（DHPLC）乳腺癌专题

乳腺癌和乳腺癌基因

乳腺癌是人类最常见的一种恶性肿瘤，也是女性主要恶性肿瘤之一。在北美、两欧等发达国家，女性乳腺癌的发病率居女性恶性肿瘤发病率的首位。我国近年来乳腺癌的发病率明显增高 ，尤其沪、京、津及沿海地区是我国乳腺癌的高发地 区。 城市职业妇女生育年龄推后，生育减少和饮食西方化是造成发病率迅速增高的主要原因。 上 海 1997 年女性乳腺癌发病率高达 46/10 万人，是 1972 发病率的近三倍，成为当地 威胁女性健康的首位恶性肿瘤。据中国抗癌协会 2005 年提供的数据显示：中国女性的乳腺癌患病率为 35 ～ 45/10 万人，从年龄上看， 35% 左右的中国女性处于 35 ～ 70 岁，而这个年龄段的乳腺癌发病人数占总发病人数的 85% 。

研究证明，乳腺癌是遗传倾向最显著的癌症之一， 5% ～ 10% 的乳腺癌来自家族遗传。 乳腺癌易感基因 BRCA1 、 BRCA2 突变与乳腺癌高度相关，并具有显性遗传特征，在家族性乳腺癌症候群中，一旦此基因产生突变，其家族子代中所有成员皆有 50% 之机会带有此突变基因。家族性乳腺癌患者罹患乳腺癌之平均年龄比一般妇女提早约十年，机率高达普通女性的九倍。以下是 BRCA 突变导致的随年龄增长的患乳腺癌累计风险：

乳腺癌基因筛查及时探查隐患

近数十年来，国内外在乳腺癌的治疗方法上取得了多方面的改进，但其病死率未见明显下降，究其原因，最主要的乃由于就诊较晚，在所治疗的病人中，中晚期病例占多数所致。

乳腺癌具有高遗传倾向。在一般家族里，约有 0.5% 的人带有 BRCA1 或 BRCA2 基因之突变，而在具有家族性或早发性乳腺癌之家族中， BRCA1 或 BRCA2 基因之突变发生率，高达 50% ，几乎注定未来患癌的命运。

爱康提供 BRCA 基因筛查服务，可以 检测出患乳腺癌的风险，以尽早采取预防和治疗措施，是为减少患癌及提高生存率的有效途径。

哪类人群必须进行乳腺癌基因筛查

遇到以下因素，会增大罹患乳腺癌的风险，强烈建议接受乳腺癌 BRCA1 、 BRCA2 基因检验：

1 、一个或两个直系亲属有乳腺癌史，如您的母亲或姐妹
2 、您母亲或父亲的旁系亲属有早发乳腺癌（ 50 岁以前）的家庭成员
3 、一个或更多后代有乳腺癌史
4 、男性乳腺癌亲属
5 、家庭成员双侧乳腺发病
6 、家族内的卵巢癌发病率
7 、亲属成员有 BRCA 基因突 变，即遗传学检测阳性

DHPLC 检测的准确率

对突变最直接的遗传学检测就是对 DNA 片段进行直接测序，但这种方法过于昂贵，而且极为耗费劳动力。因此在实际应用中进行 BRCA1 和 BRCA2 基因的流行病学研究时，倾向于选择相对快捷而便宜的筛选方法。 DHPLC 是目前此类基因筛选常规方案中最精确的，在 BIC （美国 乳腺癌信息中心 ）组织的实验中，只有 DHPLC 检测出全部的基因突变，而其它的方案， SSCP （ DNA 单链构象多态性分析）、 CSGE （构象敏感凝胶电泳）、 TDGS （二维基因扫描技术）的突变检出率分别只有 72% 、 76% 、 91% 。

DHPLC 检测以其 自动、快速、高通量、准确、稳定、重复性好、检测精度高、低消耗、操作简单（样品准备容易）等优势，在众多筛查方法中脱颖而出，成为目前理想的乳腺癌筛查方法，突变 检测的首选技术。

BRCA1 and BRCA2 Hereditary Breast/Ovarian Cancer

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Authors: Julie Bars Culver, MS; Fred Hutchinson Cancer Research Center, SeattleJudith Hull, MS; Memorial Sloan Kettering Cancer Center, New York Ephrat Levy-Lahad, MD; Shaare Zedek Medical Center, Tel AvivMary Daly, MD, PhD; Fox Chase Cancer Center, Philadelphia Wylie Burke, MD, PhD; University of Washington, Seattle

Posted: 4 March 2000

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Summary

Disease characteristics. Mutations in BRCA1 and BRCA2 are characterized by predisposition to breast cancer and ovarian cancer as well as prostate cancer, colon cancer (BRCA1), and other cancers (BRCA2). The risk of developing cancer that is associated with BRCA1 and BRCA2 cancer-predisposing mutations is not known and appears to be variable even within families of similar ethnic background with the same mutation. Estimates of breast cancer and ovarian cancer risks have been derived from families with multiple affected individuals as well as from families with few affected individuals and from population-based studies. Prognosis for breast cancer survival depends upon the stage at which breast cancer is diagnosed and may not be different among individuals with BRCA1 or BRCA2 cancer-predisposing mutations and controls.

Diagnosis/testing. DNA-based testing for BRCA1 and BRCA2 cancer-predisposing mutations is available on a clinical basis for probands who are identified to be at high risk for having a BRCA1 or BRCA2 cancer-predisposing mutation and for at-risk relatives of an individual with an identified BRCA1 or BRCA2 cancer-predisposing mutation. No currently available technique can guarantee the identification of all cancer-predisposing mutations in the BRCA1 gene or in the BRCA2 gene. Furthermore, mutations of uncertain clinical significance may be identified.

Management. Management of individuals with a BRCA1 or BRCA2 cancer-predisposing mutation includes discussion of cancer screening protocols and options for prophylactic surgery.

Genetic counseling. Cancer-predisposing mutations in the BRCA1 and BRCA2 genes are inherited in an autosomal dominant manner. It is necessary to identify the specific cancer-predisposing mutation in an affected family member before molecular genetic testing of either the BRCA1 or BRCA2 gene can be used in genetic counseling and testing of asymptomatic at-risk family members. Offspring of individuals with a BRCA1 or BRCA2 cancer-predisposing mutation have a 50% chance of inheriting the gene mutation. Prenatal testing is possible for fetuses at 50% risk; however, requests for prenatal diagnosis of adult-onset diseases require careful genetic counseling.

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Diagnosis

Clinical Diagnosis

BRCA1 or BRCA2 hereditary breast/ovarian cancer is suspected in an individual who has a family history of breast cancer or breast and ovarian cancer consistent with autosomal dominant inheritance. Other features include an early age of onset, increased incidence of bilateral (or multifocal) disease, and, occasionally, the occurrence of male breast cancer. The diagnosis of BRCA1 or BRCA2 hereditary breast/ovarian cancer is established when cancer-predisposing mutations are identified in the BRCA1 gene (chromosomal locus 17q21) or in the BRCA2 gene (chromosomal locus 13q12) using DNA-based molecular genetic testing. Such testing is currently available on a clinical basis in two settings:

For probands who are identified to be at high risk for having a BRCA1 or BRCA2 cancer-predisposing mutation (see Breast Cancer Overview - Genetic Counseling and Testing Risk Assessment)

For at-risk relatives of an individual with an identified BRCA1 or BRCA2 cancer-predisposing mutation

Testing of at-risk individuals and/or at-risk relatives can help determine management options. Pre-test education and counseling should always be provided prior to ordering molecular genetic testing (see Breast Cancer Overview - Genetic Counseling and Testing Risk Assessment).

Molecular Genetic Testing

Laboratory technique. Molecular genetic testing for BRCA1 and BRCA2 cancer-predisposing mutations is now offered both as a clinical service and within research protocols. The specific DNA-based testing techniques performed by various laboratories may include: allele specific oligonucleotide (ASO) testing, protein truncation testing (PTT), conformation sensitive gel electrophoresis (CSGE) [Ganguly et al 1993], single stranded conformational polymorphism (SSCP), complete sequencing, Southern blot analysis, or a combination of these and/or other techniques. It is important to note that no currently available technique can guarantee the identification of all cancer-predisposing mutations in the BRCA1 or BRCA2 genes [Ford et al 1998].

Sensitivity. The sensitivity of tests for detecting BRCA1 or BRCA2 cancer-predisposing mutations is dependent on the method used for DNA analysis and the a priori risk of the person tested to have a mutation in either gene based on the person's cancer history, family history, and ethnic background.

Table 1. Molecular Genetic Testing Used in BRCA1 and BRCA2 Hereditary Breast/Ovarian Cancer % of Patients Genetic Mechanism Test Type Sensitivity of Molecular Method Test Availability

BRCA1 BRCA2

All patients Mutations affecting the BRCA1 gene or the BRCA2 gene Exon scanning 63%

[Ford et al 1998] Clinical Clinical

Direct sequencing 1 63%

[Ford et al 1998]

PTT ?

Ashkenazi Jews 185delAG mutation in BRCA1 ; 5382insC mutation in BRCA1 ; 6174delT mutation in BRCA2 ASO >99%

Dutch 2804delAA in BRCA1

Icelanders 999del5 in BRCA2

1. When full gene sequencing is used, mutations of uncertain clinical significance may be identified and intronic mutations or mutations involving genomic rearrangement may not be identified. Some mutations involving genomic rearrangement can be detected by Southern blot techniques [Petrij-Bosch et al 1997].

Testing strategy in a family. It is strongly recommended that the genetic test be offered to an affected individual prior to offering it to at-risk unaffected family members. Testing an affected individual is the most effective way of determining if a BRCA1 or BRCA2 mutation is causative of breast or ovarian cancer within a family. After a cancer-predisposing mutation has been identified in an affected family member, BRCA1 or BRCA2 mutational analysis is more informative for unaffected relatives.

Interpretation of positive test results for mutations of uncertain clinical significance. When full gene sequencing is the method used for testing, mutations of uncertain clinical significance may be identified, such as previously undescribed missense mutations that are not predicted to result in a loss of protein function. Such alterations present a dilemma for physicians who are making patient management decisions. Three methods are available to determine the clinical significance of these mutations: 1) family studies to determine whether the mutation segregates with cancer in family members (which is the most practical and clinically useful method); 2) allele frequency analysis to determine whether the allele has a higher frequency in cancer patients than in the general population; and 3) protein function assays to measure the effect of the mutation on the protein [Shattuck-Eidens et al 1997]. Since these approaches are difficult to implement and usually not feasible as part of a clinical study, the clinical significance of these ambiguous mutations may remain unclear indefinitely.

Interpretation of negative test results.

When a cancer-predisposing BRCA1 or BRCA2 mutation cannot be identified in an affected individual from a family with an increased risk of an inherited predisposition to breast/ovarian cancer, negative results are uninformative, and the possibility of a false negative test must be considered [Geller et al 1997]; thus, the affected individual may still have an inherited cancer-predisposing mutation in the BRCA1 or BRCA2 gene. She may also have a mutation in some other gene that predisposes to breast/ovarian cancer.

If the affected family member has no identifiable BRCA1 or BRCA2 cancer-predisposing mutation or is unavailable for testing, all negative BRCA1 and BRCA2 test results in other family members must be considered uninformative.

An at-risk relative who does not have the BRCA1 or BRCA2 cancer-predisposing mutation identified in an affected family member is considered to have a true negative result. It is appropriate to advise the individual that a negative result does not reduce her cancer risk below that of the general population. Furthermore, if that person is from a high-risk ethnic group, e.g., of Ashkenazi Jewish descent, it may be prudent to test for all the cancer-predisposing mutations known to be common in that population, even if a single cancer-predisposing mutation has already been identified in an affected family member.

Clinical Description

Cancer Risk Assessment

The penetrance of BRCA1 or BRCA2 cancer-predisposing mutations - or likelihood of cancer when a cancer-predisposing mutation is present - is the most significant clinical aspect of BRCA1 and BRCA2 mutations. The penetrance is uncertain and probably variable. The strongest evidence for variable risk comes from studies of multiple families with the same cancer-predisposing mutation within defined ethnic populations (see Prevalence). The accumulated evidence indicates that some individuals with cancer-predisposing mutations survive to an elderly age without developing cancer. Among those who develop cancer, the age of onset, as well as type of cancer, varies. No clear explanation exists for the observation that some individuals with a cancer-predisposing mutation may have multiple primary cancers before age 50 years, while others with the same cancer-predisposing mutation may not develop cancer until after age 70 years [Abeliovich et al 1997, Levy-Lahad et al 1997]. These uncertainties are important to consider in pretest counseling.

The following is a summary of cancer risk in individuals identified with cancer-predisposing mutations in the BRCA1 and BRCA2 genes.

Breast Cancer Risk Estimates Derived from Families Ascertained for High Penetrance

BRCA1. The initial studies of the penetrance of cancer-predisposing mutations in the BRCA1 gene involved BRCA1 mutation-positive families ascertained by the presence of multiple individuals (usually four or more) affected with breast cancer or ovarian cancer at any age. The cancer risks seen in these families are high and may over-estimate the risk within all families with BRCA1 cancer-predisposing mutations. The estimates of the cumulative risk of breast cancer for women with a BRCA1 cancer-predisposing mutation from these high-risk families are summarized in Table 2 [Easton et al 1995].

Table 2. Cumulative Risk by Age of Breast Cancer in Women from Families with BRCA1 Cancer-Predisposing Mutations Age (Years) Cumulative Risk

30 yrs 3.2%

40 yrs 19.1%

50 yrs 50.8%

60 yrs 54.2%

70 yrs 85%

Male breast cancer is rarely associated with a BRCA1 cancer-predisposing mutation; however, several families with male breast cancer have shown linkage to the BRCA1 locus [Ford et al 1998].

BRCA2. Women with BRCA2 cancer-predisposing mutations appear to have a breast cancer risk similar to that of women with BRCA1 cancer-predisposing mutations [Ford et al 1994, Easton et al 1997, Ford et al 1998]. Current risk estimates are based on observations from high-risk families participating in research studies. The average age at which cancer occurs in women with BRCA2 cancer-predisposing mutations may be later than for women with BRCA1 cancer-predisposing mutations [Krainer et al 1997, Ford et al 1998]. The estimates of the cumulative risk of breast cancer for women with a BRCA2 cancer-predisposing mutation from high-risk families are summarized in Table 3.

Table 3. Age and Cumulative Risk by Age of Breast Cancer in Women from Families with BRCA2 Cancer-Predisposing Mutations Age Cumulative Risk

30 yrs 4.6%

40 yrs 12%

50 yrs 46%

60 yrs 61%

70 yrs 86%

Male breast cancer has been observed in families with BRCA2 cancer-predisposing mutations, including some families with multiple cases of male breast cancer and no cases of female breast cancer [Couch et al 1996, Thorlacius et al 1996, Thorlacius et al 1997]. Of 26 high-risk families with at least one case of male breast cancer, 77% showed linkage to the BRCA2 locus [Ford et al 1998]. However, among male breast cancer cases who were not selected on the basis of family history, only 4-14% of cases were positive for a germline BRCA2 mutation [Couch et al 1997, Friedman et al 1997].

Ovarian Cancer Risk Estimates Derived from Families Ascertained for High Penetrance

BRCA1. Cumulative risk of ovarian cancer is high but appears to be variable, and is presumed to differ with the specific BRCA1 cancer-predisposing mutation [Neuhausen et al 1996]. One model based on data from high-risk families estimates an average risk of 30% by age 60 years and 63% by age 70 years for women with a BRCA1 cancer-predisposing mutation [Easton et al 1995]. A second genetic trait may be involved; risk of ovarian cancer was twofold higher in women with a BRCA1 cancer-predisposing mutation who also had one or two rare alleles of the HRAS1 VNTR locus [Phelan et al 1996]. Among a consecutive series of women with ovarian cancer, the mean age of cancer diagnosis was 50 years for those with the 185delAG mutation [Levy-Lahad et al 1997].

BRCA2. Based on data from high-risk families, cumulative risk of ovarian cancer in women with BRCA2 cancer-predisposing mutations is estimated to be less than 27% by age 70 years [Ford et al 1998]. Ovarian cancer has been seen in up to 48% of families with BRCA2 cancer-predisposing mutations [Thorlacius et al 1995, Couch et al 1996, Tavtigian et al 1996]. Among a consecutive series of women with ovarian cancer, the mean age of cancer diagnosis was 68 years for those with the 6174delT mutation [Levy-Lahad et al 1997].

Other Cancer Risk Estimates Derived from Families Ascertained for High Penetrance

BRCA1. The risk of prostate cancer is estimated to be threefold higher in men who have a BRCA1 cancer-predisposing mutation than in the general population. The cumulative risk is 8% by age 70 years [Ford et al 1994]. The risk of colon cancer may be fourfold higher, with an estimated cumulative risk of 6% by age 70 years [Ford et al 1994]. Colon cancer does not appear to occur earlier than in individuals who do not have a known BRCA1 cancer-predisposing mutation [Ford et al 1994].

BRCA2. An increased risk of prostate cancer and pancreatic cancer may also occur in individuals with BRCA2 cancer-predisposing mutations [Berman et al 1996, Easton et al 1997, Gayther et al 1997]. Furthermore, cancers of the larynx, esophagus, colon, stomach, gallbladder, bile duct, and hematopoietic system, as well as melanomas, have been observed in families with BRCA2 cancer-predisposing mutations [Berman et al 1996, Easton et al 1997, Breast Cancer Linkage Consortium 1999].

Cancer Risk in Individuals with a BRCA1 or BRCA2 Cancer-Predisposing Mutation: Estimates Derived from Less Selected Families and Population-Based Studies

The risk for cancer in individuals positive for a BRCA1 cancer-predisposing mutation has been derived from a volunteer survey [Struewing et al 1997], a clinical series [Fodor et al 1998], and a population-based study [Thorlacius et al 1998]. A study of Ashkenazi Jewish families based on a volunteer survey found that individuals with the 185delAG mutation (BRCA1) or the 5382insC mutation (BRCA1) or the 6174delT mutation (BRCA2) had a 56% risk of breast cancer, a 16% risk of ovarian cancer, and a 16% risk of prostate cancer (by age 70 years); the incidence of colon cancer was not elevated [Struewing et al 1997]. There were no significant differences in the risk of breast cancer between women with BRCA1 cancer-predisposing mutations and women with BRCA2 cancer-predisposing mutations [Struewing et al 1997]. In the families of Ashkenazi Jewish women with breast cancer who were ascertained through clinical studies and who had the 6174del mutation (BRCA2), the lifetime risk of breast cancer was calculated to be 36% [Fodor et al 1998]. In a study of Icelandic individuals, women with the 999del5 mutation (BRCA2) had a 37% risk of breast cancer by age 70 years [Thorlacius et al 1998]. The cancer risks in these studies were derived indirectly by genotyping the proband and using the family history to estimate the penetrance of the cancer-predisposing mutation. Not all family members were genotyped and examined. In addition, a number of women with a BRCA1 cancer-predisposing mutation have now been reported who have a minimal family history of breast cancer [Langston et al 1996, Abeliovich et al 1997, Levy-Lahad et al 1997, Ozcelik et al 1997, Richards et al 1997].

Prognosis

Breast Cancer

The distinct pathological features of BRCA1 -related tumors (and perhaps BRCA2 -related tumors) coupled with the relative paucity of somatic BRCA1 / BRCA2 mutations in sporadically occurring breast cancer suggest that breast cancer in individuals with BRCA1 or BRCA2 cancer-predisposing mutations has a specific pathogenetic basis, which could lead to differences in prognosis. Accurate estimates of breast cancer prognosis in individuals with BRCA1 / BRCA2 cancer-predisposing mutations would require prospective longitudinal studies with large numbers of women. Such studies are yet to be reported. Most available data, derived from retrospective or indirect data, are based on small numbers (< 50 cases) and are probably confounded by different biases and by lack of appropriate controls (which should be matched not only for age and stage of cancer at diagnosis, but also for calendar year of diagnosis because survival has improved over time). For example, in most studies of breast cancer prognosis, mutation analysis was not performed in the control group and controls were not matched to cases for stage at diagnosis. Some investigators have suggested that matching for stage at the time of diagnosis may mask real biological differences between BCRA1 -/ BRCA2 -related tumors and sporadic tumors, e.g., if tumors in individuals with cancer-predisposing mutations indeed presented at more advanced stages. However, this would first require firm evidence (which is lacking) that stage at diagnosis is indeed different in women with BRCA1 or BRCA2 cancer-predisposing mutations [Pharoah et al 1999]. Given these limitations, most studies on prognosis of breast cancer have not found a significant difference in survival between individuals with BRCA1 or BRCA2 cancer-predisposing mutations and controls [Gaffney et al 1998, Johannsson et al 1998, Verhoog et al 1998, Lee et al 1999, Verhoog et al 1999], but conflicting reports of both better [Marcus et al 1996, Porter et al 1994] and worse [Foulkes et al 1997, Ansquer et al 1998] prognosis exist. For reviews of survival studies, see Phillips et al 1999 and Chappuis et al 1999.

Ovarian Cancer

Studies on ovarian cancer survival in women with BRCA1 / BRCA2 cancer-predisposing mutations have yielded conflicting results, at least in part because of the same methodological issues encountered in studies on breast cancer prognosis (see section above). The first study in which women with BRCA1 cancer-predisposing mutations were identified by mutation analysis found improved survival in 43 women with BRCA1 cancer-predisposing mutations (median survival of 77 months compared to 29 months in controls) [Rubin et al 1996]. This study was criticized for selection bias, lead-time bias (increased surveillance leading to earlier diagnosis in familial cases) [Burk 1997, Whitmore 1997] and differences in treatment received by individuals with cancer-predisposing mutations compared to historical controls [Cannistra 1997]. Similar improved survival was noted in a study of 25 women with BRCA1 cancer-predisposing mutations with stage III ovarian cancer [Aida et al 1998]. A population-based study in Sweden (n=38) and a Canadian study (n=44) found no differences in survival between women with BRCA1 cancer-predisposing mutations and controls [Brunet et al 1997, Johannsson et al 1998]. A population-based study in the UK including 133 women with BRCA1 cancer-predisposing mutations and 26 women with BRCA2 cancer-predisposing mutations with ovarian cancer found no difference in survival between individuals with cancer-predisposing mutations and women with ovarian cancer in whom genetic testing was negative or unavailable. Survival was worse in familial cases (five year survival of 20%) compared to sporadic cases (five year survival of 30%), but this difference was not observed after controlling for tumor stage at diagnosis. The relative prognosis of ovarian cancer in women with BRCA1 cancer-predisposing mutations is therefore unclear, but recent studies suggest that it is probably not better than that of women without such mutations.

Pathology

Breast Cancer

To summarize, BRCA1 -related tumors show an excess of medullary histopathology, are of higher histological grade, and are more likely to be estrogen receptor-negative and progesterone receptor-negative. At the molecular level there is a higher frequency of p53 mutations, and less HER2/c-erbB-2/neu overexpression. These features include both favorable and adverse prognostic factors. Information regarding BRCA2 -related tumors is more limited, but they do not seem to have a characteristic histopathology, and are at least as likely to be hormone receptor-positive as control tumors. (More details on pathology).

Ovarian Cancer

An excess of serous adenocarcinomas has been observed in women with BRCA1 cancer-predisposing mutations compared to controls. Over 90% of tumors in women with BRCA1 cancer-predisposing mutations are serous, compared to approximately 50% in women without a BRCA1 cancer-predisposing mutation [Rubin et al 1996, Aida et al 1998, Berchuck et al 1998, Lu et al 1999]. Serous adenocarcinomas are generally of higher grade and are more frequently bilateral. (More details on pathology).

Prevalence

BRCA1 Cancer-Predisposing Mutations

The prevalence of cancer-predisposing BRCA1 mutations in the general population is estimated to be between 1/500 and 1/1000. A comprehensive discussion of BRCA1 and BRCA2 population genetics is available [Szabo and King 1997]. The following describes specific BRCA1 cancer-predisposing mutations in two ethnic groups:

Ashkenazi Jews. The 185delAG mutation in BRCA1 occurs with a frequency of about 1% in individuals of Ashkenazi Jewish descent [Struewing et al 1995, Oddoux et al 1996, Roa et al 1996, Struewing et al 1997]. Another BRCA1 mutation, 5382insC, has an estimated prevalence of 0.1-0.15% [Roa et al 1996]. All were initially observed in high-risk families. The 185delAG (BRCA1) and 6174delT (BRCA2) mutation have subsequently been found in 20-30% of Jewish women diagnosed with early breast cancer [FitzGerald et al 1996, Abeliovich et al 1997] and in 45-60% of Jewish women diagnosed with ovarian cancer [Abeliovich et al 1997; Levy-Lahad, unpublished data]. The frequency of the 185delAG and 6174delT mutations is higher among individuals with a family history of breast or ovarian cancer [Modan et al 1996, Levy-Lahad et al 1997], but individuals with the mutation who have little or no family history of cancer have been identified [Langston et al 1996, Abeliovich et al 1997, Levy-Lahad et al 1997, Richards et al 1997].

Dutch. Although most cancer-predisposing BRCA1 mutations described involve only a few base pairs, studies in the Dutch population have identified two large deletions within BRCA1. These deletions were detected using Southern blot analysis and accounted for 36% of mutations on a Dutch sample of high-risk families [Petrij-Bosch et al 1997]. Large deletions of BRCA1 may occur in other populations, but may not be identified by the more commonly used PCR-based mutation screening approaches, such as single-stranded conformation polymorphism (SSCP), protein truncation testing (PTT), and direct sequencing.

BRCA2 Cancer-Predisposing Mutations

The prevalence of cancer-predisposing BRCA2 mutations in the general population is unknown. From the prevalence of cancer-prone families, BRCA1 and BRCA2 cancer-predisposing mutations have been estimated to occur in approximately one to two persons per thousand. The following describes specific BRCA2 cancer-predisposing mutations in two ethnic groups:

Icelanders. The BRCA2 cancer-predisposing mutation 999del5 occurs in 0.6% of the Icelandic population and in 7.7% of women and 40% of men patients with breast cancer from Iceland [Thorlacius et al 1996, Thorlacius et al 1997]. The mutation was seen in 17% of women diagnosed with breast cancer by age 50 years, but was also seen in 4% of women diagnosed at later ages. Among individuals with the 999del5 mutation, 17 of 44 (39%) had no first or second degree relatives with cancer, suggesting incomplete penetrance of the mutation [Thorlacius et al 1996].

Ashkenazi Jews. The BRCA2 mutation 6174delT occurs with a frequency of about 1% in individuals of Ashkenazi Jewish descent [Struewing et al 1995, Oddoux et al 1996, Roa et al 1996, Struewing et al 1997]. This mutation was initially observed in high-risk families.

Management

Several strategies have been suggested to reduce cancer risk in individuals who have a BRCA1 or BRCA2 cancer-predisposing mutation. These include cancer screening, prophylactic mastectomy and/or oophorectomy, and chemoprevention. None of these strategies has been assessed by randomized trials or case control studies in high-risk women, and no studies have evaluated the effects of these interventions in individuals with a BRCA1 or BRCA2 cancer-predisposing mutation. As a result, current recommendations are made on the basis of expert opinion.

Cancer Screening

Recommendations for cancer screening of individuals with a BRCA1 or BRCA2 cancer-predisposing mutation have been made by a task force convened by the Cancer Genetics Consortium (CGSC), an NIH-sponsored consortium of researchers assessing the ethical, legal, and social implications of genetic testing for cancer risk [Burke et al 1997]. The CGSC statement emphasized that recommendations are based on presumed benefit and may change as new evidence becomes available; therefore, patients should be counseled regarding the limited knowledge about strategies to reduce risk and patient preference should be taken into account in decisions about follow-up. Recommendations similar to those of the CGSC are in use in 16 European family cancer centers [Vasen et al 1998]. The CGSC recommendations for individuals with a BRCA1 or BRCA2 cancer-predisposing mutation are [Burke et al 1997]:

Breast Cancer Screening

No studies have evaluated the outcome of early breast cancer screening. The breast cancer screening recommendations are based on data from families with cancer-predisposing BRCA1 or BRCA2 mutations, which indicate that elevated breast cancer risk begins in the late 20s or early 30s [Burke et al 1997]. [ Mammography guidelines, Cancernet]

Monthly breast self-examination starting in early adulthood

Annual or semi-annual clinical breast examination beginning at age 25-35 years

Annual mammography beginning at age 25-35 years

Men with BRCA2 cancer-predisposing mutations may also be at increased risk for breast cancer, and evaluation of any breast mass or change is advisable; however, there are insufficient data to recommend a formal program of surveillance [Burke et al 1997].

Ovarian Cancer Screening

The ovarian cancer screening measures available (transvaginal ultrasound examination and serum CA-125 concentration) have limited sensitivity and specificity and have not been shown to reduce ovarian cancer mortality.

Annual or semi-annual pelvic examination beginning at age 25-35 years

Annual or semi-annual transvaginal ultrasound examination with color Doppler beginning at age 25-35 years

Annual serum CA-125 concentration beginning at age 25-35 years

Other Cancer Screening

Colon cancer. Individuals with BRCA1 cancer-predisposing mutations may have a small increased risk for colon cancer [Ford et al 1994]; however, this risk does not appear to occur at an early age. The same recommendations proposed for people of average risk are recommended for people with BRCA1 mutations [Burke et al 1997]. Two articles summarize recommendations for colorectal cancer screening [Winawer et al 1997, Byers et al 1997]. Several different screening strategies are available as outlined in these documents; the most commonly recommended screening program includes the following:

Annual stool occult blood testing beginning at age 50 years

Flexible sigmoidoscopy every five years beginning at age 50 years

Prostate cancer. Men with cancer-predisposing BRCA1 and BRCA2 mutations appear to have an increased risk of prostate cancer and therefore should be informed about options for prostate cancer screening [Burke et al 1997].

Prophylactic Surgery

The CGSC task force recommended that women with BRCA1 or BRCA2 mutations be offered prophylactic mastectomy and oophorectomy, with the decision determined by patient preference [Burke et al 1997].

Prophylactic surgeries (mastectomy and oophorectomy) have been proposed as a means of reducing cancer risk in people with genetic susceptibility to breast and ovarian cancer. An expert panel concluded there was insufficient evidence to recommend for or against these surgeries for individuals with BRCA1 or BRCA2 cancer-predisposing mutations, but women with cancer-predisposing mutations should be informed of the option [Burke et al 1997]. Cancers have been observed after both procedures. A retrospective cohort study of all women receiving prophylactic mastectomy at the Mayo Clinic in the state of Minnesota over a 30-year period estimated a 90% reduction in breast cancer risk from the procedure. One-third of the women in the Mayo Clinic study were considered to have a strong family history of cancer and experienced a risk reduction similar to that of the whole [Hartmann et al 1999]. Nonetheless, the experience is that few women from high-risk families choose this procedure [Stefanek et al 1995].

A National Cancer Institute study of women from families at high risk for ovarian cancer found that the rate of peritoneal cancer after oophorectomy was not significantly lower than the rate of ovarian and peritoneal cancer in women who had not had the procedure [Struewing et al 1995]. One study found a significant reduction in breast cancer risk following prophylactic oophorectomy among women with cancer-predisposing BRCA1 mutations [Rebbeck et al 1999].

Chemoprevention

No data are yet available concerning the efficacy of chemoprevention in women with BRCA1 or BRCA2 cancer-predisposing mutations.

Tamoxifen. A randomized clinical trial of treatment with tamoxifen (a partial estrogen antagonist) in women identified by the Gail model to have an increased breast cancer risk reported a 49% reduction in breast cancer in the treated group [Fisher et al 1998]. Gail et al 1999 concluded that tamoxifen prophylaxis was most beneficial in women with an elevated risk of breast cancer who were under age 50 years. However, tamoxifen reduced the incidence of breast cancers that were estrogen receptor-positive, but not estrogen receptor-negative. Since breast cancers occurring in women with BRCA1 mutations are more likely to be estrogen receptor-negative (see Pathology), it is difficult to estimate the benefit of tamoxifen prophylaxis without specifically testing the effect in women with BRCA1 or BRCA2 cancer-predisposing mutations. Interim results from two European tamoxifen prevention trials failed to observe benefit from tamoxifen [Veronesi et al 1998, Powles et al 1998]. Significant adverse consequences of tamoxifen treatment included a higher rate of endometrial cancer and thromboembolic episodes, including pulmonary embolism. The women most likely to benefit from tamoxifen treatment, and the optimal treatment protocol, have not yet been determined.

Oral contraceptives and hormone replacement therapy. The CGSC task force concluded that there were insufficient data to make recommendations concerning hormone replacement therapy for women with BRCA1 or BRCA2 mutations [Burke et al 1997]. General population studies suggest that long-term estrogen replacement therapy in postmenopausal women may increase breast cancer risk, but that short-term use to treat menopausal symptoms does not. One case control study found a decreased risk of ovarian cancer in women with BRCA1 or BRCA2 cancer-predisposing mutations who took oral contraceptives for more than three years [Narod et al 1998]. These data are consistent with data from general population studies, which indicate a reduced risk of ovarian cancer with oral contraceptive use. The case control study did not assess other outcomes such as the effect of oral contraceptives on breast cancer risk.

Genetic Counseling

Genetic counseling is the process of providing individuals and families with information on the nature, inheritance, and implications of genetic disorders to help them make informed medical and personal decisions. The following section deals with genetic risk assessment and the use of family history and genetic testing to clarify genetic status for family members. This section is not meant to address all personal or cultural issues that individuals may face or to substitute for consultation with a genetics professional. To find a genetics or prenatal diagnosis clinic, see . -ED.

Mode of Inheritance

Cancer-predisposing mutations in the BRCA1 and BRCA1 genes are inherited in an autosomal dominant manner.

Risk To Family Members

Parents of a proband. Virtually all individuals with a cancer-predisposing mutation in BRCA1 or BRCA2 have inherited it from a parent. The parent may or may not have had a cancer diagnosis depending upon the penetrance of the mutation, the gender of the parent with the mutation, the age of the parent with the mutation, and other variables. It is appropriate to offer mutation analysis to both parents of an individual with a BRCA1 or BRCA2 cancer-predisposing mutation. Occasionally neither parent will be identified as having the BRCA1 or BRCA2 cancer-predisposing mutation. Reasons for this include a de novo mutation in the proband, alternate paternity, or adoption. The number of individuals with a BRCA1 or BRCA2 cancer-predisposing mutation that has occurred as a de novo event is not known.

Sibs of a proband. The risk that a sib of an index case will inherit the cancer-predisposing BRCA1 or BRCA2 mutation is 50% if one parent has the BRCA1 or BRCA2 cancer-predisposing mutation. The risk of developing cancer, however, depends upon numerous variables including the penetrance of the mutation, the gender of the individual, and the age of the individual.

Offspring of a proband. The offspring of an individual identified as having a BRCA1 or BRCA2 cancer-predisposing mutation have a 50% chance of inheriting the mutation. The risk of developing cancer, however, depends upon numerous variables such as the penetrance of the mutation, the gender of the individual, and the age of the individual.

Related Genetic Counseling Issues

At-risk asymptomatic adult relatives. In general, relatives of an individual who has a cancer-predisposing BRCA1 or BRCA2 mutation should be counseled about their risk to have inherited the same mutation, their options for molecular genetic testing, their cancer risk, and recommendations for cancer screening and prophylactic surgery.

For those who choose to know more about molecular genetic testing, it is suggested that pre-test education include discussion of [ASCO 1997, McKinnon et al 1997, Geller et al 1997]:

The individual's motivation for requesting testing and preconceived beliefs about the test. Some at-risk asymptomatic adult family members may seek testing in order to make personal decisions regarding such issues as reproduction, financial matters, and career planning. Other may simply "need to know."

The individual's perceptions of his/her risk of developing cancer

The individual's readiness for testing and optimal timing for testing

Alternatives to testing, such as DNA banking

Inability of genetic testing to detect the presence or absence of cancer

The individual's support systems, and possible need for additional psychological support

The individual's need for privacy and autonomy

The possible effects of positive, negative, or uninformative test results on:

Cancer risk

Cancer screening protocols

Risk status for other family members

Insurance coverage and employment: An individual found to have an inherited susceptibility to cancer could face discrimination in access to health insurance and/or employment.

Individual's emotional status: depression/anxiety/guilt

Relationships with partner, children, extended family, friends

At-risk relatives who have not inherited the cancer-predisposing mutation identified in the proband are presumed, except in special circumstances, to be at the general population risk of developing cancer. Appropriate cancer screening such as that recommended by the National Cancer Institute for individuals of average risk is recommended. Note: this presumption cannot apply to individuals who test negative for a BRCA1 or BRCA2 cancer-predisposing mutation if the proband in the family has either not undergone molecular genetic testing of BRCA1 or BRCA2 or did not have an identified BRCA1 or BRCA2 cancer-predisposing mutation.

Testing of at-risk asymptomatic relatives who are children. Legitimate concerns about testing at-risk children under the age of 18 years for adult-onset conditions - including BRCA1 or BRCA2 cancer-predisposing mutations - exist, including issues of informed consent among minors, the lack of proven surveillance or prevention strategies, and concerns about stigmatization and discrimination. This testing is typically unavailable. (See also the National Society of Genetic Counselors resolution on genetic testing of children and the American Society of Human Genetics and American College of Medical Genetics points to consider: ethical, legal, and psychosocial implications of genetic testing in children and adolescents.)

Prenatal Testing

Prenatal testing for BRCA1 or BRCA2 mutations is technically possible using the same techniques described in Molecular Genetic Testing. DNA can be extracted from fetal cells obtained by amniocentesis at 16-18 weeks' gestation or chorionic villus sampling (CVS) at about 10-12 weeks' gestation. Requests for prenatal diagnosis of (typically) adult-onset diseases require careful genetic counseling.

Molecular Genetics

Table 4. Molecular Genetics of BRCA1 and BRCA2 Hereditary Breast/Ovarian Cancer Disease Name Gene Symbol Locus Normal Gene Product Genomic Databases

BRCA1 BRCA1 17q21 BRCA1 susceptibility protein

BRCA2 BRCA2 13q12 BRCA2 susceptibility protein

BRCA1

Gene: BRCA1

Abnormal product: Most BRCA1 mutations lead to frameshifts resulting in missing or non-functional protein. In all cancers that have been studied from individuals with a disease-causing mutation, the wild-type allele is deleted, strongly suggesting that BRCA1 is in the class of tumor suppressor genes, i.e., genes whose loss of function can result in neoplastic growth [Smith et al 1992]. Additional evidence that BRCA1 is a tumor suppressor gene is that overexpression of the BRCA1 protein leads to growth suppression similar to the paradigmatic tumor suppressors p53 and the retinoblastoma gene [Holt et al 1996].

Pathologic variants: More than 600 different mutations have been identified in BRCA1. While a small number of these mutations have been found repeatedly in unrelated families, the vast majority have not been reported in more than a few families. Although some research studies have suggested differences in cancer risk associated with different BRCA1 mutations, no definitive data on this point are yet available. About a third of mutations identified in BRCA1 and BRCA2 sequencing studies are of uncertain clinical significance [Shattuck-Eidens et al 1997]. As research proceeds, some of these mutations will likely be proven to be normal variants without clinical significance, while others may be associated with an increased cancer risk.

Gene Involved: BRCA1

Locus: 17q21

Normal allele: BRCA1 spans more than 80 kb of genomic DNA and encodes a 7.8 kb transcript composed of 22 coding exons [Miki et al 1994].

Normal product: BRCA1 codes for a protein of 1863 amino acids, producing a protein of about 220kd. It is normally located in the nucleus and contains phosphorylated residues [Chen et al 1996]. It contains only two recognizable protein motifs, a RING finger domain near the N-terminus and a BRCT domain at the C-terminus. RING fingers are cysteine-rich sequences that coordinate the binding of two zinc ions and are found in a number of diverse proteins. This type of domain may facilitate both protein-protein and protein-DNA interactions [Boddy et al 1994]. The RING finger in BRCA1 appears to specifically interact with another similar RING finger protein known as BARD1 that was identified based on this interaction [Wu et al 1996]. Both BARD1 and BRCA1 also share another conserved sequence known as the BRCT domain, a phylogenetically conserved sequence found in proteins involved in DNA repair and cell cycle regulation [Bork et al 1997, Callebaut and Mornon 1997]. BRCA1 is expressed in most tissues and cell types analyzed, suggesting that it is not gene expression pattern that leads to the tissue restricted phenotype of breast and ovarian cancer. The transcription of BRCA1 is induced late in the G1 phase of the cell cycle and remains elevated during the S phase, indicating some role in DNA synthesis [Gudas et al 1996, Rajan et al 1996]. A variety of evidence now points to BRCA1 as being directly involved in the DNA repair process. The BRCA1 gene product interacts with the RAD51 protein, a key component in homologous recombination and double-strand break repair [Scully et al 1997]. BRCA2 also interacts with RAD51; perhaps through this mutual association with RAD51, BRCA1 and BRCA2 associate with each other at sites of DNA synthesis after the induction of DNA damage [Chen J et al 1998]. In order to study the function of BRCA1, homozygous knockout mice have been developed. In most cases, the complete loss of function of BRCA1 results in embryonic lethality characterized by a lack of cell proliferation [Hakem et al 1996, Ludwig et al 1997]. Cells derived from mouse embryos lacking BRCA1 are defective in their repair of DNA damage [Gowen 1998]. Finally, BRCA1 knockout mice can be partially rescued by crossing with a p53 knockout strain suggesting that these genes interact with the p53-mediated DNA damage checkpoint [Brugarolas and Jacks 1997]. Therefore, the available evidence indicates that BRCA1 serves as a "caretaker," like p53, helping to maintain genomic integrity [Zhang et al 1998]. When this function is lost, it probably allows for the accumulation of other genetic defects that are themselves directly responsible for cancer formation. BRCA1 contains regions that are capable of inducing transcription [Monteiro et al 1996, Chapman and Verma 1996]. One of the targets of BRCA1 transcriptional activation appears to be the p21 cyclin dependent kinase inhibitor, itself a potent suppressor of growth at the G1/S checkpoint [Somasundaram et al 1997, Ouchi et al 1998]. It is likely that these large proteins will eventually be implicated in a variety of cellular processes, only some of which will be related to their role in the etiology of breast and ovarian cancer. BRCA1 function appears to be similar to BRCA2 function, and the two proteins may interact.

BRCA2

Gene: BRCA2

Abnormal product: Most BRCA2 mutations reported to date consist of frameshift deletions, insertions, or nonsense mutations leading to premature truncation of protein transcription, consistent with the loss of function that is expected with clinically significant mutations in tumor suppressor genes.

Pathologic variants: As with BRCA1, hundreds of BRCA2 mutations have been identified, although the number is somewhat lower than for BRCA1 (~450 v. >600). However, BRCA2 was cloned later than BRCA1 and is more difficult to screen. It is likely that the range of mutations in BRCA2 will be comparable to that in BRCA1.

Mutations of unknown clinical significance: About a third of mutations identified in BRCA1 and BRCA2 sequencing studies are of uncertain clinical significance [Shattuck-Eidens et al 1997]. As research proceeds, some of these mutations will likely be proven to be normal variants without clinical significance, while others may be associated with an increased cancer risk.

Gene Involved: BRCA2

Locus: 13q12 [Wooster et al 1995].

Normal allele: The BRCA2 gene encodes a 10.4 kb transcript composed of 27 exons.

Normal product: BRCA2 codes for a protein of 3,418 amino acids, making a 380kd protein. The BRCA2 protein is normally located in the nucleus and contains phosphorylated residues [Bertwistle et al 1997]. BRCA2 protein has no recognizable protein motifs and no apparent relation to BRCA1 protein. Nonetheless, BRCA1 protein and BRCA2 protein appear to share a number of functional similarities that may suggest why mutations in these genes lead to a specific hereditary predisposition to breast and ovarian cancer. Like BRCA1, BRCA2 is expressed in most tissues and cell types analyzed, indicating that gene expression does not account for the tissue-restricted phenotype of breast and ovarian cancer. BRCA2 transcription is induced late in the G1 phase of the cell cycle and remains elevated during the S phase, indicating some role in DNA synthesis [Rajan et al 1996, Vaughn et al 1996]. BRCA2 appears to be involved in the DNA repair process. The BRCA2 protein interacts with the RAD51 protein, a key component in homologous recombination and double-strand break repair [Sharan et al 1997, Wong et al 1997]. Perhaps through this mutual association with RAD51, BRCA1 and BRCA2 associate with each other at sites of DNA synthesis after the induction of DNA damage [Chen J et al 1998]. In order to study the function of BRCA2, homozygous knockout mice have been created. In most cases, the complete loss of function of BRCA2 results in embryonic lethality characterized by a lack of cell proliferation [Sharan et al 1997, Suzuki et al 1997, Ludwig et al 1997]. Cells derived from mouse embryos lacking BRCA2 are defective in their repair of DNA damage, [Chen PL et al 1998, Connor et al 1997] and are hypersensitive to radiation and radiomimetics [Abbott et al 1998, Chen PL et al 1998, Morimatsu et al 1998, Biggs and Bradley 1998] which may have implications for both mammographic screening and treatment modalities. Finally, BRCA2 knockout mice can be partially rescued by crossing with a p53 knockout strain suggesting that these genes interact with the p53-mediated DNA damage checkpoint [Brugarolas and Jacks 1997]. Therefore, the available evidence indicates that BRCA2 is a "caretaker", like p53, which serves to maintain genomic integrity [Zhang et al 1998]. When this function is lost, it probably allows for the accumulation of other genetic defects that are themselves directly responsible for cancer formation. Additional studies have attempted to attribute specific biochemical functions to the BRCA2 gene product. The BRCA2 protein contains regions that are capable of inducing transcription [Milner et al 1997] and has histone acetyltransferase activity potentially supporting its role in DNA repair and/or RNA transcription [Siddique et al 1998]. It is likely that the BRCA2 protein will eventually be implicated in a variety of cellular processes, only some of which will be related to their role in the etiology of breast and ovarian cancer.