By.P.B.TIRUPATHI PICHIAH.Dept Of Animal Sciences.

BHARATHIDASAN UNIVERSITY

An Introduction with Breast cancer.

BRCA1 and BRCA2 and the genetics of Breast Cancer

The Clinical Significance of Epidermal Growth FactorReceptor (EGF-R) in Human Breast Cancer.

Causes

Diagnosis

Treatment

Conclusion

In western countries, up to 20% of women diagnosed with breast cancer have at least one relative who is also affected by the disease.

Part of this familial clustering shows autosomal dominant inheritance with high penetrance and is due to mutations in the BRCA1 and BRCA2breast cancer genes.

Germline mutations in the BRCA1 and BRCA2 genes confer a strongly elevated risk of breast cancer.

Women who inherit a mutated copy of the BRCA1 gene have an estimated lifetime risk of breast cancer ranging from 56% to 87%, which is comparable to the lifetime Risk for BRCA2 mutation carriers.

INTRODUCTION

Why mutation in BRCA1 and BRCA2 lead specifically to Breast and Ovarian cancer ?

BRCA1 AND BRCA2 AND THE GENETICS OF BREAST CANCER

The function of BRCA1 and BRCA2 suggested that they are involved in two fundamental cellular processes : DNA damage repair and transcriptional regulation.

BRCA1 & BRCA2 genes contain high densities of repetitive elements.

Somatic alteration of BRCA 1 & 2 are common and driven by rearrangement between repetitive elements (Mary.C.K., et.al 2001)

The breast and ovary are estrogen responsive tissue. Beginning in puberty, the breast epithelium proliferate rapidly in response to fluctuating level of estrogen.

BRCA – mediated tumorigenesis are estrogen-mediated proliferation of breast and ovarian epithelium and distinctive genomic context of the BRCA genes.

The normal protein products of BRCA1 and BRCA2 are involved in the fundamental cellular process of maintaining genomic integrity and transcriptional regulation.

Tumorigenesis in individuals with germline BRCA mutation requires somatic inactivation of the remaining wild-type allele, suggesting that the BRCA genes are tumor suppressors. (Smith S.A. et.al 1992 and Collins. et. al 1995)

BRCA1 mutation carrier has a lifetime risk of breast cancer that is greater than 80%.

BRCA1 & BRCA2 proteins are involved in control of homologous recombination (HR)

And double-strand break repair in response to DNA damage.(Hall,J.M. et,al 1990, Miki, Y. et,al 1994, Wooster,R., et,al 1994 Deng,C.X, et, al 2000)

Within minutes of DNA damage, the histone H2A family member H2AX become extensively phosphorylated and forms foci at break sites (Paull,T.T., et,al 2000)

BRCT domain of BRCA1 bind double-stranded breaks in DNA (Yamane, et,al 2000)

BRCA1 & BRCA2 also function as transcriptional co-regulators through direct interaction with sequence specific transcription factors with components of the transcriptional machinery.

Chromatin remodeling function have been attributed to both BRCA1 and BRCA2.

BRCA1 is a component of the human SW1/SNF- related chromatin remodeling complex(Bochar et,al 2000)

BRCA1 has the ability to co-activate endogenous p53-dependent stimulation of p21and p53 promoter is dependent on its physical association with the SWI/SNF complex.

It remains to be determine whether the function of BRCA1 in the SWI/SNF complex is to direct chromatin remodeling to sites of DNA damage, allowing repair protein to function , and/or whether this complex is essential for activation of genes critical tothe DNA damage response pathway.

Among sporadic breast tumors, 50-70% have lost an allele of BRCA2 (Futreal, et.al 1994 and Cleton, et.al 1995)

DISTINCTIVE GENOMIC INSTABILITY OF BRCA GENES

The genomic region of BRCA1 and BRCA2 contain very high densities of repetitive DNA elements that may contribute to genetic instability.

The BRCA1 region consists of 42% Alu sequences and 5% non-Alu repeats (Smith et.al1996)

The BRCA2 region consists of 47% repetitive DNA, consisting of 20% Alu sequences and 27% LINE and MER repetitive DNA

[ Alu sequences:- The Alu family is a set of dispersed, related sequences, each ~300 bp long, in the human genome. The individual members have Alu cleavage sites at each end (hence the name)]

[LINE elements have two reading frames, one coding for a nucleic acid-binding protein, the other for reverse transcriptase and endonuclease activity.]

Alu mediated genomic rearrangement within BRCA1 and genomic rearrangement in BRCA2 in as yet unexplained high risk breast cancer families will reveal other such complex rearrangements (T.D. Walsh, Unpublished results)

Chromatin organized into topologically constrained loop that are anchored to the matrix attachment regions (MARs) (Getzenberg et.al 1991 , Jack et.al 1992 and Roberge et.al 1992 )

ESTROGEN AND SURVIVAL OF BRCA-DEFICIENT CELLS

Generally, failure of DNA repair leads to growth arrest or cell death. BRCA defficient mice die early in embryogenesis as the result of cellular responces .(Mary.C.K., et.al 2001)

The first paradox of BRCA biology is that BRCA-deficient breast or Ovarian epithelial cells develop tumours instead of under going cell death.

The longer the women is exposed to estrogen either endogenously or exogenously the higher her risk of developing breast cancer; both early onset of menarche and late menopause are associated with increase risk

Human breast tissue begin develop to develop very early, usually during the sixth week of fetal development. Fetal Breast tissue is responsive to circulating maternal hormones. After early infancy, no developmental changes occur in breast until puberty.

INHERITED BREAST CANCER

All breast epithelial cell of a BRCA mutation carrier have one inactivated allele of BRCA mutation carrier have one inactivated allele of BRCA1 and BRCA2.

During puberty, in direct response to estrogen surges, these cells rapidly proliferate.

There is a dramatic increase in the rate of cellular replication strain the DNA repair capacity of breast epithelia cells.

The BRCA-null cells are deficient in repair, they would sustain DNA damage at many sites, often including genes essential to cell checkpoint activation.

Mutation of a check point gene would enable a BRCA-null cell to escape death permanently and to proliferate.For BRCA1-mediated tumorigenesis, one of the key check-point gene is p53.

Evidence from conditional knock-out mice suggests that loss of BRCA1 in mammary cells leads to incomplete proliferation, apoptosis and tumors at low frequency (Xu et.al 1999)

Tumors in patients with germline BRCA1 or BRCA2 mutation are frequently associated with somatic mutation of p53.

If somatic inactivation of wild-type BRCA allele and mutation of critical check point genes occur during puberty, then breast tumorigenesis would be an early events in the life of these women.

SPORADIC BREAST CANCER

Analysis of sporadic tumor may suggest that either one or two alleles can be lost, yielding similar patterns of allelic imbalance.

Repeat mediated loss of chromatin loops formed at different points in the cell cycle will yield deletion of different sizes. If this process occur in both copies of the BRCA1 region or BRCA2 region , deletion including deletion overlap , they may incorrectly appear to define one region of LOH.

Presence of single BRCA1 or BRCA2 allele is sufficient for normal growth & development, because person heterozygous for BRCA mutations have normal phenotype apart from their cancer predisposition.

Transcriptional silencing may inactivate BRCA allele by loss of protein that regulate the expression or by an increase in negative regulatory protein (Id4 inhibitor of DNA binding 4) (Beger et.al 2001).

Thus far, only one protein has been identified as a regulator of BRCA2 expression. This is NF-ќ B, which upregulates BRCA2 transcription by binding the BRCA promoter (Wu et.al 2000)

NF-ќ B, regulates expression of a number of genes withcritical role in apoptosis, tumorigenesis and inflammation. NF-ќ B stimulates cell cycle progression in estrogen receptor breast cancer.

Methylation of CpG islands also cause transcriptional silencing.

The role of ATM, ATR and Chk2 in phosphorylating BRCA1 in response of DNA damage.[The ATR kinase exists in a complex with the another protein (ATRIP).ATRIP is recruited to single-stranded DNA by the RPA protein complex (a component of the replication fork that binds to free single-stranded DNA). This localizes ATR at the single-stranded DNA, where it acts on Chk enzymes to trigger the damage response (Zou and Elledge, 2003).]

The C-terminus of BRCA2 is phosphorylated by hBUBR1, a mitotic check point gene (Futamura et.al 2000)

THE CLINICAL SIGNIFICANCE OF EPIDERMAL GROWTH FACTOR RECEPTOR (EGF-R) IN HUMAN BREAST CANCER.

Understand the role of the Epidermal Growth Factor.

EGF can stimulate the growth of normal mammary epithelium and human breast cancer cells in vitro . Receptors for EGF have been demonstrated on several breast cancer cell lines, especially on estrogen receptor (ER)-negative tumor cells, and in human primary tumors and metastases (4, 13, 14).

ER-positive rates ranging from 34-82%. In general, EGF-R positivity was observed in 29-91% (mean: 59%) of ER-negative and in 4-51% (mean: 29%) of ER-positive tumors reported by 21 different laboratories .

Relationship with other prognostic factors

Relationship with age or menopausal status

With respect to menopausal status, Sainsbury et al reported 45% and 30% EGF-R positivity in premenopausal and postmenopausal patients, respectively. Six other groups found no relationship between menopausal status and EGF-R levels (20,35, 39, 41, 44, 66).

Relationship with tumor size.

Nicholson (25) reported a significant positive correlation between EGF-R and increasing tumor size. In addition, Spitzer et al. (56) found higher EGF-R l

Based on 40 separate studies comprising 5232 patients, the mean percentage of EGF-R positivity reported in breast cancer is 45% (range 14-91%). (J. G. M. KLIJN,1992)

A few studies indicate a positive correlation between EGF-R and higher rates of breast cancer proliferation

CAUSES

1. Getting Older

Like most cancers, breast cancer becomes more common the older women are

2. A Previous Breast Cancer

Awoman who has had breast cancer has an increased risk of getting another in the other breast.

3 .Significant Family History

The National Institute for Clinical Excellence have issued guidelines in 2004 that set out the criteria for a family history that means she may have an increased risk of breast cancer .

A women may have an increased risk of breast cancer if she have ONE of the following in her family:

• Mother or sister diagnosed with breast cancer before the age of 40 .

2 Close relatives from the same side of the family diagnosed with breast cancer - at least one must be a mother, sister or daughter

3. Close relatives diagnosed with breast cancer at any age

4. Father or brother diagnosed with breast cancer at any age

5. Mother or sister with breast cancer in both breasts -  the first cancer diagnosed before the age of 50

6. One close relative with ovarian cancer and 1 with breast cancer, diagnosed at any age - at least one must be a mother, sister or daughter

4. Breast Cancer Genes

BRCA1 and BRCA2.  Your lifetime risk of getting breast cancer ifshe carry either of these breast cancer gene faults could be as much as 85 %. 5. Benign Breast Disease

If a women have a history of benign breast lumps then she have a slightly increased risk of breast cancer

6. Hormone Replacement Therapy

Cancer Research UK looked specifically at HRT and risk of breast cancer.  They have shown, for the first time, that combined HRT (oestrogen and progesterone) is more likely to cause breast cancer than oestrogen only HRT. 

7. Having Dense Breasts

This really relates to how your breast tissue shows up on a mammogram. Women with dense breast tissue have less fat and more breast cells and connective tissue in their breasts.  Because they have a greater proportion of breast cells, the risk of breast cancer is higher.

8. Alcohol

Alcohol intake has been linked to breast cancer.  A large study looked at about 80% of the worldwide research on alcohol.  They found there was an increase in risk of breast cancer with the amount of alcohol that women regularly drink.  With each additional daily measure - a glass of wine for example - there is an increase in risk of breast cancer of about 7%.

9. Body Weight

This is a bit confusing.  If a women is overweight before she had her menopause, her risk of breast cancer is lower than average.  But if she is overweight after menopause, her risk of breast cancer is higher than average.This is because women who are overweight ovulate less than average.  So their breast cells may be exposed to lower levels of oestrogen.  But once she is post menopausal,her oestrogen levels are linked to the amount of body fat she have.  The more fat, the higher your oestrogen levels are likely to be.

10. Being Tall

Taller women may have more breast tissue, which may mean a higher risk of breast cancer.

11. Lobular Carcinoma In Situ (LCIS)

LCIS is not cancer.  It means there are some changes to the cells in a women’s breast, but the cells are not yet cancer cells.  Having LCIS does increase the risk of getting breast cancer.  But, even so, most women with LCIS will not have cancer.

 12. Radiation And Breast Cancer

13. Past Treatment For Hodgkin's Lymphoma

If a women had radiotherapy to her chest for Hodgkin's lymphoma in the past, you could be at increased risk of getting breast cancer.  This applies to all those women treated as children or in their 20s. 

Features of Breast.

Each breast has 15 to 20 sections called lobes, with many smaller sections called lobules.

Within each lobe are many smaller lobules.

Lobules end in dozens of tiny bulbs that can produce milk.

The lobes, lobules, and bulbs are all linked by thin tubes called ducts.

These ducts lead to the nipple in the center of a dark area of skin called the areola.

Fat surrounds the lobules and ducts.

There are no muscles in the breast, but muscles lie under each breast and cover the ribs.

Types of Breast Cancer.

The most common type of breast cancer is the one that affects these tubes. called ductal cancer, it is found in the cells of the ducts.

Cancer that begins in the lobes or lobules is called lobular carcinoma. Lobular carcinoma is more often found in both breasts than are other types of breast cancer

The swelling of the breast is an uncommon type of breast cancer. This rare type of breast cancer is called inflammatory breast cancer which symptoms include an increase in the skin temperature, redness, and swelling of the breast. The skin may show signs of ridges and welts or it may also have a pitted appearance. This type of cancer tends to spread quickly.

Pictures of Types of Breast Cancer

Ductal Carcinoma in situ (DCIS)

Lobular Carcinoma in situ (LCIS)

Invasive Ductal Carcinoma (IDC)

Invasive Lobular Carcinoma (ILC)

Diagnosis.

1.Self Examination.

2.Ultra Sound guided Breast biopsy.

Si RNA induced silencing of defective BRCA genes.

My personal hypothesis.

2.Ultra Sound guided Breast biopsy

Ultrasound is an excellent way to evaluate breast abnormalities detected by mammography.

Ultrasound-guided breast biopsy is a highly accurate way to evaluate suspicious masses within the breast that are visible on ultrasound, whether or not they can be felt on breast self-examination or clinical examination.

The procedure prevents the need to remove tissue surgically, and also eliminates the radiation exposure that comes from using x-rays to locate a mass.

After placing an ultrasound probe over the site of the breast lump and using local anesthesia, the radiologist guides a biopsy needle directly into the mass.

Tissue specimens are then taken using either an automatic spring-loaded or vacuum assisted device (VAD).

Stages in Breast Cancer.

Tumor Size.

STAGE I - The cancer is no wider than 2 centimeters (about 1 inch) and has not spread outside the breast.

Stage II - The tumor is more than 2 cm but less than 5 cm in the greatest dimension.

Stage III - Tumor is more than 5 cm in the greatest dimension.

Stage IV- Tumor of any size with growth extending to the chest wall or skin.

Breast Cancer Treatment Methods

1. Surgery

2. Radiation therapy

3. Chemotherapy

4. Hormonal therapy

5. Biological therapy

Surgery

An operation to remove the cancer but not the breast is called breast-sparing surgery or breast-conserving surgery .

Lumpectomy and segmental mastectomy (also called partial mastectomy)are types of breast-sparing surgery. After breast-sparing surgery, most women receive radiation therapy to destroy cancer cells that remain in the area.

An operation to remove the breast (or as much of the breast as possible) is a mastectomy.Breast reconstruction is often an option at the same time as the mastectomy, or later on.

In lumpectomy, the surgeon removes the breast cancer and some normal tissue around it. Often, some of the lymph nodes under the arm are removed.

Radiation therapy

Side viewFront view Cross–sectional view

During radiation therapy, patients may become extremely tired, especially after several treatments.

Resting is important, but doctors usually advise their patients to try to stay reasonably active

Chemotherapy

chemotherapy affects normal as well as cancer cells .

In general, anticancer drugs affect rapidly dividing cells .

These include blood cells, which fight infection, help the blood to clot, and carry oxygen to all parts of the body.

When blood cells are affected, patients are more likely to get infections, may bruise or bleed easily, and may feel unusually weak and very tired.

Rapidly dividing cells in hair roots and cells that line the digestive tract may also be affected.

Some anticancer drugs can damage the ovaries. If the ovaries fail to produce hormones, the woman may have symptoms of menopause She may not be able to become pregnant

Hormonal Therapy

Tamoxifen is the most common hormonal treatment

This drug blocks the cancer cells' use of estrogen but does not stop estrogen production.

It can cause blood clots in the veins, especially in the legs and in the lungs. tamoxifen can cause cancer of the lining of the uterus

Tamoxifen Blocking Estrogen ReceptorsEstrogen Receptors

Biological Therapy

Is a treatment designed to enhance the body's natural defenses against cancer. For example

Herceptin® (trastuzumab) is a monoclonal antibody that targets breast cancer cells that have too much of a protein known as human epidermal growth factor receptor-2 (HER-2).

By blocking HER-2, Herceptin slows or stops the growth of these cells. Herceptin may be given by itself or along with chemotherapy.