BRCA-1 and BRCA-2 mutations as prognostic factors in clinical practice and genetic counselling

CANCER TREATMENT REVIEWS 2001; 27: 295±304 doi: 10.1054/ctrv.2001.0233, available online at http://www.idealibrary.com on

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LABORATORY-CLINIC INTERFACE

BRCA-1 and BRCA-2 mutations as prognostic factors in clinical practice and genetic counselling M. O. Nicoletto, M. Donach, A. De Nicolo, G. Artioli, G. Banna and S. Monfardini Departments of Medical Oncology and Experimental Oncology, Padua City Hospital, Padova, Italy Women in general have a 10% risk of developing breast cancer and a 2±3% chance of ovarian cancer in their life-times. Mutations in BRCA-1 and BRCA-2 are present in only a small portion (5±10%) of all breast cancers. Carriers of mutations in these genes have a greater risk of cancer, especially before menopause in the case of BRCA-1 carriers. In addition, their risk of contralateral breast cancer is significantly higher than for the general population (4.2±53% vs. 2%). The grade of contralateral tumours in these patients is more aggressive. BRCA-2 hereditary breast cancer seems more heterogeneous than the BRCA-1 phenotype, and not clearly different from sporadic forms. However, since 20±30% of carriers of BRCA mutations never develop breast or ovarian cancer, there must be other `risk modifiers'. Survival is better for carriers of hereditary ovarian cancer. Patients with these mutations are referred for genetic counselling, a complex process which includes: an informative dialogue between the proband and the geneticist, drawing up a family history, informed consent, evaluation of risk, genetic testing and possible involvement of healthy family members. & 2001 Harcourt Publishers Ltd Key words: Review; BRCA-1; BRCA-2; mutations; prognostic factors; clinical practice; genetic counselling.

INTRODUCTION Breast cancer is the most frequent form of cancer in women in Western Europe and the U.S.A. (affecting 1 in 10 women) and is the most common cause of death from cancer in women. While the great majority of cases are sporadic, the various types of breast cancer include a small fraction (5±10%) of hereditary forms, half of which are due to mutations in the BRCA-1 or BRCA-2 genes (1). Similarly, BRCA genes seem to be responsible for a share (5%) of hereditary forms of ovarian cancer, which are less frequent and often asymptomatic. A small percentage of the remaining forms of hereditary breast cancer are due to mutations in the Correspondence to: S. Monfardini MD PhD, Division of Medical Oncology, Azienda Ospedaliera Via Giustiniani 2 Padova, 35128 Italy. Tel: ‡39 49 8212970 0305-7372/01/027295 ‡ 10 $35.00/0

genes TP53 (responsible for Li-Fraumeni syndrome), ATM (responsible for ataxia-telangectasia) and PTEN (responsible for the Cowden syndrome). Additionally, 10±15% of all hereditary ovarian cancer cases are attributable to the genes responsible for DNA mismatch repair (MSH2 and MLH1), which are associated with non-polyposis colorectal cancer (HNPCC). Li-Fraumeni syndrome is a disease which is characterised by early onset of various types of neoplasm including: soft tissue sarcomas, leukaemia, brain tumour and breast cancer, and is secondary to germline mutations in the TP53 gene (2), which is also the most frequently altered gene at the somatic level in human tumours. The p53 protein has an important biologic function because it intervenes in the response to DNA damage: it can block the cell cycle in G1, allowing the cell to repair genomic damage, or it can induce apoptosis (programmed cell death). When mutations in p53 render the protein biologically & 2001 HARCOURT PUBLISHERS LTD

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ineffective, growth of the damaged cells continues unchecked and these cells become genomically unstable (3). Ataxia-telangectasia is a rare autosomicrecessive hereditary condition associated with a mutation in the ATM gene, located on the short arm of chromosome 17 and involved in maintaining genomic integrity. Homozygotes for alterations in ATM present with progressive cerebral ataxia, hypersensitivity to ionising radiation, immune dysfunction and increased predisposition to neoplasm. It has also been shown that heterozygote women are at increased risk for breast cancer (4). Germline mutations in the PTEN gene, which encodes a protein that regulates the interaction between the cytoskeleton and the microenvironment, are reported in Cowden syndrome. This disease is characterised by multiple hamartomas and is often associated with increased susceptibility to breast cancer and thyroid cancer (5, 6). Much information regarding the nucleotide sequence, the various mutations of BRCA genes, the structure and the intracellular localisation of the proteins encoded is already known. Still, many aspects of their biological functions have yet to be understood. They are known to be involved in regulating the process of transcription and in maintaining gene integrity by monitoring or repairing DNA lesions. They therefore play a role as indirect tumour suppressor genes, or `caretakers' (7), rather than as `gatekeepers'. This might explain the high, but incomplete, penetrance of their mutations. In fact, a subject with constitutive mutations in a BRCA gene has a high probability of developing a tumour, however, this does not happen in 100% of the cases. Penetrance for breast cancer is estimated at 70±80%, while the estimates of penetrance for ovarian cancer are less certain (8). In the population at large, women have a 10% risk of developing cancer during their life-times while healthy subjects born to BRCA positive patients have a 50% risk of being carriers of genetic mutations, which is 3±4 times higher than the general population. Healthy carriers have a much higher risk of developing cancer, especially before menopause. Today, hundreds of BRCA mutations (9) are known: these are mostly nonsense or frameshift mutations which can be found throughout the entire DNA sequence and which produce truncated proteins. They are `private' mutations in that they are specific to each family, even though in certain restricted ethnic groups (e.g. Ashkenazi Jews, Icelandic women) recurrent mutations have been noted which can be traced to a single ancestor. Furthermore, it has been noted that in high risk families the frequency of mutations, documented by the analysis of the entire coding sequence, is less than expected. This might be due to: (1) inadequate

M. O. NICOLE T TO ET A L .

technique used to identify regulatory mutations or deletions of large DNA fragments; or (2) possible involvement of other, not yet identified genes. BRCA-1 has been localised on chromosome 17 (17q21) and it codes for a protein of 1863 amino acid proteins. This gene has a role in transcription regulation (it can be refined with holoenzyme RNA polymerase) and on DNA repair (through an interactive action with RAD 51). It has thus been hypothesised that BRCA-1 is involved in maintaining gene integrity and that alterations in this gene are capable of inducing a phenotypic transformation through the accumulation of mutations in the same cell. This is why BRCA-1 mutations have not been observed in sporadic cases of breast cancer and are present in not more than 10% of sporadic ovarian cancer. According to this theory, the process of carcinogenesis mediated by BRCA-1 requires a large number of mutations, including the inactivation of both alleles of BRCA-1 itself, thus favouring a series of genetic alterations in areas coding for proteins responsible for controlling proliferation and differentiation of breast and ovarian epithelia. The probability that these alterations accumulate in the same cell is extremely low, unless a constitutive BRCA-1 mutation is also present. BRCA-2 has been localised to chromosome 13q12, shares structural and functional similarities with BRCA-1 and codes for a 3418 amino acid protein, which does not have structural analogies with the BRCA-1 protein. BRCA-2 has a role in transcription regulation too, interacting with RAD51, for cellular response to DNA damage, and with p53. In recent years, a high number of cell parameters such as oncogenes, tumour suppressors, hormone receptors, growth factors, and secretory proteins have been identified which seem to influence the course of tumours by altering their rate of growth, apoptosis, cell differentiation, metastasis formation and the development of drug resistance.

CLINICAL ASPECTS Age at onset Mutations in BRCA-1 and BRCA-2 are present in 5.9±9.4% of women under 35 years diagnosed with breast cancer (10,11), and BRCA-1 mutations are found in 12±13.2% cases of breast cancer diagnosed before the age of 45 with one first degree relative already affected by breast cancer (10,12). Following menopause, the risk of contralateral breast cancer in BRCA-1 carriers decreases (12,13), while the risk for carriers of BRCA-2 mutations continues to increase (14). Similarly, the average age at diagnosis of hereditary ovarian cancer (defined as

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2 or more relatives affected by ovarian cancer) is 47 years (15), nearly 14 years less than for sporadic ovarian cancer.

Pathology Numerous studies have underlined the fact that the forms of BRCA-1 associated hereditary breast cancer (HBC) generally present a specific phenotype characterised by: poor differentiation, high proliferation index, aneuploidy and negative oestrogen receptors (16). This characteristic phenotype of BRCA-1 associated tumours can constitute a further test in selecting affected individuals for analysis and in differentiating them from BRCA-2 carriers (17). The hypothesis that a BRCA-1 mutation can be predicted on the basis of the morphology of the tumour remains to be confirmed (16). In patients with BRCA-1 or BRCA-2 mutations, the risk of contralateral breast cancer is significantly higher with respect to the general population (2%) (18) (Table 1). The histological grade of a contralateral tumour is also more aggressive (3,23±25). Medullary carcinoma with lymphocyte infiltration is normally rare (2%). More frequently than not it is associated with BRCA-1 alterations. Despite its high grade and its negative receptor status, it often presents a lymphoplasmocytic reaction and has a good prognosis (26,32). Preclinical data suggest that p53 might have a role in the pathogenesis of BRCA-associated tumours. The proteins encoded by BRCA-1 and

BRCA-2 interact with Rad 51 which, in turn, is involved in DNA repair. Cells with BRCA mutations have a reduced repair capacity, which is responsible for a state of genomic instability. The presence of functioning p53 assures the arrest of the cell cycle and DNA repair (provided the cell retains this capacity) or apoptosis. Thus the loss of control by p53 might be necessary for malignant transformation in cells with altered BRCA-1 or BRCA-2 (27). Various studies (28±36) have demonstrated a greater frequency of TP53 mutations in BRCA-1 positive patients (Table 2). The HER2 protein is a 185-kd transmembrane tyrosine kinase with homology to the epidermal growth factor receptor. Genetic amplification of the c-erbB-2/ TABLE 1 Incidence of contralateral breast cancer Reference

Year

Incidence

Population

Albano (19)

1982

Robson (20)

1998

20% 53% 40%

Frank (22)

1998

Verhoog (21) Verhoog (13)

1999 2000

2,8% 5,6%/year 4,2% 28.6% 40%

Narod (18)

2000

35%

HBC HBC <45 years BRCA (vs 8.2% in women without mutations) No mutation BRCA-1 mutation BRCA-2 mutation BRCA-2 mutation <50 years, BRCA-1 (164 cases) BRCA-1 & BRCA-2 mutation

HBC ˆ hereditary breast cancer.

TABLE 2 Characteristics of invasive breast carcinomas arising in carriers of BRCA1 mutations (reprinted with kind permission of KA Phillips) Reference

BRCA 1

Controls

Medullary

Histologic grade 3

ER negative

PrR negative

p53 mutation

N erbB-2 Overexpression

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

90 40 17 12 15 10 22 114 27 24 32 26 49 19 13

187 112 128 105 108 21 20 528 8948 61 200 20 196 100 33

Y Y NS NR NR Y NS Y NR NS Y NR NS NS NR

Y Y Y Y Y Y Y Y NR Y Y Matched NR Y Y

NR Y Y NR Y Y Y NR Y Y Y NR Y Y Y

NR Y Y NR Y NS Y NR Y NS Y NR Y NR NR

NR NSa NR NR NR Ya Ya NR NR NR Ya Y NR Ya Y

NR Y NR NR NR Y NS NR NR NR NS NR NR Y NR

Marcus (23) Johannsson (28) Karp (47) Foulkes (48) Ansquer (49) Armes (29, 30) Lynch (31) Lakhani (50) Loman (40) Robson (20) Eisinger (32,33) Crook (34) Verhoog (51) Noguchi (35) Phillips (36)

Y ˆ feature found significantly more frequently in BRCA1-HBC (P > 0.05 for the difference between BRCA1-HBC associated tumours and controls); NR ˆ not reported; NS ˆ no significant difference in frequency of factors between BRCA1-HBC and controls (P > 0.05). adefined by HIC and not according to DNA sequencing.

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neu oncogene, which encodes HER2, is responsible for its over-expression by tumours. Over-expression is an adverse prognostic factor in early stage breast cancer, also because tumours over-expressing the c-erbB-2 gene are often associated with a higher tumour grade, increased tumour proliferation, tumour aneuploidy and lack of hormone receptor expression (37,38). However, the fact that overexpression of c-erbB-2 is not a constant finding in cases of BRCA-1-HBC (28±33,35) would lead to the conclusion that it is not indispensable in the pathogenesis of BRCA-1-HBC. Unlike the BRCA-1 phenotype, BRCA-2-HBC seems more heterogeneous, and is not clearly distinguished from non-hereditary forms (17) (Table 3). In cases of ovarian cancer, the most frequent type found in women with BRCA mutations is serous papillary, 20% of whom show the presence of psammoma bodies (as opposed to only 3% in sporadic cases). Several studies have examined the receptor status in BRCA-2-HBC (21,31,35,39,41), however, only one (41) noted the expression of receptors. Only one immunohistochemistry study found a greater expression of p53 alterations in BRCA-2 patients with respect to controls (31), while erbB-2 expression was no different from other studies. Ovaries removed in prophylactic oophorectomies from 21 women with a family history (several were BRCA-1 positive) showed cytological differences with respect to controls (42). The figures vary according to whether two or three alterations were observed in association in the same ovary; 85% or 75% of the cases presented two or three alterations, respectively, as compared to 30% or 10% in sporadic cases. These alterations

consisted of the following:       

pseudostrati®cation of surface epithelia with intercalated `peg' cells surface papillomatosis deep invagination of the epithelial cortex frequently with microscopic papillary cystadenoma epithelial inclusion cysts frequently with hyperplasia and papillary formation stromal hyperplasia and hyperthecosis increased follicular activity hyperplasia of corpus luteum and hylar cells

Parity Fathalla hypothesises that the aetiology of ovarian cancer is due to incessant ovulation (43). Childbirth would therefore be considered a protective factor in sporadic ovarian cancer (43,44). Preliminary data, which refer to the risk of ovarian cancer in carriers of BRCA-1 mutations (45,46), do not confirm that reduced ovulation due to pregnancy reduces risk. Higher parity is not a protective factor for ovarian cancer, while an older age at last pregnancy seems to reduce risk (45).

Survival implications Despite the fact that BRCA-associated tumours present with the aforementioned histopathologic characteristics, the five-year survival according to

TABLE 3 Characteristics of invasive breast carcinomas arising in carriers of BRCA2 mutations (reprinted with kind permission of KA Phillips) Reference

1. Marcus (23) 2. Armes (29,30) 3. 4. 5. 6. 7. 8. 9. 10. a

Lakhani (50) Lynch (31) Gretarsdottir (52) Loman (40) Crook (34) Verhoog (21) Noguchi (35) Agnarsson (41)

BRCA 2

Controls

17

75

9

21

73 13 34 14 22 28 14 40

528 20 368 8948 72 112 100 160

Histologic subtype

Histol. grade 3

Less tubule formation

ER positive

PgR positive

p53 mutation

N erbB-2 Overexpression

Y (tubularlobular group) Y(pleomorphiclobular) NR NS NR NR NR NS NS NS

NSa

NR

NR

NR

NR

NR

NS

NS

NR

NR

NSb

NS

Ya Y NR NR NR NR NS Y

Y NS NR NR NR NR NS Y

NR NS NR NS NR NS NS Y

NR NS NR NS NR NS NR Y

Y Yb NS NR NS NR NSb NR

NS NS NR NR NR NR NS NR

BRCA2-HBC have significantly lower mitotic counts with respect to controls. Y ˆ Feature found significantly more frequently in BRCA1-HBC (P > 0.05 for the difference between BRCA1-HBC associated tumours and controls); NR ˆ not reported; NS ˆ no significant difference in frequency of factors between BRCA1-HBC and controls (P > 0.05). bdefined by HIC and not according to DNA sequencing.

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299

TABLE 4 5-year survival in BRCA associated hereditary breast cancer Author

Year

(5-year Survival %) BRCA Cases

Non-Carriers

BRCA 1 Porter (54) Marcus et al. (23) Foulkes (61) Gaffney et al. (55) Ansquer (49) Verhoog (51) Johannsson (28)

1994 1996 1997 1998 1998 1998 1998

35 72 25 30 15 49

(83) (67) (70.8) (75) (68) (63) (83)

(61) 187 (59) (85.9) (69) (94) (69) (59)

BRCA 2 Sigurdsson (60) Verhoog (21) Gaffney (55) Lee (53)

1996 1999 1998 1999

42 28 20 23

(40) (74) (73) (65)

115 (55) (75)

BRCA 1 or BRCA 2 Albano et al. (19) Robson (20) Pierce (62) Lee (53) Hamann (63)

1982 1998 1999 1999 2000

(78)

106 (67) 20 (73) 73 (86) (70±87) 36 (84)

(45) (69) (91) (78) (87)

TABLE 5 5-year survival in hereditary ovarian cancer Author

Rubin et al. (56) Buller et al. (57) Boyd et al. (58)

Year

1996 1993 2000

5-year Survival % Cases

Controls

77 67 45

29 17 25

several authors would seem similar to patients who do not present alterations of BRCA-1 or BRCA-2; this seems true for both breast cancer (23,53±55) (Table 4) and better for ovarian cancer (56±58) (Table 5). Albano and Lynch (19) were the first to describe a better survival in families with high incidence of breast cancer. However, later studies demonstrated the presence of phenotype factors which are traditionally associated with worse prognosis (59,60). Ansquer (49) described 15 cases of BRCA positive patients with worse prognosis with respect to 108 non-carriers affected by breast cancer having an onset before the age of 36; the disease-free survival was good, but in the group of BRCA positive patients there was a higher incidence of secondary neoplasms such as contralateral breast cancer and ovarian cancer. Gaffney et al. (55) did not find any difference in survival between BRCA positive and sporadic patients. Robson (20), who examined 91 Ashkenazi BRCA1/2 mutation carriers diagnosed with breast

cancer before the age of 42, and a group of Ashkenazi women without BRCA alterations, reported that there was no difference in event-free survival. A tendency toward better survival with respect to controls has been noted in cases of hereditary colonrectal cancer without polyposis when there were constitutive mutations in genes involved in DNA repair (64,65). These considerations demonstrate the importance of dealing with family members who are healthy carriers of constitutive mutations in BRCA, through use of genetic counselling, in order to arrive at an early diagnosis, provide information about primary and secondary prevention, including surgery, chemotherapy, hormone therapy and/or other possibilities.

BREAST AND OVARIAN CANCER: RISK AND RISK MODIFIERS Breast cancer risk in BRCA-1 or BRCA-2 mutation carriers initially was estimated at 80±90%. It now varies from 7±84% (1,41,66±68), with respect to 10% for the general population (Table 6). Likewise, risk of ovarian cancer in women who are carriers of BRCA-1 or BRCA-2 mutations varies from 0.4±44%, increasing with age, as opposed to 1±2% for the general population (8,67) (Table 7). Moreover, it would seem that carriers of BRCA-1 mutations have a substantially higher risk of developing ovarian cancer with respect to carriers of BRCA-2 mutations. Ford estimated that most families with hereditary ovarian or breast cancer (81%) are carriers of BRCA-1 mutations, while only 14% are carriers of BRCA-2 alterations (14). The risk of hereditary ovarian cancer in BRCA-1 carriers was estimated at approximately 30% by age 60 and 63% by age 70 (8) while it is calculated at 7% by age 60 and 27% by age 70 in BRCA-2 carriers (14). Gayther (70) suggested the existence of one Genotype/phenotype for BRCA-2: mutations located in the OCCR region and which are associated with increased risk of ovarian cancer. The fact that 20±30% of carriers of constitutive mutations in BRCA do not develop either breast or ovarian cancer in their life-times demonstrates that there must be additional factors at work, namely other risk `modifiers'. These modifiers may consist of: (1) qualitative and quantitative differences in constitutive BRCA mutations and possible association with other constitutive mutations in other genes and/or loci; (2) factors that act directly on the metabolism of carcinogens as in the case of the NAT2 class of genes: Brunet et al. (71) have reported that the risk of breast cancer in smokers and rapid acetylators who are BRCA carriers is reduced by 50%, while the same

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TABLE 6 Risk of breast cancer in carriers of BRCA mutations Author

Year

Incidence

Population

Ford (67)

1994

Easton (8)

1995

28% 64% 54% 62%

< 50 years (BRCA 1) < 70 years (BRCA 1) < 60 years BRCA 1 (total) < 60 years due to BRCA 1, allele 1 < 60 years due to BRCA 1, allele 2 < 50±60 years < 50 years < 70 years < 50 years (BRCA 2) < 70 years (BRCA 2)

38% Claus (68) Struewing (69)

1996 1997

Ford (14)

1998

7% 33% 56% 28% 84%

TABLE 7 Risk of ovarian cancer in carriers of BRCA mutations Author

Year

Incidence

Population

Ford (67)

1994

Easton (8)

1995

29% 44% 30% 11%

< 50 years < 70 years < 60 years < 60 years allele 1 < 60 years allele 2 < 59 years < 50 years < 70 years < 50 years < 70 years < 60 years < 70 years

42% Claus (68) Struewing (69)

1996 1997

Ford (14)

1998

10% 7% 16% 0.4% 27% 7% 27%

(BRCA 1) (BRCA 1) BRCA 1 (total) due to BRCA 1, due to BRCA 1,

(BRCA (BRCA (BRCA (BRCA

2) 2) 1) 1)

is not true for slow acetylators; (3) corticosteroid regulation, as in the case of polymorphism for `GAG repeats' in the gene encoding the androgen receptor (AR): women with alleles containing 29±30 GAG repeats are diagnosed with breast cancer 4, 7 and 10 years earlier than women who do not have this polymorphism; and (4) interference with the mechanism involved in apoptosis (p53, p21, cyclin D1, cyclin E). Relatively few studies have been carried out on factors which modify risk of ovarian cancer and the results are contradictory. Narod reported that in 207 women with BRCA-1 or BRCA-2 mutations (179 and 28, respectively) the use of oral contraceptives for 6 years reduced the risk of ovarian cancer by 60% for both BRCA-1 and BRCA-2 carriers, while if they were used for only 3 years the risk reduction was 20% (72). In his study on the usefulness of fenretinide as adjuvant therapy in breast cancer (T1-2, N0, M0), De Palo noted that none of the 1422 patients assigned to the fenretinide arm presented ovarian cancer when compared to the no treatment arm P ˆ 0.0162, even

though two cases arose (at 10 and 30 months) after suspension of therapy (73). It should also be noted that bilateral prophylactic oophorectomy reduces risk of ovarian cancer by only 45% (74). In fact Tobacman described 28 women who underwent prophylactic oophorectomy, three of whom developed a peritoneal carcinomatosis which on cytological examination could be traced to an ovarian origin (74).

GENETIC COUNSELLING Genetic counselling of hereditary breast or ovarian cancer is offered to those individuals and/or apparently healthy family members who are genetically at high risk. The main objective of this counselling is to inform the subject in a clear and non-directive way about her risk status, as well as to help her understand the problem and make responsible and informed decisions (75,76). Counselling is a long articulated process which takes place over a long period of time through repeated meetings: the first informative discussion between the proband and the geneticist, gathering information to reconstruct the family history for three generations (77) and other future updates, informed consent, risk estimation, genetic testing, communicating and explaining the results (informative/ non-informative for the proband, positive/negativefor healthy subjects), follow-up surveillance, and possible involvement of healthy family members (76,77). Genetic counselling is not just for the individual in consultation, but is directed at the entire family; in extending consultation to interested family members the possible ethical-philosophical implications (informed consent, personal autonomy), psychosocial implications (perception of risk, emotional state) and legal implications must be kept in mind (75, 76). Presently, the importance which is universally given to screening healthy carriers of BRCA mutations, with the aim of an early diagnosis, is reported and reiterated by a variety of experiences in an attempt to define proper method and a timetable (78). It is important to place particular attention on the psychological effects of screening. It should be kept in mind that these diagnostic procedures are being proposed to healthy subjects and, therefore, it is necessary to clarify the significance of frequent testing, otherwise, these women might develop the idea that they are `ill', with all the suffering attached to this idea, and which may be unjustified in a particular case. The extension of genetic counselling to members who are potentially interested will have to take into consideration the fact that this choice might be

BRCA-1 AND BRCA-2 MUTATIONS TABLE 8 Timetable and mode of screening in healthy carriers of BRCA mutations From the age of 30: Every 6 months Medical examination Breast examination Gynaecologic examination Each year Medical examination Breast examination Gynaecologic examination Mammography Pelvic and Transvaginal Ultrasound Tumour Markers Breast MRI (if indicated) Breast Pap Test (if indicated)

a violation of deontalogic and legal principles of professional secrecy which are part of the doctor patient relationship. Regardless of the implications, screening should include a physical examination with a breast examination and gynaecologic examination once every 6 months. Every year the subject should undergo a mammogram and/or ultrasound of the breast, transvaginal pelvic ultrasound, and tumour marker assays (78±80). The literature presents many different time-tables for screening: a mammogram every 3 months has even been proposed by some, however, there is no consensus as to what age to begin this intensive screening (78±80) (Table 8). Ductal lavage (which is reserved for high risk subjects who are carriers of BRCA-1 and BRCA-2 mutations) can be carried out by placing a small catheter into milk ducts and introducing 2±4 cc of saline solution. The lavage liquid collected can provide 1000±2000 cells (81, 82); if these cells present alterations upon cytological examination, the lavage is repeated. If the atypical cells are confirmed, the subject should undergo mammography (78) and MRI (83,84). It should be remembered that breast cancer with BRCA mutations has an earlier onset, often diagnosed before the age of 35; in addition, high risk families often present the phenomenon of `anticipation' in which the neoplasm tends to present earlier in successive generations. Tumour markers can easily be assayed in ovarian cancer where the association of CA125 with OVX1 and M-CSF can permit a sensitivity (98%) and specificity for early diagnosis at stage I, which is greater than 85±90% (80). In breast cancer, CA15.3 and CA27.29 have not been proven useful in early diagnosis; however, IGF1 (86) appears to be potentially interesting as

301

a biological marker in early stages of breast cancer. In this regard, further studies of this category of patients should be encouraged in order to identify and standardise tumour markers. In the future, immunohistochemical assays or PCR, may be used specifically to examine molecules involved in the cell cycle (i.e. p53, p21, p27), apoptosis (Bcl-2, Bax, Bcl-x-L) or DNA repair, with the intent of identifying molecular modifications early in tumour development, and thus they may eventually play a role in primary prevention.

CONCLUSIONS BRCA-1 and BRCA-2 mutations are present in approximately 5.9±9.4% of women with breast cancer diagnosed before the age of 35 (10, 11) and in 12±13% of breast cancer before the age of 45 with one first degree relative already affected by breast cancer (10,12). Following menopause, the risk of contralateral breast cancer in carriers of BRCA1 decreases (9, 12) while the risk continues to rise for carriers of BRCA-2 mutations (14) (Table 8). Hereditary ovarian cancer (defined as two or more relatives affected by ovarian cancer) has an average age of onset of 47 years, 14 years less than for sporadic ovarian cancer. Contralateral breast cancer is more frequent in carriers (Table 1) and alterations in p53 could be fundamental for the malignant transformation. In fact, it seems to be altered in all reports (28±35) in the literature, while the over-expression of c-erbB-2 does not seem to have an indispensable role in the pathogenesis of BRCA-1-HBC. The aetiology of hereditary ovarian cancer differs from sporadic ovarian cancer, where incessant ovulation might explain the aetiology (43,44). All this might in part explain why various studies report cases with a similar or slightly better survival for BRCA-1-HBC with respect to controls (20,23,28,53,55) which is analogous to hereditary colorectal cancer without polyposis where constitutive mutations are present in genes responsible for DNA repair (64,65). The main objective of genetic counselling is to improve an affected proband's knowledge about her pedigree for three generations and thus acquire information that only she can provide for her family (76). When it is possible to contact healthy relatives, screening information should be presented (78) and screening should be proposed (Table 8). In cases of uninformative results for healthy subjects, it should be explained that the subject still has an increased risk for sporadic cancer, especially breast

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cancer, and that in the future it might be possible to identify other yet unknown genetic mutations linked to cancer.

18. 19.

ACKNOWLEDGEMENTS 20.

The authors would like to thank Dr Anna Aprile, Institute of Medicine Legal and Forensic Sciences, and S. Serpentini, Psychology Department Med. Oncol. Az. Ospedaliera of Padua, for their help in the preparation of this manuscript.

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