Breast Cancer Screening

1. Breast Cancer Screening Yvonne Wallis SCOBEC & Birmingham Training day 6th June 2007

2. Breast Cancer • Incidence 1 in 8-12 (general population) • Familial cases account 5-10% • ~1/3 caused by single genes • BRCA1 (17q21) • BRCA2 (13q12.3) • Small proportion of cases but important group as early intervention will save lives

3. Genetic disorders associated with breast cancer susceptibility • Breast/ovarian cancer (BRCA1/BRCA2) • ~5% familial cases • Li-Fraumeni syndrome • Lynch type 2 syndrome • Cowden disease • Peutz Jegher syndrome • Ruvalcaba-Myre -Smith syndrome • Klinefelter syndrome • Number of genes which increase likelihood of breast cancer (e.g. CHEK2 and BRIP1)

4. BRCA1-associated cancers: lifetime risks Breast cancer: 50%-80% Second primary: 40%-60% Ovarian cancer: 20%-40% No clear evidence for increased risk at other sites

5. BRCA2-associated cancers: lifetime risks Breast cancer(40%-80%) Male breast cancer (6%) Ovarian cancer (10%-25%) Increased risk of prostate, pancreatic, gallbladder/bile duct, gastric, head and neck cancers and melanoma

6. Breast cancer survival rates in UK Prognosis not significantly different between mutation carriers and controls

7. Risk reducing steps for BRCA1/2 gene mutation carriers • Increased Surveillance for Breast Cancer • Self, mammography, MRI from 18 years • Increased Surveillance for Ovarian Cancer • Transvaginal ultrasound, serum CA-125 from 25 years • Preventative drug therapies for BRCA & OVCA • Tamoxifen, oral contraceptive • Prophylactic bilateral mastectomy • Reduces BRCA risk by >90% • Prophylactic bilateral oophorectomy • Reduces OVCA risk by >95% • Reduces BRCA risk by 50% if performed pre-menopausally

8. BRCA1 gene & protein • Large gene with 24 exons • 1863 amino acids (220 kd) • Nuclear phosphoprotein • Two recognizable protein domains • N-terminal RING finger domain (facilitate protein-protein and protein-DNA interactions) • C-terminal BRCT domain (found in DNA repair proteins)

9. BRCA2 gene & protein • Large gene with 27 exons • 3418 amino acids (380 kd) • Nuclear protein • No recognizable motifs • Share number of functional similarities with BRCA1

10. Caretaker Function • BRCA1 involved in DNA repair and cell cycle regulation • BRCA2 involved in DNA double strand break repair in conjunction with BRCA1 • Caretakers-maintain genome integrity

12. Aim of molecular diagnostic breast cancer screening service • To confirm a clinical diagnosis • Less straightforward than other cancers, eg FAP • To offer presymptomatic testing to at-risk relatives • Therefore prevent early death from cancer • Risk reducing steps

13. Screening BRCA genes: Problems • The genes are large • Mutations are primarily “private” • Detection of mutations involves a large amount of work: technical & analysis • Before White paper funding screening was time consuming with long reporting times

14. Screening BRCA genes: requirements • NICE guidelines stipulate all women with 20% risk of carrying BRCA gene mutations must have access to testing • Mutation analysis methodology used should be close to 100% sensitivity • Screening must be performed within 8 week turnaround time (White paper)

15. In response to these requirements • Referrals through clinical genetics unit (CGU) • Designed new plate-based sequencing screening strategy for BRCA genes: • Amenable to automation at every stage • Implementation new equipment & software for HT: • Two 8-channel Beckman NX robots • 96-channel Beckman FX robot • Two 3730 48-capillary sequencers • Mutation Surveyor

16. BRCA Screening Strategy • 3 patients per plate model • Facilitate short turnaround times • TaT less dependent on patient numbers • Backlog & new samples • 63 new BRCA primer pairs designed • Amplify under identical PCR conditions • Covered coding regions of 2 genes • 2 PCR plates designed to screen 63 fragments • Primer-spotted PCR plates used to streamline the amplification process

17. Two plates for 2 genes

18. 63 BRCA PCR Fragments per patient Plate B: BRCA1 + BRCA2 exon 10 Plate A: BRCA2 (except exon 10) Fragment sizes range from 243bp to 896bp

19. DNA sample activated If needed Plate-based Sequencing Report issued MLPA PCR using primer-spotted plates Gap fill PCRs Pre-PCR NX Log sheet entry Mini gel check On-screen checks x2 EXOSAP Post-PCR NX Mutation Surveyor project & archive 2-directional Sequencing set up Post-PCR NX Sequencing analysis & archive Bead clean up Post-PCR FX 3730 sequencing BRCA gene screening workflow

20. Normal Variant Variant confirmed No Result Result management-Patient log sheets All transfers to sheet checked Report issued upon completion

21. Reports to date • 901 reports issued since September 2005 • 459 backlog • 442 new samples (since Jan ‘06) • Average turnaround time for 442 new referrals: • 23.4 days • Average turnaround time for 176 external referrals: • 21.5 days

22. Diagnostic referrals and turnaround times* * 442 new BRCA diagnosis samples from January 2006 to May 2007

23. Range of Reporting Times for BRCA Diagnostic Samples* N=442 Mean 23.4 days 48% 37% 14% 1% *All new referrals from Jan 2005 to May 2007

24. BRCA Screening Results-901 Reports Pathogenic mutations: 163 (18%) Missense/UVs: 145 (16%)

25. BRCA Presymptomatic Reports* * Issued from West Midlands Regional Genetics Laboratory

26. Unclassified Variants • Pathogenicity unclear • Missense mutations • Synonymous changes • Intronic nucleotide variants • Potentially disease-causing • Missense affect protein function • Missense/synonymous/intronic variants could cause aberrant splicing • Cannot confidently offer predictive testing

27. Interpretation of BRCA1/2 unclassified variants • Current approach: • BIC database (breast cancer information core) • Web based literature search • Segregation analysis within families • In silico splice site prediction programmes • RNA studies • Used to determine if exons lost/introns incorporated compared to normal controls • Very useful approach but only accounts for small % of UVs

28. Typical BRCA missense family UV on BIC No information on web Does not cause aberrant splicing Missense segregates Cannot offer presymptomatic testing p.Thr790Ala BRCA1-11

29. UV interpretation best practise: pathogenic or not? • Develop an evaluation form looking at multiple properties of each UV to help assess pathogenicity: • Web-based searches • Population frequency • Evolutionary conservation • Severity of amino acid substitution • Splice prediction software • Co-segregation studies • Family studies: de novo & cis or trans analysis • Tumour studies