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Editorials |
1
Departments of Medicine and
2
Pathology University of Michigan 1301 Catherine St. Ann Arbor, MI 48109-0602
a Author for correspondence
Breast cancer is a leading cause of significant morbidity and mortality for women. Methods to reliably detect the earliest stages of breast cancer have been widely sought because the success of breast cancer treatment is influenced by how early a malignancy is diagnosed. Current methods for early detection rely on either physical examination to palpate a tumor or radiography. The development of genetic tests that enable accurate risk assessment for individuals in high-risk families has been predicted to have substantial medical benefits. Significant and unprecedented media fanfare informed both medical professionals and the general public about exciting new discoveries in breast cancer genetics, namely, identification of two critically important breast cancer genes BRCA1 and BRCA2, helping to create an immediate demand for clinical tests for these genes. The identification of genetic alterations underlying the development of breast cancer has ushered in an exciting and challenging new era in oncology, genetics, and molecular diagnostics.
DNA diagnostic tests for breast cancer genes, including BRCA1, BRCA2, and TP53 (which causes breast cancer in the context of the Li–Fraumeni cancer predisposition syndrome), are currently available in most cases only for individuals who have clear family histories of one of the hereditary breast cancer syndromes. Presymptomatic DNA-based assessment for breast cancer risk has had only limited use in clinical practice to date. In cases of autosomal dominant breast cancer (~5% of all breast cancers), identification of the specific BRCA disease-causing mutation has enabled more-accurate risk-modification counseling for first-degree relatives and facilitated susceptibility testing for family members. Presymptomatic identification of a germ-line BRCA mutation provides individuals with specific, if not yet well studied, options for preventive medical management. For example, initial studies indicate that women who have inherited a mutant BRCA1 allele from an affected mother have a markedly increased risk of developing breast cancer (~85% lifetime risk) or ovarian cancer (~50% lifetime risk) or both (1)(2)(3)(4)(5). These women may choose either increased medical surveillance to detect and treat early tumors or bilateral prophylactic mastectomies and (or) oophorectomies in an attempt to eradicate the tissues that have the greatest potential of becoming malignant. Both choices are associated with some morbidity, and neither choice, unfortunately, absolutely guarantees cancer-free survival.
We anticipate that the demand for testing among individuals with a family history of breast cancer will expand rapidly. A survey of female first-degree relatives of breast cancer patients found that ~95% of respondents desired presymptomatic genetic testing (6). In addition, specific tests to detect the more commonly observed mutations within a given population, e.g., the 185delAG mutation, which has been identified in 1% of Ashkenazi Jewish women (7)(8), are being offered to women without family histories by some testing centers (9). Despite voiced concerns by some members of the medical and scientific communities regarding widespread availability and clinical application of DNA testing for BRCA mutations in women without significant family histories (9), the demand for such testing is expected to increase. Both clinicians and the public hope that specific mutation testing may provide an accurate and rapid means of determining an individual's genetic risk for the development of cancer and ultimately aid in the prevention and therapy of the disease. Therefore, several groups are developing and analyzing various DNA diagnostic methods in an effort to deliver comprehensive, sensitive, cost-effective tests. Specific recommendations regarding the appropriate utilization of DNA testing for breast cancer have been published by groups within both the genetic and oncology communities but are still being debated (10)(11)(12)(13)(14).
Although controversy surrounds the widespread clinical application of DNA testing for breast cancer, before such testing is offered, it seems clear that healthcare providers, laboratory professionals, and individuals seeking testing should have a clear understanding about the basic concepts, strategies, clinical implications, and, importantly, limitations of this relatively new application of genetic testing for presymptomatic detection of mutations. This seems particularly important given current uncertainties regarding potential problems of presymptomatic testing, including possible discrimination against genetically susceptible individuals and uncertainties surrounding the prognosis and best medical management for identified individuals, especially those with no family history of breast or ovarian cancer who are found to carry germ-line BRCA1 or BRCA2 mutations. Although >100 BRCA1 mutations have been identified to date, the molecular mechanisms underlying breast carcinogenesis and the relationship of these mutations to other known and hypothesized environmental, genetic, and hormonal risk factors for breast cancer are poorly understood. Additionally, the role of these genes in sporadic cases of breast cancer has not yet been clearly defined. Mutations in BRCA1 are seen in a small percentage of young women with breast cancer who do not have strong family histories of breast or ovarian cancer (15). Conversely, the absence of a detectable germ-line mutation in a woman should not lead to the false assumption that she will not develop the disease.
The genes that appear to account for most cases of hereditary breast cancer in North America are BRCA1 and BRCA2. Of these, mutations in BRCA1 are the more frequently implicated (16)(17). Several approaches to determining genetic risk are available. In large families with several affected individuals, linkage studies can be performed to determine whether a cancer susceptibility trait is linked to one of the known BRCA genes. Although linkage can be used to implicate a gene as a cause of disease in a family, the possibility of erroneous results—as the result of several factors, including genetic recombination, nonpaternity, or the presence of more than one cancer susceptibility gene in a family (18)—makes direct mutation testing a more attractive approach. Moreover, in small families, the statistical power attainable with linkage calculations may be insufficient to demonstrate linkage to a disease-causing gene. Mutations in BRCA1 account for ~50% of hereditary breast cancers and up to 75% of hereditary breast and ovarian cancers (19)(20)—hence the considerable interest in detecting mutations in that gene. Unfortunately, detecting mutations in BRCA1 is not straightforward because of its large size. Of the numerous mutations that have been described, many occur in only single families. The most frequent mutations reported to date are a dinucleotide deletion (185delAG), which has been frequently observed in Ashkenazi Jewish families with breast cancer, and a single nucleotide insertion (5832insC). Each of these mutations occurs in <15% of the families studied. Thus, approaches to the detection of mutations in BRCA1 must differ considerably from those used to diagnose a single-mutation disease such as sickle cell anemia.
In this issue, Rohlfs et al. (21) describe PCR-based methods to detect five mutations in BRCA1. The approach used for four of these mutations involves the use of PCR primers that contain a nucleotide alteration to incorporate a restriction enzyme site into or remove one from the PCR products. Although the PCR/restriction digestion approach is straightforward and has been used to detect point mutations in other genes, several caveats must be considered with this approach. First, restriction enzymes recognize several base pairs, typically 4–8. A change in any of these can result in failure of the restriction enzyme to recognize the site. Thus, mutation detection strategies based on the absence of restriction endonuclease cutting of PCR products or of genomic DNA cannot be considered 100% specific for a particular mutation unless one is certain that polymorphisms or other mutations do not occur at the restriction recognition sequence. The technique is more specific for detection of a mutation when the restriction enzyme cuts the mutant allele and not the wild-type allele, because the nucleotides specific for the mutation must be present for the restriction sequence to be recognized. However, at least in theory, the increased specificity comes at the expense of decreased sensitivity: A polymorphism at any nucleotide within the restriction recognition sequence may prevent cutting by the endonuclease. Second, an allele containing sequence variations or deletions that prevent primer binding will not be amplified. Sequence analysis of a large number of normal and disease-causing alleles is needed to understand the full range of both mutations and polymorphisms in BRCA1. Finally, if a laboratory result is based on the absence of cutting by a restriction enzyme, an internal control that is always cut should be present in the assay, to ensure that the restriction enzyme activity was indeed present during this incubation.
The stepwise protocol proposed by Rohlfs et al. (21), which begins with testing for more common mutations, followed, if necessary, by screening for mutations by use of other techniques, is similar to the approach used in other genetic disorders. Because of the frequency distribution of mutations in BRCA1, initial testing for even the more-common mutations will yield negative results in samples from most of the patients with BRCA1 mutations in the general population. In certain ethnic groups, a higher detection rate for specific mutations can be expected (7)(8). The fact that most BRCA1 mutations result in premature chain termination means that in vitro protein truncation assays (22) may detect the great majority of mutations. At present, however, these assays are complex to perform and are not readily amenable to automation. Some workers have used single-strand conformation polymorphism (SSCP) analysis successfully to screen for mutations in BRCA1 (15). Another technique being adopted by several laboratories is large-scale sequencing of BRCA1, which provides a more comprehensive mutation screen; with advances in the speed of DNA sequencing technology, this may be the optimal approach to the detection of mutations in numerous genes. Other techniques, e.g., microchip-based mutation detection technologies (23)(24), will almost certainly be applied to BRCA1 mutation testing. The relative efficiencies and costs of various mutation detection strategies are critically dependent on the technologies used, and comparison of available methods is a subject of much practical importance.
Even a comprehensive mutation detection scheme, however, will not eliminate difficulties of interpretation. Although mutations that result in nonsense mutations (which introduce a premature stop codon) or frame shifts can be predicted to adversely affect protein function, there may be considerable uncertainty regarding missense mutations (which change the coding from one amino acid to another). In the case of proteins such as the gene product of BRCA1, the function of which is as yet unknown, no tests at present exist to study the effect of a missense mutation in vitro. The demonstration in a family of a BRCA1 missense mutation that is not observed in the general population is consistent with, but not absolute proof of, a disease-causing mutation.
The specific type of DNA test offered to any individual depends on several factors, including an accurate clinical assessment of the most likely breast cancer gene involved, based on a detailed review of a comprehensive, well-documented family history; the individual's ethnic background; availability of affected family members' DNA samples for study if needed; an understanding of an individual's motivation for and expectations of testing; the proven cost-effectiveness and limitations of the ever-changing technology; and, importantly, knowledge about the types and locations of mutations and benign polymorphisms in the gene. DNA testing can be expected to play an increasingly important role in screening for cancer susceptibility and, potentially, in guiding and monitoring therapy for a variety of malignancies, including breast cancer. Future progress in DNA diagnostics and further elucidation of molecular mechanisms underlying breast cancer should provide powerful new tools for predicting genetic risk and for clinical management of breast cancer, which may ultimately lead to a shift in medical practice towards disease prevention. It is extremely important in the early stages of DNA diagnostics in cancer that the new tools be carefully and cautiously applied in the care of individual patients, especially in light of the many unanswered questions regarding the molecular basis of breast cancer.
References
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