Breast Cancer Res Treat (2015) 150:71–80 DOI 10.1007/s10549-015-3293-7
PRECLINICAL STUDY
Prevalence of BRCA1 and BRCA2 germline mutations in patients with triple-negative breast cancer Michelle W. Wong-Brown • Cliff J. Meldrum • Jane E. Carpenter • Christine L. Clarke • Steven A. Narod • Anna Jakubowska • Helena Rudnicka Jan Lubinski • Rodney J. Scott
•
Received: 28 January 2015 / Accepted: 31 January 2015 / Published online: 15 February 2015 Ó Springer Science+Business Media New York 2015
Abstract Triple-negative breast cancers (TNBC) lack expression of oestrogen, progesterone and HER2 receptors. The gene expression profiles of TNBCs are similar to those of breast tumours in women with BRCA1 mutations. Reports to date indicate that up to 20 % of TNBC patients harbour germline BRCA mutations; however, the prevalence of BRCA mutations in TNBC patients varies widely between countries and from study to study. We studied 774 women with triple-negative breast cancer, diagnosed on average at age 58.0 years. Samples of genomic DNA were provided by the Australian Breast Cancer Tissue Bank (ABCTB) (439 patients) and by the Department of Genetics and Pathology
Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3293-7) contains supplementary material, which is available to authorized users. M. W. Wong-Brown R. J. Scott (&) School of Biomedical Sciences & Pharmacy, Centre for Information-Based Medicine, Hunter Medical Research Institute, University of Newcastle, Lot 1 Kookaburra Circuit, New Lambton Heights, Newcastle, NSW 2305, Australia e-mail: [email protected] C. J. Meldrum R. J. Scott Division of Molecular Medicine, Pathology North, NSW Pathology, Lookout Road, Newcastle 2305, NSW, Australia J. E. Carpenter C. L. Clarke Australian Breast Cancer Tissue Bank, University of Sydney at the Westmead Millennium Institute, Westmead, NSW, Australia S. A. Narod Familial Breast Cancer Research Unit, Women’s College Research Institute, Toronto, Canada A. Jakubowska H. Rudnicka J. Lubinski Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland
of the Pomeranian Medical University (335 patients). The entire coding regions and the exon–intron boundaries of BRCA1 and BRCA2 were amplified and sequenced by nextgeneration sequencing. We identified a BRCA1 or BRCA2 mutation in 74 of 774 (9.6 %) triple-negative patients. The mutation prevalence was 9.3 % in Australia and was 9.9 % in Poland. In both countries, the mean age of diagnoses of BRCA1 mutation carriers was significantly lower than that of non-carriers, while the age of onset of BRCA2 mutation carriers was similar to that of non-carriers. In the Australian cohort, 59 % of the mutation-positive patients did not have a family history of breast or ovarian cancer, and would not have qualified for genetic testing. The triple-negative phenotype should be added as a criterion to genetic screening guidelines. Keywords Triple-negative breast cancer BRCA1 BRCA2 Germline mutations Prevalence Genetic testing List of abbreviations BRCA Breast cancer susceptibility gene DNA Deoxyribonucleic acid PARP Poly (adenosine diphosphate)-ribose polymerase TNBC Triple-negative breast cancer UV Unclassified variant pCR Pathological complete response
Introduction TNBC describes a subgroup of breast cancers that are negative for the oestrogen, progesterone and HER2 receptors. They account for between 15 and 20 % of all breast cancers diagnosed but are over-represented in young women and in black women in the United States.
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Table 1 TNBC demographic and clinical data of the Australian and Polish patient cohorts Characteristic
Australian patients, N = 439 (%)
Polish patients, N = 335 (%)
57 ± 15 years
59 ± 10 years
Australian cohort Age at diagnosis B50 years
153 (34.9)
49 (14.6)
[50 years
286 (65.1)
286 (85.4)
Breast cancer only
80 (18.2)
Information only available for mutation carriers (see Table 2)
Breast and ovarian cancer
11 (2.5)
Breast and prostate cancer
2 (0.5)
Breast and other cancer
43 (9.8)
Ovarian cancer only
5 (1.1)
Ovarian and other cancer
6 (1.3)
Other cancer
85 (19.4)
No recorded family history of cancer
207 (47.2)
Family history of cancer
Methods Study cohort
Type of primary tumour Ductal (Invasive/ in situ/not otherwise specified)
409 (93.2)
Papillary
3 (0.7)
2 (0.6)
Medullary
10 (2.3)
42 (12.5)
Apocrine
–
4 (1.2)
Lobular (Invasive/ in situ/not otherwise specified)
9 (2)
14 (4.2)
Metaplastic
–
7 (2.1)
–
3 (0.9)
8 (1.8)
36 (10.7) N = 219
Others Unknown Tumour grade
227 (67.8)
1
6 (1.4)
1 (0.5)
2
48 (10.9)
54 (24.6)
3
374 (85.2)
143 (65.3)
Not known
11 (2.5)
21 (9.6)
BRCA1 and BRCA2 are breast cancer susceptibility genes that are part of the DNA repair pathway. Pathogenic mutations in both genes confer a high risk of breast cancer [1], and together they account for approximately 5 % of all breast cancer cases [2]. A large proportion of tumours in women with a BRCA1 mutation exhibited a triple-negative phenotype. Not all women with breast cancer qualify for BRCA1 and BRCA2 testing. Testing is currently based on
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the age of onset, the family history and in some cases (e.g. Ashkenazi Jews) on the ethnic group. Currently, tumour histology is not a clear criterion for the recommendation for genetic testing, but some have suggested that all women with triple-negative breast cancer be candidates for genetic testing, regardless of age of onset or family history. With the advent of next-generation DNA sequencing (NGS), it is cost-effective to perform BRCA1 and BRCA2 testing to women outside of a familial cancer setting. Several studies have reported that up to 20 % of women with TNBC breast cancer carry a BRCA mutation [3–12]. The aim of this study was to define the prevalence of germline BRCA1 and BRCA2 mutations in two independent populations of consecutively collected TNBC cases, in Poland and in Australia, unselected for family history and age of diagnosis.
439 patients with TNBC from Australia were included. Cases were selected based on triple-negative status and not on age of onset or family history. The demographic and clinical data are described in Table 1. This study was approved by the Hunter New England Health Human Research Ethics Committee. Samples were provided by the Australian Breast Cancer Tissue Bank (ABCTB). DNA samples from 335 Polish patients with TNBC were provided by the Department of Genetics and Pathology, Pomeranian Medical University. The study was approved by the local institutional ethics review committee for participation in this study. There are three common BRCA1 founder mutations in the Polish population. It is currently recommended that all breast cancer patients in Poland be tested for these three mutations and, for the purposes of the current study, patients with one of these mutations were excluded. 49 women were diagnosed under 50 years of age and 286 were diagnosed over age 50. The average age of diagnosis of these two patient groups is shown in Table 1. BRCA1 and BRCA2 sequencing Target-specific primers were designed by Fluidigm Corp. (San Francisco, CA). Common sequence tags (CS1 and CS2) were added to the forward and reverse primers for Access Array amplicon tagging. 184 primer pairs were designed to cover all coding exons of BRCA1 and BRCA2. Genomic DNA from TNBC patients were normalised to 50 ng/ll concentration and 1 ll of the solution was loaded onto a Fluidigm Access Array, (a microfluidic array in which many concurrent PCR reactions were performed with nested primer pairs). A two-primer protocol was used
Exon
BRCA1
p.Val233Asnfs*4 p.Leu502Alafs*2 p.Gln563* p.Lys652Glufs*21
p.Lys654Serfs*47
c.213-11T[G
c.514delC
c.697_698del
c.1504_1508del
c.1687C[T
c.1952dup
c.1961del
int 5
8
11
p.Gln172Asnfs*62
Results in frameshift
Activates cryptic splice acceptor site. Leads to aberrant splicing and insertion of 59 bp
p.Cys61Gly
Skip of exon 5 very likely
c.181T[G
5
Intronic retention of 58 bp
p.Cys24Serfs*13
c.70_80del
c.135-2A[G
p.Glu23Valfs*17
Amino acid change
c.68_69del
Nucleotide change
int 3
2
Australian cohort
Gene
rs80357747
rs80357522
rs80357885
rs80356898
rs80357888
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
rs80358061
rs80357872
Y/class 5—pathogenic
Y/UV
Y/class 5—pathogenic
Y/class 5—pathogenic
BIC (Y/N—class)
rs28897672
rs80358065
rs80359877
rs80357713
dbSNP
45
03-11-043
02-12-076
02-08-102
05-08-009
05-11-096
04-08-214
08-12-105
01-10-155
04-10-067
07-12-007
01-10-014
47
36
62
68
43
41
69
41
37
47
40
55
03-09-148
03-11-100
26
Age of diagnosis
01-07-014
Patient
Mother, ovarian, 66
Paternal uncle, bowel, 60
Maternal grandmother, breast, unknown
Mother, breast cancer, 45
Paternal cousin, breast, 40 s
Paternal aunt, breast, 50 s
Paternal aunt, breast, 50 s
Maternal niece, breast, 39
Sister, colorectal, 55
Sister, ovarian, 53
Father, pancreatic, 76
Mother, breast, 73
Brother, bowel, unknown
Grandfather, prostate, unknown
Maternal aunt, stomach, 75
Mother, cervical, 38
Maternal cousin, breast, 50 s
Paternal grandmother, breast, 90 s
Mother, breast, 38
Maternal grandaunt, ovarian, 20 s
Maternal grandfather, brain, 30
Paternal grandaunt, stomach, 50
Paternal grandmother, pancreas, 77
Paternal grandfather, leukaemia, 52
Paternal aunt, breast, 36
Father, non-Hodgkins lymphoma and colon, 75
Maternal 2nd cousin, breast, 45
Maternal aunt, breast, 47
Maternal cousin, ovarian, 45
Family history (relationship, type of cancer, age of diagnosis)
Table 2 Deleterious mutations detected in the Australian and Polish cohorts and any recorded family history of diseases associated with the mutation carriers
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123
123
BRCA2
Gene
c.5266dup
c.5272A[T
20
p.Cys419Trpfs*11 p.Phe620Leufs*24 p.Thr630Asnfs*14
c.1257del
c.1860del
c.1889del
10
Classified as pathogenic according to kConFab
Skip of exon 7 is very likely
c.631?2T[G
int 7
p.Leu105*
c.314T[G
p.Leu1764*
p.His1732Phefs*5
p.Thr1677Ilefs*2
3
c.5289del
p.Arg1758*
c.5194-12G[A
int 19
21
p.Gln1756Profs*74
c.5030_5033del
17
p.Trp1508*
p.Leu1404*
p.Ser1253Argfs*10
c.3756_3759del
c.4523G[A
p.Gln1200*
c.3598C[T
c.4210del
p.Ile946Tyrfs*6
c.2835dup
15
Amino acid change
Nucleotide change
13
Exon
Table 2 continued
rs80359315
rs80359272
rs81002899
rs80358561
rs80357906
rs80358079
rs80357862
rs80357765
rs80357868
rs62625307
rs80357519
dbSNP
Y/class 5—pathogenic
N
Y/class 5—pathogenic
Y/UV
Y/class 5—pathogenic
N
N
Y/class 5—pathogenic
N
Y/class 5—pathogenic
N
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
BIC (Y/N—class)
05-10-073
03-07-065
01-08-025
01-06-021
48
56
84
34
89 46
05-09-110
55
36
47
45
55
45
04-11-051
01-11-082
03-09-059
04-10-126
01-08-061
02-08-30S
01-12-021
29
33
04-11-088 03-06-041
30
43
44
Age of diagnosis
02-08-124
08-10-069
02-08-049
Patient
Maternal grand-aunt, breast, 70
Paternal grandfather, unknown, 80
Mother, breast, 39
Maternal uncle, colorectal, 60
Maternal uncle, colorectal, 60
Maternal uncle, liver, 23
Maternal 2nd cousin, breast, 50 s
Maternal grandfather, colorectal, 68
Maternal cousin, breast, 33
Mother, breast, 58
Paternal cousin, breast, unknown
Paternal aunt, breast, unknown
Maternal aunt, ovarian, 40
Mother, breast, 40
Mother, bladder, 74
Maternal great-aunt, breast, 50
Maternal grandmother, breast, 60
Mother, breast, 60
Mother, breast, 47
Paternal aunt, breast, 50
Maternal cousin, breast, 35
Maternal aunt, breast, 40
Paternal aunt, breast, 60 s
Paternal great-grandmother, breast, 80 s
Maternal aunt, breast, unknown
Cousin, breast
Cousin, breast
Father, throat, 40 s
Maternal aunt, ovarian, 42
Family history (relationship, type of cancer, age of diagnosis)
74 Breast Cancer Res Treat (2015) 150:71–80
Exon
11
16
BRCA1
p.Gln1273*
c.3817C[T
BRCA2
p.Gln1096*
c.3286C[T
c.4689C[A
c.2886dup
p.Tyr1563*
p.Ile963Tyrfs*19
p.Gln1396*
p.Gln1090*
c.3268C[T
c.4186C[T
p.Gly563*
c.1687C[T
13
p.Gly284*
c.80?2T[C
c.850C[T
i2
11
Amino acid change
p.Thr2766Asnfs*11
Nucleotide change
c.8297del
18
p.Ile2315Lysfs*12
p.Val2228Glyfs*5
c.6944_6947del
13
c.6682dup
p.Asn1784Thrfs*7
c.5351del p.Leu1908Argfs*2
p.Tyr1710*
c.5130_5133del
p.Ser1970*
p.Asp1469Lysfs*11
c.4405_4409del
c.5909C[A
p.Gln1452*
c.4354C[T
c.5722_5723del
p.Ala938Profs*21
c.2808_2811del
11
Amino acid change
Nucleotide change
Exon
BRCA1
a
BRCA1
Polish cohort
Gene
Gene
Table 2 continued
rs80357433
rs80357011
rs80357208
rs80357485
rs80357402
rs80356898
rs80358128
dbSNP
rs80359705
rs80359629
rs80359621
rs80358824
rs80359531
rs80359509
rs80359485
rs80359352
dbSNP
Y/class 5—pathogenic
N–UV
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
N
N
BIC (Y/N – Class)
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
N
N
Y/class 5—pathogenic
BIC (Y/N—class)
01-08-037
08-11-063
04-08-036
05-10-118
04-09-164
08-10-074
02-09-527
01-12-019
03-11-109
04-12-030
Patient
43
136,755
160,333#
220,693
161,467
36
41
54
53
62
55
349,099 192,682
33 59
342,887
40
Y
Y
N
Y
Y
N
N
Y
N
N
Family history of breast and/or ovarian cancer (Y/N)
Brother, melanoma, 35
Paternal cousin, breast, 40
Mother, multiple myeloma & lung, 60
Maternal grandmother, melanoma, 90 s
Brother, skin, 40 s
Father, skin, 60 s
Father, prostate, 65
Daughter, thyroid, 54
Maternal grandmother, gastrointestinal, unknown
Brother, prostate, 59
Father, bowel, 54
Maternal aunt, breast, 50 s
Mother, breast, 50 s
Family history (relationship, type of cancer, age of diagnosis)
Age of diagnosis
285,146
228,532
146,990
Patient
44
44
39
63
57
83
60
78
58
59
Age of diagnosis
Breast Cancer Res Treat (2015) 150:71–80 75
123
123
a
c.9097dup
c.9253dup
24
p.Glu2089Aspfs*2
c.6267_6269delinsC
c.8946del
p.Tyr1894*
c.5682C[G
23
p.Ser1882*
c.5645C[A
22
p.Asn1784Lysfs*7
c.5352del
c.7558C[T
p.Asn1747*
c.5237dup
15
p.Lys1025Asn;Lys1026*
c.3075_3016delinsTT
11
p.Thr3085Asnfs*26
p.Thr3033Asnfs*11
p.Asp2983Ilefs*5
p.Arg2520*
p.Asn433Glnfs*18
p.Val220Ilefs*4
c.1296_1297del
p.Arg1835*
c.658_659del
c.5503C[T
24
p.Thr1677Ilefs*2
10
c.5278-2A[T
i20
Intronic retention of 65 bp
8
c.5030_5033del
17
p.Met1663Valfs*14
p.Glu1661*
c.4981G[T
c.4986?3G[C
Amino acid change
Nucleotide change
i16
Exon
Patient 160,333 harbours both BRCA1 and BRCA2 mutations
BRCA2
Gene
Table 2 continued
rs80359752
rs80359747
rs80358981
rs41293497
rs80358785
rs80359499
rs80358552
rs80359276
rs80359604
rs41293465
rs80357862
rs80358023
rs80357401
dbSNP
Y/class 5—pathogenic
Y/class 5—pathogenic
N–UV
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic COSMIC—distribution in ovary and endometrium
N/UV
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
COSMIC—distribution in ovary and large intestine
Y/UV
N–UV
Y/class 5—pathogenic
Y/Class 4—likely pathogenic
Y/class 5—pathogenic
BIC (Y/N – Class)
33 41
73,032 353,587
53
174,157
192,325
285,224
266,895
99,173
151,271
45
68
73
62
52
58 50
195,860
68
349,466
346,635
63
273,641
49
52
171,190 87,035
58
71
77
58
66
38
54
135,383
119,568
334,861
173,119
156,407
166,662
94,836
62
53
101,115
136,084
Age of diagnosis
Patient
N
N
Y
Y
Y
N
Y
Y
Y
N
Y
N
Y
N
N
Y
Y
Y
N
Y
N
Y
Y
Family history of breast and/or ovarian cancer (Y/N)
76 Breast Cancer Res Treat (2015) 150:71–80
Breast Cancer Res Treat (2015) 150:71–80
A
Distribution of age of diagnosis of BRCA mutation carriers 40
% of patients within mutation status
Fig. 1 Distribution of age of diagnosis of A. BRCA mutation carriers (Australian average = 50.3 ± 15 years, Polish average = 53.6 ± 11.5 years). The average age of Australian and Polish BRCA1 mutation carriers is 47.2 ± 11.8 years and average age of BRCA2 mutation carriers is 58.8 ± 13.2. B. The distribution of the age of diagnosis of the non-mutation carriers (average age of nonmutation carriers is 58.7 ± 12.7 years)
77
35
BRCA1 mutation carriers
30
BRCA2 mutation carriers
25 20 15 10 5 0
25-34
35-44
45-54
55-64
65-74
75-84
≥85
Age of diagnosis (years)
% of patients within non-mutation status
B
Distribution of age of diagnosis of patients without mutations 40 35
Non-mutation carriers
30 25 20 15 10 5 0
25-34
35-44
45-54
55-64
65-74
75-84
≥85
Age of diagnosis (years)
to first amplify the target sequence followed by a second PCR reaction that included the sequencing tags such that ultimately each amplified product contained the target-specific sequence, a tag sequence and a sample-specific unique barcode. The exons were amplified and the PCR products were harvested from each sample, pooled, and purified using Ampure XP beads. The libraries were then sequenced on an Illumina MiSeq instrument. The sequencing data were analysed with NextGENe 2nd Generation Sequencing Software v.2.3.3. (SoftGenetics, Philadelphia). Raw data were converted into FASTA files and aligned to BRCA1 and BRCA2 reference sequences from the human reference sequences; GRCh37 Primary Assembly, BRCA1 NC_000017.10 and BRCA2 NC_00013.10 (http://www.ncbi.nih.gov). The CS1 and CS2 sequences were trimmed, as well as the primer sequences to eliminate the dilution of base calling from those regions. The mutations were described using the nomenclature guidelines of the Human Genome Variation Society (http:// www.hgvs.org). The DNA sequence numbering is based on
the cDNA sequences for BRCA1 (NM_ 007294.3) and BRCA2 (NM_ 000059.3). BRCA1 or BRCA2 mutations were only included in the analyses if classified as deleterious or possibly deleterious according to Breast Cancer Information Core (BIC) criteria or if present in the BIC database [13]. The criteria are all frameshift and nonsense variants (except those resulting in neutral or polymorphic stop codons), all intronic variants in the consensus splice acceptor or donor sites (within 2 bp of exon–intron junctions or if shown to result in aberrant mRNA transcript processing, or non-synonymous variants shown to have deleterious effect on known functional regions (based on functional or biochemical assays, or linkage analysis of high-risk families) [14].
Results We identified 74 mutations in the 774 triple-negative breast cancer patients (9.6 %). Mutations were present in 9.3 %
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78
40.0
Proportion of BRCA mutationpositive patients (%)
Fig. 2 The proportion of patients with or without recorded family histories of breast and/or ovarian cancer in each age group in those with germline BRCA mutations. In patients with family histories of disease: \40 years—17.1 %, 40–60 years—22 %, C60 years—2.4 %. In patients without recorded family histories of disease: \40 years—4.9 %, 40–60 years—36.6 %, C60 years—17.1 %
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Distribution of age of diagnosis of patients with versus without recorded family history of breast and/or ovarian cancer
35.0 30.0
With family history
25.0
Without recorded family history
20.0 15.0 10.0 5.0 0.0 60
Age of diagnosis (years)
Table 3 Percentage of patients with BRCA1 or BRCA2 mutations compared to non-mutation carriers diagnosed with breast cancer Age of diagnosis (years)
BRCA1 mutation carriers
BRCA2 mutation carriers
Non-mutation carriers
Australian breast cancer cohort
(%, n = 26)
(%, n = 15)
(%, n = 398)
\40
26.9
13.3
12.6
40–60
57.7
60
43.7
C60
15.4
26.7
43.7
(%, n = 18)
(%, n = 15)
(%, n = 302)
\40
22.2
0
2.3
40–60
61.1
60
54
C60
16.7
40
43.7
Polish breast cancer cohort
of the Australian patients and in 9.9 % of the Polish patients (see Table 2 for a complete list of deleterious mutations and Supplementary Table 1 for all other alterations identified in BRCA1 and BRCA2). In Australia, 63 % of the mutations were in BRCA1; in Poland 54.5 % of the mutations were in BRCA1. The distribution of the age of diagnoses is shown in Fig. 1. In the Australian cohort, four of the 41 mutations are novel and have not been reported in BIC, Leiden Open (source) Variation Database (LOVD), Clinvar, or any published literature. Of these, two are in BRCA1 (c.4523G[A, c.5272A[T) and two in BRCA2 (c.1860del, c.4354C[T). Among the 41 Australian patients with a mutation, 17 (41.5 %) had a family history of breast or ovarian cancer (Table 2). In the Australian cohort, the mean age of disease onset for BRCA1 mutation carriers is 46.0 years of age and for BRCA2 mutation carriers is 57.6 years of age, and for non-carriers, it was 57.8 years of age (p = 0.004). A deleterious mutation was detected in 33 of 335 patients in Poland (9.9 %). Two mutations have not been reported in BIC, Leiden Open (source) Variation Database (LOVD) or Clinvar. These include one in BRCA1 (c.80?2T[C) and one in BRCA2 (c.2886dup).
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Among the 33 Polish patients with a mutation, 19 (57.5 %) had a family history of breast or ovarian cancer (Table 2). In the Polish cohort, the mean age of onset for BRCA1 mutation carriers is 48.9 years of age, whereas for BRCA2 mutation carriers, it is 59.9 years of age and for non-mutation carriers, it is 60 ± 9.8 years of age (p = 0.004). The distribution of age of diagnosis of our patient cohort according to age groups in those with and without BRCA mutations is shown in Fig. 2.
Discussion In both Poland and in Australia, approximately 10 % of the patients with triple-negative phenotype harbour a germline BRCA1 or BRCA2 mutation. Our study highlights the importance of genetic testing for patients with TNBC regardless of age and in the absence of a family history. In Poland, genetic testing for three founder mutations is already recommended for all breast cancer patients. We recommend that in the absence of one of the founder mutations, Polish patients with triple-negative breast cancer be considered for additional genetic testing, which should include full sequencing of the coding regions of both genes.
Breast Cancer Res Treat (2015) 150:71–80
In both countries, BRCA2 mutations were less common than BRCA1 mutations, in agreement with other studies [3, 8, 15, 16]. These results may reflect the propensity for BRCA1 carriers to develop primarily triple-negative cancers, whereas among BRCA2 carriers, the majority of breast cancers are oestrogen-receptor positive. BRCA2 mutation carriers were shown in our study to be diagnosed with TNBC at an older age than BRCA1 mutation carriers and similar to the age of onset of non-carriers. Recent guidelines by the National Comprehensive Cancer Network (NCCN Guidelines in Oncology Version 3.2013 Genetic/Familial High-Risk Assessment: Breast and Ovarian) recommends that TNBC diagnosed before or at age 60 is considered to be sufficient to meet the threshold of risk assessment for genetic testing [17]. In contrast, the Cancer Institute New South Wales guidelines recommend that patients with TNBC be offered testing if diagnosed under the age of 40 years. [18]. From our results, 33.3 % of BRCA2 mutation carriers in both the Australian and Polish cohorts are over 60 years of age, therefore according to the NCCN guidelines, they would not qualify for genetic testing (see Table 3). Our results suggest that the age of diagnosis should not be a limitation to genetic testing eligibility. The purposes of genetic testing of breast cancer patients are to identify additional family members who might benefit from preventive interventions and to guide the choice of treatment of the breast cancer patients. Under optimal circumstances, the genetic test results would be made available within 2 weeks of diagnosis. BRCA1 mutations carriers have been shown to have enhanced benefit from chemotherapy, in particular to neoadjuvant cisplatin chemotherapy. In a recent study, we found that 61 % of 107 patients with a BRCA1 mutation had a complete pathologic response when given cis-platinum [19, 20] and all who experienced a pCR are currently alive. In addition, women with a BRCA1 mutation have been shown to benefit from bilateral mastectomy [21] and from oophorectomy [22]. Acknowledgments DNA samples were received from the Australian Breast Cancer Tissue Bank, which is generously supported by the National Health and Medical Research Council of Australia (NHMRC), the Cancer Institute NSW (CINSW) and the National Breast Cancer Foundation (NBCF). The tissues and samples are made available to researchers on a non-exclusive basis. This work was supported by the National Breast Cancer Foundation (NBCF), Australia. Dr Michelle Wong-Brown is supported by the Hunter Translational Cancer Research Centre with funding from the Cancer Institute New South Wales. Conflict of interest The authors of this article declare no competing interests related to the study and no commercial associations that may pose a conflict of interest.
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References 1. Dite GS, Jenkins MA, Southey MC, Hocking JS, Giles GG, McCredie MR, Venter DJ, Hopper JL (2003) Familial risks, early-onset breast cancer, and BRCA1 and BRCA2 germline mutations. J Natl Cancer Inst 95(6):448–457 2. Nathanson KL, Wooster R, Weber BL (2001) Breast cancer genetics: what we know and what we need. Nat Med 7(5):552–556 3. Gonzalez-Angulo AM, Timms KM, Liu S, Chen H, Litton JK, Potter J, Lanchbury JS, Stemke-Hale K, Hennessy BT, Arun BK et al (2011) Incidence and outcome of BRCA mutations in unselected patients with triple receptor-negative breast cancer. Clin Cancer Res 17(5):1082–1089 4. Lakhani SR, Van De Vijver MJ, Jacquemier J, Anderson TJ, Osin PP, McGuffog L, Easton DF (2002) The pathology of familial breast cancer: predictive value of immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in patients with mutations in BRCA1 and BRCA2. J Clin Oncol 20(9):2310–2318 5. Turner N, Tutt A, Ashworth A (2004) Hallmarks of ‘BRCAness’ in sporadic cancers. Nat Rev Cancer 4(10):814–819 6. Anglian Breast Cancer Study Group (2000) Prevalence and penetrance of BRCA1 and BRCA2 mutations in a populationbased series of breast cancer cases. BJC 83(10):1301–1308 7. Peto J, Collins N, Barfoot R, Seal S, Warren W, Rahman N, Easton DF, Evans C, Deacon J, Stratton MR (1999) Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer. JNCI 91(11):943–949 8. Hartman AR, Kaldate RR, Sailer LM, Painter L, Grier CE, Endsley RR, Griffin M, Hamilton SA, Frye CA, Silberman MA et al (2012) Prevalence of BRCA mutations in an unselected population of triple-negative breast cancer. Cancer 118(11):2787–2795 9. Fostira F, Tsitlaidou M, Papadimitriou C, Pertesi M, Timotheadou E, Stavropoulou AV, Glentis S, Bournakis E, Bobos M, Pectasides D et al (2012) Prevalence of BRCA1 mutations among 403 women with triple-negative breast cancer: implications for genetic screening selection criteria: a Hellenic Cooperative Oncology Group Study. Breast Cancer Res Treat 134(1):353–362 10. Robertson L, Hanson H, Seal S, Warren-Perry M, Hughes D, Howell I, Turnbull C, Houlston R, Shanley S, Butler S et al (2012) BRCA1 testing should be offered to individuals with triple-negative breast cancer diagnosed below 50 years. Br J Cancer 106(6):1234–1238 11. Young SR, Pilarski RT, Donenberg T, Shapiro C, Hammond LS, Miller J, Brooks KA, Cohen S, Tenenholz B, Desai D et al (2009) The prevalence of BRCA1 mutations among young women with triple-negative breast cancer. BMC Cancer 9:86 12. Phuah SY, Looi LM, Hassan N, Rhodes A, Dean S, Taib NA, Yip CH, Teo SH (2012) Triple-negative breast cancer and PTEN (phosphatase and tensin homologue) loss are predictors of BRCA1 germline mutations in women with early-onset and familial breast cancer, but not in women with isolated late-onset breast cancer. Breast Cancer Res 14(6):R142 13. An open access on-line breast cancer mutation data base [http:// research.nhgri.nih.gov/bic/] 14. Borg A, Haile RW, Malone KE, Capanu M, Diep A, Torngren T, Teraoka S, Begg CB, Thomas DC, Concannon P et al (2010) Characterization of BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance in unilateral and bilateral breast cancer: the WECARE study. Hum Mutat 31(3):E1200–E1240 15. Evans DG, Howell A, Ward D, Lalloo F, Jones JL, Eccles DM (2011) Prevalence of BRCA1 and BRCA2 mutations in triple negative breast cancer. J Med Genet 48(8):520–522
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Breast Cancer Res Treat (2015) 150:71–80 et al (2010) Pathologic complete response rates in young women with BRCA1-positive breast cancers after neoadjuvant chemotherapy. J Clin Oncol 28(3):375–379 21. Metcalfe K, Gershman S, Ghadirian P, Lynch HT, Snyder C, Tung N, Kim-Sing C, Eisen A, Foulkes WD, Rosen B, Sun P, Narod SA (2014) Contralateral mastectomy and survival after breast cancer in carriers of BRCA1 and BRCA2 mutations: retrospective analysis. BMJ 11(348):g226. doi:10.1136/bmj.g226 22. Huzarski T, Byrski T, Gronwald J, Go´rski B, Domagala P, Cybulski C, Oszurek O, Szwiec M, Gugala K, Stawicka M, Morawiec Z, Mierzwa T, Janiszewska H, Kilar E, Marczyk E, Kozak-Klonowska B, Siolek M, Surdyka D, Wisniowski R, Posmyk M, Sun P, Lubinski J, Narod SA (2013) Ten-year survival in patients with BRCA1-negative and BRCA1-positive breast cancer. J Clin Oncol 31(26):3191–3196
PRECLINICAL STUDY
Prevalence of BRCA1 and BRCA2 germline mutations in patients with triple-negative breast cancer Michelle W. Wong-Brown • Cliff J. Meldrum • Jane E. Carpenter • Christine L. Clarke • Steven A. Narod • Anna Jakubowska • Helena Rudnicka Jan Lubinski • Rodney J. Scott
•
Received: 28 January 2015 / Accepted: 31 January 2015 / Published online: 15 February 2015 Ó Springer Science+Business Media New York 2015
Abstract Triple-negative breast cancers (TNBC) lack expression of oestrogen, progesterone and HER2 receptors. The gene expression profiles of TNBCs are similar to those of breast tumours in women with BRCA1 mutations. Reports to date indicate that up to 20 % of TNBC patients harbour germline BRCA mutations; however, the prevalence of BRCA mutations in TNBC patients varies widely between countries and from study to study. We studied 774 women with triple-negative breast cancer, diagnosed on average at age 58.0 years. Samples of genomic DNA were provided by the Australian Breast Cancer Tissue Bank (ABCTB) (439 patients) and by the Department of Genetics and Pathology
Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3293-7) contains supplementary material, which is available to authorized users. M. W. Wong-Brown R. J. Scott (&) School of Biomedical Sciences & Pharmacy, Centre for Information-Based Medicine, Hunter Medical Research Institute, University of Newcastle, Lot 1 Kookaburra Circuit, New Lambton Heights, Newcastle, NSW 2305, Australia e-mail: [email protected] C. J. Meldrum R. J. Scott Division of Molecular Medicine, Pathology North, NSW Pathology, Lookout Road, Newcastle 2305, NSW, Australia J. E. Carpenter C. L. Clarke Australian Breast Cancer Tissue Bank, University of Sydney at the Westmead Millennium Institute, Westmead, NSW, Australia S. A. Narod Familial Breast Cancer Research Unit, Women’s College Research Institute, Toronto, Canada A. Jakubowska H. Rudnicka J. Lubinski Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland
of the Pomeranian Medical University (335 patients). The entire coding regions and the exon–intron boundaries of BRCA1 and BRCA2 were amplified and sequenced by nextgeneration sequencing. We identified a BRCA1 or BRCA2 mutation in 74 of 774 (9.6 %) triple-negative patients. The mutation prevalence was 9.3 % in Australia and was 9.9 % in Poland. In both countries, the mean age of diagnoses of BRCA1 mutation carriers was significantly lower than that of non-carriers, while the age of onset of BRCA2 mutation carriers was similar to that of non-carriers. In the Australian cohort, 59 % of the mutation-positive patients did not have a family history of breast or ovarian cancer, and would not have qualified for genetic testing. The triple-negative phenotype should be added as a criterion to genetic screening guidelines. Keywords Triple-negative breast cancer BRCA1 BRCA2 Germline mutations Prevalence Genetic testing List of abbreviations BRCA Breast cancer susceptibility gene DNA Deoxyribonucleic acid PARP Poly (adenosine diphosphate)-ribose polymerase TNBC Triple-negative breast cancer UV Unclassified variant pCR Pathological complete response
Introduction TNBC describes a subgroup of breast cancers that are negative for the oestrogen, progesterone and HER2 receptors. They account for between 15 and 20 % of all breast cancers diagnosed but are over-represented in young women and in black women in the United States.
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Table 1 TNBC demographic and clinical data of the Australian and Polish patient cohorts Characteristic
Australian patients, N = 439 (%)
Polish patients, N = 335 (%)
57 ± 15 years
59 ± 10 years
Australian cohort Age at diagnosis B50 years
153 (34.9)
49 (14.6)
[50 years
286 (65.1)
286 (85.4)
Breast cancer only
80 (18.2)
Information only available for mutation carriers (see Table 2)
Breast and ovarian cancer
11 (2.5)
Breast and prostate cancer
2 (0.5)
Breast and other cancer
43 (9.8)
Ovarian cancer only
5 (1.1)
Ovarian and other cancer
6 (1.3)
Other cancer
85 (19.4)
No recorded family history of cancer
207 (47.2)
Family history of cancer
Methods Study cohort
Type of primary tumour Ductal (Invasive/ in situ/not otherwise specified)
409 (93.2)
Papillary
3 (0.7)
2 (0.6)
Medullary
10 (2.3)
42 (12.5)
Apocrine
–
4 (1.2)
Lobular (Invasive/ in situ/not otherwise specified)
9 (2)
14 (4.2)
Metaplastic
–
7 (2.1)
–
3 (0.9)
8 (1.8)
36 (10.7) N = 219
Others Unknown Tumour grade
227 (67.8)
1
6 (1.4)
1 (0.5)
2
48 (10.9)
54 (24.6)
3
374 (85.2)
143 (65.3)
Not known
11 (2.5)
21 (9.6)
BRCA1 and BRCA2 are breast cancer susceptibility genes that are part of the DNA repair pathway. Pathogenic mutations in both genes confer a high risk of breast cancer [1], and together they account for approximately 5 % of all breast cancer cases [2]. A large proportion of tumours in women with a BRCA1 mutation exhibited a triple-negative phenotype. Not all women with breast cancer qualify for BRCA1 and BRCA2 testing. Testing is currently based on
123
the age of onset, the family history and in some cases (e.g. Ashkenazi Jews) on the ethnic group. Currently, tumour histology is not a clear criterion for the recommendation for genetic testing, but some have suggested that all women with triple-negative breast cancer be candidates for genetic testing, regardless of age of onset or family history. With the advent of next-generation DNA sequencing (NGS), it is cost-effective to perform BRCA1 and BRCA2 testing to women outside of a familial cancer setting. Several studies have reported that up to 20 % of women with TNBC breast cancer carry a BRCA mutation [3–12]. The aim of this study was to define the prevalence of germline BRCA1 and BRCA2 mutations in two independent populations of consecutively collected TNBC cases, in Poland and in Australia, unselected for family history and age of diagnosis.
439 patients with TNBC from Australia were included. Cases were selected based on triple-negative status and not on age of onset or family history. The demographic and clinical data are described in Table 1. This study was approved by the Hunter New England Health Human Research Ethics Committee. Samples were provided by the Australian Breast Cancer Tissue Bank (ABCTB). DNA samples from 335 Polish patients with TNBC were provided by the Department of Genetics and Pathology, Pomeranian Medical University. The study was approved by the local institutional ethics review committee for participation in this study. There are three common BRCA1 founder mutations in the Polish population. It is currently recommended that all breast cancer patients in Poland be tested for these three mutations and, for the purposes of the current study, patients with one of these mutations were excluded. 49 women were diagnosed under 50 years of age and 286 were diagnosed over age 50. The average age of diagnosis of these two patient groups is shown in Table 1. BRCA1 and BRCA2 sequencing Target-specific primers were designed by Fluidigm Corp. (San Francisco, CA). Common sequence tags (CS1 and CS2) were added to the forward and reverse primers for Access Array amplicon tagging. 184 primer pairs were designed to cover all coding exons of BRCA1 and BRCA2. Genomic DNA from TNBC patients were normalised to 50 ng/ll concentration and 1 ll of the solution was loaded onto a Fluidigm Access Array, (a microfluidic array in which many concurrent PCR reactions were performed with nested primer pairs). A two-primer protocol was used
Exon
BRCA1
p.Val233Asnfs*4 p.Leu502Alafs*2 p.Gln563* p.Lys652Glufs*21
p.Lys654Serfs*47
c.213-11T[G
c.514delC
c.697_698del
c.1504_1508del
c.1687C[T
c.1952dup
c.1961del
int 5
8
11
p.Gln172Asnfs*62
Results in frameshift
Activates cryptic splice acceptor site. Leads to aberrant splicing and insertion of 59 bp
p.Cys61Gly
Skip of exon 5 very likely
c.181T[G
5
Intronic retention of 58 bp
p.Cys24Serfs*13
c.70_80del
c.135-2A[G
p.Glu23Valfs*17
Amino acid change
c.68_69del
Nucleotide change
int 3
2
Australian cohort
Gene
rs80357747
rs80357522
rs80357885
rs80356898
rs80357888
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
rs80358061
rs80357872
Y/class 5—pathogenic
Y/UV
Y/class 5—pathogenic
Y/class 5—pathogenic
BIC (Y/N—class)
rs28897672
rs80358065
rs80359877
rs80357713
dbSNP
45
03-11-043
02-12-076
02-08-102
05-08-009
05-11-096
04-08-214
08-12-105
01-10-155
04-10-067
07-12-007
01-10-014
47
36
62
68
43
41
69
41
37
47
40
55
03-09-148
03-11-100
26
Age of diagnosis
01-07-014
Patient
Mother, ovarian, 66
Paternal uncle, bowel, 60
Maternal grandmother, breast, unknown
Mother, breast cancer, 45
Paternal cousin, breast, 40 s
Paternal aunt, breast, 50 s
Paternal aunt, breast, 50 s
Maternal niece, breast, 39
Sister, colorectal, 55
Sister, ovarian, 53
Father, pancreatic, 76
Mother, breast, 73
Brother, bowel, unknown
Grandfather, prostate, unknown
Maternal aunt, stomach, 75
Mother, cervical, 38
Maternal cousin, breast, 50 s
Paternal grandmother, breast, 90 s
Mother, breast, 38
Maternal grandaunt, ovarian, 20 s
Maternal grandfather, brain, 30
Paternal grandaunt, stomach, 50
Paternal grandmother, pancreas, 77
Paternal grandfather, leukaemia, 52
Paternal aunt, breast, 36
Father, non-Hodgkins lymphoma and colon, 75
Maternal 2nd cousin, breast, 45
Maternal aunt, breast, 47
Maternal cousin, ovarian, 45
Family history (relationship, type of cancer, age of diagnosis)
Table 2 Deleterious mutations detected in the Australian and Polish cohorts and any recorded family history of diseases associated with the mutation carriers
Breast Cancer Res Treat (2015) 150:71–80 73
123
123
BRCA2
Gene
c.5266dup
c.5272A[T
20
p.Cys419Trpfs*11 p.Phe620Leufs*24 p.Thr630Asnfs*14
c.1257del
c.1860del
c.1889del
10
Classified as pathogenic according to kConFab
Skip of exon 7 is very likely
c.631?2T[G
int 7
p.Leu105*
c.314T[G
p.Leu1764*
p.His1732Phefs*5
p.Thr1677Ilefs*2
3
c.5289del
p.Arg1758*
c.5194-12G[A
int 19
21
p.Gln1756Profs*74
c.5030_5033del
17
p.Trp1508*
p.Leu1404*
p.Ser1253Argfs*10
c.3756_3759del
c.4523G[A
p.Gln1200*
c.3598C[T
c.4210del
p.Ile946Tyrfs*6
c.2835dup
15
Amino acid change
Nucleotide change
13
Exon
Table 2 continued
rs80359315
rs80359272
rs81002899
rs80358561
rs80357906
rs80358079
rs80357862
rs80357765
rs80357868
rs62625307
rs80357519
dbSNP
Y/class 5—pathogenic
N
Y/class 5—pathogenic
Y/UV
Y/class 5—pathogenic
N
N
Y/class 5—pathogenic
N
Y/class 5—pathogenic
N
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
BIC (Y/N—class)
05-10-073
03-07-065
01-08-025
01-06-021
48
56
84
34
89 46
05-09-110
55
36
47
45
55
45
04-11-051
01-11-082
03-09-059
04-10-126
01-08-061
02-08-30S
01-12-021
29
33
04-11-088 03-06-041
30
43
44
Age of diagnosis
02-08-124
08-10-069
02-08-049
Patient
Maternal grand-aunt, breast, 70
Paternal grandfather, unknown, 80
Mother, breast, 39
Maternal uncle, colorectal, 60
Maternal uncle, colorectal, 60
Maternal uncle, liver, 23
Maternal 2nd cousin, breast, 50 s
Maternal grandfather, colorectal, 68
Maternal cousin, breast, 33
Mother, breast, 58
Paternal cousin, breast, unknown
Paternal aunt, breast, unknown
Maternal aunt, ovarian, 40
Mother, breast, 40
Mother, bladder, 74
Maternal great-aunt, breast, 50
Maternal grandmother, breast, 60
Mother, breast, 60
Mother, breast, 47
Paternal aunt, breast, 50
Maternal cousin, breast, 35
Maternal aunt, breast, 40
Paternal aunt, breast, 60 s
Paternal great-grandmother, breast, 80 s
Maternal aunt, breast, unknown
Cousin, breast
Cousin, breast
Father, throat, 40 s
Maternal aunt, ovarian, 42
Family history (relationship, type of cancer, age of diagnosis)
74 Breast Cancer Res Treat (2015) 150:71–80
Exon
11
16
BRCA1
p.Gln1273*
c.3817C[T
BRCA2
p.Gln1096*
c.3286C[T
c.4689C[A
c.2886dup
p.Tyr1563*
p.Ile963Tyrfs*19
p.Gln1396*
p.Gln1090*
c.3268C[T
c.4186C[T
p.Gly563*
c.1687C[T
13
p.Gly284*
c.80?2T[C
c.850C[T
i2
11
Amino acid change
p.Thr2766Asnfs*11
Nucleotide change
c.8297del
18
p.Ile2315Lysfs*12
p.Val2228Glyfs*5
c.6944_6947del
13
c.6682dup
p.Asn1784Thrfs*7
c.5351del p.Leu1908Argfs*2
p.Tyr1710*
c.5130_5133del
p.Ser1970*
p.Asp1469Lysfs*11
c.4405_4409del
c.5909C[A
p.Gln1452*
c.4354C[T
c.5722_5723del
p.Ala938Profs*21
c.2808_2811del
11
Amino acid change
Nucleotide change
Exon
BRCA1
a
BRCA1
Polish cohort
Gene
Gene
Table 2 continued
rs80357433
rs80357011
rs80357208
rs80357485
rs80357402
rs80356898
rs80358128
dbSNP
rs80359705
rs80359629
rs80359621
rs80358824
rs80359531
rs80359509
rs80359485
rs80359352
dbSNP
Y/class 5—pathogenic
N–UV
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
N
N
BIC (Y/N – Class)
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
N
N
Y/class 5—pathogenic
BIC (Y/N—class)
01-08-037
08-11-063
04-08-036
05-10-118
04-09-164
08-10-074
02-09-527
01-12-019
03-11-109
04-12-030
Patient
43
136,755
160,333#
220,693
161,467
36
41
54
53
62
55
349,099 192,682
33 59
342,887
40
Y
Y
N
Y
Y
N
N
Y
N
N
Family history of breast and/or ovarian cancer (Y/N)
Brother, melanoma, 35
Paternal cousin, breast, 40
Mother, multiple myeloma & lung, 60
Maternal grandmother, melanoma, 90 s
Brother, skin, 40 s
Father, skin, 60 s
Father, prostate, 65
Daughter, thyroid, 54
Maternal grandmother, gastrointestinal, unknown
Brother, prostate, 59
Father, bowel, 54
Maternal aunt, breast, 50 s
Mother, breast, 50 s
Family history (relationship, type of cancer, age of diagnosis)
Age of diagnosis
285,146
228,532
146,990
Patient
44
44
39
63
57
83
60
78
58
59
Age of diagnosis
Breast Cancer Res Treat (2015) 150:71–80 75
123
123
a
c.9097dup
c.9253dup
24
p.Glu2089Aspfs*2
c.6267_6269delinsC
c.8946del
p.Tyr1894*
c.5682C[G
23
p.Ser1882*
c.5645C[A
22
p.Asn1784Lysfs*7
c.5352del
c.7558C[T
p.Asn1747*
c.5237dup
15
p.Lys1025Asn;Lys1026*
c.3075_3016delinsTT
11
p.Thr3085Asnfs*26
p.Thr3033Asnfs*11
p.Asp2983Ilefs*5
p.Arg2520*
p.Asn433Glnfs*18
p.Val220Ilefs*4
c.1296_1297del
p.Arg1835*
c.658_659del
c.5503C[T
24
p.Thr1677Ilefs*2
10
c.5278-2A[T
i20
Intronic retention of 65 bp
8
c.5030_5033del
17
p.Met1663Valfs*14
p.Glu1661*
c.4981G[T
c.4986?3G[C
Amino acid change
Nucleotide change
i16
Exon
Patient 160,333 harbours both BRCA1 and BRCA2 mutations
BRCA2
Gene
Table 2 continued
rs80359752
rs80359747
rs80358981
rs41293497
rs80358785
rs80359499
rs80358552
rs80359276
rs80359604
rs41293465
rs80357862
rs80358023
rs80357401
dbSNP
Y/class 5—pathogenic
Y/class 5—pathogenic
N–UV
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic COSMIC—distribution in ovary and endometrium
N/UV
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
Y/class 5—pathogenic
COSMIC—distribution in ovary and large intestine
Y/UV
N–UV
Y/class 5—pathogenic
Y/Class 4—likely pathogenic
Y/class 5—pathogenic
BIC (Y/N – Class)
33 41
73,032 353,587
53
174,157
192,325
285,224
266,895
99,173
151,271
45
68
73
62
52
58 50
195,860
68
349,466
346,635
63
273,641
49
52
171,190 87,035
58
71
77
58
66
38
54
135,383
119,568
334,861
173,119
156,407
166,662
94,836
62
53
101,115
136,084
Age of diagnosis
Patient
N
N
Y
Y
Y
N
Y
Y
Y
N
Y
N
Y
N
N
Y
Y
Y
N
Y
N
Y
Y
Family history of breast and/or ovarian cancer (Y/N)
76 Breast Cancer Res Treat (2015) 150:71–80
Breast Cancer Res Treat (2015) 150:71–80
A
Distribution of age of diagnosis of BRCA mutation carriers 40
% of patients within mutation status
Fig. 1 Distribution of age of diagnosis of A. BRCA mutation carriers (Australian average = 50.3 ± 15 years, Polish average = 53.6 ± 11.5 years). The average age of Australian and Polish BRCA1 mutation carriers is 47.2 ± 11.8 years and average age of BRCA2 mutation carriers is 58.8 ± 13.2. B. The distribution of the age of diagnosis of the non-mutation carriers (average age of nonmutation carriers is 58.7 ± 12.7 years)
77
35
BRCA1 mutation carriers
30
BRCA2 mutation carriers
25 20 15 10 5 0
25-34
35-44
45-54
55-64
65-74
75-84
≥85
Age of diagnosis (years)
% of patients within non-mutation status
B
Distribution of age of diagnosis of patients without mutations 40 35
Non-mutation carriers
30 25 20 15 10 5 0
25-34
35-44
45-54
55-64
65-74
75-84
≥85
Age of diagnosis (years)
to first amplify the target sequence followed by a second PCR reaction that included the sequencing tags such that ultimately each amplified product contained the target-specific sequence, a tag sequence and a sample-specific unique barcode. The exons were amplified and the PCR products were harvested from each sample, pooled, and purified using Ampure XP beads. The libraries were then sequenced on an Illumina MiSeq instrument. The sequencing data were analysed with NextGENe 2nd Generation Sequencing Software v.2.3.3. (SoftGenetics, Philadelphia). Raw data were converted into FASTA files and aligned to BRCA1 and BRCA2 reference sequences from the human reference sequences; GRCh37 Primary Assembly, BRCA1 NC_000017.10 and BRCA2 NC_00013.10 (http://www.ncbi.nih.gov). The CS1 and CS2 sequences were trimmed, as well as the primer sequences to eliminate the dilution of base calling from those regions. The mutations were described using the nomenclature guidelines of the Human Genome Variation Society (http:// www.hgvs.org). The DNA sequence numbering is based on
the cDNA sequences for BRCA1 (NM_ 007294.3) and BRCA2 (NM_ 000059.3). BRCA1 or BRCA2 mutations were only included in the analyses if classified as deleterious or possibly deleterious according to Breast Cancer Information Core (BIC) criteria or if present in the BIC database [13]. The criteria are all frameshift and nonsense variants (except those resulting in neutral or polymorphic stop codons), all intronic variants in the consensus splice acceptor or donor sites (within 2 bp of exon–intron junctions or if shown to result in aberrant mRNA transcript processing, or non-synonymous variants shown to have deleterious effect on known functional regions (based on functional or biochemical assays, or linkage analysis of high-risk families) [14].
Results We identified 74 mutations in the 774 triple-negative breast cancer patients (9.6 %). Mutations were present in 9.3 %
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78
40.0
Proportion of BRCA mutationpositive patients (%)
Fig. 2 The proportion of patients with or without recorded family histories of breast and/or ovarian cancer in each age group in those with germline BRCA mutations. In patients with family histories of disease: \40 years—17.1 %, 40–60 years—22 %, C60 years—2.4 %. In patients without recorded family histories of disease: \40 years—4.9 %, 40–60 years—36.6 %, C60 years—17.1 %
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Distribution of age of diagnosis of patients with versus without recorded family history of breast and/or ovarian cancer
35.0 30.0
With family history
25.0
Without recorded family history
20.0 15.0 10.0 5.0 0.0 60
Age of diagnosis (years)
Table 3 Percentage of patients with BRCA1 or BRCA2 mutations compared to non-mutation carriers diagnosed with breast cancer Age of diagnosis (years)
BRCA1 mutation carriers
BRCA2 mutation carriers
Non-mutation carriers
Australian breast cancer cohort
(%, n = 26)
(%, n = 15)
(%, n = 398)
\40
26.9
13.3
12.6
40–60
57.7
60
43.7
C60
15.4
26.7
43.7
(%, n = 18)
(%, n = 15)
(%, n = 302)
\40
22.2
0
2.3
40–60
61.1
60
54
C60
16.7
40
43.7
Polish breast cancer cohort
of the Australian patients and in 9.9 % of the Polish patients (see Table 2 for a complete list of deleterious mutations and Supplementary Table 1 for all other alterations identified in BRCA1 and BRCA2). In Australia, 63 % of the mutations were in BRCA1; in Poland 54.5 % of the mutations were in BRCA1. The distribution of the age of diagnoses is shown in Fig. 1. In the Australian cohort, four of the 41 mutations are novel and have not been reported in BIC, Leiden Open (source) Variation Database (LOVD), Clinvar, or any published literature. Of these, two are in BRCA1 (c.4523G[A, c.5272A[T) and two in BRCA2 (c.1860del, c.4354C[T). Among the 41 Australian patients with a mutation, 17 (41.5 %) had a family history of breast or ovarian cancer (Table 2). In the Australian cohort, the mean age of disease onset for BRCA1 mutation carriers is 46.0 years of age and for BRCA2 mutation carriers is 57.6 years of age, and for non-carriers, it was 57.8 years of age (p = 0.004). A deleterious mutation was detected in 33 of 335 patients in Poland (9.9 %). Two mutations have not been reported in BIC, Leiden Open (source) Variation Database (LOVD) or Clinvar. These include one in BRCA1 (c.80?2T[C) and one in BRCA2 (c.2886dup).
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Among the 33 Polish patients with a mutation, 19 (57.5 %) had a family history of breast or ovarian cancer (Table 2). In the Polish cohort, the mean age of onset for BRCA1 mutation carriers is 48.9 years of age, whereas for BRCA2 mutation carriers, it is 59.9 years of age and for non-mutation carriers, it is 60 ± 9.8 years of age (p = 0.004). The distribution of age of diagnosis of our patient cohort according to age groups in those with and without BRCA mutations is shown in Fig. 2.
Discussion In both Poland and in Australia, approximately 10 % of the patients with triple-negative phenotype harbour a germline BRCA1 or BRCA2 mutation. Our study highlights the importance of genetic testing for patients with TNBC regardless of age and in the absence of a family history. In Poland, genetic testing for three founder mutations is already recommended for all breast cancer patients. We recommend that in the absence of one of the founder mutations, Polish patients with triple-negative breast cancer be considered for additional genetic testing, which should include full sequencing of the coding regions of both genes.
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In both countries, BRCA2 mutations were less common than BRCA1 mutations, in agreement with other studies [3, 8, 15, 16]. These results may reflect the propensity for BRCA1 carriers to develop primarily triple-negative cancers, whereas among BRCA2 carriers, the majority of breast cancers are oestrogen-receptor positive. BRCA2 mutation carriers were shown in our study to be diagnosed with TNBC at an older age than BRCA1 mutation carriers and similar to the age of onset of non-carriers. Recent guidelines by the National Comprehensive Cancer Network (NCCN Guidelines in Oncology Version 3.2013 Genetic/Familial High-Risk Assessment: Breast and Ovarian) recommends that TNBC diagnosed before or at age 60 is considered to be sufficient to meet the threshold of risk assessment for genetic testing [17]. In contrast, the Cancer Institute New South Wales guidelines recommend that patients with TNBC be offered testing if diagnosed under the age of 40 years. [18]. From our results, 33.3 % of BRCA2 mutation carriers in both the Australian and Polish cohorts are over 60 years of age, therefore according to the NCCN guidelines, they would not qualify for genetic testing (see Table 3). Our results suggest that the age of diagnosis should not be a limitation to genetic testing eligibility. The purposes of genetic testing of breast cancer patients are to identify additional family members who might benefit from preventive interventions and to guide the choice of treatment of the breast cancer patients. Under optimal circumstances, the genetic test results would be made available within 2 weeks of diagnosis. BRCA1 mutations carriers have been shown to have enhanced benefit from chemotherapy, in particular to neoadjuvant cisplatin chemotherapy. In a recent study, we found that 61 % of 107 patients with a BRCA1 mutation had a complete pathologic response when given cis-platinum [19, 20] and all who experienced a pCR are currently alive. In addition, women with a BRCA1 mutation have been shown to benefit from bilateral mastectomy [21] and from oophorectomy [22]. Acknowledgments DNA samples were received from the Australian Breast Cancer Tissue Bank, which is generously supported by the National Health and Medical Research Council of Australia (NHMRC), the Cancer Institute NSW (CINSW) and the National Breast Cancer Foundation (NBCF). The tissues and samples are made available to researchers on a non-exclusive basis. This work was supported by the National Breast Cancer Foundation (NBCF), Australia. Dr Michelle Wong-Brown is supported by the Hunter Translational Cancer Research Centre with funding from the Cancer Institute New South Wales. Conflict of interest The authors of this article declare no competing interests related to the study and no commercial associations that may pose a conflict of interest.
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