1. The impact of genetics on breast cancer
William D. Foulkes MBBS PhD FRCPC
Department of Human Genetics
McGill University
2015 Joint Congress on Medical Imaging and Radiation Sciences
May 28, 2015
Montreal, QC, Canada

2. Preamble
This presentation will discuss the relevance of genetic evaluation in the prevention, diagnosis and treatment of breast cancer
Learning objectives
Consider the importance of a genetic evaluation for women with breast cancer
Identify some of the genetic tests on offer for breast cancer susceptibility

3. Outline of this presentation
Who gets referred to genetics and why?
Genetic evaluation – what does it involve
- standard model
- newer approaches
3. Can genetics be used to prevent breast cancer?
4. Can genetics be used to help diagnose breast cancers early?
5. Can genetics assist in treatment decisions?
6. What new genetic tests are on offer and how should they be evaluated?

4. 1. Practical breast cancer genetics
Who to refer to genetics …why…and who test….
1. Practical breast cancer genetics

5. Familial Breast Cancer
Women can be classified as
average (population) risk, (<17%)
moderate risk (2-3x higher than pop. risk) (17-30%)
high risk (> 3 times population risk) (>30%)
family history is an important predisposing factor for development of breast cancer
However, for most women, increasing age is the greatest risk factor

6. Who to test? Risk assessment Provincial standards of practice
Computer models
Empiric models
Clinical judgement
Provincial standards of practice
Consensus guidelines
Commercial testing
The “10% rule”

7. How frequent are BRCA1/2 mutations in young women with breast cancer?
Depends on how young you are
Where you live, but more importantly…
Your ethnicity/population group membership

8. Genetic evaluation pitfalls
Things to look out for…that might obscure a genetic diagnosis….

10. Inability to confirm diagnosis

11. Premature death in a gene carrier

12. Non-penetrance

13. Male transmission of a condition affecting mainly women

14. Prophylactic surgery in gene carriers

15. Small families
Founder effects in ethnic groups
Adoption
Non-paternity
Family conflicts/estrangement

16. Cancers on both sides of family Ethnic origin
Be aware of:
Male transmission
Cancers on both sides of family
Ethnic origin
Small families/preponderance of males
No living affected relatives

17. Barriers to eliciting an accurate family history
Lack of information
Geographical proximity to affected relatives
Deceased relatives
Lack of communication in family
Unresolved family tension (can often surround the cancer-related death of a parent or close relative)
Estrangement from relatives
Relatives unwilling to provide consent for ROI
Issues of confidentiality (e.g. insurability)
Adoption
Lack of background information
Can lead to complicated ethical dilemmas

18. Summary: why is an accurate family history important?
In order to:
“Correct” inaccurate risk perception
Provide accurate risk assessment
Determine eligibility for genetic testing
Make appropriate recommendations re screening/ cancer risk management

19. Key information to elicit
Key screening questions:
Has anyone on EITHER SIDE of your family had breast and/or ovarian cancer?
Has anyone been diagnosed with breast and/or ovarian cancer at a young age (<50yrs)?
How big is your family? How many men vs. women in the family?
What is your ethnicity?
Sending pathology with referral can help us a lot!

20. Asking the right types of questions
‘40 y.o. female with Breast Cancer. Strong family history: 2 aunts with breast cancer. ?BRCA testing Please assess.’

21. Asking the right types of questions: Relatedness
Br 40
Br 40

22. Asking the right types of questions: Relatedness
Br 40
Br 40

23. Asking the right types of questions: Number of affected vs non affected females3 Br Br
Br 40

24. Asking the right types of questions: Age considerations73 80 75 70 65 60 58 3
Br 53
Br 56
Br 40

25. Asking the right types of questions: Age considerations
Br 40 59 56 50
Br 56 47
Br 53 61 55 60 46 40

26. Asking the right types of questions
‘28 y.o. woman with BrCa. No family history. BRCA testing? Please assess.’

27. Asking the right types of questions: Male to Female Ratio75 84 82 2
Br 28

28. Asking the right types of questions: Male:male transmission75 84 82 2
Br 45
Br 37 Ov 37
Br 28

29. Summary Expand family history to a minimum of 3 generations
Ask about :
both maternal and paternal sides of family
total number of cases of breast/ovarian cancer
age-at-onset of diagnoses
number and ages of unaffected females
family structure (size, male/female ratios)
Confirm all reported diagnoses where possible

30. Multiple generations affected Autosomal dominant
Early age of diagnosed (under 50y)
80s
78y
70y
Br 48
MI 75
80y
82y
70s
MI 79
Br 52
62y
42y
55y
58y
Br 42
Br 60
Br 38
40y
Br 40

31. 2. Genetic evaluation – what does it involve?
Standard model vs newer approaches
2. Genetic evaluation – what does it involve?

32. Germline breast cancer genetic testing : the standard model
Well-established in clinical practice for specific genes
Generally applied with reasonably clear clinical criteria
Most involve sequencing of BRCA1 and BRCA2 to identify a putative deleterious variant
Even with genes such as BRCA1 and BRCA2 that are well characterised there can be problems
Pathogenicity of specific variants often cannot be established
Assumption of pathogenicity based on class of variant

33. The process Patient or physician initiate discussion
Physician refers the patient to genetics service
Genetics service perform some kind of triage
Often then request more information to clarify diagnoses in patient and/or relatives
Depending on triage, urgent or routine
Routine appointments might be 12 months or more later, in the public system

34. Genetic evaluation After gathering relevant information
Appointment is made
45mins -90 mins interview with genetic counsellor and/or MD
Decision on genetic testing – or more info. needed
Send blood for genetic testing, as appropriate
Wait for results
Call patient back in for results
Follow-up, depending on results….

35. 3. Can genetics be used to prevent breast cancer?
If so, how?
3. Can genetics be used to prevent breast cancer?

36. BRCA1/2 – the most important breast cancer genes
The basics

37. The terrain
Foulkes, NEJM, 2008

38. BRCA1 First identified in 1994 Thousands of different mutations
Numerous founder mutations
High lifetime risk for breast and ovarian cancer
Risks at other sites less certain
Characteristic pathology
Implicated in key molecular processes esp. DNA repair

39. BRCA2 First identified in 1995
Thousands of different mutations identified
Several founder mutations identified
High lifetime risk for breast and ovarian cancer
High risks also for pancreas and prostate cancer, and possibly CMM and stomach ca
Few characteristic pathological findings
Implicated in DNA repair

40. BRCA1 and BRCA2
Approximately 3-5% of breast cancer is due to highly penetrant autosomal dominant genes
BRCA1 and BRCA2, together account for around 85% of families with four or more cases of breast/ovarian cancer
Mutations in BRCA1 and BRCA2 are spread throughout the gene
~0.11% of women in the general population carry a mutation in BRCA1
~0.12% carry a mutation in BRCA2
2.5% of individuals of Ashkenazi Jewish descent harbour one of three common BRCA1/BRCA2 founder mutations

41. Risks to age 70 breast BRCA1 ovary BRCA2 breast ovary
Antoniou et al 2003

42. BRCA1 and BRCA2 – we know a lot….

43. The FAMOUS FIVE BARD1/BRCA1/PALB2/BRCA2/RAD51
Livingston, Science, 2009
The FAMOUS FIVE
BARD1/BRCA1/PALB2/BRCA2/RAD51

44. 4. Can genetics be used to help diagnose breast cancers early?
MRI, mammography, ultrasound?
4. Can genetics be used to help diagnose breast cancers early?

45. Mammography and MRI
We know it works…

46. 5. Can genetics assist in treatment decisions?
Chemotherapy and beyond….
5. Can genetics assist in treatment decisions?

47. BRCA1/2 mutations result in specific vulnerabilities
Hoeijmakers, J.H. Nature, 411; , 2001

48. Sensitivity of Brca1 or Brca2 null cells to
platinum agents
Bhattacharyya, A. et al. J. Biol. Chem. 2000;275:
Tutt Cold Spring Harbour Symposia Quant Biol 2005

51. Breast cancer: metastatic studies using platinum
In a phase II, open-label study, 20 patients with metastatic breast cancer who carried a mutation in BRCA1 were treated with cisplatin 75mg/m2 intravenously every three weeks as part of a 21-day cycle for six cycles.
Restaging studies to assess response were performed after cycles 2 and 6, and every 3 months thereafter.
Between July 2007 and January 2009, 20 patients were enrolled.
65% had prior adjuvant chemotherapy, 55% prior chemotherapy for metastatic breast cancer; mean age 48 years (ranges ); 30% ER or PR +, 70% ER/PR/HER2 - , and 0% HER2+.
Overall response rate was 80%; nine patients experienced a complete clinical response (45%) and seven experienced a partial response (35%). One-year survival was 93%.
Cisplatin-related adverse events, including nausea (50%), anemia (5%) and neutropenia (35%) were mostly mild to moderate in severity. One patient discontinued therapy due to grade 4 neutropenia
Byrski et al, BRCT

52. What about newer agents?
PARP inhibitors…basic principles…

53. Mechanism of LOH and inactivation of WT copy of a tumor suppressor gene
Foulkes, NEJM, 2008

54. Turner, N et al. Nature Reviews Cancer, 4;1-6, 2004

55. x X x Tumour Selective Killing Lethal DNA DAMAGE normal tumour A B C
Exploitation of tumour specific DNA repair defects
by targeting “back up” DNA repair
DNA DAMAGE
normal
tumour A B C x
REPAIR MECHANISMS x X
Lethal
Slide courtesy Andrew Tutt, MD PhD

56. Tumour Selective Killing
Hypothesis
normal
BRCA1 or BRCA2 deficient
DNA DAMAGE
DNA DAMAGE x
HR NHEJ SSA BER NER etc
HR NHEJ SSA BER NER etc x
Slide courtesy Andrew Tutt, MD PhD

57. So how does PARP inhibition work?

58. Mechanism of LOH and inactivation of WT copy of a tumor suppressor gene
Foulkes, NEJM, 2008

59. BRCA1 deficient vs functional 1000 fold difference SF50
Kudos/AZ PARP inhibitor
Parp Inhibitor
BRCA1
functional
defective
Farmer et al Nature 2005 KU
PARP-1 IC50 = 3.4nM KU
PARP-1 IC50 = 3.2nM
450 fold difference SF50
BRCA1 deficient vs functional
1000 fold difference SF50
BRCA2 deficient vs functional
Farmer et al Nature :
Slide courtesy Andrew Tutt, MD PhD

60. Response to drugs that force cells to repair by HR
Slide courtesy Andrew Tutt, MD PhD
Farmer, H et al. Nature, 434; , 2005

61. Slide courtesy Andrew Tutt, MD PhD
Farmer, H et al. Nature, 434; , 2005

62. Parp1 inhibitors in clinical practice…

63. Waterfall plots…in BRCA carriers

64. Strikingly different results depending disease and on BRCA status

65. PARP inhibitors: comparison with other targeted therapies

66. PI3KCA inhibitors in BRCA1-related breast cancer
BKM120 delayed tumor doubling in a mouse model of BRCA1-related breast cancer
BKM120 reduced RAD51 foci
Adding BKM120 to olaparib had a synergistic effect in mouse model-derived tumors and in human xenotransplanted BRCA1-deficient tumors
Juvekar et al, Cancer Discovery, 2012

67. Beyond BRCA1 and BRCA2?
6. What new genetic tests are on offer and how should they be evaluated?

68. Gene variants and breast cancer risk
Outliers?
Adapted from a slide created by Peter Devilee and Doug Easton

69. Fraction of familial risk explained- high, medium and low risk alleles….
F J Couch et al. Science 2014;343:

70. 20 years of decreasing costs: data per $100

71. That court case
In June 2013, ruling on “Association for Molecular Pathology v. Myriad Genetics, Inc.”, the Supreme Court of the Unites States, unanimously invalidated specific claims made by Myriad, with respect to the patenting of the genomic DNA sequence of BRCA1 and BRCA2……

72. What panels are available now?
A Rough Guide to Panels
What panels are available now?

73. What is a gene panel test?
New sequencing technologies reduce costs substantially
Sequencing of multiple genes in a single assay possible to identify disease-associated variants
The use of a panel in itself is not a problem
The specific content of the panel may be a problem
Panels vary enormously in their content

74. Genes tested
AKT1, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, GEN1, MRE11A, MUTYH, NBN, PALB2, PIK3CA, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2   

76. So should we test for more than BRCA1/2??
Yes No
Maybe So…

77. Genes with an established association between protein-truncating variants and breast cancer risk
Risk associated truncating variants
Risk associated
missense variants
Estimated relative risks
(90% CI)
P-value
Absolute risk by age 80
Comments
Other associated cancers
>2 fold risk
>4 fold risk
BRCA1
Yes
11.4
75%
Estimates based on the BOADICEA model for woman born in 1960.
Ovary
BRCA2
11.7
76%
Ovary, prostate, pancreas
TP53
105
(62-165)
Most published risk estimates subject to ascertainment bias
Childhood sarcoma, adrenocortical carcinoma, brain tumours
PTEN
Unknown -
Published risk estimates subject to ascertainment bias
Thyroid, endometrial
CDH1
Likely
6.6
( )
.004
47%
Lobular breast cancer specific
Diffuse gastric

78. NF1 Gene >2 fold risk >4 fold risk STK11 PALB2 ATM CHEK2 NBN
Genes with an established association between protein-truncating variants and breast cancer risk part 2
Gene
Risk associated truncating variants
Risk associated
missense variants
Estimated relative risks
(90% CI)
P-value
Absolute risk by age 80
Comments
>2 fold risk
>4 fold risk
STK11
Unknown -
Published risk estimates subject to ascertainment bias
PALB2
Likely
5.3 ( )
4x10-10
40%
ATM
Unlikely
Yes
2.8 ( )
5 x 10-11
24%
c.7272G>T is associated with higher risk
NF1
2.6 ( )
2.3x10-13
26%
CHEK2
3.0 ( )
8x10-37
25%
Most data are limited to c.1100delC
p.I157T associated with ~1.3- fold risk
NBN
2.7 ( )
5 x 10-7
23%
Almost all data pertain to c.657del5 in Slavic populations

79. Other genes for which protein-truncating variants have been suggested to be associated with breast cancer, or present on breast cancer testing panels, but where the association has not been established
Gene
Comments
Estimated RR (90%CI)
P-value
Other associated cancers
AKT1
Germline AKT1 mutations predispose to rare form of Cowden like syndrome. Breast cancer risk unknown -
APC
No published evaluation of risk
Colorectal
ATR
AXIN1
BAP1
Case reports of breast cancers in families segregating germline BAP1 mutations – no systematic study
Uveal / cutaneous melanoma
BARD1
Deleterious mutations found ~9/1824 triple negative cases.
BLM
Evidence relates to p.Q548X in Slavic populations and c.2207_2212delATCTGAinsTAGATTC in Ashkenazim. Evidence of increased breast cancer risk in homozygotes
2.4 ( )
0.0002
BMPR1A
Germline mutations predispose to Juvenile Polyposis Syndrome. No published evaluation of breast cancer risk
BRIP1
Single case-control study of familial cases Most data for R798X
2.0 ( )
0.012
Ovary
CDK4
Case reports in families – no published evaluation of risk
Melanoma
CDKN2A
Melanoma, pancreas
CTNNB1
No published evidence
EPCAM
No evidence on truncating mutations. Suggestive evidence for association for missense variant p.Thr115Met
FAM175A
No evidence of truncating mutations in high-risk families. No published evaluation of risk
FANCC
Evidence from one exome sequencing study plus replication (4/1395 cases vs. 0/2210 controls)
0.02

80. Other associated cancers 0.002 0.63 0.11 2x10-5
Other genes for which protein-truncating variants have been suggested to be associated with breast cancer, or present on breast cancer testing panels, but where the association has not been established
part 2
Gene
Comments
Estimated RR (90%CI)
P- value
Other associated cancers
FANCM
Evidence from one exome sequencing study plus targeted genotyping of nonsense variant (p.Q1701X)
1.9 ( )
0.002
GEN1
Most data relate to polymorphic truncating mutation c.2515_2519delAAGTT, ~4% frequency
1.1 ( )
0.63
HOXB13
Analyses relate to p.G84E prostate cancer susceptibility variant
1.6 ( )
0.11
Prostate
MEN1
Suggestive evidence from cohort MEN1 carriers
2.0 ( )
2x10-5
Pituitary, parathyroid and pancreatic neuroendocrine tumors
MLH1
Evidence from cohort analyses in lynch-syndrome families inconclusive ( ), P=.001 for mismatch repair gene mutations combined, in one prospective study -
Colorectal, endometrial, ovary
MRE11A
Two mutations in 8 multiple case breast cancer families with tumors that showed loss of all three MRN proteins. Combined analysis of truncating and rare missense variants affecting key functional domains in MRE11A, NBN and RAD50: OR 2.88 ( ) P=.02
MSH2
see MLH1
MSH6
See MLH1
MUTYH
Suggestive evidence for increased breast cancer risk in MAP patients homozygote for MUTYH mutations One case-control study found no evidence of increased risk
1.3 ( )
0.26
Gastro-intestinal

81. Other genes for which protein-truncating variants have been suggested to be associated with breast cancer, or present on breast cancer testing panels, but where the association has not been established part 3
Gene
Comments
Estimated RR (90%CI)
P-value
Other associated cancers
PALLD
No published evaluation of risk -
PIK3CA
Germline PIK3CA mutations predispose to rare form of Cowden-like syndrome. Breast cancer risk unknown
PMS2
See MLH1
Colorectal, endometrial, ovary
PPM1D
Association in one case-control study. Genotypes mosaic lymphocytes, not inherited
15.3 ( )
0.0002
Ovary
RAD50
Analyses based on four case-control studies, three of Finnish founder variant c.697delT
2.20 ( )
0.11
RAD51
No evidence of association. No truncating variants found in large case- control study
RAD51C
Initial evidence for association through breast-ovarian cancer families, but little evidence for breast cancer risk after adjustment for ovarian cancer risk in family-based analysis
0.91 ( )
0.79
RAD51D
Evidence for association in breast-ovarian families but no evidence of breast cancer association after adjustment for ovarian cancer risk
1.3 ( )
0.49
RINT1
Suggestive evidence from exome sequencing and targeted replication
3.2 ( )
0.013
SMAD4
Germline mutations predispose to Juvenile Polyposis Syndrome. No published evaluation of breast cancer risk
VHL
No published evaluation of breast cancer risk
XRCC2
Suggestive evidence exome sequencing followed by replication case-control study (truncating + rare likely deleterious missense)
0.02
XRCC3

82. What could possibly go wrong?

83. The results, she said, were “surreal
The results, she said, were “surreal.” She did not have mutations in the breast cancer genes, but did have one linked to a high risk of stomach cancer. In people with a family history of the disease, that mutation is considered so risky that patients who are not even sick are often advised to have their stomachs removed. But no one knows what the finding might mean in someone like Jennifer, whose family has not had the disease.
It was a troubling result that her doctors have no idea how to interpret.

84. Conclusions on panel testing – Proceed with Caution
Multi-gene panels are the inevitable consequence of falling costs and changing laws
They are in principle “a good thing”
But look before you leap
BRCA1, BRCA2 still the major players
TP53, PALB2 and possibly ATM and CHEK2 deserve consideration
Other genes probably more trouble than they are worth, at least under the current model of pre-test counselling
Somatic cancer gene panels will create their own challenges
Newer delivery models may change things once again

85. Further reading on panel testing for breast cancer -
Published on-line at nejm.org on 27 May, 2015

86. Comments? Questions?
Thank you!