By Louise Lazear

dm01.jpg (14678 bytes)For most women in our country, developing breast cancer is an ever-present fear compelling us to submit to the necessity for routine mammograms and annual clinical breast examinations. For others, about one in eight over the course of our lifetimes, the fear is a reality. In 2002, it is estimated that 203,5000 new cases of invasive breast cancer and 54,000 cases of in situ disease will be diagnosed. And despite great strides in treatment and early detection, close to 40,000 deaths from this disease are projected to occur. But as the molecular mechanisms of breast cancer development are unraveled, new treatments targeting these pathways hold promise for longer survival rates and the hope for a cure.

After melanoma, breast cancer is the most commonly diagnosed cancer in women, and is the second leading cause of cancer death after lung cancer. Males also are impacted by this disease, accounting for 1 percent of breast cancer deaths in this country. From 1973 to 1980, incidence rates experienced slight elevation from 82.6 to 85.5 per 100,000. Coincident with the acceptance of screening mammography, from 1990 to 1998, incidence rates increased from 110.4 to 118.1 per 100,000. Age-adjusted mortality rates from 1973 to 1990 remained almost constant, increasing only 1.5 percent over that period. Since 1991, mortality has decreased at the rate of about 2 percent per year, possibly due to widespread screening and earlier detection, improved treatment regimens and changes in demographics.

Incidence increases much faster with age than does mortality, due in part to the fact that rates of death from causes other than breast cancer increase sharply as women age. According to the NCI’s Surveillance, Epidemiology, and End Results (SEER) Cancer Statistics Review, the risk of being diagnosed with breast cancer increases from one out of 252 for women ages 30 to 40, to 1 out of 27 for women ages 60 to 70. While age is an important risk factor for breast cancer, other risk factors are thought to exist, including familial history of breast cancer, previous breast disease, early age at menarche, late age at first childbirth, estrogen therapy, and prior thoracic radiation, which especially for women under 30 can increase the risk at about 1 percent per year, beginning at 10 years post radiation. Behavioral factors, including hormone and alcohol intake, and certain socio-economic factors also have been linked to increased risk. According to SEER, racial/ethnic groups with the highest cancer risk are non-Hispanic whites, Hawaiian, and black women, followed by Hispanic and other Asian/Pacific island groups, with Korean and Vietnamese women demonstrating the lowest risk of developing breast cancer.

Hereditary risk factors continue to propel research to determine susceptibility genes for breast cancer. It has been found that about 5 to 10 percent of breast cancer cases show a pattern of autosomal dominant inheritance, and the syndromes most associated with this are ovarian and breast cancer related to mutations of BRCA1 and BRCA2, Li-Fraumeni syndrome related to mutations of p53, and Cowden syndrome due to mutations of PTEN. Other genetic disorders that may be associated with breast cancer include ataxia telangiectasia, Peutz-Jeghers syndrome and Fanconi amemia.

In 1994, scientists discovered that women who carried BRCA1 and BRCA2 had a higher risk of developing both ovarian and breast cancer than women without these mutations. Both BRCA1 and BRCA2 are tumor suppression genes that repair damage to DNA, and it is estimated that women with these mutations have a three to seven times higher risk of developing the disease. About 2.3 percent of women with Ashkenazi Jewish ancestry have been found to have alterations in either one or both of these genes, a frequency of close to five times higher than that of the general population. Mutations in BRCA2 also have been associated with increased risk of lymphoma, melanoma, and certain cancers of the digestive system.

Testing for BRCA1 and 2 is available, and can help assess a women’s risk for breast cancer development. In families with known mutations, a positive test for mutations indicates an increased risk. For women testing positive, several options for managing risk are available, including surveillance with mammography and clinical breast examinations, lifestyle changes, chemoprevention with tamoxifen and other drugs, and prophylactic mastectomy or oophorectomy. Recently, researchers have begun to fear that cancer risk based upon positive BRCA gene mutations has been overestimated, causing women to select more aggressive approaches to managing risk. Analysis of previous studies now seems to indicate that other genetic and environmental factors also may have contributed to the development of the disease, and that many studies do not take into account risk modifiers, such as diet and exercise, that can reverse predisposition.

Most breast cancer is diagnosed when women seek medical attention due to a mass discovered during self-examination or for other symptoms including soreness. Routine mammography has been shown to identify masses too small to detect upon physical examination, and numerous studies document the ability of this modality to detect early stage disease. Surprisingly, its sensitivity is dependent upon a number of factors, including lesion size and conspicuity, and the age and hormonal status of the patient. Sensitivity factors range from 54 to 58 percent in women under 40 years of age, to between 81 percent and 94 percent in women 65 and older.

The interpretive skills of the radiologist and experience levels also have been shown to influence sensitivity. In a study assessing the correlation of interpretations between radiologists in low, medium and high volume mammography settings, sensitivity ranged from 70.3 percent to 78.6 percent in low and high volume settings respectively, while specificity ranged from 83.6 to 88 percent for same respective settings.

Despite its limitation, mammography remains a sentinel force in the early detection of breast cancer. In an attempt to limit radiation dosage, improve techniques and provide better training for personnel, in 1992 Congress enacted the Mammography Quality Standards Act requiring FDA approval for all facilities performing mammography examinations. Since that time, image contrast also has improved with the use of specialized grids and higher film densities. The FDA also has approved several digital mammography systems, which utilizes detectors instead of film to generate computer images that are interpreted by radiologists. To date, digital mammography has been shown to be as sensitive as conventional techniques, and may reduce the number of repeats due to improper technique. Further research in improved digital techniques, telemedical interpretation at expert centers and computer-aided diagnosis may add to mammography’s ability to detect breast disease. Other modalities currently used are ultrasound, which can differentiate between cystic and solid lesions, and contrast-enhanced MRI, which can assist in the evaluation of silicone breast implants, and the assessment of lesions post surgical and/or radiotherapeutic intervention.

Patients with suspect imaging studies usually undergo incisional or excisional biopsy or surgical needle localization using radiographic guidance. Some clinicians opt to perform core needle biopsy using stereotactic x-ray or ultrasound, which in one study proved to result in fewer surgical interventions and a more defined surgical margin during the initial excision. Because scarring from these biopsy procedures presents problems for future mammography studies, MRI, sestamibi scanning and PET imaging may subsequently be used to differentiate scar from lesion. Currently, researchers are assessing several other techniques to obtain breast tissue cells for pathological examination, which include ductal lavage, fine needle aspiration and nipple aspiration, with the eventual goal of developing these techniques into independent screening tools.

Upon diagnosis, treatment for breast cancer involves a complex algorithm of surgical, radiation, hormonal and chemical intervention based upon many factors including the type and stage of the carcinoma. Survival rates correlate to the stage of the disease: five-year survival rates for Stage I are 98 percent, compared with 16 percent at Stage IV. At seven years post diagnosis, survival rates continue to decrease for each stage: 92 percent for Stage 1 and 11 percent for Stage IV.

Almost daily updates in the treatment against cancer help to improve the patient’s odds in defeating the disease. However, none of these techniques are targeted specifically to the tumor, and can cause collateral damage of healthy tissue. Perhaps the most exciting news today stems from the exponential growth in research of the molecular pathways leading to cancer development, and the search for therapeutic methods targeting these mechanisms. The discovery of the HER-2(human epithelial growth factor receptor2)/neu oncogene, which encodes for a glycoprotein functioning as a growth factor receptor, led to the development of a drug specifically targeted to the HER2 protein. Called Herceptin, this monoclonal antibody has been shown to be effective as a therapeutic agent in the treatment of metastatic breast cancer that is failing to respond to treatment with chemotherapy. While HER2 overexpression has been associated with poor prognosis, it also appears to be useful as a predictive factor in determining systemic regimens for breast cancer patients. Recent studies suggest that HER2 overexpression can result in increased sensitivity to a group of chemotherapeutic agents called anthracyclines, while indicating possible resistance to hormonal therapies. The use of both trastuzumab (Herceptin) and anthracyclines have been associated with cardiac toxicity, and researchers are now investigating the use of cardiac markers, including troponins and natriuretic peptides to identify patients at risk for myocardial damage caused by treatment with these agents.

The assessment of HER2 status in breast cancer patients is typically performed with immunohistochemistry (IHC) to assess protein overexpression and fluorescence in situ hybridization (FISH) to determine HER2 gene amplification. Current studies comparing the two methods indicate an overall concordance rate of about 80 to 95 percent. Advantages and disadvantages are inherent in both techniques: IHC is relatively inexpensive and can be performed with a light microscope, but the technique lacks a standard threshold for positivity. Although technically more difficult and requiring a fluorescence microscope for interpretation, FISH has standardized methods for positivity and produces quantitative results. Consequently, clinicians and pathologists are divided as to which technique is more appropriate for routine testing.

Currently, several products are available for HER2 testing, including the PathVysion HER-2 DNA Probe Kit from Vysis, Inc. a division of Abbott Laboratories. This kit uses FISH methodology to detect amplification of the HER-2/neu gene. “There are several reasons why we decided to focus on FISH to detect amplification of this gene. The first is that this method is an in situ technology, which means that the tissue architecture is left more or less intact. One can see where the cells are in relation to the other cells, for example if they are part of duct or outside of a duct, which is the type of information that pathologists are used to seeing. With quantitative PCR, isolation of nucleic acid necessitates the destruction of this tissue architecture. The other very powerful feature of the in situ methodology is that one can view the genetics of a small select group of cells to determine amplification. PCR amplification requires considerably more cells to obtain a measurement. The third element is that we don’t perform amplification with the FISH assay. Our results are directly read from the slide, and are not prone to errors related to the amplification process,” explains Steven Seelig, M.D. Ph.D. and vice president of research and development at Vysis. “There are several in situ technologies currently available, including immunohistochemistry methods that detect proteins, and RNA detection strategies which are not used very often due to the fact that RNA is not a very robust target in the clinical setting. While immunohistochemistry is used more frequently, for HER2/neu the clinical data clearly suggests that FISH is a better marker than either of these methods,” he adds. Vysis also offers several ASRs that target genes thought to be related to breast cancer pathogenesis, including TOPIIA, Cyclin D1, and EGFR. According to Seeling, Vysis has additional FISH-based ASR and RVO products under development including a product for cytological examination of cells obtained from ductal lavage.

An increased understanding of the mechanism of breast cancer pathogenesis has led to a proliferation of potential new targets and new therapeutic agents, says Carolyn Sartor, M.D., associate professor in radiation oncology at University of North Carolina Chapel Hill. According to Sartor, there are a number of different molecular mechanisms that have both prognostic and therapeutic potential in managing breast cancer. “The first and best known is the epidermal growth receptor family, which includes EGFR, HER-2, and two lesser-known members, HER-3 and HER-4. For both EGFR and HER-2, there is a growing body of data that inappropriate activation of HER-2, which can occur with amplification of the gene and overexpression, indicates a worse prognosis overall, and a worse response to chemotherapy and more than likely radiotherapy. For HER-2 inhibitors like Herceptin, the focus from this point forward is to extend the use from the metastatic cancer setting to earlier stages of cancer, and perhaps someday to prevention, especially with some of the newer agents targeting this family,” says Sartor.

Other mechanisms are being studied for therapeutic potential, including factors that inhibit angiogenesis. According to Sartor, while there are several new agents in clinical trials, none of them are as far along as Herceptin, and none are targeted specifically for breast cancer. And while preliminary data suggest that these agents can affect tumor growth, scientists also are discovering that redundant pathways often compensate for the inhibited agent, ultimately producing new blood vessels. “There are many ways that tumors are able to circumvent normal regulatory mechanisms, and different tumors may do this in different ways. There is a blurring of distinction between these pathways in that they all interplay in tumor pathogenesis. Clearly, we need to understand more about the basic pathophysiology of tumor cells, how to combine different agents, and how to select tumors to respond to combinations of these agents,” explains Sartor.

While molecular targets hold promise for new therapies, scientists are developing new techniques including DNA microarrays to assist them in unraveling the complexities of cancer pathogenesis. It is likely that these new tools will eventually find their way to the clinical laboratory, where the dream of personalized medicine will someday become reality.

Louise Lazear is a freelance writer based in Charlotte, N.C.