Predisposition for Breast Cancer

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1 C H A T E R 4 redisposition for Breast Cancer Marion Kiechle, MD and Alfons Meindl, hd Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany Etiology Breast cancer is the most frequent carcinoma in women. In more developed countries, the probability of contracting breast cancer between the ages of 20 and 80 is approximately 7.8%, that is, 1 in 13 women. 1 A comparison of familial and sporadic cases shows that familial cancer diagnosed at a younger age is frequently bilateral and frequent in men. 2 In recent years it has been estimated that 5 15% of breast cancer cases involve genetic events. However, careful epidemiologic investigations now indicate that only one third (5%) might be caused by monogenic factors. Most inherited breast cancer cases are now assumed to be polygenic, and most diagnosed breast cancer cases are sporadic without familial clustering (FIG. 4.1). 1,3 In contrast, it can no longer be excluded that at least some of the sporadic cases may be caused through a combination of polygenic variants and exogenous factors. 3 The penetrance of inherited mutated alleles is modulated by additional low penetrant genetic variants (modifier genes), environmental factors, or both. Factors such as nulliparity, physical exercise, diet, obesity, and birth age influence the age at onset in gene mutation carriers in both sporadic and familial cancer. 4 Indeed, it can be observed in pedigrees from families with multiple occurrences of breast and/or ovarian cancer that the age at onset of the disease is decreasing. 4,5 It is still controversial whether the use of oral contraceptives increases the risk of developing breast cancer in BRCA mutation carriers. Some studies calculate a small but statistically significant risk, 6 whereas other studies have demonstrated no greater risk. 7 The penetrance of predisposing genes for breast cancer is also dependent on age and tissue type. For the two known breast cancer predisposition genes, BRCA1 and BRCA2 (see High enetrance Genes, below), risks have been determined 4 (Table 4.1). In the case of mutation carriers in the BRCA1 or BRCA2 genes (see below), only breast and ovarian tissues have a relatively high risk to transform into malignant cells. In addition, the probability to change into an aberrant phenotype is age dependent. Likewise, the probability for a BRCA1 mutation carrier to develop breast cancer at the age of 50 is approximately 29%. However, women with a carrier status and born after 1940 may have higher risks. 4,5 Only slightly increased risks for transformation are described for such tissues as prostate, colon, and pancreas. 8,9 The known forms of hereditary breast cancer are inherited in an autosomal dominant mode. It should be noted, however, that inactivation of one allele is only a prerequisite for tumor generation and is not sufficient to initiate cancer in normal epithelial breast cells. Indeed, for tumor initiation, the second allele has to be inactivated. This can happen either by loss of heterozygosity in the according regions 10,11 or by epigenetic effects such as methylation (see Chapters 26 and 27). 12 Tumor progression requires the accumulation of additional aberrations in the cellular genome, including the disturbance of T53 expression that allows cell escape from apoptosis. 13 Based on histologic parameters and gene expression profiling, BRCA1 tumors can be distinguished from BRCA2 and sporadic breast cancer samples. Tumor samples from BRCA1 mutation carriers are normally high grade, infiltrating ductal breast cancers 14 and more often estrogen and HER-2/neu receptor negative than sporadic samples. 14,15 In contrast, BRCA2 tumors are more difficult to separate from sporadic cases by histologic classification only, but it should be feasible through more validated expression profiling data; for example, tumors from BRCA2 mutation carriers show an 48

2 Chapter 4 redisposition for Breast Cancer 49 15% hereditary breast carcinoma 85% sporadic breast carcinoma 2% BRCA1 1% BRCA2 2% BRCAX 5-10% Familial Clustering FIGURE 4.1 Origins of breast carcinoma. Table 4.1 Risks for BRCA1 or BRCA2 Mutation Carriers to Develop Breast and/or Ovarian Cancer BRCA1 BRCA2 Age (yr) BCa OvCa BCa OvCa 40 21% 3% 17% 2% 50 39% 21% 34% 4% 60 58% 40% 48% 6% % 60% 80% 30% expression profile that is different from BRCA1 but also from sporadic tumors. 16 redisposition Markers It is estimated that 10 15% of all breast cancer cases may be caused by genetic predisposition. The inheritance of a single defective allele is most likely in a family exhibiting clustering of breast cancer cases, especially if it includes cases diagnosed before age 50. Consequently, a dominant monogenic trait can be demonstrated in families with multiple occurrences of breast cancer (high penetrance genes). In contrast, the segregation of multiple deficient alleles is most likely in families with fewer cases of late onset breast cancer (low penetrance genes). The genes associated with hereditary forms of breast cancer are listed in Table 4.2 and are discussed in the following paragraphs. High enetrance Genes To date, two genes that feature frequent mutations associated with breast cancer predisposition in high risk families have been isolated and validated Other genes also involved in the DNA repair or apoptosis pathways have been characterized but only rarely were shown to be mutated. Some of these genes are restricted to site-specific breast cancer, whereas others are associated with other cancer entities. BRCA1 The first gene found to be mutated in families with multiple occurrences of breast and/or ovarian cancer was the BRCA1 gene. 17,19 It has been classified as a classic tumor suppressor gene, and consequently inactivation of the second allele in the resulting tumor could be demonstrated (see above). It is located at 17q21; comprises 24 exons, 22 of them coding; and encodes for a protein consisting of 1863 amino acids. A typical pedigree of a family with a BRCA1 mutation is given in FIGURE 4.2. Currently, more than 2000 different deleterious mutations in the gene are listed in the international Breast Cancer Information Core (BIC) database ( Most of the putative pathogenic mutations found in the gene are either frame-shift or stop mutations that similarly cause the production of truncated proteins. 5,17 Only a few missense mutations, causing single amino acid substitutions, could be demonstrated to be disease causing. 20,21 Recently, a high prevalence of large gene rearrangements in the BRCA1 gene has been reported for some populations. 22,23 Mutation profiles for different countries have been published, 5,24 26 and it has become apparent that a mosaic of founder- and populationspecific mutations exists. Founder mutations are often of Ashkenazi Jewish origin and were shown to be present in different white populations. 5,24 26 Only a few missense mutations in the BRCA1 gene can be clearly associated with familial breast cancer. These include amino acid residues that are part of the zinc finger

3 50 MOLECULAR ONCOLOGY OF BREAST CANCER Table 4.2 List of Genes Associated with Hereditary Breast Cancer Syndrome Gene/Region rimary Carcinoma Secondary Carcinoma High enetrance Genes Familial breast/ BRCA1 Breast and rostate,colon ovarian carcinoma 17q21 ovaries leukemia Familial breast/ BRCA2 Breast and rostate, pancreas ovarian carcinoma 13q13 ovaries male breast Familial breast/ BACH1 Breast None bilateral b.c. 17q22 Familial breast/ RAD51 Breast None bilateral b.c. 15q15 Li-Fraumeni T53 Sarcomas, Brain, leukemia syndrome 17p13 breast Cowden TEN Hamartomatous olyps syndrome 10q23 lesions eutz-jeghers STK11 Gastrointestinal Melanin spots syndrome 19q polyps on lips, b.c. Low enetrance Genes Ataxia telangiectasia ATM Lymphomas Heterozygous: 11q22 gliomas breast cancer Li-Fraumeni/ CHEK2 Breast Brain, leukemia breast cancer 13q21 sarcomas b.c., breast cancer. I:1 I:2 OvarialCa 54J II:2 II:1 MammaCa bilateral Dx: 42/42J OvarialCa bilateral Dx: 60/60J verst: 62J II:4 II:3 OvarialCa 51J III:2 III:1 MammaCa bilateral Dx: 34/42J III:3 III:4 27J IV:1 9J IV:2 14J For individual: III:1 Calculated risk of cancer (34) Heteozygote risk: 98.1% FIGURE 4.2 A mutation in the BRCA1 gene, causing a truncated protein (1364X).

4 Chapter 4 redisposition for Breast Cancer 51 1 Sensing/signaling DNA damage 2 ATM ATR CHK2 Kinases MRE11, RAD50, Nbs1 Sensors BRCA1 MSH2 MSH6 BLM BARD1 MRE11 RAD50 Nbs1 3 DSB resection SWI/SNF BACH1 HDAC 300/CB ZBRK1 Ctl 4 Transcriptional regulation of GADD45 6 Ubiquitin ligation 5 MSH2 MSH6 RNA pol. II 300/CB Altering DNA topology at damage sites FIGURE 4.3 Role of the BRCA1 protein in the DNA repair process. (From Ref. 28.) Transcription, transcription-coupled repair (Cys61Gly, Cys64Y) and amino acids located in the BCRT domain that have been shown to interact with different proteins (e.g., W1837R). Several strategies have been established to evaluate the character of such unclassified variants. These include segregation analysis, evolutionary methods to identify functionally important amino acid sites, loss of heterozygosity analysis in the corresponding tumor samples, and functional assays. 20,21 A variety of assay approaches has now become available, enabled by the advanced characterization of the BRCA1 protein. Based on the initial observation that the BRCA1 protein is a caretaker of chromosomal stability, several studies demonstrated that it is involved in the repair of double-stranded DNA breaks (for details, see Chapter 25). Such genomic lesions can be repaired by nonhomologous end joining, recombination between homologous DNA sequences, or single-strand annealing. 27,28 In addition, other than its local activities at sites of DNA damage, BRCA1 seems to be involved in processes that are located upstream and downstream of DNA damage responses. On the one hand, BRCA1 is part of protein complexes that apparently have functions intrinsic to the sensing and signaling of different types of DNA lesions; on the other hand, it operates as a transcriptional regulator of genes, whose expression affects downstream biologic responses (FIG. 4.3). The local activities of BRCA1 at sites of DNA damage have been elucidated by several experiments. Sites of DNA damage are rapidly marked by the phosphorylation of the histone species H2A-X, and BRCA1 interacts with the MRE11/RAD50/Nbs1 protein complex to migrate to such sites and resect double-stranded DNA breaks. 28,29 It may be necessary to alter the DNA topology at the damage sites via recruiting the protein complexes SWI/SNF, BACH1, HDAC, and 300/CB for effective DNA repair as well as the regulation of downstream processes. Included in these downstream processes are the transcriptional regulation of GADD45 and transcription coupled repair (FIG. 4.3). Surprisingly, to date, only the BACH1 gene has been associated with hereditary breast cancer (see Infrequently

5 52 MOLECULAR ONCOLOGY OF BREAST CANCER Mutated Genes, below). Upstream processes of these local activities require the phosphorylation of the BRCA1 protein via the kinases ATR, ATM, and CHK2 proteins (FIG. 4.3) Interestingly, these two latter kinases could be associated with hereditary breast cancer (see The ATM Gene and The CHK2 Gene, below). Apart from the breast and ovary, compared with many organs in which there is no cancer predisposition risk, several other organs such as the pancreas, prostate, and colon have a slightly increased risk of cancer development in BRCA1 mutation carriers. 8,9 This observation indicates that BRCA1 disruption may have tissue-specific effects that favor malignant transformation. It has been suggested that BRCA1 functions as an inhibitor of estrogen receptor signaling. 33 Studies have substantiated this observation. 34,35 Evidence that BRCA1 mediates repression of the estrogen receptor has been reported, and this process is correlated with the down-regulation of the transcriptional coactivator p Further characterization of this pathway may help to explain the tissue specificity of cancer development in BRCA1-deficient cancers. The clinical outcome of patients with germline mutations in BRCA1 has been evaluated in several studies. Most informative is the comprehensive study of Robson et al. 15 in which two retrospective cohorts of Ashkenazi Jewish women undergoing breast-conserving treatment for invasive cancer were genotyped and the clinical characteristics of the women with and without BRCA founder mutations were compared. In brief, BRCA1 mutation carriers demonstrated a significantly shorter overall survival. This adverse prognosis could not be linked to tumor size or axillary nodal status. This study also demonstrated that women with BRCA1 mutations are at substantially increased risk for developing metachronous contralateral breast cancer, whereas no increased risk could be seen for the development of ipsilateral breast cancer when compared with noncarriers. BRCA2 A second high penetrance gene found to be mutated in families with multiple occurrences of breast cancer is the BRCA2 gene. It is located at 13q12.3 and consists of 27 exons, 26 of them coding for a protein of 3418 amino acids. 18,19 A typical pedigree with a mutation in the BRCA2 gene is given in FIGURE 4.4. Similar to the BRCA1 gene, most of the defined cancer-causing mutations are either frameshift or stop mutations that produce truncated proteins. More than 1000 deleterious mutations in the BRCA2 gene are listed in the international BIC database. However, compared with BRCA1, more missense mutations can be seen in the BRCA2 gene. Because the BRCA2 protein is not as well characterized as the BRCA1 protein, it is necessary to evaluate the character of the observed unclassified variants carefully. Overall, it appears that most of the listed unclassified variants in the BRCA2 gene are rare polymorphic variants (unpublished data) rather than pathogenic. Analogous to the BRCA1 gene, founder mutations are presented frequently in most populations. 36 I:1 I:2 II:1 II:2 Breast cancer diagn: 50ys II:3 unclassified carcinoma II:4 Breast cancer diagn: 70ys II:5 III:5 III:3 rostate cancer III:4 Breast cancer diagn: 52ys III:6 III:7 III:2 III:1 Breast cancer diagn: 63ys IV:4 IV:6 IV:7 IV:5 IV:9 IV:8 IV:10 IV:11 IV:1 Breast cancer diagn: 46ys V:1 V:2 V:3 FIGURE 4.4 A mutation in the BRCA2 gene, causing a truncated protein (S1882X).

6 Chapter 4 redisposition for Breast Cancer 53 Nucleus FA complex DNA damage/ replication A G C F E DNA-repair BRCA1 BRCA2 RAD51 RAD51 RAD51 D2 K561 D2 K561 Ub DNA-repair foci FIGURE 4.5 BRCA2 and FA proteins in DNA repair. (From Ref. 44.) Mutations in the BRCA2 gene are normally found in site-specific breast cancer families, including male breast cancer. However, families with a clustering of pancreatic cancer were also shown to harbor BRCA2 mutations. 37,38 In one study, European families in which at least two first-degree relatives had a histologically confirmed diagnosis of pancreatic ductal adenocarcinoma were screened for mutations in the BRCA2 gene, and three deleterious and missense aberrations were found. In addition, it could be shown that 2% of men with early onset prostate cancer harbor germline mutations in the BRCA2 gene. Male BRCA2 mutation carriers under the age of 60 have a 26-fold risk increase of developing prostate cancer compared with control subjects. 39 However, further studies are required to confirm that BRCA2 mutations are associated with a higher penetrance in familial cancers of the pancreas and prostate than are BRCA1 mutations. Of further interest is the recent finding that biallelic mutations in the BRCA2 gene have been found in two Fanconi complementation groups. 40 Fanconi anemia (FA) is an autosomal recessive disorder affecting all bone marrow elements and is associated with cardiac, renal, and limb malformations as well as dermal pigmentation changes. Fanconi patients also have an increased cellular sensitivity to DNA cross-linking agents. FA has eight identified complementation groups, and for the complementation groups B and D1, mutations in the BRCA2 gene can be identified. This finding has now linked the FA pathway to the BRCA1/ BRCA2 mediated DNA repair process (FIG. 4.5). Several FA proteins, including FANCA, FANCC, FANCE, FANCF, and FANCG, form a constitutive complex in the nucleus of human cells that in response to DNA damage mediates the monoubiquitination of FANCD2. The activated FANCD2 protein is then translocated to chromatin and the DNA-repair foci that contain BRCA1 and BRCA2 (FANCD1). BRCA2 is known to bind directly to RAD51 41 and, similar to BRCA1, is involved in homology-directed DNA repair. Also, similar to the BRCA1 protein, the BRCA2FA pathway can be linked to ATM and to another upstream activator, the MRE11 complex. A study indicates that in response to cellular exposure to ionizing radiation, the ATM protein directly phosphorylates FANCD2. 42 Thus, the FANCD2 protein functions in mediating two signaling pathways by undergoing two different types of posttranslational modifications. A link to the MRE11 complex (NBS-MRE11-RAD50) can be established through the demonstration that the activated form of FANCD2 and NBS1 are colocalized in nuclear foci. Either the disruption of the MRE11-binding carboxy terminal end of NBS1 or the disruption of the FANCD2 monoubiquitination site results in mitomycin hypersensitivity. 43 In summary, both BRCA proteins are involved in DNA repair, a cellular mechanism activated in response to irradiation or chemically induced lesions. As well as the ATM and NBS pathways, respectively, the FA pathway has now linked to this cellular network system that maintains genomic integrity and stability. 44

7 54 MOLECULAR ONCOLOGY OF BREAST CANCER As in the case of BRCA1 mutation carriers, the clinical outcome for BRCA2 mutation carriers has been evaluated. 15 In contrast to females with predisposing mutations in the BRCA1 gene, females with mutations in the BRCA2 gene have no reduced survival after breast cancer therapy. However, similar to BRCA1 carriers, these patients carry an approximately 30% risk of developing contralateral breast cancer after 10 years of posttreatment follow-up has elapsed. Further follow-up studies are required to confirm these initial data obtained in Ashkenazi Jewish women. BRCAX Although most of the high risk families with multiple occurrences of site-specific breast cancer or breast and ovarian cancer (six or more cases) map either to 13q13 or 17q21, no further predisposing loci have been determined for most families exhibiting fewer cases of breast cancer. Different models have been developed to determine the genetic transmission modes for the relatively large number of BRCA1/BRCA2 negative families. Based on such models, in most of the families showing familial clustering, polygenic traits may segregate. For only a limited number of families, further high penetrance genes acting in an autosomal dominant form are expected. 45 However, one published study has predicted the involvement of an as yet unknown autosomal recessive gene in a significant portion of such families. 46 Infrequently Mutated Genes Based on the observation that BRCA1 and BRCA2 are involved in double-stranded DNA break-repair mechanisms, several groups have searched for aberrations in other genes located in this pathway. The BACH1 gene (BRCA1-associated C-terminal helicase-1) codes for a nuclear protein that acts directly with the highly conserved C-terminal BRCT repeats of the BRCA1 protein. The predicted 1249 amino acid long protein, encoded by 20 exons, contains the seven helicase-specific motifs that are conserved among members of the DEAH helicase family. Cantor et al. 47 identified a germline mutation affecting the helicase domain in 2 of 65 patients with either early onset or familial breast cancer. In subsequent experiments the same group demonstrated that these two and a novel third mutation impair the function of the BACH1 protein. 48 This novel protein is both a DNAdependent ATase and a 5 to 3 -DNA helicase. The helicase activity is strictly AT dependent, and when a ro47ala missense mutation is present, this activity is abrogated. Due to another missense mutation (Lys52Arg), ATase activity is disrupted as the unwinding of DNA and RNA hybrids, an additional function associated with the wild-type. Finally, one missense mutation maintains the ATase activity but disturbs the unwinding process. Further studies will show whether other variants in the gene will have at least a minor effect on hereditary breast cancer, for example, in combination with CHEK2 (see The CHK2 Gene, below). Mouse experiments demonstrated that BRCA2 interacts with RAD51 (see BRCA2, above). Recently, ellegrini et al. 49 showed, on the basis of crystal structures, that the direct binding is mediated by the BRC repeat (BRCA2) and the RecA-homology domain (RAD51). The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament that is essential for strand-pairing reactions during DNA recombination. In parallel to the still ongoing functional works on BRCA2 and RAD51, several groups looked for aberrations in the RAD51 gene. In a study with 20 patients from breast cancer families, Kato et al. 50 identified a missense mutation (Arg150Gln) in two unrelated females, both presenting with bilateral breast cancer. In addition, a single nucleotide polymorphism in the 5 -untranslated region of the RAD51 gene (135g c) has been associated with an increase for BRCA2 mutation carriers. 51 However, further studies, including quantitative reverse transcriptase-polymerase chain reaction, are required to substantiate this finding. Genes Associated with Cancer Syndromes Breast cancer is also a component of several other cancer syndromes, including Li-Fraumeni syndrome (OMIM ), Cowden syndrome (OMIM ), and eutz- Jeghers syndrome (OMIM ). All these syndromes are rare, and mutations in the genes causing them contribute only a small fraction of hereditary breast cancer cases. The T53 gene is mutated in Li-Fraumeni syndrome, which is characterized by the occurrence of multiple cancers, including childhood sarcoma and breast cancer (see Chapter 23). 52 Mutations in the T53 gene

8 Chapter 4 redisposition for Breast Cancer 55 are found in approximately 70% of the Li- Fraumeni families. A higher cancer risk in females than in males was observed by using follow-up studies. 53 Quite recently, mutations in the CHEK2 gene have also been associated with Li-Fraumeni syndrome (see The CHK2 Gene, below). Although only a few germline mutations in T53 contribute to hereditary breast cancer, somatic inactivation of T53 in BRCA mutation related tumors is frequently identified (see above). 54 It is now accepted that inactivation of T53 prevents the initiation of apoptosis, prevents the depletion of cells that have accumulated genomic lesions, and thus contributes to the establishment and progression of malignancy. Multiple hamartomatous lesions, especially of the skin, mucous membranes, breast, and thyroid, and craniomegaly are characteristic lesions of the Cowden syndrome. In 1997, mutations in the TEN (phosphatase and tensin homolog) gene were identified in families with the Cowden syndrome phenotype. 55 Although germline mutations in TEN are rare, TEN is frequently inactivated in sporadic cancers, including glioblastomas and prostate cancer (see Chapter 23). 56 Recent data indicate that TEN is involved in the regulation of apoptosis 56 and angiogenesis. 57 During both processes, the TEN protein acts through the well-recognized tumor progression associated phosphoinositol-3-kinase and Akt-dependent pathways. A serine-threonine kinase, the STK11/ LKB1 gene, was found to be mutated in several families with eutz-jeghers syndrome. 58 This is an autosomal dominant disorder characterized by melanocytic macules of the lips, multiple gastrointestinal hamartomatous polyps, and an increased risk for various neoplasms, including gastrointestinal and breast cancer. Carrier females with a mutation in the STK11 gene also have an elevated risk for breast cancer, as has been shown in a British study in which 33 eutz-jeghers families were evaluated by follow-up data. 59 Whether the STK11 gene also plays a role in sporadic breast cancer, as suggested in a recent study, 60 requires validation with larger patient groups. Low enetrance Genes Currently, three genes are considered to act as low penetrance genes for hereditary breast cancer. In contrast to high penetrance genes, such genes act in a concerted way, either in connection with variants in other predisposing genes or in combination with epigenetic or environmental factors. In addition, several modifier genes in breast cancer have been described and are summarized elsewhere. 61 The ATM gene A first gene in which variants have been associated with breast cancer is the ATM gene (OMIM ). The ATM gene is mutated on both alleles in ataxia telangiectasia and codes for a member of the phosphatidylinositol-3 kinase family of proteins. Similar to the two BRCA proteins, ATM plays a key role in monitoring genomic integrity and regulating cell cycle checkpoints, DNA repair, and apoptotic pathways induced by doublestranded DNA breaks. After the occurrence of double-stranded breaks in the cell, ATM interacts with and phosphorylates a number of different targets, including p53, Nibrin, CtI, BRCA1, and CHEK2 (FIGS. 4.3 and 4.6). 62 Given that there are several confirmed direct functional links between the ATM and BRCA1 proteins, the ATM gene also has been implicated in breast carcinoma susceptibility. In two larger mutation screening reports, variants in the ATM gene have been linked to familial breast cancer. 62,63 In both studies, missense mutations in the ATM gene were shown to be specific for familial cases but were absent from normal control subjects (Table 4.2). However, additional studies are required to confirm that ATM mutations confer increased susceptibility to breast cancer. The CHK2 gene Cell cycle checkpoint kinase 2 (CHEK2 [OMIM ]) plays an important role in the maintenance of genome integrity and in the regulation of the G2/M cell cycle checkpoint. It has been shown to interact with other proteins involved in DNA repair processes, such as BRCA1 and T53. 32,64 These findings suggest that CHK2 could be an attractive candidate gene associated with susceptibility for a variety of cancers (FIG. 4.6). atients with Li-Fraumeni syndrome were analyzed, and in a small proportion of families a single mutation in exon 10 of CHK2 was identified. 65 This del1100c variant causes a stop codon after amino acid 380 abrogating the kinase activity. The CHK2 Breast Cancer Consortium analyzed 718 families with breast and/or ovarian cancer that harbor neither a BRCA1 nor a BRCA2 mutation. The 1861delC mutation was identified in 5.1% of the families

9 56 MOLECULAR ONCOLOGY OF BREAST CANCER DNA damage ATM or ATR p53 CHEK2 BRCA1 p53 Cell-cycle arrest Apoptosis DNA repair FIGURE 4.6 ossible role of CHK2 in breast cancer development. (From Ref. 64.) compared with a prevalence of 1.2% in a healthy control group, indicating a low penetrance of this mutation. 66 To establish the relevance of CHK2 mutations in the German population, all 14 coding exons were analyzed in 516 families negative for mutations in both the BRCA1 and BRCA2 genes. As in other studies, a statistically significant association for the exon 10 mutations was observed ( 0.006), and carriers had an almost fourfold increased risk for familial breast cancer (relative risk, 3.9). However, the prevalence of the 1100delC mutation in both familial breast cancer patients (1.6%) and control subjects (0.5%) in the German population is significantly lower than reported for other white populations. It shows independent segregation for breast cancer in two of four families analyzed. Furthermore, no increased breast cancer risk was found for the most common missense mutation I157T or the splice site mutation IVS2 1G A, which added together for more than half of the variants (12 of 21) observed in patients. 67 The low prevalence of the exon 10 deletion mutations together with no or an uncertain elevation in risk for other CHK2 mutations suggests only a limited relevance of mutations in the CHK2 gene for familial breast cancer. Therefore, at present it appears premature to include CHK2 in genetic counseling for patients with an unexplained high familial incidence of breast cancer. Androgen Receptor Gene Mutations in the androgen receptor (AR) gene have been linked to different disease entities. Expansions of CAG repeats within the coding region can cause spinal and bulbar muscular dystrophy (Kennedy disease) and different forms of androgen insensitivity. 68 The first link to breast cancer for AR was confirmed by the identification of germline mutations in two brothers with breast cancer and Reifenstein syndrome. 69 More recently, a polymorphic CAG repeat in the AR gene has been associated with a poorer prognosis for BRCA1 mutation carriers. Comparison of 165 women with and 139 women without breast cancer revealed that women carrying at least one allele with 29 or 30 CAG repeats manifested the disease earlier. 70 In summary, an evaluation of genes localized in either apoptosis, DNA repair, or endocrine signaling will provide further low penetrance or modifier genes. Ongoing mapping and cloning studies will show whether a gene segregates recessively or dominantly in high risk families.

10 Chapter 4 redisposition for Breast Cancer 57 redisposition Testing and Risk Management Because of the complexity of genetic counseling in hereditary breast cancer, referral to a subspecialized multidisciplinary clinic staffed by practitioners with expertise in this field is generally recommended. In this setting, a new client can be educated by an experienced genetic counselor about the a priori risks associated with breast cancer predisposing germline mutations. At the same visit, the client can also receive information from medical and surgical/gynecologic oncologists concerning screening, diagnosis, and treatment options. Finally, this approach also allows the client to meet with a psychologist/psychiatrist to consider the important psychologic and emotional issues associated with breast cancer predisposition testing and the implications for the client and her family. In such a multidisciplinary counseling setting, the sensitivity and specificity of the applied molecular techniques, the indications for testing, and possible risk managements in BRCA1/2 mutation carriers and/or BRCA1/2 negative females from putative BRCAX families can be thoroughly considered. Methods A variety of laboratory techniques has been used for screening for genetic mutations in hereditary breast cancer. In addition, protein truncation tests have been developed as prescreening methods 71 and can be applied for the rapid identification of the likely presence of underlying pathogenic mutations. However, it is now accepted that screening of index patients in families with either multiple cases or early onset manifestation of breast cancer should be applied by investigating the entire gene to also detect missense mutations and putative splice sites. Two independent methods that are applied currently in routine diagnosis include the direct double-stranded sequencing approach, as performed by the company Myriad Genetics, 19,24 and denaturing high-performance liquid chromatography (DHLC) analysis of all coding exons with the adjacent intronic sites. 5,36 In families with a significant prior probability of a BRCA mutation, testing for large gene rearrangements 22,23 also has to be included. For this approach, a kit based on multiplex ligation-dependent probe amplification is available now. 72 Overall, the sensitivity of this technique is greater than 90%, and when either complete direct double-stranded sequencing or comprehensive DHLC analysis is combined with multiplex ligation-dependent probe amplification, the specificity is greater than 90%. Indications for Testing Genetic testing is recommended only after multidisciplinary genetic counseling. The criterion for testing is a 20% probability of the presence of a mutation Candidates for BRCA1/2 genetic testing are as follows: Two breast cancer cases under age 40 or three under age 50 years; Four cases of breast cancer under age 60 years; More than four cases of breast cancer at any age; Ovarian and breast cancer in a family (breast cancer under age 50 years if only one ovarian and one breast cancer case); Early onset female breast cancer at under age 60 years and male breast cancer at any age; Single case of breast cancer at under age 40 years if the patient is of Ashkenazi Jewish decent. Risk Management Three categories of options for management of women with BRCA mutations are available: screening and surveillance, prophylactic surgeries, and chemoprevention. These options should be discussed with women in detail to allow them to make informed decisions regarding their health care. References 1. Collaborative Group on Hormonal Factors in Breast Cancer. Familial breast cancer: collaborative reanalysis of individual data from 52 epidemiological studies including 58,209 women with breast cancer and 101,986 women without the disease. Lancet 2001;358: Hall JM, Lee MK, Newman B, et al. Linkage of earlyonset familial breast cancer to chromosome 17q21. Science 1990;250: haroah D, Antoniou A, Bobrow M, et al. olygenic susceptibility to breast cancer and implications for prevention. Nat Genet 2002;31: King MC, Marks JH, Mandell JB. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 2003;302: Meindl A. German Consortium for Hereditary Breast and Ovarian Cancer. Comprehensive analysis of 989

11 58 MOLECULAR ONCOLOGY OF BREAST CANCER patients with breast and ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population. Int J Cancer 2002; 97: Narod SA, Dube M, Klijn J, et al. Oral contraceptives and the risk of breast cancer in BRCA1 and BRCA2 mutation carriers. J Natl Cancer Inst 2002; 94: Marchbanks A, McDonald JA, Wilson HG, et al. Oral contraceptives and the risk of breast cancer. N Engl J Med 2002;346: Thompson D, Easton DF, Breast Cancer Linkage Consortium. Cancer incidence in BRCA1 carriers. J Natl Cancer Inst 2002;94: Breast Cancer Linkage Consortium. Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst 1999; 91: Smith TM, Easton DF, Evans DG, et al. Allele losses in the region 17q12-21 in familial breast and ovarian cancer involve the wild-type chromosome. Nat Genet 1992;2: Osorio A, de la Hoya M, Rodriguez-Lopez R, et al. Loss of heterozygosity analysis in tumor samples in patients with familial breast cancer. 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