC, an important gap in our knowledge is identification of moderately penetrant genetic risk factors whose presence in the germline would justify increased surveillance for cancer. Because they are moderately penetrant and potentially rare, the classic genetic linkage and the GWAS studies have missed them. They present challenges in that they may be without a strong phenotype, require interplay of other risk factors, and may overlap with sporadic cases. MET T992I serves as a good example of how these moderately penetrant genes could work.

Programme

Thursday 23rd

Session 5:

Plantation Room

Chairperson: Gillian Mitchell

“ CANCER 2015” (VIC CANCER GENOME COHORT): TRANSLATING DISCOVERIES INTO CURES

John Parisot 1, Heather Thorne1, Neil Watkins8, Paul Waring8, Thomas John10, Melissa Southey7, John McNeil3, Paula Lorgelly4, Stephen Fox2, Lara Lipton6, David Ashley5, Gary Richardson11, David Thomas1, Joe Sambrook1. 1Cancer Research Division; 2Molecular Pathology, Peter MacCallum Cancer Centre, East Melbourne, 3Department of Epidemiology and Preventative Medicine, Alfred Centre, Monash University, Prahan, 4Centre for Health Economics, Monash University, Clayton, 5The Andrew Love Cancer Centre, Geelong Hospital, Barwon Health, , 6The Royal Melbourne Hospital, Melbourne Health, Parkville, 7Cancer Genomics; 8The Department of Pathology, The University of Melbourne, Parkville, 9Monash Medical Centre, Clayton, 10Austin Health, Heidelberg, 11Cabrini Health, Malvern

According to the American Society of Clinical Oncology’s “Blueprint for Transforming Clinical and Translational Cancer Research”, the goal of improving cancer survival and outcomes will be achieved by translating advances in molecular genomics into the effective design, clinical research, and use, of the new wave of molecularly targeted therapies across the entire health system. It will also necessitate development of sustainable, economically sound models for future cancer care that will involve the key partners in health: government, clinicians and researchers, the pharmaceutical industry, and the community. The “Cancer 2015” Cohort is an epidemiological study funded by the Victorian Cancer Agency (VCA) to help ascertain the benefits of such a targeted, “personalised genomic medicine” approach to cancer care and treatment in Victoria. As one of the world’s largest prospective, longitudinal population-based molecular studies, it will re-classify cancers and allow more targeted patient treatments using molecular genomic data to improve outcomes. It involves a unique collaboration between researchers, pathologists, treating clinicians and cancer patients using shared molecular information, validated clinical trial methodology and data input from both clinicians and consumers. In the UK, Norway, US and elsewhere, large-scale population-based cohorts enrolling tens of thousands of patients, are being developed to elaborate an evidence base for affordable health care in the new genomic era. The study will run over 5 years and include recruitment of 10,000 new cancer patients independent of cancer histopathology type, representing both metropolitan, regional and rural health services across Victoria, with the study to be implemented in 2 phases. The current, phase 1 pilot aims to recruit 1000 patients from 4 hospitals in 2012 and establish the necessary infrastructure and processes needed for upscaling of the study for the recruitment of a further 9000 patients over 2013-14 from upto an additional 10- 12 hospitals.

Cancer 2015 is building on upon existing research and clinical infrastructure and will ultimately provide a high-resolution map of cancer patterns, survival outcomes and health economic metrics in Victoria. As one of the world’s largest prospective, longitudinal population-based molecular studies, Cancer 2015 will make a major contribution to cancer re-classification using molecular pathology, and link this data to treatments and outcomes. Data presented herein will demonstrate progress with the cohort to date and some preliminary results obtained with molecular genotyping and health economics evaluations as proof-of-principle sub-studies.

CASCADE: A CANCER TISSUE COLLECTION AFTER DEATH PROGRAMME TO IMPROVE OUR UNDERSTANDING OF THE PROGRESSION FROM PRIMARY STAGE CANCER TO METASTATIC, TREATMENT=RESISTANT DISEASE.

Kathryn Alsop, Heather Thorne, Paul Waring, Gillian Mitchell, Robin Anderson, Odette Spruyt, Anne Hamilton, Mark Shackleton and David Bowtell

The Peter MacCallum Cancer Centre, East Melbourne VIC and the University of Melbourne, Parkville VIC

With the support of the Victorian Institute of Forensic Medicine (VIFM) and Tobin Brothers Funerals.

Cancer tissue collected for research purposes is most often obtained from primary tumours that are deemed resectable - comparatively few samples are collected from metastatic deposits because patients with disseminated disease usually do not undergo surgery. However, it is becoming apparent that multiple cancer genomes can exist within a single patient and even within a single tumour. The relationships between inter- and intra-tumoural genetic heterogeneity and cancer evolution are presently unclear but likely profoundly influence patient outcome. For example, although brain metastases from melanoma are a major cause of morbidity and mortality, it is not known what evolutionary changes occur in melanoma cells to facilitate cerebral spread. Similarly, although acquired resistance to cancer therapy is a major event that leads inexorably to death, in most cases the mechanisms of resistance are obscure. Fundamentally, the determinants of these outcome-defining events are mostly unknown because metastatic and/or therapy-resistant tumor tissues are almost never available for study in research projects. Understanding of these determinants would be greatly facilitated by collecting multiple, spatially- and temporally-separated tumour samples from individual patients.

Recent work has highlighted the value of rapid autopsy in pancreatic and prostate cancer. Autopsies provide an opportunity to obtain a comprehensive survey of tumour deposits and relatively large amounts of material for multiple assays. We are piloting a programme, called CASCADE, of rapid autopsy in cancer patients, with an initial focus on breast, ovarian, prostate cancer, and melanoma. CASCADE leverages existing Kathleen Cunningham Foundation Consortium (kConFab), Australian Ovarian Cancer Study (AOCS) and Melbourne Melanoma Project (MMP) studies, and involves senior pathologists, palliative care specialists, medical oncologists, familial cancer clinicians, and scientists with extensive experience in biobanking and cancer cohort studies. The aim of CASCADE is to create a bank of metastatic tumour tissue and clinical data to investigate mechanisms of resistance, metastasis, and cancer evolution using genomic and biological tools. Our initial experience of the logistic and scientific challenges in establishing the CASCADE program will be discussed.

1. Campbell, P.J., et al., The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature, 2010. 467(7319): p. 1109-13. 2. Grasso, C.S., et al., The mutational landscape of lethal castration-resistant prostate cancer. Nature, 2012.

INTRODUCING AN ONLINE BOWEL CANCER RISK CALCULATOR TO PROMOTE COLORECTAL CANCER AWARENESS IN THE COMMUNITY

Jody Simmons 1, Prof Finlay Macrae2, 3, Dr Elizabeth Chow2, 3, Stuart Wells1, Philippa Davis1, Chris Enright1 1 Cancer Council Victoria 2 Department of Colorectal Medicine and Genetics, The Royal Melbourne Hospital 3 The Victorian Cooperative Oncology Group

Background One in twelve males and one in seventeen females in Australia will develop colorectal cancer in their lifetime, and the number of new cases per year is increasing in Australia. If detected early it is up to 90% curable, yet it remains the second most common cause of cancer-related death in Australia. NHMRC Guidelines for colorectal cancer screening and surveillance in Australia have been in place since 1991. However, there is poor understanding in the general public of risk factors and the need for screening/surveillance for colorectal cancer. There needs to be an increased awareness and education of bowel cancer risk factors, in order to implement appropriate screening and surveillance for bowel cancer in the community.

Aim To develop an online interactive risk calculator for colorectal cancer that can be used by individuals and their doctors to establish the individual’s colorectal cancer risk. The aim is to accurately define an individual’s risk for bowel cancer and provide advice regarding screening/surveillance practices based on the individual’s specific risk for colorectal cancer development (thereby rectifying both over- and under-screening).

Method The assessment from the online questionnaire captures three aspects of the individual’s risk for bowel cancer: 1. modifiable lifestyle factors 2. personal history of colonic neoplasia and inflammatory bowel disease 3. family history of colorectal neoplasia

Once the patient’s lifestyle factors have been assessed and specific risk category for bowel cancer according to NHMRC guidelines has been calculated, educational feedback in a user friendly format tailored to the subject’s responses will be provided. This will include general recommendations for screening/surveillance for bowel cancer in accordance with current NHMRC guidelines, and education on aspects of lifestyle factors that could be improved upon. A printable synopsis and letter to the patient’s GP summarising their risk category and screening recommendations will be provided on completion.

Results We will introduce this new online tool and discuss the development and implications of use in aiding understanding of colorectal cancer in the community. We will also discuss any preliminary statistics and the perception of the general public.

A CASE OF TWO MUTATIONS IN TRANS IN A WOMEN DIAGNOSED WITH BREAST CANCER AT THE AGE OF 3+0 YEARS

Margaret Gleeson1, Allan D. Spigelman1,2,3, Cliff J. Meldrum4, Susan Dooley4, Michelle Wong5, Barbara Young4, Stephen Braye4 and Rodney J. Scott4,5,6

1. Hunter Family Cancer Service, , HNELHD Cancer Services, Newcastle, NSW 2. Hereditary Cancer Clinic, Cancer Services, St Vincent’s LHN, Sydney, NSW. 3. UNSW St Vincents Clinical School, Sydney, NSW 4. Hunter Area Pathology Service, John Hunter Hospital, Newcastle, NSW 5. School of Biomedical Sciences and Pharmacy, Faculty of Health, University of Newcastle, NSW 6. Hunter Medical Research Institute, University of Newcastle, NSW.

Biallelic mutations in BRCA1 are generally considered to be incompatible with neonatal development as judged by studies in mice. In the presence of an additional TP53 mutation there is partial rescue but the resultant offspring are compromised. In humans, biallelic mutations are considered to be embryologically lethal, based on the absence of biallelic mutation carriers. Although a single case has been reported previously of a woman harbouring a biallelic c.2681_2682delAA mutation in BRCA1, this report has subsequently been shown to be erroneous.

In this report, we have documented the presence of two BRCA1 mutations (c.2681_2682delAA and c.594-2A>C) in a woman diagnosed with unilateral breast cancer at age 30 years. The c.2681_2862delAA results in a frameshift resulting in a premature termination codon. The c.594-2A>C change is a splice site mutation that has been reported to result in the skipping of exon 10, resulting in a frameshift and consequently a premature termination codon.

The breast tumour was a triple negative, node negative, poorly differentiated invasive carcinoma with squamous metaplasia. The c.2681_2862delAA was maternally inherited and the splice site change c.594-2A>C was paternally inherited. While the proband’s mother is currently unaffected at 59 years of age, the probands maternal aunt was diagnosed with breast cancer at 48 years and subsequently with a parotid gland tumour at 53 years. The cancer status in the paternal side of the family is sparse. The sibling of the proband has been tested for the two mutations and thus far, the proband is the only biallelic case within the family.

This is the first report, to our knowledge, of an unequivocal biallelic mutation carrier. A possible explanation of this unusual finding is that the splice site mutation may not be as severe as previously thought. An investigation into the expression of the allele harbouring the c.594-2A>C slice site change will be reported in the context of this case.

EVALUATION OF BRCA1/2 VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE USING A COMBINATION OF MULTIFACTORIAL LIKELIHOOD ANALYSIS, BIOINFORMATICS AND IN VITRO APPROACHES

Whiley, P.J.1, Parsons, M.1, Pettigrew, C.2, Beesley, J.1 , Tavtigian, S.V.3, Vallee, M.3, kConFab4 ,Tucker, K.5, Goldgar, D.E.6, Brown, M.A.2, Spurdle, A.B.1 1Genetics and Population Health Division, Queensland Institute of Medical Research, Brisbane, Queensland 2School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 3Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA 4Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria 5Hereditary Cancer Clinic, Prince of Wales Hospital, Randwick, New South Wales 6University of Utah School of Medicine, Salt Lake City, UT, USA

Evaluation of unclassified variants in breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of patients and families carrying these variants. Multifactorial likelihood analysis combines a prior probability of pathogenicity based on bioinformatic predictions with likelihood ratios based on clinical data. It is often limited by a paucity of relevant clinical data, and there is need to improve specificity and sensitivity of bioinformatic predictions. In vitro analysis of the effect of variants on mRNA/protein structure and function can contribute to assessing the clinical significance of variants, and is also valuable for testing and refining bioinformatic prediction tools. We have undertaken multifactorial likelihood analysis, bioinformatic and in vitro studies of BRCA1/2 variants located in coding, intronic, 5’ untranslated or 3’ untranslated regions. Multifactorial analyses categorised ring domain BRCA1 His41Arg missense variant as pathogenic, consistent with publications reporting abrogated BARD1 binding for the 41Arg protein. Transactivation domain (TAD) assays for three missense variants, Met1775Lys, Val1833Met and Val1838Glu in the BRCA1 c- terminus indicate that all three substitutions to result in abrogated BRCA1 TAD function. Multifactorial analysis indicates that Met1775Lys and Val1838Glu are pathogenic. mRNA splicing assays undertaken for 19 exonic and intronic variants identified splicing aberrations for 3 variants, all of which were located in splicing recognition motifs: BRCA1c.4484G>C , at the last base of exon 14; BRCA2c.67+3G>A, exon 2 donor motif; BRCA2c.8954-5A>G, exon 23 acceptor motif. The BRCA2 c.67+3 G>A resulted in deletion of exon 2 and thus the ATG translation start site, so GFP reporter assays were undertaken to test levels of translation initiation at downstream alternative ATG start sites at codon 107 and 123. Results indicated that the start site at codon 107 is preferentially used to initiate translation in the absence of exon 2, resulting in out-of frame translation. In addition, to assess the utility of bioinformatic predictions for refining prior probability estimates, MaxEntScan-based predictions of de novo donor/acceptor creation were compared to splicing results, and have been used to design minigene assays of variants likely to create donors. Lastly, bioinformatic analysis has prioritised variants in the 5’UTR, 3’UTR and intronic regions for in vitro investigation of altered gene regulation. A panel of approaches incorporating clinical and molecular information can facilitate the clinical classification of variants in BRCA1 and BRCA2, and may help refine bioinformatic modelling and estimation of the prior probability of pathogenicity for future studies of rare variants of uncertain significance.

Programme

Thursday 23rd

Session 6:

Plantation Room

Chairperson: Prue Cowin

CLONING OF THE GENE FOR MIXED POLYPOSIS SYNDROME: THE END OF A 50- YEAR JOURNEY

Ian Tomlinson, The Wellcome Trust, Centre for Human Genetics, Nuffield Department of Clinical Medicine, University of Oxford, UK

Hereditary mixed polyposis syndrome (HMPS) was first described in the early 1960s by clinicians from St Mark's Hospital who were looking after a Jewish family with members throughout the world, including a branch in London. Affected individuals developed unusual, large-bowel polyps of several different types and individual polyps could have more than one morphology. Many individuals had colorectal cancer. The family was large and the disease had Mendelian dominant inheritance. The family was thought to be unique. After a few false leads in the 1990s, by the early 2000s, we had mapped the HMPS gene to the long arm of chromosome 15. We had also identified a few more HMPS families. However, all affected individuals were of Ashkenazi origin and shared a chromosome 15 haplotype of about 2Mb on 15q13.3. Conventional sequencing found no protein-coding mutations to be present in the genes within the region. We then turned our attention to the possibility of more unusual mutations, consistent with the apparent monophyletic origin of the disease-causing variant. As part of a copy number screen, we found a constitutional 40kb copy number gain unique to HMPS patients. This resulted from a tandem duplication that encompassed part of the gene SCG5 and lay upstream of the GREM1 (gremlin) gene. We have recently shown that the HMPS duplication affects a genomic control region, leading to massive over-expression of gremlin and relocation of its expression from the colorectal mesenchyme to the epithelium. The HMPS mutation provides a rare example of a Mendelian condition caused by non-coding variation that has profound effects on gene expression. In the next 50 years, we hope to work out how the HMPS duplication causes the aberrant gremlin expression and how in turn that causes colorectal cancer to develop.

FAVR (FILTERING AND ANNOTATION OF VARIANTS THAT ARE RARE): METHODS TO FACILITATE THE ANALYSIS OF RARE GERMLINE GENETIC VARIANTS FROM SOLID AND ILLUMINA DATASETS

Tú Nguyen-Dumont 1, Bernard J Pope2, Fabrice Odefrey1, Andrew Lonie2, Melissa C Southey1, Daniel J Park1*, Genetic Epidemiology Laboratory, Department of Pathology, Medical Building, The University of Melbourne, Victoria 3010, Australia, Victorian Life Sciences Computation Initiative, The University of Melbourne, 187 Grattan Street, Carlton, Melbourne, Victoria 3010

Massively parallel sequencing (MPS) is a powerful tool to facilitate research on the genetic basis for observed phenotypes, including predisposition to and progression of human diseases. However, interpretation of MPS data is challenging. A typical single whole-human exome analysis outputs thousands of genetic variant signals. Only a fraction of these are real, the remainder being artefacts from the processes of sequencing and mapping to the reference genome. Many of these are not readily distinguishable from true variants on the basis of ‘quality metrics’. Of the authentic variants, only a very small proportion might be relevant to a given phenotype or disease. Here, we present FAVR, a suite of new methods designed to assist the shortlisting of genetic variants under a rare variant-phenotype/disease model. The methods are designed to work with commonly used MPS analysis pipelines, such as the GATK or ANNOVAR, and have been made publically available as a suite of software tools (https://github.com/bjpop/favr). The FAVR methods use signatures in comparator sequence alignment files to facilitate the filtering of mapping artefacts and common genetic variants, and annotation of genetic variants based on evidence of co-occurrence in individuals. As relevant, FAVR methods can also be used to filter out artefacts derived from imbalanced paired-end sequencing. By applying FAVR methods to whole-exome sequencing datasets generated on a SOLiD4 platform, we demonstrate a 3-fold reduced single nucleotide variant shortlist compared to a conventional analysis pipeline with no detected reduction in sensitivity. This analysis included Sanger sequencing of rare variant signals not evident in dbSNP131, assessment of known variant signal preservation, and comparison of observed and expected rare variant numbers across a range of first cousin pairs. Preliminary analysis of Illumina platform-generated whole- exome sequencing datasets indicates that similar benefits will be realised across MPS systems.

The principles described herein were applied in our recent work identifying that rare mutations in XRCC2 are associated with early-onset breast cancer risk.

IDENTIFICATION OF RARE DNA COPY NUMBER VARIANTS OVERLAPPING MISMATCH REPAIR PATHWAY GENES IN ENDOMETRIAL CANCER PATIENTS AND THEIR POTENTIAL CONTRIBUTION TO DISEASE RISK GL Moir-Meyer1, F Lose2, JF Pearson1, Y Tan2, The Australian National Endometrial Cancer Study Group2, RJ Scott3,4, The Hunter Community Study5, Studies of Epidemiology and Risk Factors in Cancer Heredity6, DJ Thompson7, PD Pharoah6,7, AM Dunning4, DF Easton6,7, AB Spurdle2, LC Walker 1 1Department of Pathology, University of Otago, Christchurch, 2Genetics and Population Health Division, Queensland Institute of Medical Research, Queensland, Australia, 3John Hunter Hospital, NSW, Australia, 4Discipline of Medical Genetics, University of Newcastle, NSW, Australia, 5University of Newcastle, NSW, Australia, 6Department of Oncology, University of Cambridge, Strangeways Research Laboratory, Cambridge, UK, 7Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratory, Cambridge, UK.

Endometrial cancer is the most common gynaecological cancer in New Zealand and the incidence is increasing as the population ages1. Genetic predictors of endometrial cancer risk that allow early detection of the disease are important for prevention and improved management strategies. Mutations in the mismatch repair genes MLH1, MSH2, MSH6 and PMS2 are known to confer increased risk in a proportion of endometrial cancer cases, and the mutation spectrum includes copy number variants (CNVs). There are several other genes encoding proteins that act in the mismatch repair pathway, but to date the evidence for their involvement in endometrial cancer predisposition is limited. We have utilised an existing large genetic dataset to screen for CNVs in all genes for which there is evidence for a role in the mismatch repair pathway. Genome-wide scanning of CNVs was performed using Illumina610k single nucleotide polymorphism data from a large cohort of 1336 endometrioid endometrial cancer cases and 619 healthy female controls. Fine-mapping of CNVs was carried out using four CNV calling algorithms to increase both sensitivity and specificity of predictions and novel CNVs were validated by quantitative PCR. By interrogating the array data, we identified and confirmed deletions that disrupted known endometrial cancer susceptibility genes, MSH2 and MSH6. This genetic information can now be used to facilitate counselling and clinical management of these families. We also identified novel variants in several other mismatch repair pathway genes, including duplications overlapping TGFBR3 and MUTYH, and a deletion in RPA3, that are predicted to disrupt the coding sequence of TGFBR3 and RPA3. Thus, we have identified novel aberrations in mismatch repair pathway genes that fall outside the standard testing panel for hereditary endometrial cancer. Furthermore, pathway analysis of all rare CNVs (CNVs found in cases but not controls or the Database of Genomic Variants) showed genes predicted to be disrupted by CNVs belong to several biological networks that may be relevant to endometrial cancer pathogenesis. These results suggest further investigation is warranted to better understand rare CNVs as susceptibility factors in endometrial cancer.

SCOPE VS HOPE: WHEN SHOULD STUDIES OF “MISSING HERITABILITY” GO WHOLE-GENOME?

Khalid Mahmood 1, Chol-Hee Jung1, Joyce Teo2, Tu Nguyen-Dumont2, Fleur Hammet2, Daniel J. Park2, Miroslaw Kapuscinsji3, David E. Goldgar4,5, Bernard Pope1, Graham G. Giles6, John L. Hopper3, Andrew Lonie1, Gianluca Severi6, Melissa C. Southey2.

Victorian Life Sciences Computation Initiative (VLSCI), Carlton, Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Centre for Molecular, Environmental, Genetic and Analytical Epidemiology, School of Population Health, The University of Melbourne, Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City,, Department of Dermatology, University of Utah School of Medicine, Centre for Cancer Epidemiology, The Cancer Council Victoria, Carlton

Massively parallel sequencing (MPS) methodologies now offer robust platforms to support a diverse array of genetic analyses. This technology is supporting a rapid transition from single gene studies to genome-wide studies of Mendelian disorders/syndromes to complex diseases, including cancer.

Until very recently there has been compelling justification for undertaking exome capture followed by MPS (XC-MPS) in studies seeking to identify “missing heritability”. This justification has centred around the research community’s more advanced ability to interpret genetic variation in the exome and flanking intronic regions, and considerably lower cost. Indeed, application of XC-MPS has already identified a number of genes to be involved in cancer predisposition and progression.

There is now considerably more justification for whole-genome MPS (WG-MPS) in this setting. The cost differential is now minimal and the bioinformatics tools for exploring genetic variation in non-coding regions are increasing in number and sophistication. There is also increasing interest and ability to integrate genome-wide datasets such as transcriptomes and methylomes with whole-genome sequencing data. Limited use of this technology has already proven its capacity to characterise genetic syndromes and challenge the bioinformatician.

We have conducted some pilot work applying WG-MPS (at 30 times average genome coverage) to a family that had already undergone an array of other genetic analyses including XC-MPS. As the variations identified by WG-MPS approach are highly concordant with those identified by XC-MPS or other approach, this has provided us with more evidence to support the value and potential of a WG-MPS approach. In the context of our breast cancer research program, we are particularly interested in applying WG-MPS to examine the genomic regions that have provided signals in other settings (eg linkage, GWAS) to avoid having to apply repeated upstream targeted capture methodologies that involve a restricted genetic distance.

We will present our findings and discuss the current and future potential of WG-MPS to support identification of missing heritability in different contexts.

MAPPING THE GENETIC BASIS OF SARCOMAS IN HIGH RISK KINDRED

David Thomas 1, Mandy Ballinger1, Ian Campbell1, Ella Thompson1, Paul James1, Gillian Mitchell1, and the International Sarcoma Kindred Study (ISKS) Consortium 1Peter MacCallum Cancer Centre, Melbourne, Australia

Sarcomas have contributed tremendously to understanding the genetic basis of cancers although only a small fraction of key cancer genes in sarcoma have been mapped including TP53, RB1 and NF1/2. We aim to identify causal genes in highly penetrant sarcoma families. ISKS has enrolled families with an unexplained increased risk for sarcomas. We have utilized massively parallel sequencing (MPS) to analyze the exomes of first degree relatives and affected distant relatives to identify shared alleles in those with cancer. We have refined our candidate list of cancer genes by complementary MPS analysis of associated tumors, and by medium-throughput screening of additional high- risk sarcoma families using a modified BROCA strategy. Family 1 is comprised of two unaffected parents and two affected children with four cancers by age 18 (rhabdomyosarcoma, Ewing sarcoma, germ cell tumor and therapy-associated myelodysplasia). A fifth degree relative developed a high-grade sarcoma at age 19. Several putative cancer genes were identified in the nuclear family by MPS using whole exome capture, including Nibrin (NBN) and DNA Ligase 1 (LIG1). Mutations in NBN cause the Nijmegen Breakage Syndrome, a radiosensitivity-associated cancer predisposition condition, not previously linked to sarcoma. In a medium throughput screen of 34 other families, a second frameshift mutation (219fsX) in NBN was identified in another high-risk family. LIG1 germline mutations have been associated with radiosensitivity and increased cancer risk. Both the NBN and LIG1 mutations in Family 1 were paternally transmitted. The LIG1 mutation was subject to LOH in the myelodysplastic peripheral blood of one proband, and a complementary somatic mutation was identified in the tumor of the other affected, consistent with biallelic inactivation of this gene in two tumors. Several other mutations (homozygous TP53, BAX & PTEN; heterozygous NRAS & AML1) were also detected in the leukemia of the first proband that were not shared by the other sibling, consistent with somatic tumor evolution. The genetic screening of highly penetrant families will provide insights into the biological mechanisms of sarcoma leading to better outcomes by guiding early detection strategies and informing therapeutic approaches.

Programme

Thursday 23rd

Session 7:

Plantation Room

Chairperson: Anna De Fazio

INVASIVE EPITHELIAL OVARIAN CANCER AND BRCA MUTATIONS: THE SOUTH AUSTRALIAN PERSPECTIVE

Nicola K Poplawski 1, 2 and Graeme Suthers1,2 1Familial Cancer Unit, SA Pathology (WCH Site), North Adelaide, South Australia; 2University of Adelaide, Adelaide, South Australia

Background: Two recent studies report detecting BRCA1/2 mutations in more than 10% of women with invasive, non-mucinous epithelial ovarian cancer in prospective unselected series.1, 2 Both studies also reported their mutation detection rate was influenced by the age at which the ovarian cancer was diagnosed suggesting that the relative risk of ovarian cancer due to a BRCA mutation declines with age. Aim: To audit the BRCA1/2 mutation detection rate in women with invasive, non- mucinous epithelial ovarian cancer, who have been referred to the South Australian Clinical Genetics Service (SACGS) with a personal family history of ovarian cancer with or without a family history of breast/ovarian cancer. Method: Audit of the SACGS KinTrak database from 01/01/1995 – 30/06/2012. Results: A total of 188 women who experienced epithelial ovarian cancer had BRCA1 and BRCA2 testing. The average age at diagnosis of ovarian cancer was 56.3 years (range 22-83; median 58.5). Genetic testing identified 47 BRCA1 variants (46 pathogenic; 1 variant of uncertain significance or VUS) and 25 BRCA2 variants (16 pathogenic; 9 VUS). Testing was normal in 103 and is not yet completed in 13. Most women with a pathogenic mutation had a family history of breast and/or ovarian cancer. The results of testing by age at diagnosis are show in the table.

Zhang AOCS SACGS BRCA1/ 2 age at numbe BRCA1/2 numbe BRCA1/2 numbe mutatio diagnosis r tested mutation r tested mutation r tested n <30 years 9 0 4 0 3 0 30-39 years 65 9 (13.8%) 28 5 (17.9%) 11 1 (9%) 40-49 36 17 years 258 67 (26%) 145 (24.8%) 43 (39%) 50-59 56 19 years 363 50 (13.8) 326 (17.2%) 46 (41%) 60-69 36 33 21 years 306 (11.8%) 322 (10.2%) 56 (37%) 70+ years 229 12 (5.2%) 173 11 (6.4%) 27 3 (11%)

Conclusion: Despite the difference in ascertainment, our study documents a BRCA mutation detection rate which is similar to the two recently published larger studies. The higher detection rate in older women in our study may be explained by a bias towards referral of women with a family history of breast and/or ovarian cancer. References 1. Zhang et al. Gynecologic Oncology 2011;121:353-357 2. Alsop et al. J Clin Oncol 2012;30:(10.1200/JCO.2011.39.8545)

LRPIB DELETION IN HIGH-GRADE SERIOUS OVARUAN CANCERS IS ASSOCIATED WITH ACQUIRED CHEMOTHERAPY RESISTANCE TO LIPOSOMAL DOXORUBICIN

Prue A Cowin 1,2,3, Joshy George1,4, Sian Fereday1, Elizabeth Loehrer1, Peter Van Loo5,6, Carleen Cullinane1,3, Dariush Etemadmoghadam1,3, Sarah Ftouni1, Laura Galletta1, Michael S Anglesio7, Joy Hendley1, Leanne Bowes1, Karen E Sheppard4,8, Elizabeth L Christie1, Australian Ovarian Cancer Study1,9,10, Richard B Pearson2,4,,8, Paul R Harnett11, Viola Heinzelmann-Schwarz12, Michael Friedlander13, Orla McNally14, Michael Quinn14, Peter Campbell4, Anna deFazio9 and David DL Bowtell1,2,3,4

1. Cancer Genomics and Genetics Program, Peter MacCallum Cancer Centre, 2. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville 3. Department of Pathology, University of Melbourne, Parkville. 4. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville. 5. Cancer Genome Project, Wellcome Trust Sanger Institute, Cambridge, UK 6. Department of Human Genetics, VIB and University of Leuven, Leuven, Belgium 7. University of British Columbia, BC Cancer Research Centre, Vancouver, Canada 8. Oncogenic Signalling and Growth Control Program, Peter MacCallum Cancer Centre 9. Department of Gynaecological Oncology and Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, New South Wales. 10. Queensland Institute of Medical Research, Brisbane, Queensland. 11. Crown Princess Mary Cancer Centre Westmead and Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute. 12. Ovarian Cancer Group, Lowy Cancer Research Centre, University of New South Wales, School of Women’s and Children’s Health/ Prince of Wales Clinical School, Sydney. 13. Prince of Wales Hospital, Randwick, Sydney, NSW. 14. Department of Obstetrics and Gynaecology, Royal Women’s Hospital, Parkville.

High-grade serous cancer (HGSC), the most common subtype of ovarian cancer, often becomes resistant to chemotherapy, leading to poor patient outcomes. Intratumoral heterogeneity occurs in nearly all solid cancers, including ovarian cancer, contributing to the development of resistance mechanisms. In this study, we examined the spatial and temporal genomic variation in HGSC using high-resolution SNP arrays. Multiple metastatic lesions from individual patients were analyzed along with 22 paired pre- treatment and post-treatment samples. We documented regions of differential DNA copy number between multiple tumor biopsies that correlated with altered expression of genes involved in cell polarity and adhesion. In the paired primary and relapse cohort, we observed a greater degree of genomic change in tumors from patients that were initially sensitive to chemotherapy and had longer progression-free interval, compared to tumors from patients that were resistant to primary chemotherapy. Notably, deletion or downregulation of the lipid transporter LRP1B emerged as a significant correlate of acquired resistance in our analysis. Functional studies showed that reducing LRP1B expression was sufficient to reduce the sensitivity of HGSC cell lines to liposomal doxorubicin, but not to doxorubicin, whereas LRP1B overexpression was sufficient to increase sensitivity to liposomal doxorubicin. Together, our findings underscore the large degree of variation in DNA copy number in spatially and temporally-separated tumors in HGSC patients, and they define LRP1B as a potential contributor to the emergence of chemotherapy resistance in these patients.

CLINICALLY, MOLECULARLY AND FUNCTIONALLY ANNOTATED MODELS OF HG- SOC IN WHICH TO PERFORM PRE-CLINICAL ANALYSIS OF TARGETED THERAPIES.

TOPP M*%, Hartley L*, Cook M*, Rutkowski R*, A Dobrovic#, Hutt K%, McNally O^, Pyman J^, Bowtell D#, Wakefield M, Scott CL* and the Australian Ovarian Cancer Study (AOCS). Walter and Eliza Hall Institute of Medical Research*, Peter MacCallum Cancer Centre#, The Royal Women’s Hospital^, Monash University%, Australian Ovarian Cancer Study.

Background: Altered DNA repair capability in epithelial ovarian cancer (EOC) may underlie response to treatment1,2,3. Molecular sub-classification of serous and endometrioid EOC, may uncover potential drug targets and possible mechanisms of drug resistance3,4,5,6. In order to realize the potential of these discoveries, pre-clinical molecularly-characterized in vivo models of EOC are essential for therapeutic analysis. Methods: A novel xenograft cohort derived from consecutive cases of human high- grade serous ovarian cancer (HG-SOC) has been generated and characterized according to histologic, functional and molecular features (at baseline and following xenotransplantation). Analysis includes documentation of in vitro Homologous Recombination (HR) DNA repair and drug response capabilities (using novel - irradiation and explant drug assays); classification according to molecular subtype (Tothill classifier4); documentation of NHEJ pathway status7, BRCA1/2 status8 and other DNA repair gene status9. This in-depth characterization allows meaningful stratification for in vivo drug treatment studies with the choice of treatment targeted to specific molecular characteristics. Each individual chemotherapy naïve HG-SOC is analysed before and after in vivo treatment with cisplatin and novel therapeutics, an invaluable model for investigating mechanisms of drug response and resistance. Clonal evolution studies will allow novel discovery. Resistance to platinum is driven by re-treating relapsed tumours in vivo or harvesting material for molecular analysis and serial transplantation with re-treatment. This approach will also be adopted with novel therapies, including PARP inhibitors. Results: 14 consecutive clinically annotated HG-SOC have been collected. Analyses carried out to date on the novel xenograft cohort (at baseline and following xenotransplantation) include histological review; preliminary documentation of in vitro Homologous Recombination (HR) DNA repair capability including IHC for markers of DNA damage (H2AX) and DNA repair (RAD51AP1); classification according to molecular subtype (Tothill classifier) and preliminary RNA sequencing analysis. Seven of the first 9 HG-SOC transplanted have successfully xenografted, with cryo- preservation, serial transplantation and phenotyping of xenograft derivatives underway. In vivo platinum response data has been obtained for 6 individual HG-SOC. Five HG-SOC are platinum sensitive, with 2 showing early relapse, and one HG-SOC appears to be primary treatment refractory. Cohorts of recipient tumor-bearing mice are being generated for investigation of PARP inhibitor therapy in vivo, based on HR status as defined by preliminary IHC, germline BRCA1/2 status (where available) and RNA sequencing analysis. Conclusions: A novel xenograft cohort of human HG-SOC has been generated, with characterization of important prognostic features at baseline panel and in subsequent xenografts.

This resource will enable us to test molecular hypotheses and provide outstanding utility for the development of improved therapeutic approaches.

1. Audeh, Lancet. 2010 Jul 24;376(9737):245-51 2. Ledermann et al., N Engl J Med. 2012 Apr 12;366(15):1382-92. 3. Cancer Genome Atlas Research Network Nature. 2011 Jun 29;474(7353):609-15. 4. Tothill et al, Clinical Cancer Research.2008 (14), 5198-5208 5. Helland et al, Plos one. 2011 Apr 13;6(4):e18064. 6. Etemadmoghadam D et al, PLoS One. 2010 Nov 12;5(11):e15498. 7. Patel A et al, Proc Natl Acad Sci 108(8):3406-11. 8. Noquist B et al, J Clin Oncol. 2011 Aug 1; 29 (22):3008-15 9. Walsh T et al, Proc Natl Acad Sci 108(44):18032-7.

MOLECULAR PROFILING OF PRE-INVASIVE SEROUS OVARIAN TUMOURS Sally M. Hunter 1, Kylie L. Gorringe1,2, Michael S. Anglesio3, David G. Huntsman3,4, C. Blake Gilks3,4, Raghwa Sharma5, Anna deFazio6,7, Yoke-Eng Chiew6,7, Simone M. Rowley1, Maria Doyle1, Australian Ovarian Cancer Study1, Ian G. Campbell1,2. 1Centre for Cancer Genomics and Predictive Medicine, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia; 2The Department of Pathology, University of Melbourne, Parkville, Australia. 3The Department of Pathology and Laboratory Medicine, University of British Columbia; 4Genetic Pathology Evaluation Centre of the Prostate Research Centre and Department of Pathology, Vancouver General Hospital and University of British Columbia, Vancouver, British Columbia, Canada; 5Anatomical Pathology, University of Sydney and University of Western Sydney at Westmead Hospital, Australia; 6Department of Gynaecological Oncology, Westmead Institute for Cancer Research, Westmead Hospital, Sydney, New South Wales, Australia, 7University of Sydney at Westmead Millennium Institute, Westmead Hospital, Sydney, New South Wales, Australia Ovarian low grade serous carcinoma (LGSC) are relatively rare compared to their high grade counterparts, but are nonetheless a clinically relevant subset of epithelial ovarian carcinoma (EOC). Correct diagnosis of LGSC is integral to maximising patient benefit from treatment, as LGSC has better outcomes from debulking surgery prior to chemotherapy and patient surgery can be compromised by use of neo-adjuvant chemotherapy that is effective for high grade serous carcinomas. Serous borderline tumours (SBTs), the presumed precursors to LGSC, are relatively common and are considered to be of low malignant potential, typically following an indolent clinical course. Long-term clinical follow-up has demonstrated that SBTs can recur, most frequently as SBTs but also as LGSCs, inspiring a search for factors that may identify which tumours are likely to recur. Despite significant progress in the past two decades in differentiating the major histological types of epithelial ovarian carcinoma (EOC), much remains to be determined and understood regarding the pre-invasive counterparts to these lesions. Understanding precursor biology contextualises the molecular pathogenesis of these tumours, informing tumour progression models, and assisting identification of biomarkers and biologically relevant molecular targets. We have undertaken molecular characterisation of benign (N=35) and borderline (N=57) serous ovarian tumours using high-resolution genome-wide copy number analysis, mutation hotspot screening and exome sequencing. A novel finding of this study was that only 2.9% serous cystadenomas and cystadenofibromas had detectable copy number aberrations in the epithelial component of the tumour. In contrast, copy number aberrations were identified in the stromal component in 34% of cases. In contrast to the benign serous tumours, 96.5% of SBTs have molecular evidence of epithelial neoplasia. Consistent with previous reports, 79% of SBTs had oncogenic mutations in the RAS/RAF/ERBB2 pathway, but appear relatively heterogeneous at the copy number level. Intriguingly, specific clinical features and copy number events were found to be significantly associated with either KRAS or BRAF mutation status, indicating distinct biological consequences arising from these oncogenic events. Contrary to the hypothesis that low grade serous tumours arise through a cystadenoma- borderline tumour-adenocarcinoma sequence, our findings suggest that only a small subset of serous borderline tumours are likely to be arising from serous cystadenomas and cystadenofibromas. The vast majority of serous cystadenomas and cystadenofibromas are not epithelial neoplasms and a significant proportion have evidence of stromal neoplasia with molecular events consistent with a fibroma. Serous borderine tumours were found to be a molecularly heterogeneous group that can be stratified based on clinical features that correlate with oncogenic mutation events, which may prove informative for identifying tumours that may progress or recur.

GENOMIC PROFILING OF BENIGN, BORDERLINE AND INVASIVE MUCINOUS OVARIAN TUMOURS IDENTIFIES KEY AND NOVAL GENETIC EVENTS

Sally M. Hunter*1, Georgina L. Ryland*1,2*, Maria A. Doyle3, Simone M. Rowley1, Michael Chri stie5, Prue E. Allan4, Australian Ovarian Cancer Study Group3#, Ian G. Campbell†1,5,6 and Kyli e L. Gorringe 1,5,6

1. Victorian Breast Cancer Research Consortium (VBCRC) Cancer Genetics Laboratory, Pet er MacCallum Cancer Centre, East Melbourne, Victoria, Australia 2. Centre for Cancer Rese arch, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australi a 3. Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia 4. Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia 5. Depart ment of Pathology, University of Melbourne, Parkville, Victoria, Australia 6. Sir Peter MacC allum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. *Th ese authors contributed equally.

Mucinous carcinomas represent a distinct morphological subtype at several organ sites including the ovary, and their genetic characteristics remain under-described. While some invasive mucinous carcinomas at the ovary are metastases from distant sites, primary ovarian mucinous carcinomas may arise through a stepwise progression from benign cystadenoma to borderline tumour to invasive carcinoma. To explore the spectrum of genomic alterations common to mucinous tumours we performed high resolution genome-wide copy number analysis, mutation screening, and immunohistochemistry on a series of primary ovarian mucinous cystadenomas (n=20), borderline tumours (n=22) and invasive carcinomas (n=12).

Integration of copy number data, targeted mutation screening of RAS/RAF pathway members and IHC revealed that p16 loss and RAS/RAF pathway alterations are highly recurrent events that occur early during mucinous tumour development. The frequency of concurrence of these events was 40% in benign cystadenomas, 68% in borderline tumours and 75% in invasive carcinomas. Pilot exome sequencing of 12 tumours to comprehensively investigate molecular events underlying tumour progression identified truncating mutations in a novel tumour suppressor gene, which was the fourth most frequently mutated gene after KRAS, BRAF and TP53. Further screening of this gene in more cases identified additional deleterious mutations, with a final frequency of 2/22 (9%) in mucinous ovarian borderline tumours and 6/30 (20%) in mucinous ovarian carcinomas. The gene was mutated at <1% in TCGA serous carcinomas, suggesting a mucinous-specific tumour-suppressive role.

This study is the largest and highest resolution analysis of mucinous tumors performed to date and provides strong support for benign and borderline tumours as precursors of primary ovarian mucinous adenocarcinoma. The prevalence of the observed genetic alterations in mucinous tumours from extra-ovarian sites remains to be explored in a larger data set but is a key step in identifying common pathways that may suggest targeted treatments specific for this subtype.

Programme

Thursday 23rd

Plantation Room

Illumina Presentation.

FFPE BASED MOLECULAR ANALYSIS - THE ILLUMINA STORY Brett Kennedy, Product Marketing Manager, Australia and New Zealand

FFPE is a key sample material of interest with a raft of technical challenges. This talk will outline products and applications from Illumina with proven performance to anaylse FFPE material. From genotyping and CNV analysis to methylation and sequencing.

Programme

Friday 24th

Session 8

Plantation Room

Chairperson: Michael Bogwitz

IMPROVING EARLY DIAGNOSIS OF MEN2B: RAIDING AWARENESS AND STIMULATING DEBATE

Hilda High 1, Kathy Tucker 1, Anne Turner 2, Mary-Louise Freckmann 2 1. Hereditary Cancer Clinic, Prince of Wales Hospital, Randwick, Australia 2. Clinical Genetics, Sydney Childrens Hospital, Randwick, Australia

The patient: As a child, our patient had been reviewed by genetic services for dysmorphic features, low tone, delayed speech and toe walking. Extensive workup was done to exclude a connective tissue disorder. Over the 18 months prior to diagnosis he had had severe diarrhoea, requiring intermittent hospitalisations for rehydration There was no family history. His uncle has a similar facial appearance The diagnosis of MEN2B seems obvious now that he has medullary thyroid cancer. However, due to the missed diagnosis, previous photos were shown to a panel of clinical geneticists and none considered the diagnosis. Why is MEN2B hard to pick?  50% of patients present with de novo mutations  the typical phenotype becomes more obvious with age  referrals to genetic centres for investigation for a connective tissue disorder are common while MEN2B has an estimated prevalence of 1: 600,000. Should targeted RET testing be performed in children when a diagnosis of Marfan’s is considered?  There is high clinical utility in testing. Thyroid cancer presents young, metastasises early and treatment are limited, prolonged and expensive. Thyroidectomy is preventive when performed early.  Often young siblings and other family members are at risk  >98% of the mutations in MEN2B are RET c.2753T>C, RET c.2647-2648 del ins TT  Batch testing for these could be performed inexpensively. We postulate that referrals for connective tissue disease may provide an opportunity to exclude the diagnosis of MEN2B. Criteria for proposed RET testing:  Medullated corneal nerve fibres, detectable as prominent thickened corneal nerves on slit lamp examination  mucosal neuromas of the anterior dorsal surface of the tongue, palate, or pharynx.  enlarged “blubbery” lips  submucosal nodules of the lips  ganglioneuromatosis of the gastrointestinal tract, often associated with constipation in childhood. In one case series, >80% reported gastrointestinal symptoms in infancy / early childhood.  asthenic ‘Marfanoid’ body habitus in 75%. Remembering MEN2B as a differential diagnosis will facilitate detection of the mucosal and corneal lesions, since investigated for a connective tissue disease includes a full examination including of the eyes and palate. A question on constipation could easily be included.

MEN2B is a devastating condition if not diagnosed and treated with early thyroidectomy. Modelling studies are underway to determine the economic validity of genetic screening.

VARIABILITY OF CLINICAL FEATURES IN MEN2

Charlotte Slade, Ingrid Winship

Multiple Endocrine Neoplasia 2 (MEN2) is an autosomal dominant syndrome characterized by a very high prevalence of medullary thyroid cancer (MTC). It occurs in approximately 1 in 30,000 individuals, and accounts for approximately 25% of cases of MTC. The condition is caused by ‘gain of function’ mutations in the RET proto-oncogene, located on chromosome 10.

MEN2 is characterized by a spectrum of features, and can be categorized as MEN2A, MEN2B or Familial MTC. MEN2A includes the diagnoses of pheochromocytoma and hyperparathyroidism. MEN2B features an earlier age of onset of MTC than MEN2A, as well as marfanoid body habitus, mucosal ganglioneuromatosis and medullated corneal nerves, but lacks primary hyperparathyroidism. FMTC is defined as 2 or more generations with a RET mutation and MTC, without other features.

The most common RET mutation is a missense change at codon 634, and is associated with almost complete penetrance of medullary thyroid cancer. However the discovery of many other mutations has revealed a spectrum of disease with strong genotype/phenotype correlation. On the basis of the strength of that association the specific RET mutation is now used to guide screening and management decisions. The American Thyroid Association has published guidelines regarding the timing of prophylactic thyroidectomy based upon genotype.

We describe 2 families, seen at Royal Melbourne Hospital’s Familial Cancer Clinic, in whom the variable penetrance of clinical features is demonstrated, and is consistent with the published evidence for geneotype/phenotype correlation.

REVIEW OF MUTATION DETECTION RATE FOR WOMEN DIAGNOSED WITH BREAST CANCER AGED 30 YEARS AND YOUNGER Annabelle Ng 1, Camron Ebzery1, Judy Kirk2, Michelle Bowman2, Leonie Noon2, Hilda High2, Kathy Tucker3, Jessica Duffy3, Sian Greening3, Michael Field4, Joanne Perkins4 and Annabel Goodwin1, 1Cancer Genetics Clinic, Royal Prince Alfred and Liverpool Hospitals, NSW, 2Familial Cancer Service, Westmead Hospital NSW, 3Hereditary Cancer Centre, Prince of Wales Hospital, Randwick, NSW, 4Hereditary Cancer Clinic, Royal North Shore Hospital, St Leonards NSW

Background Population based data (UK) found less than 10% of women diagnosed with breast cancer under 30 have an inherited mutation in BRCA1 or BRCA2 unless there is additional family history or triple negative pathology1,2. Testing in NSW is usually offered only if there is additional family history. Aim To review the mutation detection rate in women diagnosed with breast cancer < 31 who were tested after attending 4 NSW Family Cancer Clinics. To describe the clinical characteristics of the BRCA1 & BRCA2 mutation carriers identified. Methods Retrospective study involving 4 NSW Family Cancer Clinics (FCC). Women diagnosed with breast cancer < 31 were identified from each service, databases and files were searched for additional clinical information. Results There were 172 women diagnosed with breast cancer < 31 identified who had a mutation search of BRCA1 and BRCA2. Eleven women diagnosed with breast cancer < 31 had positive predictive testing for a known family mutation (TP53=1, BRCA1=4, BRCA2=6). Due to additional family history, 13/172 had testing of TP53 and 4/13 (31%) had a mutation identified. An inherited BRCA mutation was detected in 55/172 women (32%). Of the 38/172 with no additional family history, three had an inherited BRCA mutation (8%), (BRCA2=2, BRCA1=1) and one a TP53 mutation. Pathology information was available for 29 (76%) of this group: Eight were known to be triple negative and two of the eight (25%) had a BRCA mutation. Of those with a family history of breast cancer only, 29.3% (29/99) had an inherited BRCA mutation (BRCA1=16, BRCA2=13) and three a TP53 mutation. There was a high mutation detection rate in the presence of breast/ovarian cancer family history, 66% (23/35). Triple negative pathology was present for 9/38 BRCA carriers (23.7%) and an additional two had HER2 status unknown (pathology information was available for 38/55). There were more women with BRCA1 mutations (n=42) diagnosed with breast cancer under age 31 than BRCA2 (n=23). Risk of contralateral breast cancer for BRCA1 carriers was higher, 50% (21/42) than for BRCA2 carriers, 22% (5/23). Conclusion These results support performing a mutation search of BRCA1 and BRCA2 for young women diagnosed with triple negative cancers < 31 without any family history (25%), those with a family history of breast cancer only (29%) or breast/ovarian cancer (66%). We found a high rate of contralateral breast cancer for BRCA1 mutation carriers.

1 Evans DG, Moran A, Hartley R, J Dawson et al. Long-term outcomes of breast cancer in women aged 30 years or younger, based on family history, pathology and BRCA1/BRCA2/TP53 status. British Journal of Cancer (2010) 102, 1091 – 1098.

2 Evans, D. G., A. Howell, D. Ward, et al. "Prevalence of BRCA1 and BRCA2 mutations in triple negative breast cancer." 2011 J Med Genet 48(8):520-522

THE EVOLUTION OF LI-FRAUMENI SYNDROME AND CHALLENGES FOR COUNSELLING

Linda Cicciarelli and Alexandra Lewis, Peter MacCallum Familial Cancer Centre

Historically, TP53 mutation searching was offered to families who had a strong family history, of sarcoma and other Li Fraumeni syndrome (LFS) defining cancers, which met traditional LFS criteria. These families had often experienced significant trauma as a result of their experiences of cancer in their family and therefore could be more vulnerable in the genetic counselling setting. Many patients appeared uncertain about the benefits of genetic testing for themselves and their families. Clinicians also demonstrated uncertainty and apprehension when considering genetic testing for TP53 as there were limited evidence-based screening and clinical management options available; clinicians wished to minimise any additional adverse psychological consequences in this already vulnerable group. This emphasised the importance of comprehensive counselling for patients considering a TP53 test. For many clinicians, LFS is a condition that continues to cause appreciable apprehension when discussing the possibility of and consequences of a TP53 gene mutation.

More recently, research studies have been identifying germline TP53 mutations in patients who do not meet traditional LFS criteria, but have been ascertained for other reasons. This includes patients diagnosed with sarcoma regardless of their family history (through the International Sarcoma Kindred Study), early-onset HER2 positive breast cancer, young age of breast cancer onset (<30 years) or multiple tumours diagnosed in a single individual. Appreciation of these features, and also because we have new advances in screening and pre-implantation diagnosis (PGD), TP53 testing is now being considered for a wider group of patients. This position is facilitated by access to massively parallel sequencing (next generation sequencing) for clinical testing. This technology will allow testing for several cancer genes in parallel.

As testing for LFS becomes more commonplace, the challenge in genetic counselling will be using a model that encompasses those families presenting with traditional LFS who are traumatized as a result of their family history and those who do not have a family history and view LFS as a less burdensome familial cancer syndrome.

In this presentation we will review the literature about genetic counselling practice in LFS and provide recommendations to inform future genetic counselling practice in the increasingly variable clinical landscape of germline TP53 mutations.

WHEN DO WE TEST FOR COWDEN SYNDROME?

Leonie Noon 1, Michelle Bowman1 and Judy Kirk1,2., 1Familial Cancer Service, Westmead Hospital. 2Sydney Medical School – Westmead, University of Sydney.

Approximately 10% of all breast cancer diagnoses are due to an underlying genetic predisposition1. Known genes in which a germline mutation causes an increased risk of breast cancer include BRCA1 and BRCA2 (Hereditary Breast and Ovarian Cancer Syndrome), TP53 (Li-Fraumeni Syndrome), STK11 (Peutz-Jeghers Syndrome) and PTEN (Cowden Syndrome). Cowden syndrome is a part of the PTEN-related hamartoma syndrome spectrum. Germline mutations in PTEN are inherited in an autosomal dominant manner. PTEN mutations are rare with a prevalence of at least one in 200,000-250,0002 and penetrance as high as 80%3. Characteristic phenotypic features of Cowden Syndrome include benign skin lesions (trichilemmomas, papillomatous papules and acral keratosis), which are present in up to 99% of individuals by the age of 303. Other clinical features include multinodular goitre, macrocephaly, gastrointestinal hamartomatous polyps, benign breast fibrocystic disease and multiple endometrial leiomyomas. Variable developmental delay has also been reported in some cases. Cowden Syndrome is also associated with an increased risk of breast cancer (up to 85% lifetime risk4), thyroid cancer, endometrial cancer and dysplastic gangliocytoma of the cerebellum. An association between Cowden syndrome and an increased risk of renal cancer, bowel cancer and melanoma has also been reported4. JC was referred following a diagnosis of multiple bilateral breast fibroadenomas at 48 years of age. In addition, JC has a strong family history of breast cancer, as well as numerous additional relatives with benign breast disease and several cases of mild developmental delay. On clinical examination, JC was found to have a striking number of pigmented keratoses on her back, as well as sebaceous lesions on her face. She reported a personal history of a multinodular goitre, uterine fibroids and mild developmental delay. No macrocephaly or oral lesions were noted. Clinical BRCA1/2 germline testing had previously been carried out on both JC’s affected mother and JC’s affected maternal aunt. The family is also a part of the kConFab study. All BRCA1/2 genetic testing to date was uninformative. Without biopsy confirmation of her trichilemmomas , JC had an Online Cleveland Clinic PTEN score of 8, corresponding to a 2% chance of a mutation5. Germline PTEN testing has been initiated in JC and results are anticipated in late July 2012. The contribution of PTEN mutations to hereditary breast cancer may have been underestimated until now, as genetic testing has focussed primarily on BRCA1/2. The increasing use of next generation sequencing may reveal the role of PTEN in families such as the one described above.

CLINICAL GENETIC TESTING FOR HEREDITARY CANCER USING NEXT GENERATION SEQUENCING

Lesley Andrews1, Bettina Meiser2, Ben Storey3, Belinda Rahman2 1Hereditary Cancer Clinic, Prince of Wales Hospital, Randwick NSW 2Psychosocial Research Department, Prince of Wales Hospital, Randwick NSW 3University of New South Wales, Kensington NSW

Genetic testing is undergoing a major transformation with the introduction of Next Generation Sequencing (NGS) to laboratories around the world. A number of Australian laboratories providing mutation searching for hereditary cancer syndromes are introducing this technology over the next year, however many of the issues associated with this type of testing are yet to be resolved. NGS will enable testing of genes which are part of current practice at lower cost and greater speed. This raises the question as to whether we should change established criteria for testing. Decisions will need to be made regarding testing of isolated cases of rarer tumours, and whether testing of unaffected relatives in families with no living affected members is appropriate. Secondly, testing for cancer predisposition is likely to involve a large panel of cancer genes. Some results may be unexpected on the clinical presentation. In addition, data regarding the risks associated with mutations in some of these genes and their optimal management may not yet be robust. Thirdly, it is likely that standard testing may soon involve exome sequencing, where the outcome may involve  identification of incidental genetic disorders of clinical and/or personal significance

 identification of possibly multiple genetic variations of unknown significance in a single genome

 identification of pathogenic variations in newly identified genes with little clinical data available to guide management.

The major challenges facing us are the provision of informed consent for these outcomes, institutional policy regarding the returning of incidental findings and how to manage the vastly increased the work load for genetic counselling services generated by the increased complexity of potential results, as well as the capacity for higher volumes of testing . In order to determine the attitudes of Australian patients to these challenges, we are conducting semi-structured telephone interviews with patients of the Prince of Wales Hereditary Cancer Clinic who have previously undergone mutation searching with an inconclusive result, as this is a cohort who may be offered NGS in the near future. This study aims to explore patients’ hypothetical interest and motivations in genomic testing, perceived advantages and disadvantages, information and communication preferences regarding incidental findings, and need for assistance regarding decision making using qualitative research methods. Results of the study will be presented.

Programme

Friday 24th

Session 9:

Plantation Room

Chairperson: Mandy Spurdle

MODELLING GENETIC SUSCEPTIBILITY TO BREAST AND OVARIAN CANCER: UPDATES TO THE BOADICEA RISK PREDICTION MODEL, AND RESULTS ON GENETIC MODIFIERS OF CANCER RISK FOR BRCA1 AND BRCA2 MUTATION CARRIERS FROM THE ICOGS CUSTOM GENOTYPING ARRAY

Antonis C. Antoniou, Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, UK

The BOADICEA risk prediction model can be used to compute the probability that an individual carries a BRCA1 or BRCA2 mutation and estimates their probability of developing breast or ovarian cancer in the future. A new version of BOADICEA is expected to be made available in September 2012 which will incorporate a number of novel features. The presentation will describe the work on extending BOADICEA to incorporate breast tumour pathology and how this impacts both the predicted mutation carrier probabilities and cancer risks. The incremental predictive value of incorporating tumour pathology is currently being evaluated in 7,353 families from the German Hereditary Breast and Ovarian Cancer Consortium. Additional novel features include the inclusion of population specific cancer incidences, allowing BOADICEA to be customised to specific populations, and the inclusion of population cancer incidences from more recent calendar periods, thus allowing for more accurate future cancer risk predictions. Ongoing work on incorporating the explicit effects of the common breast cancer susceptibility alleles identified through genome-wide association studies (GWAS), and modelling the residual, non-BRCA1/2 familial clustering of ovarian cancer, will also be described.

Although BOADICEA incorporates a modifying component of breast cancer risk for BRCA1 and BRCA2 carriers, the explicit effects of the known genetic modifiers are not currently incorporated. Work by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) aims to identify and characterise genetic modifiers of cancer risk for BRCA1 and BRCA2. CIMBA studies have demonstrated that several common alleles modify the risks of developing breast or ovarian cancer for BRCA1 and BRCA2 mutation carriers. CIMBA participated in the design of the iCOGS custom array containing 211,155 SNPs selected by several international consortia, primarily on the basis of evidence from GWAS of breast, ovarian and prostate cancer. Approximately 52,000 of those SNPs were selected on the basis of the BRCA1 and BRCA2 GWAS. Analysis of the CIMBA iCOGS SNPs in 11,705 BRCA1 and 8,211 BRCA2 carriers has identified 4 novel loci that are associated with breast cancer risk of BRCA1 and/or BRCA2 mutation carriers and 2 novel loci associated with ovarian cancer risk for mutation carriers. Two of these loci appear to be specifically associated with risk for mutation carriers, the first of their kind. The presentation will review the latest analyses using the iCOGS array and the implications for risk prediction in BRCA1 and BRCA2 mutation carriers.

THE BREAST CANCER RISK PREDICTION MODEL BOADICEA: CALIBRATED FOR AUSTRALIAN WOMEN AND A NEW EASY-TO-USE BATCH PROCESSOR

Robert MacInnis (1,2), Adrian Bickerstaffe (2), Carmel Apicella (2), Gillian Dite (2), James Dowty (2), Kelly Aujard (2); Kelly-Anne Phillips (2,3), Prue Weideman (3), Antonis Antoniou (4), John Hopper (2), Cancer Epidemiology Centre, The Cancer Council, Centre for MEGA Epidemiology, University of Melbourne, Division of Cancer Medicine, Peter MacCallum Cancer Centre, 4 CR-UK Genetic Epidemiology Unit, University of Cambridge, UK Risk prediction algorithms are an important tool for identifying individuals at increased risk of developing the disease who can then be offered individually tailored clinical management. BOADICEA (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm, Antoniou et al., 2004, 2008) is a risk model for familial breast and ovarian cancer that can be used to compute BRCA1 and BRCA2 mutation carrier probabilities and age specific risks for breast and ovarian cancer. Two criticisms of the model include: Although the carrier prediction algorithm has been shown to be well calibrated (ref Antoniou 2008), it is still uncertain how well the age- specific risks perform, particularly in an Australian setting and the web based version can be difficult for users to input data and is currently not set up to batch process multiple pedigrees. Firstly, to evaluate the age-specific risk estimates of BOADICEA, we used 1438 families of ABCFS case probands, diagnosed from 1991 to 1998 and aged less than 80 years. Cumulative breast cancer risks over 10 years of follow-up until 2010 were calculated for 4182 unaffected female relatives. Australian age-specific breast cancer incidences from 1985 to 2000 were used as the population reference rates. A total of 136 incident breast cancers were reported. The ratios of expected to observed number of breast cancers was 0.86 (95% CI 0.73-1.02), while the test for discrimination (ROC curve) was 0.60 (95% CI 0.55-0.65). Secondly, a set of new software programs were designed to automate the process of using BOADICEA in batch mode. These include setting up the pedigree files in the correct format, validating the pedigrees against the requirements of BOADICEA, data cleaning and estimation of missing values (using information from relatives), processing the submission of the data to BOADICEA and collating the results. These programs were written in Java and can be easily run on a variety of platforms. We have shown that BOADICEA is easy-to-use, supports batch processing, and is well calibrated for Australian women. Work is ongoing to add additional factors to BOADICEA to improve discriminatory accuracy, which will ultimately improve targeting of clinical interventions. Multiple independent TERT variants associated with telomere length and risks of breast and ovarian cancer

MULTIPLE INDEPENDENT TERT VARIANTS ASSOCIATED WITH TELOMERE LENGTH AND RISKS OF BREAST AND OVARIAN CANCER.

Georgia Chenevix-Trench1, Stig E Bojesen2, Karen A Pooley3, Sharon E Johnatty1, Jonathan Beesley1, Stacey L Edwards4, Hilda A Pickett5, Howard C Shen6, Chanel E Smart7, Simon A Gayther6, Paul D P Pharoah3, Roger R Reddel5, Ellen L Goode8, Mark H Greene9, Douglas F Easton3, Andrew Berchuck10, Antonis C Antoniou3, and Alison M Dunning3 on behalf of BCAC, OCAC and CIMBA

(1)Genetics Department, Queensland Institute of Medical Research, Brisbane, Australia. (2) Copenhagen General Population Study, Herlev Hospital, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark, (3) Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK., (4) School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia, (5) Cancer Research Unit, Children’s Medical Research Institute, Westmead, NSW, Australia, (6) Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA, (7) The UQ Centre for Clinical Research, The University of Queensland, The Royal Brisbane and Women's Hospital, Herston, Brisbane, QLD. 4029 Australia, (8) Department of Health Science Research, Division of Epidemiology, Mayo Clinic, Rochester, MN, USA, (9) Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA (10) Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC, USA

Common variants around the TERT-CLPTM1L locus and differences in mean telomere length in blood cells are both reported to be associated with risk of multiple cancer types. To identify variants associated with telomere length, breast or ovarian cancer risk, ~480 genotyped and imputed TERT-CLPTM1L locus single nucleotide polymorphisms (SNPs) were analysed in 103,991 breast cancer cases and controls, 44,308 ovarian cancer cases and controls, and 11,705 BRCA1 mutation carriers as part of the ‘iCOGS’ chip. Whole blood telomere length was determined in 53,724 individuals from the UK and Denmark. Associations with all phenotypes fall mainly into three independent peaks of association: in the TERT promoter (Peak 1), the minor alleles of SNPs correlated with rs2736108 associate with longer telomeres (P=5.8×10-7), reduced estrogen receptor negative breast cancer (P=1.2×10-9), reduced breast cancer risk in BRCA1 mutation carriers (P=1.0×10-5) and altered signal in promoter assays. In Peak 2, the minor allele of SNP rs7705526 associates with longer telomeres (P=2.3×10-14), increased low malignant potential ovarian cancer risk (P=1.3×10-15) and increased activity in promoter assays. In Peak 3, minor alleles of highly correlated SNPs, rs10069690 and rs2242652, increase risk of estrogen receptor negative breast cancer (P=1.2×10-12), breast cancer risk for BRCA1 mutation carriers (P=1.6×10-14) and serous invasive ovarian cancer (P=1.3×10-11), but are not associated with telomere length. The cancer-risk allele of rs2242652 increases the strength of a silencer regulatory element, while that of rs1006960 generates a novel, truncated TERT splice variant. Both SNPs lie within an open chromatin site, discovered in stromal and myoepithelial cells from reduction mammoplasty samples. Only Peak 1, of the three, supports the hypothesis that increased cancer risk is mediated through shorter telomeres. These strong and complex associations indicate multiple TERT roles in controlling telomere length and cancer development.

RISK PREDICTION MODELS FOR MISMATCH REPAIR GENE MUTATIONS: A SYSTEMATIC REVIEW AND A META-ANALYSIS Aung Ko Win 1, Mark A. Jenkins1 1 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Parkville, Victoria, Australia.

One of the strongest predictors of colorectal cancer risk is carrying a germline mutation in a DNA mismatch repair gene. Identifying people carrying a mismatch repair gene mutation is important because they are at high risk of colorectal, endometrial and several other cancers and they can benefit from screening. Conversely, identifying non- carriers of mismatch repair gene mutation who are at the population risk of cancer is important as they can be relieved of the burden of this intensive screening program. Prediction models for who is likely or not likely to be a mutation carrier are needed for cost-effective targeted genetic testing. Current methods for predicting risk of carrying a mutation in a mismatch gene depend primarily on whether a person had a colorectal cancer. For those with colorectal cancer, mutation prediction is currently done by testing the tumour for mismatch repair protein deficiency and guided by age of diagnosis, other pathology features and/or family history. For those without a personal history of colorectal cancer, mutation prediction is currently done using models that incorporate family history. Several prediction models for mismatch repair gene mutation status have been developed: Leiden, Amsterdam-plus, MMRpro, PREMM1,2,6, MMRpredict, AIFEG and the Myriad Genetics Prevalence table. A meta-analysis of the 17 validation studies on the discrimination performance of these models (area-under curves or c-statistics) revealed 0.84 (95% confidence interval, CI 0.81-0.88) for PREMM, 0.80 (95% CI 0.72-0.88) for MMRpro, 0.81 (95% CI 0.73-0.88) for MMRpredict, and 0.85 (95% CI 0.78-0.91) for Leiden model. I will present an overview of the various published models to predict risk of mismatch repair gene mutation and their performance.

TUMOUR MORPHOLOGY OF EARLY-ONSET BREAST CANCERS PREDICTS BREAST CANCER RISK FOR FIRST DEGREE RELATIVES

Gillian S. Dite1, Enes Makalic1, Daniel F. Schmidt1, Graham G. Giles1, 2, John L. Hopper 1, Melissa C. Southey3

1Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Australia; 2Cancer Epidemiology Centre, Cancer Council Victoria, Australia; 3Department of Pathology, The University of Melbourne, Australia.

Background: We previously found that tumour morphological features predict BRCA1 mutation carriers among women with early-onset breast cancer better than family history and standard immunohistochemistry. We now hypothesised that risk of breast cancer could be predicted by tumour morphological features for relatives of women with early-onset breast cancer, including relatives of women without a detectable germline mutation in a known breast cancer susceptibility gene.

Methods: We studied mothers and sisters of a population-based sample of 452 index cases with a first primary invasive breast cancer diagnosed before the age of 40 years and for whom a standardised tumour morphology review had been conducted. Standardised incidence ratios (SIR) were calculated by comparing the number of relatives with breast cancer to the number expected based on Australian incidence rates. Hazard ratios were calculated using Cox proportional hazards modelling.

Results: Absence of extensive sclerosis, extensive intraductal carcinoma, absence of acinar and glandular growth patterns, and presence of trabecular and lobular growth patterns were independent predictors with between 1.8 and 3.1–fold increased risk for relatives (all p < 0.02). Excluding the 77 (17%) index cases with genetic or epigenetic causes, independent predictors included minimal sclerosis, circumscribed growth, extensive intraductal carcinoma and lobular growth pattern, all with between 2.0 and 3.3–fold increased risk for relatives (all p < 0.02). Relatives of the 128 (34%) index cases with none of these features were at population risk (SIR = 1.03, 95% CI = 0.57– 1.85), while relatives of the 37 (10%) index cases with two or more features were at high risk (SIR = 5.18, 95% CI = 3.22–8.33).

Conclusion: There is such a wide variation in risks for relatives based on tumour characteristics that early-onset cases without a known genetic predisposition can be divided into three groups for whom the breast cancer risks for relatives are the same as for the three categories in the Guide for Health Professional on Familial Aspects of Breast and Ovarian Cancer developed by the National Breast and Ovarian Cancer Centre. This should be important for genetic counselling, prevention and screening. It adds support to the concept that morphology review of a few key measures could be used to better inform families of their risks of breast cancer, and therefore be considered as an integral part of a breast cancer family genetics service. The identification of a group of relatives at high risk of breast cancer could also help discover new breast cancer susceptibility genes.

Programme

Wednesday 22nd

Poster Session

6.00 – 8.00pm

In the main Foyer of Mantra

THE HETEROGENEITY OF BIRT HOGG DUBE SYNDROME

Authors: Charlotte Slade, Ingrid Winship

Birt Hogg Dubé (BHD) syndrome is a rare, autosomal dominant genodermatosis. It is characterized by a predisposition to the development of cutaneous hamartomas (fibrofolliculomas and trichodiscomas), renal tumors, and lung cysts. The lung cysts may be complicated by spontaneous pneumothorax. Mutations within the folliculin gene, located on the short arm of chromosome 17, are identified in the majority of cases. The penetrance of clinical features is highly variable, and the spectrum of features may be seen within individual families. Management of these patients requires multidisciplinary care, with the involvement of dermatologists, nephrologists, respiratory physicians and in some cases thoracic surgeons and urologists.

We describe the clinical features of 32 affected individuals from nine families in whom we have confirmed the diagnosis of BHD clinically. Seven of those nine families have had genetic testing which confirms the presence of a folliculin mutation. The variability of clinical features within families is highlighted, with some individuals developing renal cancers at a young age, whilst some elderly mutation carriers in the same family are unaffected.

SURVEILLANCE PROTOCOL SNAPSHOT: VICTORIAN FAMILY CANCER REGISTER, CANCER COUNCIL VICTORIA

McArdle T1, Black M1, Farrugia H2, Giles GG3, Winship I4 1 Victorian Family Cancer Registry (VFCR), Cancer Council Victoria (CCV) 2 Victorian Cancer Registry, CCV 3 Cancer Epidemiology Centre, CCV 4 VFCR Clinical Consultant

Background Since 2000 the Cancer Council Victoria has managed an opt-on register for family members assessed as having a high risk of developing cancer due to a genetic predisposition (also includes re-consented members from the FAP register established in 1988). Referrals to the Victorian Family Cancer Register (VFCR) are largely from the four Victorian Family Cancer centres (FCCs) with a small number referred by doctors for familial adenomatous polyposis (FAP). The VFCR provides several functions including two important services; a cancer verification service to help FCCs with cancer risk assessment and a surveillance appointment reminder service for its members. For one third of members enrolled in the reminder service, collecting surveillance protocols from the FCCs is a priority for managing their surveillance appointment reminders. Surveillance outcomes are sought from hospital, clinics and doctors for FAP members. For other family cancer syndromes, outcomes are self-reported. Cancer diagnoses are confirmed by record linkage to the population-based Victorian Cancer Registry. The benefit of collecting surveillance and cancer outcomes is the ability to measure the efficacy of surveillance protocols recommended. Aim: To review surveillance protocols held on the VFCR and evaluate the proportion of cases that are within recommended clinical guidelines for FAP, Lynch and Breast/Ovarian cancer syndromes. Description: The selected sample for review included 715 consented and alive members up to June 30, 2012. Of these 64% had a surveillance protocol and are described by procedure, frequency and age group. Surveillance protocols on the VFCR were compared with EviQ risk management guidelines for FAP, Lynch and Breast/Ovarian cancer syndromes. Summary: Results demonstrate the proportion of cases found to have surveillance protocols within recommended guidelines. Some cases of variance from the recommended clinical guidelines could be explained by age, medical history or personal circumstances. In other cases, a complete screening picture was not available to the VFCR. The major limitation of the review was that the VFCR holds surveillance protocols for only 64% of its members. With a comprehensive data set, the VFCR may in the future have an opportunity to answer complex questions about surveillance, such as, compliance, efficacy and impact on cancer diagnoses for high cancer risk families.

DILEMMAS WITH IDENTIFICATION OF A CAUSATIVE GENE FOR MULTIPLE SELF HEALING EPITHELIOMAS

Camron Ebzery1, Annabelle Ng1, Georgina Fenton1, Cliff Meldrum2 and Annabel Goodwin1 1Cancer Genetics Clinic, Royal Prince Alfred and Liverpool Hospital, Sydney. 2 Deputy Director Genetics, Hunter Area Pathology Service

Multiple Self Healing Squamous Epithelioma (MSSE) or Ferguson Smith disease, is a rare autosomal dominant condition, first identified in patients of Scottish ancestry in 1934. The condition is characterised by the development of multiple keratoacanthoma (KA) like lesions which are malignant. The lesions can spontaneously regress without treatment but may cause scarring resulting in significant disfigurement. A family with a clinical diagnosis of MSSE was referred to our service for an opinion regarding management, due to confusion between this condition and Muir Torre Syndrome by the referring doctor. We reviewed the literature regarding MSSE and a research group had recently identified a causative gene, TGFBR1 (Gouide et al). Age of onset and severity of MSSE can vary within families and clinical discrimination of mutation carriers may be difficult. Additionally, treatment of skin cancers for individuals diagnosed with MSSE (clinical or genetic diagnosis) may be altered since there are reports of progression and disfigurement following radiotherapy. Given this knowledge, we contacted the researchers who offered to test TGFBR1 in this family. Since this condition may be diagnosed by clinical findings and there is little evidence to guide management of affected individuals, the benefit of predictive testing for this condition is not yet established. Several family members expressed interest in genetic testing to learn whether this was the cause of MSSE for their family. Research testing of TGFBR1 was then arranged for an affected family member and a novel truncating mutation was identified in exon 7 (c.1237C>T). We have since approached a clinical laboratory (HAPS), who will perform predictive genetic testing if it is considered to be of benefit to the family. We will discuss the clinical dilemmas and potential benefits associated with predictive genetic testing for this condition.

Goudie DR, D'Alessandro M, Merriman B, Lee H, Szeverenyi I, Avery S, et al. Multiple self-healing squamous epithelioma is caused by a disease-specific spectrum of mutations in TGFBR1. Nat Genet 2011;43:365–9.

HEREDITARY LEIOMYOMATOSIS AND RENAL CELL CARCINOMA SYNDROME – THE ROYAL MELBOURNE HOSPITAL EXPERIENCE Authors:

Manju Salaria, Alison Trainer, Ingrid Winship. Affiliations: Department of Genetic Medicine, Royal Melbourne Hospital, Grattan Street, Parkville, Melbourne

Background: Hereditary leiomyomatosis and renal cell cancer (HLRCC) characterized by cutaneous leiomyomas, uterine leiomyomas (fibroids), and/or a renal tumor is a rare autosomal dominant cancer predisposition syndrome. The most important clinical feature associated this condition is a predisposition to renal cell carcinoma (RCC), typically papillary type 2 on histopathology, which occurs at median age of 44 years.

Aim: The study aimed to characterize the phenotypic features and genotypes of patients with HLRCC referred to the familial cancer centre at the Royal Melbourne Hospital.

Methods: The medical records of all the patients assessed with HLRCC from 2007-2012 at the Royal Melbourne Hospital, Victoria, Australia were scrutinized for inclusion in this audit.

Results: A total of five patients were diagnosed with HLRCC during this time period. All of these patients were females. All of these patients had multiple cutaneous and uterine leiomyomas. One patient had a renal cancer diagnosed at the age of 32, whilst another patient had a family history of renal cancer in a sibling. Genetic testing of 4 patients identified potentially pathogenic mutations in the fumarase hydratase gene (FH gene).

Conclusions: Skin leiomyomas are an important clue to diagnosis of HLRCC. These skin coloured and tender lesions usually start during adolescence. Furthermore, in females with multiple uterine leiomyomas (requiring hysterectomy in 30s or 40s), a skin examination and a detailed family history may provide important clues towards diagnosis of HLRCC. The renal tumor in HLRCC is an aggressive tumor, usually requiring nephrectomy. Early diagnosis of HLRCC is important, and surveillance of at risk family members can be life saving.

INTERDISCIPLINARY PATIENT FILE REVIEWS AND FOLLOW-UP OF PEUTZ- IEGHERS SYNDROME MANAGEMENT

Kirsty Mann1, Adrienne Sexton1, Masha Slattery2, Finlay Macrae1,2 1Familial Cancer Centre, Royal Melbourne Hospital 2Bowel Cancer Surveillance Service, Royal Melbourne Hospital

Peutz-Jeghers syndrome (PJS) is caused by mutations STK11 and is characterised by: hamartomatous polyps in the gastrointestinal system (50% have onset before age 20); increased risk of cancers of the small bowel, colon, stomach, breast, and pancreas; and increased risk of adenoma malignum of the cervix, sex cord ovarian tumours with annular tubules (SCTAT) of the ovaries, and Sertoli cell tumours of the testes. Surveillance options for PJS have been modified in recent years with the availability of improved technologies such as capsule endoscopy and breast Magnetic Resonance Imaging (MRI). A consensus of best practice would aid in improved co-ordination, rather than a fragmented approach to management distributed across a range of specialists. In light of these recent developments, we reviewed the current local management guidelines for PJS. We present a standard letter created to summarise these recommendations. We jointly undertook an audit of all individuals with PJS that have attended the Royal Melbourne Hospital (RMH) Familial Cancer Centre (FCC) or Bowel Cancer Surveillance Service (BCSS). We identified 17 patients seen at the FCC and/or BCSS since year 2000 and assessed their surveillance records, and compared this to the newly agreed local guidelines. Where there was discrepancy, the relevant endoscopic surveillance was arranged. To inform these meetings, we checked genetic (FCC) files, BCSS files, and private medical records. We reviewed whether or not breast MRI had been discussed with female patients (and at-risk female relatives of male probands if applicable), issues related to cascade testing, and any other psychosocial issues. These interdisciplinary file reviews found that gastrointestinal surveillance was either not up to date or records were incomplete for seven patients. There were nine patients (female or with female at-risk relatives) with no record of breast MRI being discussed. Twelve individuals had at-risk relatives without record of being seen at a genetics service. Accordingly, the follow-up involved arranging nine endoscopic procedures, contacting five patients about cascade testing, contacting six doctors/patients about breast MRI, and contacting five doctors about general surveillance recommendations. This process highlighted the complexity of follow-up for PJS patients, and the value of reviewing files in a systematic way, especially for patients who may have not been in contact with an FCC in recent years. It may be used as a model for audits of other rare cancer syndromes, for example Hereditary Diffuse Gastric Cancer and Juvenile Polyposis.

TWO PATHOGENIC MUTATIONS IN BRCA2 GENE IN THE SAME ALLELE

Aamira Huq1, Megan Cotter1, Serguei Kovalenko2, Agnes Bankier1

1 Department of Genetic Medicine and Familial Cancer, Austin Health, Melbourne, Australia. 2 Genetic Technologies Limited, Melbourne, Australia

We present a case with two pathogenic mutations in the same allele of BRCA2 in a female with breast cancer.

Case: Mrs. LA, a 52 year old lady was referred for BRCA testing, having been diagnosed with a grade 3 ER/PR/Her2 negative basal type invasive carcinoma with associated ductal carcinoma in situ at age 51 (in 2011). Family history includes prostate cancer in her father (aged 75) and her paternal uncle (aged 66) and breast cancer at 36 in his daughter. Her BRCAPro score was 9% based on her family history and pathology. BRCA testing was offered on the basis of her pathology and the BRCAPro not being able to take into account her cousin’s young onset breast cancer diagnosis. She also developed neuroendocrine tumour of the small bowel with hepatic metastases, and underwent extensive abdominal surgery at which time she also had bilateral risk reducing salpingo oophorectomy. Past history includes hepatitis C, treated with interferon.

Results: Two separate pathogenic mutations in exon 11 and exon 21 of the BRCA2 gene were identified. The first mutation in exon 11 - c.3405C>A has not previously been reported in the Breast Cancer Information Core database but it is predicted that this mutation would result in a truncated BRCA2 protein with impaired function and/or activity. The second mutation in exon 21 - c.8673_8674delAA causes a frame shift mutation previously reported in the BIC database as clinically important. This mutation has also been reported in the dbSNP database, but validation summary is unknown.

Segregation analysis: Further genetic testing in the family identified both mutations in her father confirming the common allelic nature of both these mutations. Predictive testing in other family members is still ongoing

Discussion: Compound heterozygosity of the BRCA1 and the BRCA2 genes, as well as two mutations in an exon and a splicing site of BRCA2 have been previously described. In our case, the truncating effect of the first mutation would likely dominate over the effect of the second mutation. However, as there is paucity of such mutations found in literature, clear genotype phenotype correlations are difficult to obtain.

INDENTIFICATION OF A WHOLE BRCA2 GENE DELETION: NOW WHAT IT SEEMS!

Jan Sullivan, XXXXX

Genetic Health Service NZ – South Island Hub, Christchurch Hospital, Christchurch, NZ Large genomic rearrangements in the BRCA2 gene are reported as being rare. Routine BRCA1 and BRCA2 sequencing was performed in 2005 in a 71 year old woman with a personal history of breast cancer (G1 IDC, node positive, at age 61), and a family history of breast cancer (two sisters with IDC at ages 45 and 50 respectively, and mother at age 80). No pathogenic mutation was identified. After another at-risk family member represented in 2011, MLPA was performed on stored DNA from the original patient sample for completeness. A heterozygous deletion of the entire BRCA2 gene was detected. This was confirmed by array CGH, which showed two deletions of 53.7Mb and 354 kb on 13q, and encompassing the BRCA2 gene. With a large deletion of almost the entire 13 q arm, an acquired cause for this karyotypic abnormality was suspected. It was then confirmed that the patient had been diagnosed with myelofibrosis in 1996, and had died from AML in 2007. Therefore, this deletion was attributed to her myeloproliferative disorder and was almost certainly not a germline BRCA2 deletion. It remains unknown whether there was a germline BRCA2 gene mutation present in the deleted BRCA2 gene, however. In conclusion, haematological malignancies need to be considered when whole gene deletions or duplications are identified in BRCA1 and BRCA2.

HIGH BRCA1/2 MUTATION FREQUENCY IN WOMEN AFFECTED BY YOUNGEST ONSET BREAST CANCER WHO ATTEND AN AUSTRALIAN FAMILIAL CANCER CENTRE

Aamira Huq, Michael Bogwitz, Nicholas Pachter, Ingrid Winship, Geoffrey Lindeman, Alison Trainer The Royal Melbourne Hospital Familial Cancer Centre, Parkville, VIC 3050, Australia.

Background: Women who develop breast cancer at a young age may carry an underlying genetic predisposition and have an increased risk of developing another breast cancer. Population studies of women with young onset breast cancer suggest a BRCA1and BRCA2 mutation rate of up to 11%, with unclassified variants rate observed in up to 19%.

Objectives: Our aim was to determine the rate of BRCA1, BRCA2 and TP53 mutations detected in a cohort of women aged 35 years or under affected by breast cancer who attended the Royal Melbourne Hospital Familial Cancer Centre and to determine the associated pathology and family history.

Methods: We reviewed the medical records of 107 women referred to our service between 2000 and 2012, with breast cancer diagnosed at the age of 35 or less, in whom BRCA1/2 mutation detection was conducted. Family history and pathology data including the type and grade of the tumour as well as receptor status were collected from all cases. We also analyzed any TP53 testing that was performed in this cohort. Women who underwent predictive testing were excluded from analysis

Results: Of the 107 cases, 19 (18%) were found to carry a pathogenic BRCA1 or 2 mutation, with 7 (6%) cases carrying unclassified variants. TP53 testing was carried out on 18 (17%) of patients. No TP53 mutations were identified in this subset. Details of associated pathology information will be provided.

Conclusions: The policy at our Familial Cancer Centre has been to offer BRCA mutation detection for any woman diagnosed with breast cancer 35 years old or under, irrespective of family history. Our data suggests an 18% chance of finding a BRCA1 or BRCA2 mutation in this population. The likelihood increases where there is a family history of breast cancer. A larger cohort will be required to investigate the merits of TP53 testing, where tumour grade and HER2 status could be considered.

COMPARISON OF IMMUNOHISTOCHEMISTRY FOR MISMATCH REPAIR PROTEINS IN PRE AND POST CHEMORADIOTHERAPY TREATED RECTAL CANCER

Names: Cook BC1, Warrier SK2, Yeung J, Norris B, Mitchell C1, Lynch AC1, Heriot AG1, Trainer A*1, Mitchell G*1. *Joint senior authors 1 Peter MacCallum Cancer Centre 2 Alfred Hospital

Background Preoperative adjuvant chemoradiotherapy is commonly used in the treatment of advanced rectal cancer. Chemoradiotherapy has previously been shown to affect the reliability of microsatellite instability testing for Lynch syndrome. Immunohistochemistry (IHC) staining for mismatch repair (MMR) proteins is now routinely performed in individuals diagnosed with bowel cancer at a young age as a preliminary test for Lynch syndrome. Therefore, it is important to determine whether chemoradiotherapy also affects the reliability of IHC staining for MMR proteins.

Methods A series of patients (n=24) diagnosed with rectal cancer, where both pre-treatment biopsies and post-treatment resections were available, was obtained from the Peter MacCallum Cancer Centre.

Results All four MMR proteins were present on IHC staining of pre-treatment biopsy in 18 cases. Four pre-treatment biopsy cases had a loss of staining of MSH2 and MSH6 proteins, and 2 pre-treatment biopsy cases showed a loss of MSH6 protein on IHC staining. There was 100% concordance of IHC staining between each pre-treatment biopsy and the corresponding post-treatment resection.

Conclusion In rectal cancer, it appears that the ability of IHC staining to determine the presence of MMR proteins is not affected by chemoradiotherapy provided there is sufficient tumour material remaining in the post-chemoradiotherapy surgical resection specimen.

SDHM IMMUNOHISTOCHEMISTRY: FOUR CASES FROM CLINICAL PRACTICE

Michelle Bowman1, Leonie Noon1, Anthony Gill2, Diana E Benn2, 3, Anne Louise Richardson3, Roderick Clifton-Bligh2, 3, Hilda High1 and Judy Kirk1, 2, 1Familial Cancer Service, Westmead Hospital, 2University of Sydney, 3Cancer Genetics, Kolling Institute of Medical Research,, Royal North Shore Hospital

Until recently, a decision to undertake germline genetic testing of the succinate dehydrogenase subunits (SDHB, SDHC, or SDHD) was made on clinical criteria alone: the personal and family history of disease which indicated a likely diagnosis of a familial paraganglioma syndrome. Possible differential diagnoses include Von Hippel-Lindau, Neurofibromatosis type 1, and other renal cell cancer (RCC) predispositions (Birt-Hogg- Dubé, Hereditary Leiomyomatosis RCC, and Hereditary Papillary RCC). Tumour immunohistochemistry (IHC) for SDHB protein can be used to triage genetic testing, as negative staining is good evidence for an underlying mutation in one of the SDH subunits. This is especially helpful for situations where the differential diagnosis cannot be clarified on history alone, and/or when there is no family history to suggest an inherited predisposition. We present four cases where SDHB IHC has been utilised. Case 1: Twenty year old woman, diagnosed with chromophobe renal cancer at the age of 17. No personal or family history of cancer to suggest any of the possible inherited reasons for this diagnosis, including no pneumothoraces or the skin lesions seen in Birt-Hogg-Dubé. Chest CT was normal (no lung cysts). IHC showed positive staining for the SDHB protein. FLCN testing is pending. Case 2: Forty-seven year old woman, with personal history of bilateral multiple clear cell renal cancers and renal cysts from the age of 45. A sister has a possible retinal haemangioma. VHL genetic testing did not identify a mutation. IHC showed positive staining for the SDHB protein. Case 3: Twenty-one year old man, diagnosed with a malignant carotid body paraganglioma, with no definite family history of cancer. IHC showed absent staining for the SDHB protein. An SDHB mutation has been identified (c.689G>A, p.Arg230His). Case 4: Forty-one year old woman, with new diagnosis of breast cancer and a past history of a paraganglioma in the neck at the age of 29. Family history of paternal grandmother with breast cancer at an older age, and maternal grandmother dying from cancer in her 30s (type unknown). IHC on the original paraganglioma showed absent staining for the SDHB protein and positive staining for the SDHA protein. No point mutations or small INDELs were identified in SDHB, SDHC or SDHD by Sanger sequencing; large deletion analysis in these genes is pending. In each of these cases, SDHB IHC has provided valuable information in the clinic to guide assessment, genetic testing and management. Our study amplifies the utility of SDHB IHC for determining the likelihood of germline SDHx mutations in a familial cancer setting. The immunostain is useful both when negative (when SDHx mutations are highly likely) and when positive (which effectively excludes SDHx mutations).

A CLINICAL AUDIT OF INDIVIDUALS WITH HEREDITARY DIFFUSE GASTRIC CANCER (HDGC) AT THE FAMILIAL CANCER CENTRE AT THE ROYAL MELBOURNE HOSPITAL

B. Greyling1*, J. Taylor1*, L. Hodgkin1*, M. Slattery2, F. Macrae1, 2

*Joint first authors

1Familial Cancer Centre, The Royal Melbourne Hospital, Parkville, Victoria, Australia 2Bowel Cancer Surveillance Service, The Royal Melbourne Hospital, Parkville, Victoria, Australia

Hereditary Diffuse Gastric Cancer (HDGC) is a rare cancer condition which predisposes to diffuse gastric cancer and lobular breast cancer. In 25-30% of families, HDGC is caused by a pathogenic mutation in the CDH1 (E-Cadherin) gene. At the Royal Melbourne Hospital Familial Cancer Centre, we have several families with HDGC, three of which have a known mutation in the CDH1 gene.

We carried out a clinical audit on these families, looking at management recommendations (both surgical and/or surveillance options), screening compliance and mutation status. We also assessed the level of information disclosure and cascade testing in each family. In addition, the number, type and histopathology of all cancers were reviewed.

Of particular interest, we found a high proportion of individuals with either an occult gastric cancer or pre-invasive lesions following histopathological examination after prophylactic gastrectomy. Our audit also supported the current literature which suggests that a mutation is identified in a low number of families. Other findings of interest will be presented.

Finally, this audit will be used to streamline the future care of these families.

CONTINOUS QUALITY IMPROVEMENT PROJECT

RishaZia1 Cancer Care Centre, St George Hospital, Dr Kathy Tucker 2 HCC, Prince of Wales Hospital, Dr Patricia Bastick3 Cancer Care Centre, St George Hospital Alison Szwajcer4 Cancer Care Centre, St George Hospital, Rachel Willims5 HCC, Prince of Wales Hospital HCC-Hereditary cancer clinic Aims: Improve referrals to St George Hospital Hereditary Cancer Clinic through breast multidisciplinary meetings Method: 1. Identified problems for low referral rate from breast MDT at St George Hospital. 2. Map the process 3. Implement proposed changes to Breast MDT 4. Evaluate changes to process Project team: Risha Zia, Dr Kathy Tucker, Alison Szwajcer, Dr Patricia Bastick, Rachel Williams Time frame: November 2011 to June 2012 Identified problems: a. Discordance between number of patients discussed at breast MDT for genetic counselling referral and the number of referrals received. b. MDT Consultant focus is treatment focussed-consultants only occasionally present family history or genetic concerns. c. Delays in referrals being sent to St George Hospital Hereditary Cancer Clinic, both from MDT and Prince of Wales Hospital Hereditary Cancer Clinic to where referrals are initially faxed. Outcome: 1. Mapped the process. 2. Genetic Counsellor/Cancer Genetics Specialist started to attend every MDT and obtained number of patients discussed at Br MDT with the coordinator, if unable to attend. 3. Genetic Counsellor/Cancer Genetics Specialist now enquires about family history of relevant patients at the meeting 4. Discussed patients with the consultants/coordinator of Breast MDT and organized referral to genetic counselling. Referral recommendations recorded in MDT database. 5. New process implemented with POWH HCC-all referrals discussed and received weekly. 6. 65 patients were discussed at the Br MDT in timeframe, 42 referrals, 21 patients declined genetic counselling and 2 patients choose a service near their work. Strategies for maintaining improvement:Attend all Br MDT’s and discuss with the consultant/coordinator regarding referrals to continue raising awareness of family history, Discuss with the breast nurse coordinator significance of family history of other cancers, example, family history of any cancers below 40, family history of thyroid cancers. Continue weekly discussion with POWH HCC admin regarding all referrals received in preceding week. These are then sent to STGH HCC.

“Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland

#13 FIRST IMPRESSIONS COUNT! COMPARISON OF CLIENT SATISFACTION AND DISTRESS BETWEEN THREE DIFFERENT INTAKE PROCESSES TO A FAMILY CANCER CLINIC.

Silberbauer, L.1, Williams, R. 2, Nicholls, R.1, Wakefield, C.3, Barlow-Stewart, K.4

1. Faculty of Medicine, University of Sydney, NSW

2. Hereditary Cancer Clinic, Prince of Wales Hospital, Randwick, NSW

3. School of Women’s and Children’s Health, University of New South Wales and Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, NSW

4. Centre for Genetics Education, Royal North Shore Hospital, St Leonards, NSW

Effective genetic counselling requires establishing a good working relationship between clients and genetic counsellors. Such a relationship usually starts during intake to the clinic. There has been very little published research looking at intake processes in genetic counselling in Australia, although some data is available examining the use of family history questionnaires in the UK and Canada and has specifically examined the use of family history questionnaires. There is a clear gap in research to address the attributes of the three commonly-used intake procedures from clients’, genetic counsellors’ and clinicians’ viewpoints: face-to-face intake (interview), telephone intake, and independent completion of a family history questionnaire. In this study we compare client satisfaction and distress for cohorts of clients going through each of these intake processes. Clients referred to the Hereditary Cancer Clinic at the Prince of Wales Hospital, who consent to be part of the study, were randomly allocated to one of the three intake groups. Following intake, they were asked to complete three questionnaires - one immediately prior to their first clinic appointment, one immediately after their first clinic appointment, and one three months after their first clinic appointment. The questionnaires consisted of a five-point client distress measure (distress thermometers) and a six-point client satisfaction measure (satisfaction with genetic counselling). Demographic data were also collected with the first questionnaire. To date 45 of the planned 90 patients have been recruited to the study. Preliminary results from the first two questionnaires will be presented.

This study is part of a larger study examining clinician experience and comparing administrative factors between different intake processes.

THE FAMILY HISTORY QUESTIONNAIRE – WHO IS RESPONDING?

Melanie Boon, Katharine Regnart, Annette Hattam Genetic Health Queensland, Royal Brisbane and Women’s Hospital

The clinical benefit of family history questionnaires has been previously studied, both in terms of assisting pre-clinic risk assessment and pedigree construction as well as reducing the amount of time required for pre-clinic phone intake. However, the response rates of mailed questionnaires amongst family cancer clinics that utilise them, is consistently reported to be lower than desired. In order to determine the questionnaire response rate within Genetic Health Queensland, an internal audit was performed.

Genetic Health Queensland routinely sends out a family history questionnaire to all individuals referred to our metropolitan service with a personal or family history of cancer. The accompanying cover letter requests that this questionnaire is returned within 21 days. The returned questionnaire is then processed by our Cancer Administration Officer who generates a pedigree and gathers appropriate pathology reports prior to the patient being seen in clinic.

A retrospective analysis was performed on all cancer family history questionnaires sent out during the six month period between October 2011 and March 2012 to determine the return rate and response time. Patient demographics were analysed to identify possible future predictors of responders versus non-responders. Variables assessed included gender, age, referral source, reason for referral and personal history of cancer.

Subsequent feedback was provided by the genetic counsellors after pre-clinic patient intake on reasons given for not returning the questionnaire, if volunteered. A qualitative assessment was also performed on patient questionnaire responses relating to their expectations of the session, in order to assess themes regarding patient needs.

A detailed analysis of this information will be presented along with possible suggestions for improvement of our family history questionnaire and departmental processes in order to facilitate optimal patient care and efficient time management for clinical staff.

‘KNOWLEDGE IS POWER’: COPING WITH THE RISK AND UNCERTAINTY OF FAMILIAL BREAST CANCER

Louise E Heiniger1, Melanie A Price1, Margaret Charles1, kConFab Psychosocial Group on behalf of the kConFab Investigators, Phyllis N Butow1 1 Centre for Medical Psychology and Evidence-based Decision-making (CeMPED), School of Psychology, University of Sydney, NSW, Australia

Background: Studies of how individuals living with increased risk for disease cope with being ‘at risk’ regard optimal psychosocial functioning as evidence of adaptation, yet the strategies and processes that precede adaptation have been predominantly unexplored. This study investigated social, cognitive and behavioural efforts to adapt to familial risk of breast cancer in a sample of unaffected women.

Method: Adopting a Grounded Theory approach to data collection and analysis, semi-structured interviews with 27 women enrolled in the Kathleen Cuningham Consortium for Research into Familial breast cancer (kConFab) psychosocial study explored women’s understanding of their risk level and their experiences in adapting to the familial risk. Participants were sampled to include a range of genetic testing statuses (carriers, non-carriers, eligible untested and ineligible for testing) and patterns of distress (identified from existing quantitative data).

Results: Two themes – risk identity and managing uncertainty and risk – illustrated the approaches adopted by women in their attempts to cope with being at risk, and a number of personal, family and healthcare characteristics that facilitated and challenged adaptation were identified. Knowledge of familial risk was seen as an opportunity to engage proactive strategies, while subconscious use of social comparisons, intuition and desensitisation facilitated the integration of ‘being at risk’ into self concept. The impact of, and experiences due to, the family history of breast cancer shaped the women’s perceptions of risk and their ability to adapt, as did feelings of security and trust in relation to healthcare. Most regarded personal satisfaction with risk management efforts, acceptance and a ‘day-by-day’ attitude as crucial to maintaining a positive outlook. A range of views on the value of genetic testing emerged and adaptation was facilitated through the preservation of autonomy and provision of information in a manner appropriate to the individual’s needs.

Conclusion: At-risk women adopt strategies for coping with increased risk to match their personal and social resources. These findings will be used to guide the development of interventions for and communication with individuals at increased risk who experience difficulty adapting to this knowledge.

AN EXPLORATION OF THE LIVED EXPERIENCES OF HAVING BRCA1/2 PREDICTIVE TESTING TOGETHER WITH A SIBLING

Kitty Cho, Melody Menezes, Maira Kentwell, Yasmin Bylstra, Geoffrey Lindeman Familial Cancer Centre, Royal Melbourne Hospital, Melbourne, Australia

This study was carried out to explore the lived experiences of individuals who have had pre-test and post-test genetic counselling for BRCA1/2 predictive testing together with a sibling. The purpose of this study was to gain a better understanding of the experience of joint genetic counselling for siblings who had BRCA1/2 predictive testing together and to ascertain whether being consulted together met their needs and expectations.

The study design utilised a qualitative approach, with a phenomenology theoretical framework. Data was collected using one-on-one in-depth interviews and analysed using an interpretative phenomenological approach. Six participants who have had BRCA1/2 predictive testing with their sibling were recruited from the Royal Melbourne Hospital Familial Cancer Centre, Melbourne.

For this group of participants, the experience of attending together for genetic testing was a positive and beneficial one. Participants reported that they generally were not influenced by their sibling in their decision to have testing or to attend together. Their reasons for attending together were mainly based on practical or logistical considerations. In hindsight, participants identified numerous types of support that they were able to provide and receive from one another throughout the duration of the genetic testing process. Participants were highly satisfied with the joint genetic counselling they received and explained that their needs and expectations were evenly addressed. They recommended genetic counsellors should provide the option to see siblings together for BRCA1/2 predictive testing.

This is the first known study specifically seeking to understand the issue of siblings undergoing predictive genetic testing together. This study presents preliminary evidence that will assist in informing the genetic counselling practice when siblings request to be seen together for BRCA1/2 predictive testing.

GENETIC PREDISPOSITION TO CANCER: A BARRIER TO ADOPTION?

Gemma Correnti, Genetic Health Queensland, Toowoomba Base Hospital, Toowoomba Qld 4350 Australia

Patients attending clinical genetics services who are considering predictive testing for hereditary cancer syndromes are routinely informed about the possibility of genetic discrimination. Within Australia, this discussion has generally been focused on the implications surrounding life and income protection insurance. Although other types of potential discrimination may exist for individuals who have an increased risk of developing cancer, they are not well described and little information is available to guide professionals and their patients. One such area is the potential barrier to adopting a child. Presented here is a recent case which demonstrates that individuals with a familial predisposition to cancer could face discrimination from Australian adoption services.

RISK FACTORS FOR BREAST CANCER: HOW DO WOMEN WITH A FAMILY HISTORY OBTAIN AND INTERPRET THIS INFORMATION?

Emma J Steel, Belinda J McClaren, Carmel Apicella, John L Hopper, Louise A Keogh for the Australian Breast Cancer Family Study

Background: For women whose family history suggests an increased risk of breast cancer, but in which no mutation has been found, little is known about how they obtain, perceive and manage their risk. This study was concerned with describing how these women perceived their risk of breast cancer, their theories of the cause of breast cancer in their family, and how they obtained and interpreted information about breast cancer risk factors. We were particularly interested in lifestyle adaption.

Methods: Participants were recruited through the Australian Breast Cancer Family Study (ABCFS), which is a population-based case-control-family study of breast cancer. Data collection involved a semi-structured interview which was audio recorded and transcribed verbatim. Women were asked what they thought might increase or decrease their risk of breast cancer. They were also asked to describe the ways they adapted their lifestyle because of their family history of breast cancer. Thematic analysis explored women’s sources of knowledge about breast cancer, beliefs about the cause of breast cancer, and knowledge of and interpretation of the risk factors for developing breast cancer.

Results: We have previously reported the five risk management styles adopted by this group, summarising how they interpreted their personal risk. Here we report the analysis of women’s understanding and interpretation of risk factors. The most common source of information on breast cancer risk factors was the media (22/24) including newspapers, television programmes and magazines. Media reports either caused confusion for women or influenced them to make lifestyle changes. Only two women reported seeing a specialist about their risk, with most were relying solely on a GP for medical advice about their risk. We summarise the common theories women used to understand the cause of breast cancer, namely genetics and stress from ‘internal upsets and worries’. We summarise the various ways women interpreted and responded to what they identified as the key risk factors. Healthy eating, exercise, and not smoking were viewed as risk-reducing behaviours by some, but were also mentioned as having no effect on risk by others. There was also confusion about whether or not being overweight and drinking alcohol has an effect on risk.

Conclusions: Women need to be provided with consistent and clear messages about breast cancer risk and risk factors so they can make informed choices around preventative measures.

THE AUSTRALIAN FAMILIAL PANCREATIC CANCER COHORT: SCREENING FOR PANCREATIC CANCER IN HIGH RISK AUSTRALIANS

S. Simpson1, A. Johns1, A. Spigelman2, D. Williams3, A. Stoita3, K. Tucker4, J. Kirk5, M. Field6, R. Williams4, A. Goodwin5, S. Grimmond7, J. Humphris1, S. Mead1, D. Chang1, A. Biankin1

1Cancer Research Program, Kinghorn Cancer Centre/Garvan Institute of Medical Research, Darlinghurst, NSW, Australia 2Familial Cancer Clinic, Kinghorn Cancer Centre/St Vincent's Hospital, Darlinghurst, NSW, Australia 3Gastroenterology, St Vincent's Hospital, Darlinghurst, NSW, Australia 4Hereditary Cancer Clinic, Prince of Wales Hospital, Randwick, NSW, Australia 5Familial Cancer Service, Westmead Hospital, Westmead, NSW, Australia 6Familial Cancer Service, Royal North Shore Hospital, St Leonards, NSW, Australia 7Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, St Lucia, QLD, Australia

The majority of pancreatic cancer cases are termed sporadic, although it is estimated that 10% are familial. Known genetic conditions account for less than 20% of this familial aggregation1. The predisposing genetic basis for most families with a clustering of pancreatic cancers is unspecified, and the predicted risks to unaffected family member can be ambiguous2. Multiple international familial pancreatic cancer registries are utilising genome sequencing to collaboratively identify novel pancreatic cancer susceptibility genes, such as PALB23, however more needs to be known about the predisposing genetic aspects of pancreatic cancer in Australian families. The Australian Familial Pancreatic Cancer Cohort (AFPaCC) is a recently established registry targeting families or individuals with a history of pancreatic cancer. Registration involves consent, and collation of relevant clinical information. A blood sample is collected to search for additional pancreatic cancer susceptibility genes. Recruitment will use a multi-centred approach, with individuals identified through Family Cancer Clinics, treatment centres, cancer registries or directly by clinicians and our website. AFPaCC will also help to identify high-risk individuals who may benefit from screening trials. The overall aims of AFPaCC are to further clarify the risk in families with a history of pancreatic cancer in Australia and discover genetic and non-genetic causes of pancreatic cancer. Although AFPaCC is still in it's infancy, this poster aims to raise awareness of the project, provide an initial overview of the Australian families already involved, and outline some of the challenges already faced.

[1] Shi C, Hruban RH, Klein AP. Familial pancreatic cancer. Arch Pathol Lab Med 2009 March; 133(3):365-374 [2] Permuth-Wey J, Egan KM. Family history is a significant risk factor for pancreatic cancer: results from a systematic review and meta-analysis. Familial Cancer 2009;8(2):109-117 [3] Jones S, Hruban RH, Kamiyama M et al. Exomic sequencing Identifies PALB2 as a pancreatic cancer susceptibility gene. Science 2009;324:217

ANALYSIS OF GERMLINE VARIANTS IN DNA DAMAGE REPAIR GENES IN PANCREATIC CANCER IDENTIFIED BY NEXT-GENERATION SEQUENCING

S. Simpson1, A. Johns1, K. Kassahn2, N. Waddell2, J. Humphris1, S. Grimmond2, A. Biankin1 1Cancer Research Program, Kinghorn Cancer Centre/Garvan Institute of Medical Research, Darlinghurst, NSW, Australia 2Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, St Lucia, QLD, Australia

For the Australian Pancreatic Cancer Genome Initiative (APGI). For full list of investigators please see http://www.pancreaticcancer.net.au/apgi/collaborators.

The original localization of the BRCA2 gene was aided by its homozygous deletion in a pancreatic carcinoma, and BRCA2 is now known to be a high-risk gene that plays a role in a number of different cancers. It is estimated that BRCA2 accounts for up to 10% of familial pancreatic cancer (FPC) cases, and the proposed relative risk of pancreatic cancer in BRCA2 carriers ranges from 3.5 to 10 – fold1. In addition, it is proposed that up to 7% of apparently sporadic cases of pancreatic cancer can harbour pathogenic BRCA2 mutations2. There have also been descriptions of other DNA damage repair pathway genes, such as BRCA1, ATM and PALB2 playing a role in pancreatic pathogenicity in a familial setting.

The Australian Pancreatic Cancer Genome Initiative was formed in 2009 to comprehensively catalogue genomic, transciptomics and epigenomic changes in well characterized, large cohorts of pancreatic cancers as part of the of the International Cancer Genome Consortium (ICGC, www.icgc.org). Exome sequencing and copy number analysis in a clinical cohort of 142 prospectively accrued early stage sporadic pancreatic cancers has been performed to date. Detailed analysis of 70 informative tumour/germline pairs, over 40 germline positions were detected in the BRCA2 gene, many of which have not been previously demonstrated in this disease. Smaller but significant numbers of positions were also seen in BRCA1 and PALB2 genes. Many of these positions were recurrent across numerous patients and ranged from common previously described variants, to those of unknown clinical significance, to pathogenic variants of clinical importance.

An overview of the DNA damage repair gene mutation spectrum in a sporadic pancreatic cancer cohort will be described, with correlation to clinico-pathological data including family history. We also describe the identification of a pathogenic BRCA2 mutation present in an APGI participant which was returned via an ethically defensible plan. Processes and outcomes will be discussed, including predictive and subsequent positive testing of other family members and the enrollment in a pancreatic cancer screening trial.

[1] Naderi A, Couch FJ. BRCA2 and pancreatic cancer. Int J Gastointest Cancer 2002;31:99-106 [2] Goggins M, Schutte, M, Lu J, et al. Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas. Cancer Research 1996;56:5360-5364

ASSOCIATION OF EXPRESSION AND GENOTYPE OF THE HUMAN MIR146A AND MIR146B MICRORNA LOCI WITH SURVIVAL FROM EPITHELIAL OVARIAN CANCER

Cameron N. Johnstone, Sharon E. Johnatty, Jonathan Beesley, Lin Zhang, Georgia Chenevix-Trench

Background: Hyperactivation of the NF kappa B transcription factor has been implicated in progression of many human cancers, including ovarian cancer, and drugs that block NF kappa B activation are being explored in clinical trials as novel anti-cancer agents (1). The MIR146A and MIR146B genes encode closely related microRNAs that are targets of NF kappa B. They are thought to constitute a NF kappa B negative feedback loop through targeting of IRAK1 and TRAF6; important signaling molecules downstream of cytokine and Toll-like receptors (2). In this study, we explored the association between MIR146A and MIR146B expression level and survival in advanced epithelial ovarian cancer in a US patient cohort. In addition, we performed a case:control association study involving assessing a common MIR146A G>C single nucleotide polymorphism and risk of developing and surviving from sporadic epithelial ovarian cancer. Results: MicroRNA microarray data were interrogated for miR146a and miR-146b expression levels in a pilot cohort of 73 patients with advanced epithelial ovarian cancer and expression levels were validated by stem-loop qPCR. Univariate Cox proportional hazard regression modeling with global permutation test indicated that patients with higher miR-146b expression had a significantly (p < 0.05) worse overall survival (OS) and progression-free survival (PFS) than those with low expression. Expression of miR-146a was not associated with survival. In a preliminary case:control association study, case (n=1406) and control (n=1214) genomic DNAs from the Australian Ovarian Cancer Study (AOCS) dataset (3) were genotyped for MIR146A SNP rs2910164 using iPLEX technology (Sequenom Inc.). Modification of ovarian cancer risk by the minor allele of rs2910164 was not found [age-adjusted odds ratio (OR) per-allele 1.01 (95% CI, 0.89–1.15), p = 0.8]. However, when the 611 invasive cancer cases on whom we had clinical outcome data were analysed, a significant association was observed with PFS, whereby women with at least one variant ‘C’ allele (that is, G/C + C/C) had longer PFS than those with the G/G genotype (p = 0.05). This effect was significantly enhanced when only those patients that received sub-optimal debulking surgery (residual tumour > 1cm) were considered (n = 128, p = 0.004). The follow-up results from a larger cohort will be presented. The results to date suggest that both MIR146B expression level and MIR146A genotype may influence the progression of epithelial ovarian cancer and that further investigation is warranted. References: 1. Baud V, and Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat. Rev. Drug Discov. (2009) 8:33-40 2. Taganov KD, Boldin MP, Chang KJ, and Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci USA. (2006) 103:12481-12486 3. Johnatty SE, Beesley J, Paul J, Fereday S, Spurdle AB, Webb PM, Byth K, Marsh S, McLeod H; AOCS Study Group, Harnett PR, Brown R, DeFazio A, and Chenevix-Trench G. ABCB1 (MDR 1) polymorphisms and progression-free survival among women with ovarian cancer following paclitaxel/carboplatin chemotherapy. Clin. Cancer Res. (2008) 14:5594-5601

THE ASSOCIATION OF IDENTIFIED BREAST CANCER COMMON GENOMIC VARIANTS AND PATHOLOGICAL FEATURES IN INDIVIDUALS WITH HEREDITARY BREAST AND OVARIAN CANCER

Sawyer S1, Chenevix-Trench G2, Harris M3, Lindeman G4, Mitchell G1 and James P1,5. 1Peter MacCallum Cancer Institute, 2Queensland Institute of Medical Research, 3Monash Medical Centre, 4Royal Melbourne Hospital, 5Victorian Clinical Genetics Service

Background: Genome-wide association studies have identified numerous common genomic variants that are significantly associated with breast cancer risk. In recent times, investigators have begun to examine the association between breast cancer common genomic variants and pathological features such as histopath subtype, hormonal status and grade of tumour differentiation. Results from studies utilising breast cancer affected individuals from the general population have found several common genomic variants are associated with ER-positive (FGFR2, TOX3, 8q24, MAP3K1, 5p12 and 12q24) and ER-negative breast cancers (CASP8, RAD51L, ESR1). Here we assess the association of pathological features and with an extended panel of common genomic variants in index cases from high-risk Victorian Breast and Ovarian cancer families.

Aim: To examine the association of recently described common genomic variants and pathology features of breast cancer in the context of a family history.

Methods and Results: Detailed information of breast cancer pathology was reviewed for 200 of the index cases from the Victorian Familial Cancer Centre (FCC) Translational Breast Cancer Cohort. Genotyping was performed for 22 common genomic variants (SNPs) previously identified in breast cancer GWAS. Control genotype data was obtained from the Australian population (n=895, recruited from the electoral role for the AOCS). The association of common genomic variants with specific pathological features was examined by logistic regression and proportions compared for hormones status, HER2 expression and grade.

Conclusions: We will present data on the association of common genomic variants and breast cancer pathological features. Our study highlights the need for further research to determine if these associations can help us understand tumour heterogeneity and lead to improved treatment and prevention for high-risk breast and ovarian cancer families.

HIGH GRADE PIN DOES NOT DISPLAY LOSS OF HETEROZTGOSITY AT THE MUTATION LOCUS IN BRCA2 MUTATION CARRIES WITH AGGRESSIVE PROSTATE CANCER

Liam Kavanagh1,2,4, Amber Willems-Jones1,2, David Clouston3, Damien Bolton4, kConFab Investigators1,2, Stephen Fox2,5 and Heather Thorne1,2 1Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab), Research Department, Peter MacCallum Cancer Centre, East Melbourne, 3002, Australia; 2Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, 3010, Australia; 3Focus Pathology, South Yarra, 3141, Australia; 4Department of Urology, Austin Hospital, Heidelberg, 3084, Australia; 5Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, 3002, Australia.

Introduction & objectives: The risk of developing prostate cancer is increased for men carrying a pathogenic germline mutation in BRCA2. High-grade prostatic intraepithelial neoplasia (HG PIN) has been considered a precursor to prostate adenocarcinoma. This study aims to determine genetic hallmarks within normal tissue, HG PIN or invasive tissue identifying it as a precursor of tumour development and progression in this group of men.

Methods: From the kConFab biobank of high-risk breast cancer families, twenty participants were identified with a diagnosis of aggressive prostate cancer and presence of HG PIN, confirmed carriers of a family specific BRCA2 mutation, and where there was access to archival radical prostatectomy specimens. Using laser capture microscopy, we were able to micro-dissect prostate adenocarcinoma tissue, as well as HG PIN tissue and normal prostate tissue, and extract DNA from these samples. We examined for loss of heterozygosity (LOH) at the site of their family specific BRCA2 gene mutation from HG PIN using mutation specific PCR and sequencing of DNA. Prostate adenocarcinoma tissue DNA, plus normal prostate and HG PIN DNA, was sent for whole exome analysis of copy number changes across the genome.

Results: We were able to retrieve sufficient DNA from the HG PIN tissue of ten pathogenic BRCA2 carriers. No participant displayed LOH at the mutation locus within HG PIN, irrespective of whether or not the invasive tumour DNA displayed LOH. Further analysis will follow from whole exome analysis of prostate adenocarcinoma tissue from BRCA2 mutation carriers.

Conclusions: HG PIN does not appear to be a genetic precursor to the development of prostate cancer in patients who carry a family specific pathogenic BRCA2 mutation.

HOXB13 G84E MUTATION IN COLORECTAL CANCER CASES FROM THE AUSTRALASIAN COLORECTAL CANCER FAMILY REGISTRY

Daniel D. Buchanan1, Mark Clendenning1, Michael D. Walsh1, Christophe Rosty1,3,4,Joanne P Young1, Rhiannon Walters1, Sally Pearson1, Belinda Nagler1, David Packenas1, Aung Ko Win2, ACCFR Investigators, Sean Cleary5, Steve Gallinger5, John L. Hopper2, Mark A. Jenkins2. 1Cancer and Population Studies Group, Queensland Institute of Medical Research, Bancroft Centre, Herston QLD 4006, Australia, 2Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne, Carlton VIC 3053, Australia 3University of Queensland, School of Medicine, Herston, QLD 4006, Australia 4Envoi Specialist Pathologists, Herston Q4006 5 Cancer Care Ontario, Toronto, Ontario, Canada

Background: Recently, the G84E missense mutation within the HOXB13 gene has been shown to be associated with a significantly increased risk of hereditary prostate cancer. The HOXB13 gene encodes a homeobox transcription factor that is important in prostate development and is reportedly expressed in the distal colon. The aim of this study was to investigate the association between the HOXB13 G84E mutation and colorectal cancer (CRC).

Methods: A total of 600 CRC cases diagnosed before 60 years of age and 243 sex- and age- frequency matched controls from the population-based resources of the Australasian Colorectal Cancer Family Registry (ACCFR) were genotyped for the HOXB13 G84E missense mutation (rs138213197) using a Taqman allelic discrimination assay.

Results: Two CRC cases were identified as carriers of the G84E mutation (0.33%), with no carriers identified in the controls.

The first carrier developed a high grade adenocarcinoma in the sigmoid colon at 42 years of age. This carrier inherited the mutation from his father who was diagnosed with prostate cancer at 65 years of age. No other family history of cancer was reported nor was there DNA available from other relatives for testing.

The second carrier was diagnosed with a high grade adenocarcinoma in the caecum at 22 years of age. This CRC also demonstrated loss of expression of MSH6 and high levels of microsatellite instability (MSI-H) and was shown to carry a MSH6 c.3311_3312delTT p.Phe1104TrpfsX3 deleterious mutation. No DNA was available from relatives for further genotyping.

Conclusions: The HOXB13 G84E mutation does not account for a significant proportion of CRC suggesting the tumorigenic effects of this mutation may not play a significant role in CRC development.

TESTING FOR GERMLINE MUTATIONS IN POLYPOSIS – ASSOCIATED GENES BMPRIA, SMAD4, PTEN AND MUTYH IN PATIENTS WITH SERRATED POLYPOSIS

Joanne P. Young1, Mark Clendenning1, Kevin Sweet2, Michael D. Walsh1, Rhiannon Walters1, Sally Pearson1, Belinda Nagler1, David Packenas1, Genetics of Serrated Neoplasia Study Investigators, Christophe Rosty1,3,4, Susan Parry5,6, Daniel D. Buchanan1. 1Cancer and Population Studies Group, Queensland Institute of Medical Research, Bancroft Centre, Herston QLD 4006, Australia 2Division of Human Genetics, The Ohio State University Medical Center, USA 3University of Queensland, School of Medicine, Herston, QLD 4006, Australia 4Envoi Specialist Pathologists, Herston QLD 4006, Australia 5New Zealand Familial Gastrointestinal Cancer Registry, Auckland City Hospital, Auckland, New Zealand 6Department of Gastroenterology and Hepatology, Middlemore Hospital, Auckland, New Zealand

Background: Recent reports have made the observation that patients with serrated polyps, som e of whom meet the clinical criteria for serrated polyposis, are among those who carry germline mutations in genes associated with polyposis syndromes including; (1) genes known to underlie hamartomatous polyposes (SMAD4, BMPR1A; and PTEN), (2) MutYH-associated polyposis and (3) GREM1 in Hereditary mixed polyposis syndrome (HMPS). Therefore, the aim of this study was to screen patients fulfilling the current WHO criteria for serrated polyposis syndrome for germline mutations in these polyposis-associated genes.

Methods: Seventy-eight patients with serrated polyposis from the Genetics of Serrated Neoplasia (GSN) study, none of whom were of Ashkenazi descent, were screened for coding mutations in the MutYH, PTEN, SMAD4 and BMPR1A genes as follows. Standard PCR was performed to amplify DNA across exons 1-9 of PTEN (NM_000314.4), exons 2-12 of SMAD4 (NM_005359.5) and exons 3-13 of BMPR1A (NM_004329.2), and the common European variants of MutYH in exons 7 and 13. Only coding regions and directly flanking introns were sequenced. PCR products underwent clean-up using Millipore Multi-screen HTS PCR plates. Sequencing reactions were run on an ABI 3100 genetic analyser, and annotated using DNA Star Lasergene 8 software. Large deletions in PTEN, SMAD4 and BMPR1A were sought using the JPS MLPA kit from MRC Holland.

Results: We found no deleterious germline mutations in any patient who filled the current clinical criteria for serrated polyposis, and identified only a single patient with a novel variant in intron 2 (c.164+223T>C) of PTEN, predicted by in silico analysis to have no functional consequences. Testing for the recently reported duplication within GREM1 is currently being completed.

Conclusions: Genes involved in gastrointestinal hamartomatous polyposes and in hereditary mixed polyposis syndrome are not altered in individuals with serrated polyposis.

DETECTION OF LARGE SCALE 3’ DELETIONS IN THE PMS2 GENE AMONGST COLON-CFR PARTICIPANTS – HAVE WE BEEN MISSING ANYTHING?

Mark Clendenning1*, Michael D Walsh1, Judith Balmana Gelpi2, Stephen N. Thibodeau3, Noralane Lindor4, John D. Potter5,6, Polly Newcomb5, Loic LeMarchand7, Robert Haile8, Steve Gallinger9, Colorectal Cancer Family Registry, John L. Hopper10, Mark A. Jenkins10, Christophe Rosty1,11, Joanne P. Young1, Daniel D. Buchanan1 1Cancer and Population Studies, Queensland Institute of Medical Research, Herston, QLD, Australia 2Cancer Prevention Unit, Medical Oncology Department, Hospital Vall d’Hebron, Barcelona, Spain 3Department of Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA 4Department of Health Science Research, Mayo Clinic Arizona. Scottsdale, AZ, USA 5Cancer Prevention Program, Fred Hutchinson Cancer Research Centre, Seattle, WA, USA 6Centre for Public Health Research, Massey University, Wellington, New Zealand 7Cancer Research Centre of Hawaii, University of Hawaii at Manoa, Honolulu, HI, USA 8Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 9Cancer Care Ontario, Toronto, Ontario, Canada 10Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, Melbourne School of Population Health, The University of Melbourne, Parkville, VIC, Australia 11University of Queensland, School of Medicine, Herston, QLD, Australia Mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2 result in increased risk of colorectal, endometrial and other cancers, as part of Lynch Syndrome. Identification of MMR gene mutation carriers is of critical importance to ensure that appropriate genetic counselling, screening and prophylactic treatment options are offered to suspected Lynch patients. Immunohistochemical (IHC) analysis of an individual’s tumour is generally used to direct mutation detection in Lynch syndrome, with loss of expression of PMS2 only indicative of a mutation in the PMS2 gene. However, in a proportion of tumours that are deficient in PMS2, a mutation in PMS2 cannot be identified after exonic sequencing and MLPA. Until recently, the detection of large-scale deletions from the 3′ end of the PMS2 gene has not been possible due to technical difficulties associated with pseudogene sequences. The aim of this study was to determine the frequency of germline deletions in the 3’ end of the PMS2 gene in CRC-affected individuals whose tumours show solitary loss of PMS2 expression, but in whom no other germline aberrations of PMS2 have been observed. The study set consisted of 76 CRC-affected probands from the Colorectal Cancer Family Registry (Colon CFR) who were identified as being candidates for a PMS2 mutation based on the solitary loss of expression of PMS2 and an MSI-H phenotype following immunohistochemical (IHC) for the four MMR proteins and microsatellite instability (MSI) analyses (from a total of 4402 probands’ tumors had undergone IHC analysis of all four proteins). Standard mutation detection via long-range PCR and large deletion detection for the 5′ end of the gene identified pathogenic mutations in 59/76 (78%) cases. Of the remaining 17 cases whose tumors showed solitary loss of PMS2 expression but did not have a pathogenic mutation detected in PMS2, sufficient DNA was available to perform this new MLPA/long-range analysis for all but one of these unresolved cases. These remaining 16 probands suspected of harbouring mutations in the 3′ end of the PMS2 gene were screened using a new MLPA/long-range PCR-based approach (MRC-Holland MLPA kit P008-B1). Testing revealed no deletions in the 3’ end of PMS2 within these 16 cases. Therefore, we conclude that 3′ deletions in PMS2 are not a frequent occurrence in such families.

BRAF V600E IMMUNOHISTOCHEMISTRY – A NEW APPROACH TO SCREEN COLORECTAL CANCERS FOR LYNCH SYNDROME-

Michael D Walsh*1,2, Chris Toon*3,4, Angela Chou5, Adele Clarkson3, Keith Byron6, Scott Mead5, Loretta Sioson1, Stephen Clarke4,7 Capper D8,9, von Deimling A8,9, Rhiannon J. Walters1, Mark Clendenning1, Christophe Rosty1,10,11, Joanne P. Young1,11, Aung Ko Win12, John L. Hopper12, Mark A. Jenkins12, Anthony J Gill#3,4,13, Daniel D. Buchanan PhD#1 * and # These two authors contributed equally, 1 Cancer and Population Studies Group, Queensland Institute of Medical Research Brisbane , Department of Histopathology, Sullivan Nicolaides Pathology, Taringa QLD, Department of Anatomical Pathology, Royal North Shore Hospital, Sydney, NSW, Northern Cancer Translational Research Unit, Royal North Shore Hospital, Sydney, Department of Anatomical Pathology, SYDPATH, St Vincents Hospital Darlinghurst, University of Sydney, University of Sydney, Sydney, Department of Neuropathology, Institute of Pathology, Ruprecht-Karls University; Heidelberg, Germany, Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany, Envoi Specialist Pathologists, Herston, University of Queensland, School of Medicine, Herston, QLD, Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne, Carlton, University of Sydney, Sydney, NSW Australia 2006

Background: The presence of a somatic BRAFV600E mutation in a colorectal cancer (CRC) is an effective marker for excluding Lynch syndrome in a tumour that shows loss of expression of the MLH1 and PMS2 mismatch repair (MMR) proteins. The aim of this study was to; 1) compare a new immunohistochemistry (IHC) test for the BRAFV600E mutation to currently used PCR-based tests, 2) determine its utility in the identification of MLH1 mutation carriers and 3) assess the value of a novel approach that includes IHC testing for BRAFV600E and the DNA mismatch repair (MMR) proteins on a series of CRCs.

Methods: We compared BRAFV600E (clone VE1) IHC to BRAF determined by multiplex polymerase chain reaction (PCR) and MALDI-TOF spectrometry in 216 consecutive CRCs. Discordant cases were resolved by realtime PCR (rt-PCR). An additional 51 CRCs from the Australasian Colorectal Cancer Family Registry (ACCFR) that were characterized for MMR status (MMR IHC and MSI), MLH1 promoter methylation, germline MLH1 mutations and the BRAF V600E mutation using an allele-specific PCR (AS-PCR) assay were also tested to determine the utility of BRAFV600E IHC (sensitivity, specificity and concordance) for the identification of MLH1 mutation carriers. We then applied this new BRAFV600E IHC method to tissue microarrays comprising 1403 consecutive CRCs.

Results: Comparison of the results for both BRAF IHC (45/216 positive 20%) and BRAFV600E by MALDI-TOF spectrometry results (38/201 positive 19%) demonstrated a high sensitivity or positive percent agreement (PPA=97.4%, 95%CI=86.5-99.5%), specificity or negative percent agreement (NPA=96.3%, 95%CI=92.2-98.3%) and accuracy or overall percent agreement (OPA=96.5%, 95%CI=93.0-98.3%) between tests. Of the 7 initial discordant cases, rt-PCR was concordant with the BRAFV600E IHC result in 6. In the 51 CRCs from the ACCFR, IHC was concordant with allele-specific PCR in 50 cases, also demonstrating high sensitivity (PPA=100%, 95%CI=87.7-100%), specificity (NPA=96.4%, 95%CI=86.3-96.4%) and accuracy (OPA=98%, 95%CI=86.9-98%). Furthermore, we estimated the utility of a negative result for BRAF IHC “Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland

(BRAF-wildtype) in the identification of MLH1 mutation carriers, demonstrating a sensitivity of (PPA=90%, 95%CI=76.6-98.1%), and specificity of (NPA=41.7%, 95%CI=19.4-55.2%). We then performed BRAFV600E IHC and MMR IHC on 1403 CRCs arranged on TMAs, demonstrating the following phenotypes: BRAF+ve/MSS (97 cases, 7%), BRAF-ve/MSS (1030,73%), BRAF+ve/MSI (178,13%), and BRAF-ve/MSI (98,7%). BRAF+ve/MSS CRC presented with higher stage disease.

Conclusion: BRAFV600E IHC demonstrated a high level of concordance with two currently used PCR-based BRAF V600E assays and therefore, performs equally well as in the identification of MLH1 mutation carriers and Lynch syndrome. BRAF IHC however, has the advantage of being a cost-effective and easily deployable alternative to PCR-based BRAF V600E testing in a routine clinical diagnostic setting. It is for this reason that we propose an alternative approach to screening for Lynch syndrome encompassing reflex MMR and BRAFV600E IHC.

INCREASING FAECAL BUTYRATE CONCENTRATION BY INDESTION OF BUTYRYLATED STARCH IN PARTICIPANTS WITH FAMILIAL ADENOMATOUS POLYPOSIS

JM Clarke1, A Boussioutas2, T Lockett1, FA Macrae2 1CSIRO Preventative Health National Research Flagship; 2Department of Gastroenterology, Royal Melbourne Hospital

Introduction: Experimental evidence suggests dietary butyrylated high amylose maize starch (HAMSB) may reduce the risk of colorectal cancer (CRC) in rats (1;2). HAMSB can deliver butyrate to the colon of humans (3) and ingestion of 40 g/day increased faecal butyrate concentration in healthy adults (4). A Cancer Council Multi-State Research Grant was awarded to investigate the effect of HAMSB, a novel dietary chemopreventative agent, on polyposis in familial adenomatous polyposis (FAP) participants. The aim of this pilot study was to identify the optimal daily intake of HAMSB by FAP who have reduced large bowel microflora as a result of undergoing colectomy with ileo-rectal anastomosis (IRA), or total proctocolectomy with ileal pouch (IP).

Methods: Five participants with IRA and three with IP were recruited and randomly assigned to one of six treatment sequences consuming 2.5, 10 or 20 g HAMSB twice daily for 14 days. There was a seven day washout between each dose period. Faeces were collected (24 hours) at baseline and day 13 to enable measurement of faecal output, pH, moisture and free and esterified butyrate concentrations. Participants completed questionnaires assessing the effects of the starches on their gastrointestinal function and quality of life at baseline and end of each period, and 3 day weighed food diaries during baseline and each washout period.

Results: The starches were well tolerated. The change in free faecal butyrate concentrations between baseline and d13 increased with the dose of HAMSB ingested. Faecal output, moisture and pH were not affected by amount of butyrylated starch consumed.

Conclusions: This study confirms that a significant proportion of ingested esterified butyrate is released by the large bowel microflora in FAP with IRA and IP and supports the selection of this well recognised model of sporadic CRC for testing the efficacy of HAMSB and other potential CRC chemo-preventative agents that require an active microbiota to promote their bioavailability.

Key words: butyrate, colorectal cancer, familial adenomatous polyposis

References: 1. Clarke JM et al. Carcinogenesis 2008;29:2190-4. 2. Bajka BH, et al. Carcinogenesis 2008;29:2169-74. 3. Clarke JM, Bird AR, Topping DL, et al. Am J Clin Nutr 2007;86:1146-51. 4. Clarke JM, Topping DL, Christophersen CT et al. Am J Clin Nutr 2011;94:1276-83.

ANALYSIS OF THE START CODON VARIANT MLH1 C.1A>G P.(MET1?)

Michael Parsons1, Bryony Thompson1, Phillip Whiley1, Jonathan Beesley1, Adrian Weigmans1, John Hopper2, Mark Jenkins2, Colon Cancer Family Registry, Daniel Buchanan1, Amanda Spurdle1 Queensland Institute of Medical Research, Brisbane, Australia1; Centre for Genetic Epidemiology, University of Melbourne, Melbourne, Australia2

Mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2 result in increased risk of colorectal, endometrial and other cancers, as part of Lynch Syndrome. Identification of MMR gene mutation carriers is of critical importance to ensure that appropriate genetic counselling, presymptomatic screening and prophylactic treatment options are offered to suspected Lynch patients. However, guiding appropriate patient care becomes difficult for clinical geneticists when gene sequencing identifies variations of unknown clinical significance, such as missense, small in-frame insertions/deletions and intronic variants. Variants that alter the translation initiation site are a specific class of unclassified variant that might be amenable to investigation using bioinformatic predictions with supporting functional data, in addition to empirical risk data. We are investigating the clinical significance of the start codon variation MLH1 c.1A>G p.(Met1?). The family identified with this variant was participating in the Australian Colorectal Cancer Family Registry. There was limited clinical information available to assist with interpretation of pathogenicity, with no tumour MSI and MMR IHC data, and few family DNAs to genotype for segregation analysis. To further investigate this variant, we designed an in vitro Green Fluorescence Protein (GFP) reporter assay to assess whether the next three downstream ATG trinucleotide sequences are possible translation initiation sites. These alternative start sites at c.89A and c.122A are predicted to result in out of frame transcripts/truncated proteins while the start site at c.103A is in-frame and if translated would result in a 722aa protein lacking two of four ATP-binding domains. Current results indicate that the G variant within the start codon reduces the level of reporter protein translation initiation by ~90% relative to the wildtype A variant. There was no evidence for translation initiation at the next downstream ATG at c.89A. The second downstream ATG at c.103A appears to be recognised as the predominant alternative start site in the presence of the c.1A>G variant, resulting in 70-100% levels of reporter protein expression relative to that for the wildtype construct with start site c.1A. There was also evidence for limited translation initiation for the ATG at c.122A. We are currently assessing which transcripts are likely to be translated into protein in vivo, by conducting an in vitro transcription/translation assay with LCL-derived RNA from the c.1A>G proband. Our experimental study indicates that the molecular consequences of the MLH1 c.1A>G variant are complex, and that further data from carrier families will be necessary to determine its clinical significance. Our study importantly highlights the need for molecular investigation of start codon variants to determine the effect on protein translation, and to guide development of bioinformatic prediction models.

DNA MISMATCH REPAIR DEFICIENCE IN SEBACEOUS SKIN LESIONS: A LARGE CASE SERIES FROM SULLIVAN NICOLAIDES PATHOLOGY

Michael D Walsh1,2, Rhonda J Edwards1, Kevin J Whitehead1, Michael R Gattas3, and Daniel D Buchanan2 [1] Department of Histopathology, Sullivan Nicolaides Pathology, Taringa QLD 4068 [2] Cancer and Population Studies Group, Queensland Institute of Medical Research, Herston QLD 4006 [3] Clinical Genetics, Sullivan Nicolaides Pathology, Taringa QLD 4068

Muir Torre syndrome is characterized by the presence of sebaceous skin tumours (sebaceous adenomas, carcinomas, sebaceomas, and hyperplasia,) and/or multiple keratoacanthomas, as well as a personal or family history of internal malignancies. It is now recognized that many of these families are a phenotypic variant of Lynch syndrome in which there is an inherited genetic defect in one of the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2), and that mutation carriers may be identified through testing sebaceous skin tumours for loss of expression of these proteins. In many cases, however, loss of MMR expression in skin tumours may be sporadic in nature, and further study is required to determine whether clinicopathological features or other tumour molecular changes can refine the process of triaging patients for expensive genetic testing for Lynch syndrome. The aim of this retrospective study was to assess the results from screening sebaceous tumours for MMR protein expression in order to identify associations between clinicopathologic features of these skin tumours including subtypes and locations and loss of MMR expression (MMR deficiency). An audit of sebaceous tumours tested by Immunohistochemistry (IHC) for MMR expression identified 428 individuals (149 females, 279 males) with one or more lesions tested between January 2009 and April 2012, at Sullivan Nicolaides Pathology. Patients’ ages ranged from 17 to 100 years. A total of 450 skin lesions were examined: 232 sebaceous adenomas, 66 sebaceous carcinomas, 82 sebaceomas, 27 sebaceous hyperplasias, 12 sebaceous tumours NOS, as well as 14 squamous cell and 13 basal cell carcinomas with sebaceous differentiation, and 4 keratoacanthomas. Excluding unclassified sebaceous tumours, MMR deficiency was detected in a total of 129/438 (29%) lesions. Of the MMR deficient tumours 97 showed loss of MSH2 and MSH6 (75%), with MLH1/PMS2 loss observed in 21 (16%) cases, solitary MSH6 loss in 10 (8%), and PMS2 loss alone in one (1%). No statistical association was found between MMR deficiency and gender (81/279 (29%) males; 37/149 (25%) females), or patient age (68 yr MMR deficient vs. 65 yr MMR normal). The majority of tumours were located on the head and neck (366/447: 82%), while 52 (12%) were on the trunk and 19 (4%) on the limbs. While most MMR deficient tumours were located on the head and neck (84/366; 23%), MMR loss of expression was more commonly observed in lesions on the trunk (36/62; 58%) and limbs (9/19; 47%), and MLH1/PMS2 and MSH6 only losses were proportionally more common in non-head and neck sites. MMR loss was most commonly observed in sebaceous adenomas (84/232: 36%) and sebaceomas (22/82: 27%), and less frequently in other sebaceous lesions. We have identified MMR deficiency in a significant number of sebaceous skin lesions, yet it remains unclear from the current data what proportion of these patients have a germline mutation in an MMR gene and thus have Muir Torre (Lynch) syndrome.

MISMATCH REPAIR GENE ISOFORMS: ISSUES IN THE INTERPRETATION OF ABERRANT SPLICE TRANSCRIPTS

Bryony A Thompson1,2, Amanda B Spurdle1

Genetics and Computational Biology Division, Queensland Institute of Medical Research, Brisbane, QLD1; School of Medicine, University of Queensland, Brisbane, QLD2

Germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause the hereditary colorectal and endometrial cancer predisposition disorder known as Lynch syndrome. A significant number of MMR gene mutations result in aberrant mRNA splicing, including exon skipping, intron retention, and cryptic splice site usage. However, the clinical interpretation of splicing assays for MMR gene variants is complicated by the existence of multiple naturally occurring alternative mRNA transcripts. These isoforms can mask the detection of truly aberrant transcripts in RT-PCR assays, or may be mistaken as patient-specific splicing aberrations. In addition, some gene variants are reported to be associated with upregulation of naturally occurring isoforms, but the relationship between isoform expression level and cancer risk is unknown.

We conducted a comprehensive search to identify all reported naturally occurring isoforms in MLH1, MSH2, MSH6 and PMS2 to provide a catalogue for design and interpretation of splicing assays. A literature review was performed using PubMed (http://www.ncbi.nlm.nih.gov/pubmed/). Additional searches of mRNA expression data were also done using the Ensembl (http://asia.ensembl.org/) and UCSC (http://genome.ucsc.edu/) genome browsers. The InSiGHT database (http://insight-group.org) and PubMed were used to identify MMR gene variants reported to be associated with altered mRNA splicing. Searches identified 37 MLH1, 32 MSH2, 16 MSH6 and 5 PMS2 naturally occurring isoforms (51 from public databases only, including all MSH6 and PMS2 isoforms). Many isoforms are predicted to produce truncated protein. Level of isoform upregulation has been quantified in vitro for only the MLH1 ∆ exon 15, ∆ exon 16, and ∆ exon 17, with relative expression between 20-60% of the full-length transcript. Furthermore, 60 variants in MLH1 and 21 variants in MSH2 are reported to affect the expression of isoforms, none of which have been studied using truly quantitative assays.

This is the first systematic review conducted of alternate splicing isoforms in the MMR genes. It demonstrates that there are a large number of naturally occurring isoforms for the MMR genes, which may complicate interpretation of splicing assays. It also highlights the importance of quantifying isoform expression level in controls to interpret relevance of isoform upregulation in carriers of MMR gene variants.

DICHLOROACETATE REVERSES THE WARBURG EFFECT AND SENSITIZES BREAST CANCER CELLS TOWARDS APOPTOSIS

B.P. Gang1, P. Dilda2, P. Hogg2, A.C. Blackburn1 1Cancer Metabolism and Genetics Group, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia 2Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia

The Warburg effect occurs in 90% of tumours and causes a high rate of glycolysis even in the presence of oxygen, resulting in increased lactate production and reduced mitochondrial oxidation of pyruvate. Glucose metabolites are diverted to anabolic processes as a consequence, reducing pyruvate oxidation, hyperpolarizing the mitochondrial membrane potential, causing apoptotic resistance. Dichloroacetate (DCA) is a drug that can reverse the Warburg effect by inhibiting the pyruvate dehydrogenase kinases (PDKs), promoting oxidative metabolism of pyruvate. We are investigating in breast cancer cells (a) the effects of DCA on cell growth, (b) factors governing DCA sensitivity and (c) if DCA can enhance apoptosis induced by 4-(N-(S- penicillaminylacetyl)amino) phenylarsonous acid (PENAO), a novel anti-mitochondrial agent.

At 5 mM DCA (48 hr treatment) there were 3-40% less viable cells present in MDA-MB-231, MCF7, MDA-MB-468, MCF10AT1, and T-47D breast cancer cell cultures. Growth of MCF10A non- cancerous cells was not affected, showing DCA selectively targets cancer cells. The PDKs have different sensitivities towards DCA inhibition (PDK2>PDK4>PDK1>PDK3). To determine if PDKs governed DCA sensitivity, PDK expression was examined by western blotting. In T-47D cells the expression of PDK2 (Ki 0.2 mM) and low levels of PDK1 (Ki 1 mM) and 3 (Ki 8 mM) correlated with their high sensitivity to DCA treatment. In MCF7 and MDA-MB-468 cells, high expression of PDK3 (Ki 8 mM) may explain their relative insensitivity to DCA. Extracellular lactate was also reduced by 50% at 1 mM and 5 mM DCA in T-47D and MCF7 cells respectively after 24 hr, indicating reversal of the Warburg effect, correlating with the PDK profiles. Induction of PDK1 in MCF7 cells under hypoxia increased sensitivity to DCA, showing the PDK profile still correlated with DCA sensitivity.

The ability of DCA to enhance apoptosis induced by PENAO was also examined. The IC50 for PENAO (48 hr) was 3-13 µM for MDA-MB-468, MDA-MB-231, T-47D, MCF7 and MCF10AT1 cells, whereas 12 µM reduced cell viability by only 7% in the non-cancerous MCF10A cells. When combined with 5 mM DCA, the IC50 of PENAO for all cancer cell lines decreased by 15-70%, while toxicity to MCF10A cells was not increased. To measure apoptosis, cells were stained with annexin V and sorted by FACS. Treatment for 48 hr with 5 mM DCA and 5 µM PENAO doubled the proportion of apoptotic cells compared to PENAO alone on T-47D and MDA-MB-231 cells. DCA alone did not inhibit growth or induce apoptosis of MDA-MB-231 cells, thus showing potentiation of apoptosis.

We have shown that DCA reverses the Warburg effect, inhibiting growth and enhancing apoptosis. PDKs may be a useful biomarker in determining whether DCA alone will be effective against different tumour types.

VITAMIN D, CYP2RL AND BREAST CANCER SUSCEPTIBILITY IN MICE

Madara Ratnadiwakara1, Rohan Williams2, Anneke C. Blackburn1 1Cancer Metabolism and Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra ACT 0200. 2Molecular System Biology Group, John Curtin School of Medical Research, Australian National University, Canberra ACT 0200.

Vitamin D is a group of fat-soluble secosteroids. Apart from its main physiological role in calcium homeostasis, several studies have demonstrated a wide range of functions of vitamin D which can be associated with cancer. Even though the exact mechanism underlying the protective action of vitamin D against cancer is not clearly understood, there is strong experimental evidence showing that vitamin D participates in cell growth regulation, apoptosis and proliferation in normal and malignant breast cells. This evidence has lead to the hypothesis that high levels of vitamin D might reduce the risk of breast cancer.

In order to be biologically active, vitamin D must be converted to its active form 1,25(OH) 2D3. CYP2R1 is a major vitamin D hydroxylase that catalyzes the first step of this activation producing 25(OH)D3. Cyp2r1 is located within the SuprMam1 locus, a mammary tumour susceptibility locus identified in the BALB/c-Trp53+/- mouse model of spontaneous breast cancer (1). Affymetrix data comparing T cell gene expression have identified Cyp2r1 to be differentially expressed between BALB/c and C57BL/6 mouse strains (0.68 fold reduction in BALB/c, the mammary tumour susceptible strain). As lower CYP2R1 levels should result in lower vitamin D levels and may contribute to the higher cancer susceptibility of BALB/c mice, we studied the Cyp2r1 gene expression and plasma 25(OH)D3 levels in SM09 congenic-mice (BALB/c SuprMam loci in C57BL/6 background) in comparison to C57BL/6-controls.

A real-time quantitative PCR of Cyp2r1 in different tissues from SM09 and control mice revealed that there is a significant difference in Cyp2r1 expression in mammary glands of the two strains (2.76 fold reduction in SM09, p=0.04). But this difference was not observed in liver or lymph node tissues of the same mice. An affymetrix exon array on hormone stimulated mammary glands from SM09 and control mice confirmed the q-rtPCR result, where SM09 mice had a 2.04 fold reduction in Cyp2r1 expression compared to control mice (p=0.0003). Measurement of 25(OH)D 3 levels in plasma collected from SM09 and control mice were performed (RDDT, Melbourne) to identify the biological effects of differential expression of Cyp2r1. Surprisingly, the plasma levels of 25(OH)D3 were significantly higher in SM09 mice, the opposite of what was expected (59.03 vs 46.67 nM/L in SM09 vs control males, p=0.02, and 59.85 vs 52.75 nM/L in SM09 vs control females, p=0.018).

Further investigation of the major regulators of vitamin D pathway (serum levels of calcium, phosphate and parathyroid hormone) and 25(OH)D3 levels within the mammary gland is required to determine if vitamin D metabolism is contributing to BALB/c mammary tumour susceptibility .

1. Blackburn AC, Hill LZ, Roberts AL, et al. Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk. Am J Pathol. 2007; 170:2030–41.

EGFR MUTATIONS, TRIPLE NEGATIVE BREAST TUMOURS AND BRAIN METASTASES

Tatjana Seidens, Erik Tilch, Sibylle Cocciardi, Fares Al-Ejeh and Georgia Chenevix-Trench Queensland Institute of Medical Research, Brisbane

Triple-negative and basal-like breast cancers share many properties, including the upregulation of EGFR (epithelial growth factor receptor) expression. Teng et al., (2011) analysed 70 triple negative Asian breast cancers and reported EGFR mutations in 11% tumours , while we reported an EGFR mutation in 1/14 breast tumours that had metastasized to the brain (daSilva et al., 2010), as well as in 3/9 brain metastases from other primary tumours (lung and kidney). We have used the near-normal mammary epithelial cell line, MCF10A, to generate clones that over- express wildtype or mutatnt EGFR (EGFR_G719S and EGFR_E746-A750 del). We then assayed the clones in proliferation and morphological assays (2D and 3D cultures), as well as for their sensitivity to the tyrosine kinase inhibitor, gefitinib. Both EGFR mutations rendered the cell lines independent of EGF supplement, and also conferred resistance to gefitinib inhibition. In contrast, the parental and the wildtype over-expressing cell lines were hardly affected. We observed morphological features in 3D cultures, such as the filling of luminal spaces, that are characteristic of tumour cells for all three transfected cell lines but these were most distinctive for the EGFR_G719S mutant cell line. This cell line also developed filopodia-like structures. However, we have not yet observed tumours in mice injected with these cell lines. Given our identification of somatic mutations in tumours that have metastasized to the brain, it is possible that activation of EGFR may promote metastasis in mice and so we are now using the whole MCF10 series to investigate the metastatic potential in pre-malignant and tumorigenic cell lines.

EXPLORATORY TARGETED RESEQUENCING OF BRCA1 AND BRCA2 IN INHERITED BREAST CANCER

1 Michelle W. Wong, 2Simone Li, 2Marc Wilkins, 1 Kelly A. Avery-Kiejda, 1 Nikola A. Bowden, 1,3 Rodney J. Scott 1 Discipline of Medical Genetics and Centre for Information-Based Medicine (CIBM), The University of Newcastle and Hunter Medical Research Institute (HMRI), Newcastle, Australia 2 Department of 3 Division of Genetics, Hunter Area Pathology Service (HAPS), Newcastle, Australia

Breast cancer is the leading cause of cancer-related deaths in women worldwide, affecting about 13,000 women every year in Australia. Inherited loss-of-function mutations in BRCA1 and BRCA2 predispose to high risk of breast and/or ovarian cancer. Since the discovery of breast cancer susceptibility genes BRCA1 and BRCA2 two decades ago, there have not been any other genes identified that play a significant role in predisposition to inherited breast cancer. A large proportion of individuals with inherited breast cancer are negative for BRCA mutations and despite numerous research efforts, further breast cancer susceptibility genes still remain elusive.

We hypothesize that genetic anomalies are present in the BRCA1 and BRCA2 genes in a subset of individuals with inherited breast cancer where no genetic anomalies where identified using traditional Sanger sequencing. This study aims were to identify genetic anomalies in BRCA1 and BRCA2 by completely re-sequencing 200 kilobases surrounding BRCA1 and BRCA2 using targeted massively parallel sequencing, or next-generation sequencing.

For this study, DNA was used from 10 individuals referred for genetic testing after meeting the criteria for inherited breast cancer, and had been screened for BRCA1 and BRCA2 mutations by the Hunter Area Pathology Service (Newcastle, NSW, Australia). All individuals used for this study did not harbour causative genetic changes in the coding regions of BRCA1 or BRCA2. Targeted next-generation paired-end sequencing of regions containing BRCA1 and BRCA2 was performed using Agilent SureSelect and an Illumina GAIIx (The Ramaciotti Centre for Gene Function Analysis). An average of 50x coverage was achieved across the targeted genomic region for all samples. The sequence data was aligned to the Human Reference Sequence 37.2. Single nucleotide polymorphisms present in dbSNP or the 1000 Genomes Project were removed from further analyses. Genetic differences in the form of single nucleotide variants (SNVs) and insertions/deletions (indels) were identified in most individuals tested in regions that had previously remained unexplored, such as the non-coding regions of BRCA1 and BRCA2, the 5’UTR and promoter sites. This study has comprehensively investigated BRCA1 and BRCA2 and surrounding genomic regions in a mutation-negative inherited breast cancer population. The issue of accuracy of mutation detection by traditional methods, such as Sanger sequencing alone, has also been addressed by this study. The outcome of this study is the increase in current knowledge of the genetic variations that results in the development and/or progression of inherited breast cancer, aid in the management of individuals with breast cancers by providing a more specific diagnosis of disease risk and provide information required for the development of personalized treatment.

CLINICAL GENETIC TESTING FOR HEREDITARY CANCER USING NEXT GENERATION SEQUENCING

Lesley Andrews1, Bettina Meiser2, Ben Storey3, Belinda Rahman2 1Hereditary Cancer Clinic, Prince of Wales Hospital, Randwick NSW 2Psychosocial Research Department, Prince of Wales Hospital, Randwick NSW 3University of New South Wales, Kensington NSW

Genetic testing is undergoing a major transformation with the introduction of Next Generation Sequencing (NGS) to laboratories around the world. A number of Australian laboratories providing mutation searching for hereditary cancer syndromes are introducing this technology over the next year, however many of the issues associated with this type of testing are yet to be resolved. NGS will enable testing of genes which are part of current practice at lower cost and greater speed. This raises the question as to whether we should change established criteria for testing. Decisions will need to be made regarding testing of isolated cases of rarer tumours, and whether testing of unaffected relatives in families with no living affected members is appropriate. Secondly, testing for cancer predisposition is likely to involve a large panel of cancer genes. Some results may be unexpected on the clinical presentation. In addition, data regarding the risks associated with mutations in some of these genes and their optimal management may not yet be robust. Thirdly, it is likely that standard testing may soon involve exome sequencing, where the outcome may involve  identification of incidental genetic disorders of clinical and/or personal significance

 identification of possibly multiple genetic variations of unknown significance in a single genome

 identification of pathogenic variations in newly identified genes with little clinical data available to guide management. The major challenges facing us are the provision of informed consent for these outcomes, institutional policy regarding the returning of incidental findings and how to manage the vastly increased the work load for genetic counselling services generated by the increased complexity of potential results, as well as the capacity for higher volumes of testing . In order to determine the attitudes of Australian patients to these challenges, we are conducting semi-structured telephone interviews with patients of the Prince of Wales Hereditary Cancer Clinic who have previously undergone mutation searching with an inconclusive result, as this is a cohort who may be offered NGS in the near future. This study aims to explore patients’ hypothetical interest and motivations in genomic testing, perceived advantages and disadvantages, information and communication preferences regarding incidental findings, and need for assistance regarding decision making using qualitative research methods. Results of the study will be presented.

THE 10-YEAR FOLLOW-UP OF THE AUSTRALIAN BREAST CANCER FAMILY REGISTRY

Carmel Apicella1, Kelly Aujard1, Gerda Evans2, Gillian Dite1, Roger Milne1, Graham Giles3, Melissa Southey4, John Hopper, The Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Australia. 2 Breast Cancer Network Australia (BCNA) Community Representative 3Cancer Epidemiology Centre, Cancer Council Victoria, 4Department of Pathology, The University of Melbourne

Background: The Australian Breast Cancer Family Registry (ABCFR) is a resource of families, data, bio-specimens, researchers and community representatives established to conduct of collaborative research on breast and associated cancers. It is enriched for women above average risk for breast cancer, and includes 13,363 participants from 3,313 Australian families mostly recruited between 1992 and 2000. Ten years after recruitment, we conducted a follow-up of the cohort. Aims: To systematically update family history; to expand the pedigree to include first- and second-degree relatives of the participants other than the proband; to update the epidemiological data provided by participants; to collect more blood samples including renewal of exhausted samples; and to recruit more participants within these families. Eligibility: The 9,773 participants from 2,272 families who were informed at baseline interview that we might wish to contact them in the future. Methods: An invitation letter with information sheet and consent form and a short questionnaire was mailed. This was followed up with a phone call to expand on responses to the questionnaire and obtain a family history from the participant. Reponses to some questions triggered other activities, such as: retrieval of genetic testing results from clinics; obtaining pathology reports and tumour material for new breast cancer cases; and request for a blood sample. All participants were asked about their family history, sometimes resulting in multiple reports on the same individuals in the family. Interviewers reconciled discrepant information to obtain the best estimate of the family history. Pedigrees were expanded to include first- and second-degree relatives of the participants. Verification of cancers and deaths through linkage with the Victorian Cancer Registry, Australian Institute of Health and Welfare and pathology reports was conducted and is ongoing. Results: We were able to update the family history for 1982 (87%) families, and epidemiology data for 6651 (79%) participants. We recruited an additional 694 participants, obtained an additional 1,759 blood samples, and learnt about new clinical genetic testing for BRCA1 and BRCA2 mutations for 196 participants. Conclusions: We have demonstrated how high participation at follow-up after 10 years without contact can be achieved with a family design. The 10-year follow-up has added value to the cohort and the new dataset is being used in analyses and as a basis for successful NHMRC and NIH grant applications. Acknowledgements: We thank the ABCFR participants who continue to generously give their time, information and samples for this research. Funding has been received from the NIH (USA), NHMRC, Cancer Australia, NBCF and VBCRC.

THE AUSTRALASIAN COLORECTAL CANCER FAMILY REGISTRY 5-AND 10-YEAR FOLLOW UP

Mark A. Jenkins, Judi Maskiell, Aung Ko Win, Ingrid Winship, John L. Hopper for the ACCFR investigators. Centre for MEGA Epidemiology, The University of Melbourne

Introduction: The Australasian Colorectal Cancer Family Registry (ACCFR) is a resource of families, data, biospecimens, researchers and community representatives established for collaborative research on colorectal and associated cancers.

The ACCFR was established in 1996. It comprises 11,500 participants from 1,800 families either recruited via a population-based incident colorectal cancer (960 families from Melbourne), via a population-based control (270 families from Melbourne) or via a family cancer clinic (580 families from Brisbane, Sydney, Melbourne, Adelaide, Perth, or Auckland). 7920 participants have completed a detailed risk factor questionnaire at recruitment and donated a blood sample. These participants have been genetically characterised and comprise 972 carriers of a mutation in a mismatch repair gene (350 MLH1, 453 MSH2, 94 MSH6, 57 PMS2, 18 EPCAM) (308 families) and 88 carriers of a MutYH mutation (76 monoallelic and 12 biallelic) (48 families). In addition to participants, we have cancer status and demographic data for 64,500 relatives of participants.

Methods: We have attempted follow-up of participants every five years, by questionnaire, to update vital status, surgery, cancer screening, polypectomy, and personal and family history of cancer history. This has been done for all participants from population-based case families and clinic-based families. We have also linked all participants and non-participant relatives to the National Death Index and the National Cancer Clearing House to identify any additional cancers and deaths and to verify personal reports.

Results: We have 5-year follow-up on 1299 participants who had colorectal cancer at baseline (CRC cohort) and 6169 participants who did not have colorectal cancer at baseline (non-CRC cohort). We have 10-year follow-up on 628 of the CRC-cohort and 3059 of the non-CRC cohort. Total person years = 9635 for the CRC cohort and 46,140 years for the non-CRC cohort. Response for the 5- and 10-year follow-ups was 90% for individuals and 95% for families.

For the CRC cohort, since baseline, 103 have been diagnosed with a new primary CRC (11 per 1,000 person years), 184 with a new primary extracolonic cancer (19 per 1,000 person-years) and 422 have died (44 per 1,000 person years). For the non-CRC cohort, since baseline, 125 have been diagnosed with a CRC (3 per 1,000 person years), 455 with an extracolonic cancer (10 per 1,000 person-years) and 436 have died (9 per 1,000 person years).

Conclusion: The ACCFR is a part of the international Colon Cancer Family Registry of 35,000 participants and ~400,000 non-participating relatives from 17,000 families available to address clinical, genetic and public health research.

AN UPDATE ON THE IMPACT STUDY: IDENTIFICATION OF MEN WITH A GENETIC PREDISPOSITION TO PROSATE CANCER: TARGETED SCREENING IN BRCA1/2 MUTATION CARRIERS AND CONTROLS

Drew K1, Amor D4, Frydenberg M5, Harris M6, Kirk J7, Lam J8, Lindeman G9,10, McKinley J1, Murphy D3, Pachter N11, Petelin L1, Shackleton K9, Spigelman A12,13,14, Susman R15, Suthers G16,19, Taylor L8, Townshend S11, Tucker K17, Ward R18, IMPACT collaborators and Mitchell G1,2. Familial Cancer Centre, Peter MacCallum Cancer Centre, VIC 2. Sir Peter MacCallum Department of Oncology, The University of Melbourne, VIC 3. Division of Cancer Surgery, Peter MacCallum Cancer Centre, VIC 4. Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, VIC 5. Dept of Urology, Monash Medical Centre, VIC 6. Southern Health Familial Cancer Centre, Southern Health / Monash Medical Centre, VIC 7. Familial Cancer Service, Westmead Hospital, NSW 8. Dept of Urology, Repatriation General Hospital, SA 9. Familial Cancer Service, Royal Melbourne Hospital, VIC 10. The Victorian Breast Cancer Research Consortium Laboratory, WEHI, VIC 11. Genetic Services of WA, King Edward Memorial Hospital, WA 12. Hunter Family Cancer Service, Hunter New England Health, NSW 13. St Vincent's Clinical School, Faculty of Medicine, University of NSW 14. St Vincent’s Hospital, NSW 15. Genetic Health QLD, Royal Brisbane & Women’s Hospital, QLD 16. Department of Genetic Medicine, Women’s & Children’s Hospital, SA 17. Hereditary Cancer Clinic, Prince of Wales Hospital, NSW 18. Dept of Medical Oncology, Prince of Wales Hospital, NSW 19. University of Adelaide, SA

IMPACT is a worldwide prostate cancer screening study of men with germline BRCA mutations and the Australian arm has now been underway for over 5 years. It comprises 5 years of annual serum PSA screening with prostate biopsy triggered at a PSA >3ng/ml. At April 2012, 1457 men from 45 centres across 15 countries had been recruited (BRCA1=488, 33%; BRCA2=500, 34%; Controls=462, 32%), with the overall aim to enrol 1000 carriers and 850 controls. Australia’s contribution makes up ~20% of the cohort, having recruited 299 of these men from 10 centres across VIC, NSW, SA, QLD, TAS and WA. Of the 171 men within the worldwide cohort who have had an elevated PSA, 129 underwent a prostate biopsy. Pathology for 25 biopsies is pending, but prostate cancer was diagnosed in 42/114 giving a positive predictive value (PPV) of PSA screening in this cohort of 36.7% (39.5% for mutation carriers only; 45.5% for BRCA2 mutation carriers only). Ten of the cancers have been from the Australian cohort. Of these, 7 were in mutation carriers (1 BRCA1; 6 BRCA2), 3 in controls; 6 were clinically significant enough to treat with a prostatectomy, 1 had radiotherapy and 1 had Brachytherapy. This initial data shows that the PPV of PSA screening in BRCA mutation carriers is relatively high and supports the hypothesis that men with BRCA mutations are at increased risk of prostate cancer and that PSA screening can detect clinically significant prostate cancer in this group. These results support the rationale for prostate cancer screening in such men and the importance of the IMPACT study in developing an effective method of screening for this group. A recent addition to the study design is a voluntary end of study (EoS) biopsy after 5 years of annual PSA screening if the participant has not already required a biopsy as a result of a raised PSA. Of the 3 Australian EoS biopsies (all mutation carriers), 2 returned a cancer diagnosis and 1 showed atypical cells suspicious of (but not definitive for) cancer. No cancers have been detected in 23 other EoS biopsies performed in the overseas cohort. These biopsies will enable an evaluation of the prevalence of prostate cancer in men with lower PSA levels, determine the most appropriate PSA thresholds for biopsy and help refine prostate cancer risk estimates for this population – essential for accurate genetic counselling and risk management for this population.

THE KCONFAB EXPERIENCE – 15 YEARS OF BIOBANKING

Heather Thorne, Eveline Niedermayr, Lynda Williams, Lana Djandjgava, Kylie Ioculano, Carla Osinski, Genna Glavich, Sarah MacRaild, and the kConFab research nurses on behalf of the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab). kConFab, Research Department, Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne, VIC, 3002. kConFab, the Australian/New Zealand consortium for research into families at high risk of breast and ovarian cancer, has completed collection & recruitment of 1,487 families during the past 15 years. Biological material, genetic, epidemiological, and psychosocial data are collected from affected and unaffected, female and male participants over the age of 18. This material is available to peer reviewed, ethically approved and funded research projects. kConFab has previously and currently supplied biospecimens and/or data to 121 research projects worldwide.

The kConFab biological repository contains blood specimens from a total of 12, 642 participants and 234 best friend controls. The standardized blood processing protocol produces plasma, non lymph, blood pellet and white blood cell fractions. White blood cells undergo EBV transformation which can be used by in functional assays or as a replacement source of DNA/RNA. To date, 1560 unique EBV cell line transformations are available.

As of Jun 2012, 97% of completed kConFab families have had genetic testing; identifying 40% of families with a pathogenic, large genomic rearrangement (LGR) or splice site mutation in either BRCA1 or BRCA2. An additional 5% of completed families carry unclassified variants in BRCA1 or BRCA2; with a further 0.9 % with mutations in the ATM, CHEK2 or TP53 genes. Of the 1702 female participants who harbour a germline mutation, 56% are affected with breast or ovarian cancer. kConFab has collected a total of 923 fresh tissue collections, including prophylactic mastectomy and oophorectomy specimens; and has a large collection of archival specimens. The tissue bank consists primarily of breast, ovarian and prostate tissue (tumour and normal), with a small proportion of other tissues. Following collection, a full research pathology review is conducted, wherein features such percentage tumour, normal epithelial, lymph and necrotic components are scored. kConFab has constructed a total of 29 tissue microarrays (TMAs) (both sporadic and familial tumours) from our tissue bio bank. Where possible, tumour is matched to normal from the same archival block. kConFab are currently working to supplement our glass slide archive with a digital slide repository. This will provide researchers with high resolution, high quality whole slide digital images for ease of transport, storage, review and analysis. Currently we have >2000 slides scanned for more than 1200 participants.

The kConFab resource enables researchers to answer important questions relating to familial aspects of breast cancer. Information about the kConFab resource and the application process is available on the web site (http://www.kconfab.org). “Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland

IMMUNOHISTOCHEMISTRY FOR 616 AND THE IDENTIFICATION OF MLH1 MUTATION CARRIERS IN COLORECTAL CANCER DEMONSTRATING LOSS OF MLH1 AND PMS2 EXPRESSION

Sally Pearson1, Michael D. Walsh1, Mark Clendenning1, Rhiannon Walters1, Belinda Nagler1, David Packenas1, ACCFR Investigators, John L. Hopper2, Mark A. Jenkins2, Joanne P. Young1, Christophe Rosty1,3,4, Daniel D. Buchanan1, 1Cancer and Population Studies Group, Queensland Institute of Medical Research, Bancroft Centre, Herston QLD , 2Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, School of Population Health, University of Melbourne, Carlton , 3University of Queensland, School of Medicine, Herston, QLD , 4Envoi Specialist Pathologists, Herston

Background: Diagnostically, the presence of MLH1 methylation and/or the somatic BRAFV600E mutation in colorectal cancers (CRC) that demonstrate loss of expression of the MLH1 and PMS2 mismatch repair (MMR) proteins largely excludes the presence of a MLH1 gene mutation and the diagnosis of Lynch syndrome. Recently, an immunohistochemistry (IHC) test for p16 expression was reported to improve the effectiveness of tumour screening for Lynch syndrome. We assessed the utility of p16 IHC as a screening tool for the identification of MLH1 mutation carriers.

Methods: Individuals with CRC (n=42) recruited from both population- and clinic-based resources from the Australasian Colorectal Cancer Family Registry (ACCFR) were characterized for MMR status (MMR IHC and MSI), MLH1 promoter methylation, the BRAFV600E mutation and for germline MLH1 mutations. IHC for p16 expression was conducted on whole tissue sections. Staining results were dichotomised to represent loss of expression versus other p16 expression patterns.

Results: CRCs (n=42) were grouped into the following categories based on molecular and genetic testing: 1) MLH1/PMS2 deficient CRCs that were from MLH1 germline mutation carriers (n=18, 42.9%), 2) MLH1/PMS2 deficient CRCs with MLH1 methylation (n=13, 31%) with (9/13) and without (4/13) the BRAFV600E mutation and 3) MMR-proficient/MSS CRCs with BRAFV600E mutation (n=11, 26.1%). Loss of p16 expression was identified in 23.8% of the CRCs (10/42) and was associated with a significantly older mean age at CRC diagnosis (p<0.001, 63.1 ± 8.9 (SD) years versus 48.1 ± 11 years). CRCs from all 3 categories demonstrated loss of p16 expression: MLH1 mutation carriers (4/18, 22.2%), MLH1 methylated, MSI-H CRCs (5/13, 38.5%) and BRAF- mutated, MSS CRCs (1/11, 9.1%). We found no significant association between loss of p16 expression and MLH1 methylation (p=0.14, OR=3.0, 95%CI=0.55-16.9), BRAF V600E-mutated CRC (p=0.37, OR=1.9, 95%CI=0.37-10.4) or right-sided tumour site (p=0.31, OR=2.4, 95%CI=0.37-10.4). Comparing all MLH1/PMS2 deficient CRCs (n=31), the positive predictive value (PPV) of loss of p16 expression for identifying MLH1 methylated, MSI-H CRC was 55.6% (95%CI=25.2-82.7%) with a negative predictive value (NPV) of 63.6% (95%CI=51.2-74.7%). In comparison, BRAFV600E mutation testing demonstrated a PPV=100% (95%CI=51.2-74.7%) for MLH1 methylated CRC, and NPV =81.8% (95%CI=51.2-74.7%).

Conclusions: In this study, IHC for p16 was not associated with features of sporadic MSI-H CRC including, MLH1 methylation and the BRAFV600E mutation. BRAFV600E mutation was more effective than IHC for p16 expression in distinguishing MLH1/PMS2 deficient CRCs resulting from MLH1 methylation from those resulting from MLH1 mutation carriers. “Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland

TUMOUR MORPHOLOGY OF EARLY-ONSET BREAST CANCER CANCERS PREDICTS BREAST CANCER RISK FOR FIRST-DEGREE RELATIVES

Gillian S. Dite1, Enes Makalic1, Daniel F. Schmidt1, Graham G. Giles1, 2, John L. Hopper1, Melissa C. Southey3 1Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Australia; 2Cancer Epidemiology Centre, Cancer Council Victoria, Australia; 3Department of Pathology, The University of Melbourne, Australia.

Background: We previously found that tumour morphological features predict BRCA1 mutation carriers among women with early-onset breast cancer better than family history and standard immunohistochemistry. We now hypothesised that risk of breast cancer could be predicted by tumour morphological features for relatives of women with early-onset breast cancer, including relatives of women without a detectable germline mutation in a known breast cancer susceptibility gene.

Methods: We studied mothers and sisters of a population-based sample of 452 index cases with a first primary invasive breast cancer diagnosed before the age of 40 years and for whom a standardised tumour morphology review had been conducted. Standardised incidence ratios (SIR) were calculated by comparing the number of relatives with breast cancer to the number expected based on Australian incidence rates. Hazard ratios were calculated using Cox proportional hazards modelling.

Results: Absence of extensive sclerosis, extensive intraductal carcinoma, absence of acinar and glandular growth patterns, and presence of trabecular and lobular growth patterns were independent predictors with between 1.8 and 3.1–fold increased risk for relatives (all p < 0.02). Excluding the 77 (17%) index cases with genetic or epigenetic causes, independent predictors included minimal sclerosis, circumscribed growth, extensive intraductal carcinoma and lobular growth pattern, all with between 2.0 and 3.3–fold increased risk for relatives (all p < 0.02). Relatives of the 128 (34%) index cases with none of these features were at population risk (SIR = 1.03, 95% CI = 0.57–1.85), while relatives of the 37 (10%) index cases with two or more features were at high risk (SIR = 5.18, 95% CI = 3.22–8.33).

Conclusion: There is such a wide variation in risks for relatives based on tumour characteristics that early-onset cases without a known genetic predisposition can be divided into three groups for whom the breast cancer risks for relatives are the same as for the three categories in the Guide for Health Professional on Familial Aspects of Breast and Ovarian Cancer developed by the National Breast and Ovarian Cancer Centre. This should be important for genetic counselling, prevention and screening. It adds support to the concept that morphology review of a few key measures could be used to better inform families of their risks of breast cancer, and therefore be considered as an integral part of a breast cancer family genetics service. The identification of a group of relatives at high risk of breast cancer could also help discover new breast cancer susceptibility genes.

DOES RNA ANALYSIS MAKE A DIFFERENCE?

Christopher Manning1, Graeme Suthers1, 2, Lesley Rawlings3, ScottGrist4 and Nicola Poplawski1, 2 1. SA Clinical Genetics Service, SA Pathology (WCH site), North Adelaide, SA 5006 Australia 2. University of Adelaide, Adelaide, SA 5008 Australia 3. Molecular Pathology, SA Pathology, Frome Road, Adelaide SA 5000 Australia 4. Molecular Pathology, SA Pathology, Flinders Medical Centre SA 5042 Australia

BACKGROUND: Variants of unknown significance (VUS) are identified in many genes responsible for familial cancer syndromes. Most VUS have only been studied at the genomic level. Analysis of RNA size, sequence, and abundance can elucidate the effect of VUS on mRNA expression and hence their likely pathogenicity1–3. Since 1998 the Familial Cancer Unit has performed RNA analyses on selected cases involving splice sites, exon deletions and exon duplication events of uncertain significance.

AIMS: 1. To audit the RNA studies performed in clients of the SA Clinical Genetics Service who have a VUS in BRCA1, BRCA2, or the DNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. 2. To identify the proportion of RNA studies which altered the clinical management of the family in which the VUS was identified.

METHOD: A clinical audit of the clinical database from 1 January 1998 to 31 December 2011.

RESULTS: Study population: In the study period 398 probands were screened for MMR mutations and 38 VUS were identified. In the same period 1540 probands were screened for BRCA1/2 mutations and 163 VUS were identified.

Results: 9 (24%) of the patients with an MMR VUS had RNA studies. Studies of RNA size and abundance were normal in 4 (45%) and equivocal in 2 (22%); the classification of these variants was not altered by this analysis. However, the remaining 3 variants (33%) exhibited unequivocal abnormalities of RNA size or abundance such that the VUS was reclassified as pathogenic.

20 (12%) of the patients with a BRCA1/2 VUS had RNA studies. Studies of RNA size and abundance were normal in 8 (40%) and equivocal in 6 (30%); the classification of these variants as VUS was not altered by this analysis. However, the remaining 6 variants (30%) exhibited unequivocal abnormalities of RNA such that the VUS was reclassified as pathogenic

Overall a VUS was reclassified as pathogenic in 1/3 of cases in which RNA analysis was performed. These involved 3 splice sites, 5 deletions, and 1 duplication event.

CONCLUSION: In one third of those tested, RNA analysis clarified the interpretation of the VUS. This allowed predictive genetic testing in close relatives, confirming that there is utility associated with RNA studies. The outcome of such RNA studies should be added to the available mutation databases.

A RESOURCE FOR BREAST CANCER RESEARCH

Lisa Devereux1, Gillian Mitchell1, Anne Kavanagh2, Bruce Mann3, Stephen Fox1, Vicki Pridmore4, Rhonda Mawal1, Jaymes Charlesworth5, Kelly Aujard5, John Hopper5, Ian Campbell1

Affiliations: 1. Peter MacCallum Cancer Centre 2. Key Centre for Women’s Health, Gender & Society, University of Melbourne 3. The Royal Melbourne and Royal Women’s Hospital 4. BreastScreen Victoria 5. Centre for MEGA Epidemiology, University of Melbourne

Background Lifepool is a prospective cohort of Victorian women. The project is funded by the National Breast Cancer Foundation and is an open resource. Lifepool aims to recruit 100,000 women by the end of 2014.

The Resource Lifepool participants provide health and lifestyle information through completion of a baseline questionnaire. Data is collected on factors contributing to breast cancer risk across the following headings: Personal medical history, family history of cancer, reproductive history, menopause, surgery, use of hormone replacement therapy and other medication use, smoking habits, alcohol consumption and ionising radiation exposure. Mammographic density calculations will be available on all participants as the women give consent for lifepool to access their mammograms from BreastScreen Victoria and other providers. Cancer incidence is tracked through linkage with the Victorian Cancer Registry and participants also give written consent for linkage with the Medicare and Pharmaceutical Benefits databases to track cancer assessments and diagnoses and consequent health service utilisation. As 1 August 2012, the resource has 21,011 participants. The average age of lifepool participants is 60 years. The vast majority of participants do not have breast cancer at the time of entry into lifepool.

Participants indicate consent for future contact to allow completion of further questionnaires and also to consider donating DNA for research. To date, the lifepool resource holds 967 peripheral blood and 129 saliva samples from women on whom cancer diagnosis can be ascertained with certainty through linkage and from whom health and lifestyle information is provided. The lifepool resource is open to application from national and international researchers. Data and biospecimens are available to support research into breast cancer and other women’s health issues, subject to review by the NBCF Access Committee and appropriate Human Research Ethics Committee approvals.

For further information please contact [email protected] or visit the website www.lifepool.org

#45 CONTRALATERAL PROPHYLACTIC MASTECTOMY – WHAT MAKES A SURGEON RECOMMEND REMOVING A NORMAL BREAST?

Christobel Saunders and Toni Musiello, School of Surgery, University of Western Australia [email protected]

Background: Contralateral prophylactic mastectomy (CPM) is the removal of the opposite, unaffected breast in patients with unilateral breast cancer (BC). This may be logical in women carrying a BRCA mutation who have up to a 75% lifetime risk of a second cancer on the opposite side (1) but there is no known survival benefit in normal risk women (2) with an annual risk of contralateral BC in older women around 0.25% (3). Yet the rates of CPM are increasing significantly . Whilst increasing awareness of high risk genes and enhanced availability of genetic testing may partially explain this increase, the vast majority of CPMs are performed on women with sporadic BC without susceptibility genes (3). Patients report “doctor’s advice” as the main reason for deciding on a CPM . Few studies have examined surgeon characteristics influencing recommendations for CPM. We investigated breast surgeons’ perceptions, knowledge and attitudes towards CPM, and reasons for recommending it. Methods: A cross sectional study of 220 surgeons in BreastSurgANZ using a self-report on-line questionnaire Results: Response rate was 37% (a third female). 44% of surgeons perceived that the rates of CPMs they performed had increased over the last year, with half performing over 5 CPMs per year. CPM rates were found to be unrelated to surgeons’ age (P=0.773) or gender (P=0.941). The majority reported they discussed CPM with 5-20% of their patients and a third of discussions are with women with no risk factors for contra-lateral cancer. Interestingly 57% of surgeons felt the side effects of a second mastectomy in these low risk women outweighed any benefit, yet most surgeons felt these discussions were increasing in number. The top three reasons a surgeon recommended CPM included 2 objective risk factors: BRCA mutation and family history of breast cancer, and 2 subjective patient factors: fear/anxiety of a new breast cancer and desire for symmetry. Conclusions: Most surgeons use BRCA status and family history as the main reason for recommending CPM but the third most common reason is patients’ fear and anxiety. This raises the question of whether it is advisable for surgeons to recommend an irreversible, surgical procedure to relieve fear and anxiety, rather than using alternative management options, including treatment with preventative agents such as Tamoxifen, accurate communication of risk, or psychological interventions to reduce anxiety. Breast cancer specific survival appears to have little impact on either surgeons’ or patients’ decision making regarding CPM, in line with evidence.We intend to develop decision algorithms to assist both the patient and her surgeon in making an informed decision regarding CPM. References: 1. Blackwood M et al. JCO 1998;16(5):1969-77 2. Peralta EA et al Am J Surg 2000; 180(6):439-45 3. King TA et al JCO 2011; 29(16):2158-64 4. Yao K et al. Ann Surg Onc 2010;17(10):2554-62 Arrington AK et al. Ann Surg Onc 2009; (10):2697-704

Delegates List 2012

Aamira Huq [email protected] Adrienne Sexton [email protected] Agnes Bankier [email protected] Alex Metcalf [email protected] Alexandra Lewis [email protected] Alison Trainer [email protected] Amanda Spurdle [email protected] Amber Johns [email protected] Amy Pearn [email protected] Alina Stoita [email protected] Anna DeFazio [email protected] Anna Nash [email protected] Annabel Goodwin [email protected] Annabelle Ng [email protected] Anneke Blackburn [email protected] Annette Hattam [email protected] Antonis Antoniou [email protected] Ashley Crook [email protected] Aung Ko Win [email protected] Belinda Creighton [email protected] Belinda Nagler [email protected] Bettina Meiser [email protected] Brenda Greyling [email protected] Briony Patterson [email protected] Brett Kennedy [email protected] Bronwyn Cook [email protected] Brooke Brewster [email protected] Bruce Hopper [email protected] Bryony Thompson [email protected] Carla Osinski [email protected] Camron Ebzery [email protected] Charlotte Slade [email protected] Chol-Hee Jung [email protected] Christobel Saunders [email protected] Clare Scott [email protected] Cliff Meldrum [email protected] Daniel Buchanan [email protected] Daniel Park [email protected] David Packenas [email protected] David Thomas [email protected] Deborah Weston [email protected] Deborah Neklason [email protected] “Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland Dragana Prodanovic [email protected] Diana Moynihan [email protected] Christopher Manning [email protected] Driss Ait Ouakrim [email protected] Ella Thompson [email protected] Eveline Niedermayr [email protected] Finlay Macrae [email protected] Fiona McKenzie [email protected] Gemma Correnti [email protected] Georgia Chenevix- Trench [email protected] Geoffrey Lindeman [email protected] Gillian Mitchell [email protected] Graeme Suthers [email protected] Heather Thorne [email protected] Helen Mar Fan [email protected] Hilda High [email protected] Ian Campbell [email protected] Ian Collins [email protected] Ian Tomlinson [email protected] James Flanagan [email protected] Jacquie Armstrong [email protected] Jan Sullivan [email protected] Jan Wakeling [email protected] Jane Garrad [email protected] Jasmin Mar [email protected] Jessica Taylor [email protected] Jeff Gleeson [email protected] Jillian Hung [email protected] Joanne Young [email protected] Jodie Painter [email protected] John Heath [email protected] John Hopper [email protected] John Parisot [email protected] Jonathan Beesley [email protected] Judy Kirk [email protected] Julie McGaughran [email protected] Jun Li [email protected] Karen Crowe [email protected] Kate Crough [email protected] Kate Mahendran [email protected] Kate Drew [email protected] Kate Riley [email protected] Kate Thompson [email protected] Kathy Tucker [email protected] Kathryn Alsop [email protected]

“Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland Khalid Mahmood [email protected] Kim Riddell [email protected] Kimberley Gamet [email protected] Kirsty Mann [email protected] Kitty Cho [email protected] Kristy Barnes-Cullen [email protected] Kylie Gorringe [email protected] Kylie Shackleton [email protected] Laney Lindor [email protected] Lara Lipton [email protected] Lara Petelin [email protected] Leonie Noon [email protected] Lesley Andrews [email protected] Linda Cicciarelli [email protected] Lindsay Fowles [email protected] Lisa Devereux [email protected] Lisa Huang [email protected] Logan Walker [email protected] Louise Heiniger [email protected] Lucy Holland [email protected] Lynda Williams [email protected] Lyn Schofield [email protected] Lynne Purser [email protected] Madara Ratnadiwakara [email protected] Maira Kentwell [email protected] Mandy Ballinger [email protected] Manju Salaria [email protected] Margaret Tassell [email protected] Margaret Gleeson [email protected] Marion Harris [email protected] Mark Clendenning [email protected] Mark Jenkins [email protected] Marni Giles [email protected] Martin Delatycki [email protected] Mary McPhillips [email protected] Mary Shanahan [email protected] Mary-Anne Young [email protected] Megan Cotter [email protected] Melanie Boon [email protected] Melissa Brown [email protected] Melissa Southey [email protected] Michael Bogwitz [email protected] Michael Field [email protected] Michael Parsons [email protected] Michael Walsh [email protected] Michelle Bowman [email protected] “Familial Cancer 2012: Research and Practice” August 2012 Mantra Resort, Queensland Michelle Wong [email protected] Monique Topp [email protected] Ngaire Knight [email protected] Nicola Poplawski [email protected] Paul James [email protected] Phillip Whiley [email protected] Prue Cowin [email protected] Prue Weideman [email protected] Robyn Ward [email protected] Rachel Susman [email protected] Rachel Waller [email protected] Rachel Williams [email protected] Rebecca Driessen [email protected] Rhiannon Walters [email protected] Risha Zia [email protected] Robert MacInnis [email protected] Robyn Sayer [email protected] Rony Duncan [email protected] Sally Hunter [email protected] Sally Jackson [email protected] Sally Pearson [email protected] Sarah Jordan [email protected] Sarah Sawyer [email protected] Serguei Kovalenko [email protected] Sharon Johnatty [email protected] Shona O'Connell [email protected] Sian Fereday [email protected] Sian Greening [email protected] Simone McInerny [email protected] Skye Simpson [email protected] Sophie Walker [email protected] Sue Healey [email protected] Stephen Fox [email protected] Tatjana Seidens [email protected] Tish Silberbauer [email protected] Tony Proietto [email protected] Tony Roscioli [email protected] Toula McArdle [email protected] Tu Nguyen [email protected] Vicki Fennelly [email protected] Vivianne Geldard [email protected] Xiaoqing Chen [email protected] Yoke-Eng Chiew [email protected]