Breast Cancer Risk Factors

The American Cancer Society estimates that in 2009 there were 192,370 new cases and 40,170 deaths from breast cancer, making breast cancer the most common cancer and the second leading cause of cancer death among women in the United States.(1) According to data from the Surveillance, Epidemiology and End Results (SEER) program from the mid-1990s, the average lifetime risk of breast cancer is 1 in 8 or 12.6%.(2) Breast cancer risk is strongly influenced by age, family history,(3) the presence of a hereditary predisposition to mutations in one of several breast cancer genes,(4)(5)(6)(7)(8)(9) reproductive factors,(10) exposure to exogenous estrogen,(11) alcohol(12) and an abnormal breast biopsy showing atypical cells.(13)

Breast cancer incidence increased between 1980 and 1987 with an average annual percentage increase of 3.7%,(2) presumably due to the increasing utilization of screening mammography.(14) Between 1987 and 2001 the rate of increase dropped to 0.5% per year, and between 2001 and 2004 rates decreased by 3.5% per year.(2) This decline is thought to reflect an earlier decreased use of hormone therapy resulting from publication of the Women’s Health Initiative trial findings that showed a 26% increase in breast cancer risk associated with postmenopausal estrogen and progesterone use.(15)

In the United States, breast cancer incidence is higher among white compared to African American) women, except among younger women where the trends are reversed,(16)(17)(18) although mortality rates are higher among African American women.(16)(19)(20) The disproportionately higher mortality seen among African American women may reflect the influences of socioeconomic, cultural and behavioral factors that affect access to screening(21) and treatment,(22) both of which are highly correlated with survival. In addition, tumor characteristics that negatively influence prognosis, are more prevalent in African American women including higher nuclear grade, hormone receptor negativity, aneuploidy (abnormal number of chromosomes) and positive axillary lymph nodes.(23)

Family history is one of the strongest risk factors for breast cancer. Using data from the Breast Cancer Detection Demonstration Project, Gail et al. computed lifetime risk estimates based on age, family history of breast cancer in a first-degree relative, prior breast biopsies (higher risk associated with lobular carcinoma in-situ or atypical hyperplasia) and reproductive history. This model, known as the Breast Cancer Risk Assessment Tool, is widely used for counseling and determining eligibility for chemoprevention trials.(24)(25)(26) Claus et al. used family history data from the Cancer and Steroid Hormone (CASH) Study(27) to predict lifetime breast cancer risks for white and African American women with a family history of breast cancer, and is currently used to determine eligibility for MRI screening as an adjunct to mammography.(28) Recently, Gail et al. developed a model for projecting absolute risk of invasive breast cancer in African American women using data from the Women's Contraceptive and Reproductive Experiences (CARE) Study and found that the “CARE model” resulted in higher breast cancer risk estimates for African American women than the earlier Breast Cancer Risk Assessment Tool.(18)

Somatic Mutations

While 5-10% of women with breast cancer have mutations in one of a number of tumor suppressor genes inherited in an autosomal dominant fashion,(4)(5)(6)(7)(8)(9) most breast cancers have somatic genetic changes which act as both prognostic and predictive factors.(29) Oncotype DX® and MammaPrint are two gene-expression assays which categorize women with early stage breast cancer to either low or high risk for distant recurrence, allowing physicians to tailor their use of adjuvant chemotherapy to those who are at the highest risk of relapse.(30)

Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase protein that is over-expressed in 10-20% of women with breast cancer,(31) which when activated leads to tumor cell proliferation,(32) and malignant transformation.(33) Trastuzumab is a HER2-specific monoclonal antibody that was developed to inhibit HER2 activation.(33) Utilization of trastuzumab among women with HER2-positive breast cancer in the metastatic(34)(35)(36) and locally advanced setting(30) has led to improved clinical response and survival. Molecular sub-typing using gene expression arrays has identified four subtypes of breast cancer, two of which are derived from Estrogen Receptor (ER)-negative tumors (basal-like HER2 negative and HER2 positive) and two from ER-positive tumors (luminal A and B).(37)(38) Triple negative or basal type breast cancers are associated with worse clinical prognosis,(39) are more commonly associated with breast cancer in African American women(40) and BRCA1 mutation carrier status.(41)

Hereditary Breast and Ovarian Cancer (HBOC)

An estimated 7% of breast cancers and 10% of ovarian cancers are due to inherited mutations in two cancer susceptibility genes called BRCA1 (breast cancer 1 gene; chromosome 17q21) and BRCA2 (breast cancer 2 gene; chromosome 13q12.3).(42)(43)(44)(45) BRCA1 and BRCA2 function as tumor suppressor genes, but also appear to play a role in DNA repair. The prevalence of BRCA1 and BRCA2 mutations in the general population is estimated to be approximately 1 in 400 to 1 in 800.(42)(43)(44)(45)(46)(47) However, a number of founder mutations have been observed, particularly in the Ashkenazi (central or Eastern European) Jewish population, where three specific mutations-187delAG (formerly known as 185delAG), 5385insC (formerly known as 5382insC), and 6174delT-occur at a frequency of approximately 1 in 40.(48)(49)(50) Mutations in either BRCA1 or BRCA2 are the principal cause of hereditary breast and ovarian cancer (HBOC).

Estimates of penetrance for BRCA1 and BRCA2 female carriers range from 36% to 87% lifetime risk of breast cancer and from 16% to 60% lifetime risk of ovarian cancer, depending upon the population studied.(48)(51)(52)(53)(54)(55) The contralateral breast cancer risk in female carriers ranges from 12% to 27% within five years of the initial breast cancer diagnosis.(56) Male breast cancer is more commonly associated with BRCA2 mutations when compared to BRCA1 mutations. The cumulative probability to age 70 of male breast cancer in carriers of BRCA2 mutations has been reported as 6%(57) and 6.8%.(58) Individuals with BRCA1/2 mutations have also been shown to be at an increased risk of other cancers including cancer of the fallopian tubes, peritoneum, prostate, pancreas, gallbladder, bile duct and stomach, as well as melanoma.(59)(60) Initial reports of increased colorectal cancer risk have generally not been replicated.

BRCA1/2 gene mutations are inherited in an autosomal dominant fashion. It has been a misconception that breast cancer can only be inherited from the maternal lineage. On the contrary, half of all inherited breast cancer cases are inherited from the father’s side of the family. An autosomal dominant pattern, however, is not always apparent in these families due to small family size, limited family structure (defined as having fewer than two first- or second-degree female relatives surviving beyond age 45 years in either lineage), and/or adoption in the lineage. Although reported, the de novo mutation rate for BRCA1/2 appears to be low but is not currently known.(61)(62)(63)

According to the National Comprehensive Cancer Network Practice Guidelines: Genetic/Familial High-Risk Assessment: Breast and Ovarian,(64) management options for women with BRCA1/2 mutations include:

Regular monthly breast self-examination (BSE) beginning at age 18. Clinical breast examination (CBE) twice yearly starting at age 25. Annual mammogram and breast magnetic resonance imaging (MRI) starting at age 25 or earlier based on the earliest breast cancer in the family. Discussion of prophylactic mastectomy. Recommendation for prophylactic bilateral salpingo-oophorectomy between ages 35 and 40 or upon completion of childbearing, or earlier based on the earliest ovarian cancer in the family. For those individuals who have not elected to undergo bilateral salpingo-oophorectomy, twice-yearly transvaginal ultrasound and CA-125 screening beginning at 35 or earlier based on family history. Consideration of chemoprevention options for breast and ovarian cancer.

Male mutation carriers are recommended to:

Perform monthly BSE. Undergo twice-yearly CBE. Consider baseline mammogram, with annual repeat pending clinical findings on the baseline examination.

Finally, screening options for prostate cancer, melanoma and pancreatic cancer (despite the absence of proven clinical benefit) should be discussed as well.

Cowden Syndrome (CS)

Cowden syndrome (CS) is an autosomal, dominantly inherited syndrome otherwise known as one of the multiple hamartoma syndromes. It is poorly recognized with variable phenotypic features and therefore felt to be under-diagnosed.(65) According to Nelen and colleagues, the estimated incidence in the Netherlands is approximately one in 200,000 to one in 250,000 live births.(66) CS is characterized by multiple hamartomatous lesions in a number of body organs, as well as both benign and malignant breast, thyroid and endometrial tumors.(67)(68) Cutaneous manifestations involving predominantly the face, tongue, mouth and hands are the most consistent features which occur in as many as 99 percent of affected individuals by the third decade of life.(69) These lesions consist of papillomas (small wart-like growths), trichilemmomas (flesh-colored bumps involving a hair follicle) and lipomas (fatty tissue deposits under the skin).(70)

Other less commonly seen features include macrocephaly greater than 97th percentile with normal sized ventricles,(71) hemangiomas and hamartomas, which are largely asymptomatic, in the stomach, small bowel and colon.(72) Lhermitte-Duclos disease (LDD) is a rare central nervous system tumor and considered to be pathognomonic.(73)(74)(75) Table 1 lists clinical features of CS, and the approximate percentage that develop each feature.

The average age of female breast cancer is between 38 and 46 but has been reported to be as young as 14 and as late as 65.(76) Male breast cancer has also been reported but the exact risk is unknown.(77)Benign breast findings include: ductal hyperplasia, intraductal papillomatosis, adenosis, tubular atrophy, hamartomas, fibroadenomas and fibrocystic changes.(78) Thyroid cancers are most commonly follicular followed by papillary.(79) Other cancers without definitive risk estimates include colon, skin, kidney, small intestine, stomach, ovary and lung.(80)(81)

The International Cowden Consortium arrived at a set of consensus operational diagnostic criteria for CS based on published data and expert opinion.(82) These criteria are listed in Table 2 below and are reflected in the practice guidelines of the National Comprehensive Cancer Network Genetics/Familial High-Risk: Breast and Ovarian.

CS has been linked to mutations in the PTEN gene which has been mapped to 10q22-q23 and a mutation is identified in up to 85% of individuals who meet the clinical diagnostic criteria.(74)(83)(84) Germline mutations in PTEN have also been found in approximately 60% of individuals with Bannayan-Riley-Ruvalcaba syndrome (another multiple hamartoma syndrome).(85)(86)(87)(88)

It is recommended that individuals with a clinical or molecular diagnosis of CS should undergo screening for component cancers at ages listed below, or five years younger then the earliest onset of the cancer in the family (whichever comes first):

Annual physical examination of the neck by age 18 and a baseline thyroid ultrasound is recommended for thyroid cancer screening. Semi-annual CBE and monthly SBE should start by age 25 and annual mammography and breast MRI screening between the ages of 30 and 35 for women. Annual blind endometrial biopsy between the ages of 35 and 40 prior to menopause, and trans-abdominal ultrasound post menopause for endometrial cancer screening.(89) Consider annual dermatologic examination.

Li Fraumeni Syndrome (LFS)/ Li Fraumeni-like Syndrome (LFL)

Li Fraumeni syndrome (LFS) is an inherited cancer syndrome associated primarily with the following cancers: soft-tissue sarcoma, leukemia, brain tumors, osteosarcoma, breast cancer and adrenal cortical tumors. LFS is due to germline mutations in the p53 gene (chromosome 17p13.1), which functions as a tumor suppressor gene and also regulates the cell-cycle arrest that is required to permit repair of DNA damage.(90)

Two types of Li Fraumeni syndrome have been described: classic Li Fraumeni syndrome (LFS) and Li Fraumeni-like syndrome (LFL). LFL shares some, but not all, of the features associated with LFS. Several criteria have been developed to identify families at risk for a germline p53 mutation. (91)(92)(93)(94)(95) The alternative Chompret criteria, which were developed more recently, are the only criteria that allows for the possibility of a negative family history. These criteria are listed below in Table 3.

Approximately 70% of families meeting the classic LFS definition have a p53 mutation.(96) Studies based on LFL criteria found the percent having an identifiable p53 mutation varied from 22% using Birch’s definition to 8% using Eeles’ definition.(97)

LFS appears to be rare with fewer than 400 families reported worldwide. As a result, it has been difficult to characterize the specific cancers associated with LFS. LFS was initially referred to as sarcoma, breast, leukemia and adrenal gland (SBLA) syndrome. However, the spectrum of tumors in LFS appears to be much broader than these original tumor types and may include melanoma, germ cell tumors, gastric carcinoma and Wilms' tumor.(95) Although the exact risk of cancer in individuals with LFS is not known, age-specific cancer risks have been calculated. It is estimated that by age 40 years the cancer risk is 50% and by age 60 years is up to 90%.(98) In families with LFL, 44% were diagnosed before age 30 and 78% were diagnosed by age 50 years.(97) Early-onset breast cancer accounts for 25% of all LFS-related cancers(99) but LFS/LFL is thought to account for less than 1% of the total cases of breast cancer. Individuals with LFS are also at an increased risk to develop more than one type of cancer during their lifetime. One study found that 15% of individuals with LFS developed a second cancer, 4% a third cancer and 2% a total of four cancers. In this study, survivors of childhood cancers were found to be at greatest risk for developing additional cancers.(100)

Some families with LFS and LFL have been found to have mutations in a different gene (other than p53), known as the CHEK2 gene on chromosome 22q12.1.(101)(102) At this point, it is not clear whether mutations in the CHEK2 gene cause different cancer risks from those associated with p53. p53 and CHEK2 gene mutations are inherited in an autosomal dominant pattern. The new mutation rate for p53 was recently reported to be at least 7% and may be as high as 20% in a study of 341 individuals with early onset cancer.(103)

Breast cancer is the only LFS-related malignancy for which effective screening exists. According to the National Comprehensive Cancer Network Practice Guidelines: Genetic/Familial High-Risk Assessment: Breast and Ovarian(64), management options for women with p53 mutations include:

Monthly BSE beginning by age 18. CBE twice yearly beginning at age 20-25 or 5-10 years before the earliest known breast cancer in the family (whichever comes first). Annual mammogram and breast MRI beginning at ages 20–25 or earlier depending on the family history. Discussion of prophylactic mastectomy.

The risks and benefits of screening for other LFS malignancies in both men and women with p53 mutations have not been established. Colonoscopy should be considered every 2-5 years beginning by age 25. Additional surveillance activities may be tailored based on individual family histories. Pediatricians should be alerted to the risk of specific childhood malignancies in affected families. Finally, an annual comprehensive physical examination is encouraged with a high index of suspicion for syndromic malignancies and second cancers in previously treated patients.

Hereditary Diffuse Gastric Cancer (HDGC)

According to the World Health Organization, gastric cancer is the second leading and breast cancer is the fifth leading cause of cancer mortality worldwide. Hereditary diffuse gastric cancer (HDGC) accounts for approximately 1% of all gastric cancer cases.(104) HDGC is characterized by gastric cancer of diffuse histology, which often shows signet ring cell morphology, and an increased risk for invasive lobular breast cancer in women. Table 4 lists the clinical diagnostic criteria for HDGC. HDGC is caused by mutations in the CDH1 gene (chromosome 16q22.1), which encodes the E-cadherin protein, and is inherited in an autosomal dominant fashion. E-cadherin is a cell-to-cell adhesion molecule and the loss of expression accounts for the morphologic differences between diffuse and intestinal variants of gastric cancer.(105)

Of individuals who meet diagnostic criteria for HDGC, only 30% will have an identifiable CDH1 mutation. Penetrance data on CDH1 mutation carriers suggests the cumulative risk for lobular breast cancer in women by 80 years of age is 39%, while the cumulative risk for gastric cancer in women and men is much higher—83% and 67%, respectively.(106) The average age of onset of gastric cancer is 38 years with a range of 14-69 years, while the average age of onset for breast cancer is currently unknown. Finally, there have been reports of prostate, colon (particularly signet ring cell) and ovarian cancers in CDH1 mutation carriers; however, it remains to be determined if the risks to develop these cancers are substantially increased over the general population.(104)(107)

Although the association of diffuse gastric cancer and HDGC has been well established, lobular breast cancer has only recently gained recognition as a more significant feature of this syndrome than once thought. Approximately 5-10% of all cases of breast cancer are caused by germline mutations in single genes, and the exact proportion of those caused by CDH1 mutations is unknown. Lobular breast cancer accounts for 10% of all breast cancers, and comprises approximately 9% of the breast cancer in carriers of germline BRCA2 mutations and 3% in BRCA1 mutation carriers.(108) In 2007, Mascari and colleagues studied 23 women with documented lobular breast cancer less than 45 years of age with no personal or family history of diffuse gastric cancer and who previously tested negative for BRCA1 and BRCA2 mutations.(109) Of these 23 women, one had a germline CDH1 mutation. These results suggest that diffuse gastric cancer does not necessarily have to be present in a CDH1 carrier or their family. This may have implications for the consideration of genetic counseling and possibly testing in familial lobular breast cancer families. However, in order to determine the prevalence of CDH1 mutations in women with lobular breast cancer a large population based study must be performed.

While there are no definitive breast cancer screening or risk reduction recommendations for women with HDGC, it is suggested that they follow current recommendations for women with other hereditary breast cancer predisposition syndromes (i.e., HBOC):

Regular monthly BSE beginning at age 18. CBE twice yearly starting at age 25. Annual mammogram and breast magnetic resonance imaging (MRI) beginning at the age of 30-35 or earlier based on the earliest breast cancer in the family. Some recommend annual breast ultrasounds in place of breast MRI.(99) Discussion of prophylactic mastectomy; however the exact risk reduction has not been established.

Also, most lobular breast cancers are estrogen receptor positive, and therefore women with CDH1 mutations may potentially benefit from chemoprevention through the use of tamoxifen or raloxifene, although there are no clinical trial data to support this.(110)

Men and women with CDH1 mutations should be screened for gastric and colon cancer:

Individuals who have not yet undergone a prophylactic gastrectomy should undergo a detailed endoscopic procedure with multiple random biopsies and biopsies of suspicious lesions every 6 to 12 months beginning at the age of 16.(111) It is possible that the use of chromoendoscopy, which uses a mucosal dye to enhance subtle lesions, may improve the detection rate of early diffuse gastric cancer.(112)(113) If colon cancer is present in the family history, it is recommended to follow the National Comprehensive Cancer Network guidelines by beginning colonoscopies at age 40 or 10 years prior to the earliest diagnosis in the family. Earlier reports suggested an association of colon cancer with HDGC; however, there are conflicting data that do not support the role of CDH1 germline mutations in the development of colon cancer.(114)

The value of gastric cancer surveillance is controversial since the effectiveness of endoscopy in detecting early diffuse gastric cancer lesions has not been proven. Findings from several studies suggest that prophylactic gastrectomy, rather than regular endoscopic screening, is the best preventive measure in individuals with CDH1 mutations since the diffuse type of gastric cancer spreads submucosally and does not form a discrete mass that is readily detected with endoscopy.(115)(116) It remains unclear at what age prophylactic gastrectomy should be recommended, although several studies suggest that prophylactic gastrectomy should be considered prior to the age of 30 years since the risk of gastric cancer by that age is approximately 4%,(106)

Peutz-Jeghers Syndrome (PJS)

Peutz-Jeghers syndrome (PJS) is an autosomal dominant condition characterized by hamartomatous gastrointestinal polyps, mucocutaneous pigmentation and several types of malignancies. PJS is caused by mutations in serine-threonine-kinase 11, STK11 (also known as LKB1). STK11 is a tumor suppressor gene located on chromosome 19p13.3. Germline mutations can be detected in approximately 100 percent of patients with a positive family history, and in 91 percent of patients with clinical PJS, with no family history.(117) Family history may be negative in up to 50 percent of individuals, indicating a high de novo mutation rate.

Definitive diagnosis of PJS can be made in individuals with histopathologic confirmation of hamartomatous gastrointestinal polyps with the distinctive PJS morphology* and two of the following clinical criteria proposed by Giardiello and colleagues: (118)

Small bowel polyposis Family history of PJS consistent with autosomal dominant inheritance Pigmented macules of the buccal mucosa, the lips, fingers, toes, and external genitalia.

For individuals who do not have histopathologically confirmed hamartomatous polyps, a probable diagnosis can be made if two of the three above criteria are met.

*While PJS polyps have a distinct histology, individuals with PJS can also develop adenomatous polyps, particularly in the colon, and therefore can present similarly to familial adenomatous polyposis (FAP).

The characteristic mucocutaneous, pigmented lesions are typically more pronounced before age five years, and may fade through puberty and into adulthood. The lesions present as flat dark blue or brown lesions around the mouth, eyes, nostrils, perianal area and on the buccal mucosa.(119) Pigmented macules may also be present on fingers and toes. Pigmentation occurs in greater than 95% of individuals with PJS.(99)

PJS polyps usually become symptomatic in the second decade of life (120) and are most commonly present in the small bowel (96%), followed by colon (27%), rectum (24%) and stomach (24%).(121)(122) PJS type polyps can cause intussusception, bleeding and bowel obstruction.

Females with PJS are at risk for developing ovarian sex cord tumors with annular tubules (SCTAT), which is a benign neoplasm of the ovaries. Some studies report that these tumors are present in almost all females with PJS. Symptoms of these tumors include menorrhagia or precocious puberty. Males with PJS will occasionally develop Sertoli cell tumors of the testis, which can secrete estrogen and lead to gynecomastia.

The cumulative cancer risks are highest for cancers of the gastrointestinal tract (esophagus, stomach, small bowel, colorectum and pancreas). Females have up to a 54% chance of developing breast cancer (123), compared to a risk of up to 87% in BRCA1 and BRCA2 mutation carriers. In 2004, Lim and colleagues reported that breast cancer developed in 8% of women with PJS by age 40, and 32% by age 60.(124) Table 5 summarizes the cancers that have been associated with PJS and their cumulative lifetime risks. The majority of cervical cancers consist of a rare and highly malignant cervical adenoma malignum subtype, and ovarian tumors primarily of granulosa cell subtype.(123) Kidney, thyroid, male breast and prostate cancers have been reported in PJS families; however, their exact association with PJS is uncertain.

Optimal screening strategies for PJS have not yet been determined but there are some suggested guidelines:(125)(126)(127)

Women with PJS:

Regular monthly BSE beginning at age 18. Annual CBE beginning at age 25. Annual mammography beginning between 25-35 years of age. Annual pelvic examination, pelvic ultrasound, and Pap smear beginning at age 20.

Men with PJS:

For Men and Women with PJS:

Upper and lower endoscopy and small bowel follow-through every two years beginning at the age of 10. Some studies have suggested beginning stomach and small bowel screening at 8 years of age. Hinds and colleagues found that laparotomy for bowel obstruction was performed in 30% of individuals by age 10 years.(128) Colonoscopy every 2-3 years beginning at the age of 25. Pancreatic cancer screening by endoscopic ultrasound or abdominal ultrasound every 1-2 years beginning at the age of 30. Pancreatic cancer screening has generally not been routinely recommended in PJS, and is being evaluated in clinical trials (i.e., CAPS-3 study).

Ataxia-Telangiectasia (AT)

Ataxia-telangiectasia (AT) is a rare, autosomal recessive condition with an incidence of one in 30,000 to one in 100,000. AT is characterized by progressive neurological disease, immunodeficiency, radiosensitivity and an increased risk for malignancy, primarily leukemia (usually acute or chronic lymphocytic leukemia) and lymphoma, with onset in childhood. AT is caused by mutations in the AT-mutated (ATM) gene on chromosome 11q22.3. Cerebellar ataxia, present in 100 percent of cases, is typically present around the time a child begins to walk, and most affected individuals are wheelchair-bound by the age of 10. The expected lifespan of individuals with AT has increased, with most living beyond 30 years of age.

Interestingly, women who are monoallelic (heterozygous) for ATM mutations are at an increased risk to develop breast cancer. ATM heterozygotes in the general population are estimated at about 1%. Thorstenson and colleagues identified ATM mutations in 3.7% (10/270) of families who had previously been analyzed for BRCA1 and BRCA2 mutations.(129) Several other studies have also reported the association of monoallelic ATM mutations and the increased risk of breast cancer. Thompson and colleagues studied 169 AT patients and their families and found that the overall relative risk of breast cancer was 2.2, but in women younger than age 50 the relative risk increased to 4.9.(130) It is estimated that approximately 15% of women with ATM mutations will develop breast cancer.(131) Original studies in AT families suggested an increase in stomach, colon, pancreas, bladder and ovarian cancers in ATM heterozygotes (131)(132)(133), although this has not been substantiated by subsequent studies.(131)(134) Therefore, further studies will be needed to determine if there is an increased risk to develop cancer other than breast in ATM heterozygotes. Finally, some early studies suggest that ATM heterozygotes are at an increased risk for heart disease.(133)(135)(136)

Currently, there are no proven guidelines for managing ATM heterozygotes. Due to the possibility of increased sensitivity to ionizing radiation in ATM mutation carriers, the role of mammography at an earlier age in these individuals is unclear(99) Breast MRI may offer a better screening strategy but currently there are no data to support this.