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Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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5
Biologically Based Technologies

Advances in molecular biology are gradually revealing the biological processes underlying the individual predisposition to breast cancer, its early development, and its progression from benign to invasive to lethal. As our understanding of cancer biology grows, so does the potential to turn that knowledge into technologies that are not limited to screening, prognosis, monitoring, or even treatment, but which inform every aspect of patient care. The three areas of biologically based technologies discussed in this chapter—cancer biomarkers, molecular profiles, and molecular imaging—hold the promise of revolutionizing breast cancer detection and management.

Instead of competing with mammography, biologically based technologies for breast cancer detection are currently poised to serve as its adjuncts. Molecular biomarkers or profiles of breast cancer will need to be linked with imaging information to define tumor size and location. Among the most important recent insights into breast cancer biology is the recognition that cancer can arise through various sequences of events, and through the actions of many genes with small but additive effects.35 While some researchers are seeking these genes (or their products) one by one, investigating the most promising candidates as potential biomarkers for breast cancer, others are examining overall patterns of gene expression associated with breast cancer risk or prognosis. Whatever the method of discovery, however, the final result is likely to reflect a highly individual molecular profile, characterized by both tumor and patient heterogeneity.

A major goal of these efforts is the development of blood tests to detect specific types of cancer. It is important to recognize, however, that the value

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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of such a test depends on existing options. For example, the development of a test for ovarian cancer, even one that is not highly accurate, could save many lives because there is no existing technique to detect early stage ovarian cancer. In contrast, a comparable test for breast cancer would be unlikely to save lives unless it is more sensitive or specific than mammography and gave localization information or was paired with mammography.

Recent headlines to the contrary, it will be many years—if ever—before blood tests replace mammograms. The most obvious reason is that a blood test would measure biomarkers (usually proteins) that have been released from cancerous tissue into the general blood circulation, which means they are highly diluted in the midst of a multitude of other proteins, and are a long way from their source. A blood test would have to be able to measure trace quantities of any biomarkers and, at best, a blood test would indicate that cancer was present somewhere in the body, but not where—unless the biomarkers were found only in breast tissue, which puts yet another restriction on possible tests. For example, a problem could arise if it was so sensitive that only a few cancer cells would result in a positive test. The cancer could not be physically located with current imaging technology within the breast and thus true positives could not be distinguished from false positives.

No existing blood test—for breast or any other cancer—rivals mammography as a screening method. Mammography has an acceptable sensitivity, and despite its modest specificity, it locates the tumor for definitive biopsy. Furthermore, mammograms provide richer data than would be possible from a low-dimensional biochemical assay that measures only one or a few substances; improvements described in Chapter 3 have the potential to increase the information available from mammography.

Many different biologically based approaches to detecting breast cancer are in development, but they face many of the same challenges if they are to become truly useful for improving outcomes for breast cancer patients. Certain themes recur throughout this chapter in the discussions of the different types of biologically based cancer detection technologies:

  • Biological methods may prove to be advantageous for screening high-risk populations, but are not likely to replace mammography.

  • Nonimaging biological techniques must be linked to imaging methods that can localize the cancer.

  • Statistical methods necessary for definitive analysis of large genomic and proteomic data sets are not yet defined or standardized.

  • Assays to detect cancer must account for the variability that exists among tumor types and among patients in order to be effective for widespread use.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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  • New technologies should be developed in conjunction with experts in the current best practices for breast cancer detection and diagnosis.

  • Novel diagnostic approaches need to be validated in large-scale clinical studies.

CLUES TO BREAST CANCER: INDIVIDUAL BIOMARKERS

Broadly defined, a biomarker is an objectively measurable characteristic that can be evaluated as an indicator of normal biological processes, disease, or response to therapeutic intervention.15,65 The search for biomarkers of breast cancer should not be confused with the search for inherited, or germ-line, mutations that affect the likelihood of developing breast cancer. Although the discovery of such mutations is important to assess breast cancer risk and may, ultimately, lead to the identification of the causes of breast cancer, the presence of such mutations does not indicate or predict the presence of breast cancer in an individual.

Biomarkers are being sought—and some have been identified—across a wide spectrum of events in the development of breast cancer, as shown in Table 5-1. The clinical use of breast cancer biomarkers is currently limited largely to prognosis, predicting response to therapy, and monitoring patients with diagnosed malignancy, but biomarkers hold considerable potential for risk assessment, screening, diagnosis, and the identification of therapeutic targets.11,18,38,44,55,58,65 Fulfilling that potential will not be easy. There are considerable biological and technical challenges to both the discovery and development of assays to detect early events in cancer development.18,44,55

The search for cancer biomarkers is proceeding along parallel paths: the “hypothesis-driven” assessment of candidate genes or proteins and the “discovery-based” comparison of gene expression and proteomic profiles.55,58 The potential uses and limitations of bioassays based on individual biomarkers for breast cancer are reviewed in this chapter. Molecular profiles of breast cancer, as revealed by DNA microarrays and proteomic analysis, are also discussed later in this chapter.

Biomarker Assays May Complement Mammography

Research on cancer detection has long been inspired by the search for a single, specific biomarker: a molecule or compound produced at such high levels by newly malignant or premalignant cells that it could be detected in an easily obtained fluid or tissue sample. This ideal marker would appear in all patients with a specific type of cancer and be absent or below a definable threshold in individuals without the disease. Its concentration in the sampled

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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TABLE 5-1 Biomarkers of Events in the Development of Breast Cancer: Their Potential Uses and Limitations

Event

Potential Use for Biomarkers

Progress to Date

Key Limitations

Germ-line mutations27

Risk indicator

Several mutations identified; genetic testing available for BRCA1 and BRCA2

Account for only 10 percent of breast cancers

Genetic polymorphisms27,44

Risk indicator

Some candidate polymorphisms identified; thousands of single nucleotide polymorphisms (SNPs) have been mapped

Validation difficult due to genetic diversity among different ethnic populations and the need to measure cumulative effects of multiple SNPs

Somatic genetic alterations27

Risk indicator; screening; diagnosis; prognosis

Loss of heterozygosity (LOH) at several loci associated with premalignant disease, as well as early and late-stage breast cancer

Unknown which, if any, LOH events are specific to invasive or metastatic cancer

Epigenetic changes (e.g., methylation) in breast cells 14,57,67

Risk indicator; screening; diagnosis; prognosis; therapeutic target

Research correlating methylation patterns at key loci with breast cancer presence and stage

Validation will require large-scale longitudinal studies and comprehensive cancer registry data

Altered gene expression in breast cells18,25,36,67

Screening; diagnosis; prognosis; choosing therapy; monitoring outcome

Studies under way on several overexpressed and underexpressed genes in breast tumor tissue; estrogen receptor status predicts response to antiestrogen therapy

Validation will require large-scale longitudinal studies and comprehensive cancer registry data

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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Changes in protein signaling pathways in breast cells36

Screening; diagnosis; prognosis; choosing therapy; monitoring outcome

Clinical trials underway in breast cancer patients before, during, and after therapy

Population heterogeneity reduces sensitivity and complicates standardization; sampling involves microdissection

Changes in individual serum markers18

Screening; diagnosis; prognosis; monitoring outcome

Preliminary findings indicate prognostic benefits of monitoring a mucin, CA 15-3, which has received FDA approval for the detection of recurrent breast cancer

Typically lack sensitivity for early malignancy and organ specificity; not elevated in all patients

Changes in serum protein/peptide profile36

Secondary screening; diagnosis; prognosis; choosing therapy; monitoring outcome

Research under way to improve ovarian cancer diagnosis

Low sensitivity and specificity; population heterogeneity

Angiogenesis5,22

Risk indicator, prognosis, choosing therapy

Research on several angiogenesis-related receptors being conducted to develop a possible treatment

Validation will require large-scale longitudinal studies. Main focus is currently on developing therapeutics

Invasion and metastasis18,25,36,67

Prognosis

Candidate proteases and inhibitors have been identified; prognostic benefit of urokinase plasminogen activator for node-negative breast cancer confirmed in large prospective randomized trial

Lack of effective therapy for metastatic breast cancer

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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BOX 5-1
CA 15-3

The CA 15-3 protein is a member of the family of proteins known as mucins, whose normal function is cell protection and lubrication. It plays a role in reducing cell adhesion and is found throughout the body. Elevated levels in breast cancer tissue may be involved in metastasis. CA 15-3 levels can also be elevated in patients with other cancers (lung, colorectal, ovarian, pancreatic) or because of hepatitis or cirrhosis of the liver.

fluid would increase or decrease as the cancer progresses or regresses and could be determined by a simple, reliable, inexpensive assay.

Individual biomarkers currently in clinical and experimental use fall far short of this ideal, however. (See Table 5-1 for a summary of potential issues and limitations of biomarkers for specific events in the development of breast cancer.) Most are synthesized by normal as well as malignant tissues and are only rarely elevated in premalignant or early stage disease. For example, in cancerous breast tissue high levels of the protein CA 15-3 are produced, but usually not until the cancer has reached an advanced stage (Box 5-1).18 Few of the biomarkers in use today are found among all patients with a particular type of cancer, and with the exception of prostate specific antigen, none are organ-specific.18

Absent an ideal biomarker, it is likely that any biomarker-based assay used as a primary screen for breast cancer in normal-risk populations will produce significant numbers of false positives. However, biomarker-based screening may prove to be a practical means of screening women at high risk for breast cancer for premalignant disease and/or occult cancer. Such an assay could detect clusters of proliferating cells at a preclinical stage, as well as cell clusters that may never require treatment. With the discovery of additional or better markers, bioassays may eventually be developed that not only detect the presence of breast cancer or precancer, but also predict clinical course.

As with mammographic screens, the performance of a biomarker assay should increase as additional time points are taken, particularly if the marker(s) reflect disease burden. This is true of existing biomarker assays for prostate, ovarian, and colon cancer. Therefore, although it may be unreasonable to expect that a single assay measurement can replace mammographic screening, multiple measurements taken over time that show a consistent rise in value could be indicative of an enlarging mass. This type of algorithm is likely to be the first implementation for biomarkers in breast cancer screening.

Biomarker assays could also be used to aid the decision-making process

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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for biopsy following a suspicious mammogram, if the bioassay reduced the number of false positives (increased specificity) without sacrificing sensitivity. Reliance on such an assay in cases of questionable mammographic results (for example, BI-RADS® 3-4) would be prudent only if the positive predictive value of the assay is extremely high, particularly when the result is to forgo biopsy. This is especially critical in the United States, where biopsy is the current standard of care for virtually every suspicious lesion. Bioassays may also be performed on the sample of suspicious cells obtained at biopsy in order to inform treatment decisions, but such tests will not supplant pathological examination of the biopsied tissue sample for the primary diagnosis.

As long as biomarkers continue to have low sensitivity and specificity, their use in primary diagnosis will be limited, and histological examination of biopsied tissue will remain the gold standard. But even then, biomarkers are likely to be useful as adjunct to other procedures, including:

  • Differential diagnosis or prognosis, such as distinguishing among types of ductal carcinoma in situ;

  • Assistance in the choice of therapy and evaluation of its outcome; or

  • Monitoring patients with ongoing disease before or after therapy.

Results of preliminary studies suggest that pre-operative serum levels of CA 15-3 are as good, if not better, predictors of patient outcome than traditional measures such as tumor size and nodal status.18 Tissue levels of estrogen and progesterone receptors and the erbB2 receptor, as determined by immunohistochemical analysis, are considered in the selection of therapy.18,42 CA 15-3, approved in 1997 by the Food and Drug Administration (FDA) for the detection of recurrent breast cancer, may also prove useful in monitoring response to therapy for metastatic breast cancer.18

Roadblocks to Biomarker Discovery and Development

The path to biomarker-based assays for breast cancer, and particularly for the early detection of the disease, is far from smooth. The considerable challenge of identifying highly sensitive and specific screens that rival the effectiveness of mammography is made more difficult by biological heterogeneity among humans, as well as among cancers, and even among different cell populations within a single tumor.55 A successful bioassay for breast cancer will need to overcome variability associated with cancers of different histologic types, expression patterns within histologic types, additional (noncancerous) patient conditions, and intrinsic human biochemistry. For now, as noted by Kenneth Pritzker, “our conceptual framework of

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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cancer biology remains inadequate to recognize the ideal or optimal biomarker for most cancers.”55

Guiding principles for the validation of promising biomarkers have been developed by the National Cancer Institute’s (NCI’s) Early Detection Research Network (Box 5-2). Shown in Table 5-2, these principles define a process for selecting biomarkers with sufficient positive predictive value that they can be used for population screening.44 Navigating this process will require extensive and unprecedented collaboration among industry,

BOX 5-2
Early Detection Research Network

The NCI’s Early Detection Research Network (EDRN) was founded in 2000 to facilitate biomarker discovery and validation through the collaboration among government, academia, and industry. The EDRN was established to set standards for the development and evaluation of biomarkers and guide the process of biomarker discovery in an effort to produce a useful population-screening tool.

The goals of the EDRN include:

  • Development and testing of promising biomarkers or technologies

  • Evaluation of promising, analytically proven biomarkers or technologies

  • Collaboration among academic and industrial leaders in molecular biology, molecular genetics, clinical oncology, computer science, public health, and clinical application for early cancer detection

  • Collaboration and rapid dissemination of information among awardees

The research network consists of three components:

  • Biomarker Discovery Laboratories are responsible for the development and characterization of new biomarkers or the refinement of existing biomarkers. There are currently 18 facilities involved in this research.

  • Biomarker Validation Laboratories serve as a network resource for clinical and laboratory validation of biomarkers, which includes technological development, quality control, refinement, and high throughput. The EDRN includes three validation facilities.

  • Clinical Epidemiological Centers conduct clinical and epidemiological research regarding the clinical application of biomarkers. There are nine facilities responsible for this research.

A fourth component, the Data Management and Coordinating Center located at the Fred Hutchinson Cancer Research Center, is responsible for coordinating the EDRN research activities in order to develop a common database for network research.

For more information see: http://www3.cancer.gov/prevention/cbrg/edrn/.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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TABLE 5-2 Guiding Principles Used in Biomarker Validation44

Phase

Results

Phase 1: Preclinical exploratory

Promising directions identified

Phase 2: Clinical assay and validation

Clinical assay detects established disease

Phase 3: Retrospective longitudinal

Biomarker detects preclinical disease and a “screen positive” rule defined

Phase 4: Prospective screening

Extent and characteristics of disease detected by the test and the false referral rate are identified

Phase 5: Cancer control

Impact of screening on reducing burden of disease on population is quantified

NOTE: The phases of research are ordered according to the strength of evidence that each phase provides in favor of the biomarker, from the weakest to the strongest. In general the results of earlier phases are necessary to design later phases. In some cases, where discovery of the biomarker establishes the method of detection, such as surface-enhanced laser desorption, then Phase I is skipped.

academia, and government, each of which controls resources essential to the development of clinically significant biomarkers.44,66 New legislation may be needed to provide incentives for cooperation between the pharmaceutical industry, which has identified hundreds to thousands of potential biomarkers for early cancer detection, and medical schools and research institutes possessing tissue banks, cell lines, and other reagents necessary to test these candidates.44 Increased effort is being made to sample tissues with precancerous and early stage disease due to their crucial role in testing biomarkers for cancer screening; such specimens are currently underrepresented in tissue banks.42

Once a promising biomarker is identified, researchers must address the technical challenges of developing a viable assay. The procurement, handling, and storage of fluid or tissue sample warrants careful consideration, because minor differences in these procedures may introduce systematic but unknown biases. However, it will be difficult to specify precise parameters for handling samples until the effects of inconsistencies on a given bioassay can be determined.

Further progress toward biomarker-based screening will require large-scale, longitudinal studies to evaluate the ability of a given screen to reduce cancer deaths and/or increase survival. Existing cancer registry data are woefully inadequate for this purpose, but more extensive information gathering may be hampered by the Health Insurance Portability and Account-

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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ability Act (HIPAA) and other legislation to protect patient confidentiality. (For more about HIPAA, see Chapter 6.) There is also a need to define statistical and inferential criteria for the evaluation of biomarker candidates for cancer screening, so that their efficacy can be measured against competing technologies.65 This comparison will ultimately hinge on the ability of the candidate technologies to reduce cancer mortality through the early detection of treatable disease.

If serum markers are shown to have promise in breast cancer studies such as these, the concept that a blood test can actually reduce cancer mortality should ultimately be evaluated in a randomized trial with cancer mortality as the endpoint, as was required in the pioneering studies of radiologic screening using mammography. Such a study would require resources similar to the mammography trials, namely tens of thousands of participants and lengthy follow-up. Methodologic issues in evaluating breast cancer screening tests are further discussed in Chapter 6.

PROFILES OF BREAST CANCER: GENOMICS AND PROTEOMICS

Until the early 1990s, the search for cancer biomarkers proceeded through the one-by-one investigation of candidate genes and proteins. The advent of high-throughput techniques capable of screening thousands of genes and, more recently, proteins, has made possible broad comparisons of cancerous and normal cells, revealing new biomarker candidates and introducing the possibility that patterns of gene expression or protein profiles could themselves serve as cancer biomarkers.51,58,69 DNA microarrays, consisting of thousands of DNA oligonucleotides (short sequences of DNA) or cDNAs (complete gene sequences of DNA reverse-transcribed from RNA templates) spotted in fixed locations are used to screen samples via hybridization (joining of two complementary strands of nucleic acid). Various applications of this technique can identify cancer-related changes in gene activity and reveal qualitative and quantitative variations in genomic DNA that occur during tumor formation.

Additional high-throughput methods focus on cancer-induced changes in protein pathways and populations, both within the tumor cell and at the tumor-host interface.37,51 These techniques scan the proteome—the protein equivalent of the genome—of affected cells and tissues for cancer biomarkers. Protein microarrays, which are an analogous technology to DNA microarrays, enable researchers to screen many proteins simultaneously for function and amplification.69 Serum proteomic profiling, the analysis of disease-related changes in proteins circulating in the blood, reveals patterns that may ultimately be used to detect cancer, identify therapeutic targets, and monitor response to therapy.51

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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Expression Profiles of Breast Cancer

The disruption of cell growth and survival pathways that lead to cancer occurs through multiple, cumulative genetic and epigenetic changes that in turn alter gene expression.2 While all breast tumors reflect changes common to malignant tissue, such as disordered cell cycle control, apoptosis (programmed cell death), adhesion and motility, and angiogenesis (new blood vessel formation), each tumor presents a unique pattern of gene expression (reviewed by Chung and colleagues in 2002).13 By classifying tumors according to their expression profiles, as revealed by microarrays, researchers hope to create a taxonomy that will improve prognosis and better predict each patient’s response to available therapies.13,28

Expression microarrays can analyze gene expression levels in a single sample or compare the expression of thousands of genes between two different cell types or tissue samples, such as malignant and normal breast tissues. Although the technology is still in its infancy, expression-based classifications for many types of tumors, including breast cancers, have already been developed through microarray analysis.1,13,81 For example, researchers identified five distinct subtypes of breast tumors derived largely from patients with infiltrating ductal carcinoma.49,63 This approach to detecting tumor classes based on a priori similarities in expression signature is known as “unsupervised” analysis.

A contrasting approach, “supervised analysis,” directly examines the relationship between gene expression profiles and a clinically determined variable, such as breast cancer prognosis. Van’t Veer and colleagues determined expression patterns of 98 primary breast tumors from lymph node-negative patients less than 55 years of age using oligonucleotide microarrays of 25,000 genes.74 Based on the clinical outcome of these patients, the researchers identified a set of 70 genes with expression patterns that closely predicted patient prognosis. A poor prognosis was associated with increased expression of genes associated with cell cycle control, invasion, angiogenesis, and signal transduction. A subsequent study tested the 70-gene prognosis profile in microarrays from 295 patients under age 53 with primary breast cancers with and without lymph node involvement. In this group of patients, the prognosis profile outperformed other standard criteria—including age, tumor size and histology, and the involvement of axillary lymph nodes—in predicting outcome.73 The next step should be to conduct studies in larger and more representative groups of breast cancer patients to determine whether these encouraging initial results prove to be reliable in clinical practice.30 However, despite the lack of evidence from true prospective clinical trials, versions of this test (Oncotype DX) are already on the market in the United States and The Netherlands. The test became available in the United States in early 2004 without having been approved by the

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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FDA because the device was marketed as a laboratory service rather than a diagnostic kit.24

Although expression microarray analysis is likely to identify clinically significant diagnostic and prognostic markers for breast cancer, the technology may not be well suited to the clinic.13 Microarray analysis as currently performed is too expensive for routine clinical use, and RNA is difficult to recover from biopsy specimens, which are normally fixed in formalin and embedded in paraffin for histological analysis and preservation.33,82 However, once subsets of clinically significant genes are identified via expression microarray analysis, other faster and less expensive techniques could be used in diagnostic tests. For example, researchers at Johns Hopkins University used expression microarray analysis to identify a large number of genes that were overexpressed in breast cancers.67 This group was further narrowed to a group of 35 potential tumor suppressor genes, selected on the basis of hypermethylation, and then to a panel of 3 genes that were highly and specifically correlated with early stage breast cancer. The markers are measured, via methlyation-specific polymerase chain reaction (PCR), in breast cells obtained by ductal lavage (described in Appendix A). Large clinical trials are currently under way to test the ability of this panel of markers to detect early stage breast cancer in asymptomatic women, as well as in women who are at high risk for primary and recurrent breast cancer.17,21

Cancer Clues Throughout the Genome

Mutations in DNA repair genes that normally protect the body against cancer-related mutations often result in chromosome loss, breakage, and gene amplification.2 The number and location of these “hits” to the genome strongly influence a person’s risk for developing cancer and, if malignancy occurs, its relative aggressiveness. A technique called microarray comparative genomic hybridization (CGH), which detects and maps cancer-related changes in DNA copy number, is therefore being pursued for clinical use in cancer prognosis. Microarray CGH is performed by hybridizing large pieces of sample genomic DNA to arrays spotted with DNA from a spectrum of known chromosomal locations. Differences in hybridization between, for example, normal ductal epithelial cells and biopsied breast tumor cells indicate key regions of chromosomal damage associated with the development of individual tumors. Unlike RNA-based expression analysis, DNA-based microarray CGH can be performed on formalin-fixed tissue such as archival biopsy material and therefore is more adaptable to routine pathology practice.33

In breast tumors, microarray CGH frequently reveals the loss of whole or partial chromosome arms, gene amplification, and erosion of the ends of

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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chromosomes, characteristics that may provide prognostic and/or diagnostic markers for breast cancer.2 For example, amplification of the erbB2 gene, as detected by microarray CGH, strongly correlates with increased gene expression. ErbB2 protein levels, which have been shown to predict breast cancer response to Herceptin therapy, are currently measured by immunohistochemical analysis of biopsied tumor tissue, but erbB2 copy number may prove a simpler and more sensitive indicator of Herceptin susceptibility.2,4,48 The further identification of dysfunctional genes in breast tumors by microarray CGH may enable the design of therapies targeting those genes, as well as tests to reveal genetic changes that make tumors resistant to certain therapies, such as defects in DNA repair genes associated with cisplatin resistance. (Herceptin and cisplatin are intravenously administered anticancer drugs.2)

Researchers have also used microarrays to search the genome for disease-associated patterns of single nucleotide polymorphismsa (SNPs) and for loss of heterozygosity (LOH)b (reviewed by Singletary, 2003).62 Both characteristics reflect somatic changes that are thought to occur early in cancer development, and thus hold potential for risk assessment or early detection. As with aberrations in DNA copy number, the analysis of SNP and LOH patterns in breast tumors may provide the basis for the prognostic and therapeutic classification schemes, as well as leads for the development of targeted therapies.

Signaling Circuits Gone Haywire

Cancer alters the signaling circuitry that governs cell growth and death by changing the expression level, post-translational processing, and functional modification (such as phosphorylation) of key proteins (Box 5-3).51,69 None of these parameters can be measured reliably, if at all, by expression profiling. Thus researchers have developed versions of protein microarrays that can simultaneously measure the concentration and phosphorylation state of thousands of individual proteins. The reverse-phase protein array consists of a nitrocellulose slide onto which tumor cell proteins—potentially from hundreds of patients—are applied in a range of dilutions, then probed with antibodies to phosphorylated forms of known signaling proteins.51 The specifically bound antibodies can be located and quantified by chemiluminescent, fluorescent, or colorimetric methods.

a  

An SNP is defined as a variation of a specific nucleotide that is present in over 1 percent of the population.

b  

LOH results from a mutation resulting in the absence or loss of one of the normal two forms of a gene from one of a chromosome pair for tumor DNA as compared to nontumor DNA in the same subject.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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BOX 5-3
How Genetic Mutations Can Disrupt Control of Cellular Functions

There are many other ways that cell functions are altered, but these are especially pervasive and underlie the regulations of processes such as cell division, cell movements, and cell death—each of which are critical elements in cancer development and progression.

Alterations in gene copy number, or changes in the number of copies of individual genes, are key genetic events in the development and progression of human cancers. At least 12 percent of all the variation in gene expression among the breast tumors is directly attributable to underlying variation in gene copy number.54 Widespread DNA copy number alteration can lead directly to global deregulation of gene expression, which may contribute to the development or progression of cancer.

Post-translational processing refers to the chemical modification of a protein after it has been synthesized, or translated. These modifications impart specialized functions upon the resultant proteins. Examples include the addition of glycosyl groups (glycosylation) or acetyl groups (acetylation) to a protein.

Phosphorylation is a post-translation modification that refers to the attachment of a phosphoryl group (P04) onto a protein. Many proteins remain inactive until they are phosphorylated, thus phosphorylation is a critical element in the control of cellular functions.

A comparison of the phosphorylation states of signaling proteins involved in growth factor and apoptosis pathways in breast epithelial cells from a total of 150 patients with either normal, premalignant, or cancerous results indicated that patient survival was correlated with the phosphorylation of two key signaling proteins.36 However, the overall signaling protein profile determined for each patient was sufficiently unique to resist comparison, except possibly with subsequent profiles of the same patient, over time or following therapy.

The promise of protein microarrays lies in their potential to identify the specific, highly individual cancer-related changes in each patient’s signaling circuitry—perhaps at an earlier stage than is currently feasible—and on the basis of that information, select the best possible treatment. It may also be possible to identify and target each of several different altered signaling proteins in a single tumor. The additive effect of this “combinatorial” treatment might require smaller amounts of each drug used, thereby reducing the toxic effects of therapy.51 A drug aimed at a single molecular target to inhibit cell proliferation rather than, as is often the case currently, unselectively destruction of cells will likely also prove less toxic to the normal cells of the body. However, because the vast majority of signaling proteins remain to be identified, and their role in cell growth and death

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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characterized, the future utility of protein microarrays in cancer detection and treatment depends on an expanding knowledge of the molecular basis of cancer. Current protein microarrays can display less than 10 percent of the total cell proteome;33 however, as key diagnostic and prognostic protein markers are identified and validated, they could be incorporated into smaller, more selective arrays. In addition, the inherent instability of proteins and the difficulty of producing consistent, reproducible results in protein-binding assays present significant technical roadblocks to the development of protein microarrays for clinical use.51

Serum Signatures of Cancer

Proteins, protein fragments, and metabolites from every tissue that blood flows though, accumulate in the serum. Some of these molecules, if derived from a tumor or its host organ, could serve as markers for malignant transformation or tumor-host interactions.51 Serum proteomic profiling, a high-throughput technique, reveals patterns comprising many individual proteins, the identities of which remain unknown (described in Appendix A). As with gene expression profiles, proteomic patterns can be analyzed using unsupervised methods to reveal groups of related proteins that can form the basis of taxonomic categories, or using supervised methods that relate protein patterns to a clinically determined variable, such as survival. Often these analyses are performed by artificial intelligence systems capable of handling vast amounts of data: A typical proteomic profile can include more than 15,000 data points.

Serum proteomic profiling could be used to identify novel biomarker candidates for characterization by conventional methods, but the great promise of the technology lies in the possibility of using a discriminating pattern within a patient’s profile to diagnose cancer and monitor the results of treatment.36,50 If this were possible, a profile might be obtained following a suspicious mammogram to inform a decision to biopsy, or in the event of a positive biopsy, a proteomic profile might be used to inform therapeutic choices. With even more exacting validation, serum proteomic profiling could be used to screen high-risk patients for early signs of cancer. Serum profiles of 50 ovarian cancer patients and 50 unaffected women have already been used to develop a discriminating pattern, formed by a subset of small proteins and peptides in the serum, that could discriminate ovarian cancer from noncancer.50 Follow-up results of this study incorporating 250 patients determined the sensitivity (rate of true negatives) and specificity (rate of true positives) were both 99 percent for stage I ovarian cancer.36

Although these results are encouraging for the early detection of ovarian cancer, for which no screen exists, they are far from competitive with mammography, particularly given the much higher population prevalence

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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of breast cancer as compared with ovarian cancer. However, to explore the possibility of serum proteomic profiling as a supplement to biopsy, an initial study was performed on serum samples from 317 patients who received breast biopsies. A training set, consisting of sera from 43 patients with benign lesions and 58 with breast cancer, was used to identify a discriminating pattern, which, when blind-tested on the remaining 216 samples, detected breast cancer with 90 percent sensitivity and benign lesions with 71 percent specificity.36 These results suggest the feasibility of using this methodology as a supplement to biopsy, as well as the need for significant improvement in the accuracy of proteomic diagnosis before it could be substituted for the actual results of biopsy. However, a serum proteomic test will only reveal certain biological characteristics of a tumor; other characteristics such as the size, shape, and location of the tumor remain unknown when using only a serum test. Therefore, in using this technique, lesions will most likely still have to be imaged by modalities such as mammogram, ultrasound, and magnetic resonance imaging (MRI), for effective treatment.

Barriers to Clinical Use of Molecular Profiles

In addition to previously described technical challenges to the clinical adaptation of DNA and protein microarrays and of serum proteomic profiling, these high-throughput, biologically based technologies face several barriers to development for the detection, diagnosis, or monitoring of cancer. Two largely unmet requirements stand out: to validate a strong and reliable link between profile characteristics and clinical outcomes and to create reliable, cost-effective profiling methods that can be performed in the clinical setting.52

The accurate analysis and correct interpretation of data from high-throughput experiments, key factors in establishing the clinical significance of molecular profiles, are far from ensured. Many sources of noise can obscure the results of these experiments. Results generated by DNA microarrays, for example, may be influenced by methods of sample storage, preparation, and labeling; by spot location on the array; or by imperfections in the array itself. These problems were clearly illustrated in a recent study in which samples from the same tissue, analyzed with different DNA microarray technologies (cDNA versus oligonucleotide), produced different gene expression profiles.32 In the case of serum proteomic profiling, where the identity of the specific proteins is unknown, minor differences in specimen procurement and subsequent handling may introduce systematic but undetectable biases into profiles.

Thus it is perhaps not surprising that statisticians have warned of significant potential for error in the analysis of voluminous genomic and

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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proteomic data.52 Even experts in the field disagree on the merits of the various statistical methods employed to bring molecular profiles into focus.26,70,76 Some argue that there is no “right” way to analyze molecular profiles, only the best choice based on the characteristics of the data and the scientific questions being asked.70,76 Thus, the more detailed our understanding of cancer biology is, the better informed such choices will be.

In the meantime, work is under way to resolve and refine statistical methods applied to microarray data, including gatherings of experts and standard-setting efforts by data repositories and professional journals. The annual meeting of the Critical Assessment of Microarray Data Analysis, first held in 2000, features direct competition among analytical methods. To support public use and dissemination of gene expression data, the National Center for Biotechnology Information, an arm of the National Library of Medicine, is building an expression data repository and online resource for data storage and retrieval. To facilitate comparisons of gene expression data from different sources, submissions to the repository must meet standards for data representation, experimental controls, and analysis. Although initial publications of DNA microarray results featured little if any statistical analysis, major journals, most notably Nature, recently have begun to impose publication standards for such data.3

The resolution of technical, interpretive, and statistical issues will help move molecular profiles to the clinic, but not before these technologies have been shown to reduce cancer deaths or increase survival in large-scale clinical trials. This process is likely to be hindered by the same constraints (lack of cancer registry data, restricted information gathering under HIPAA) as the validation of individual biomarkers, but perhaps even more to the need to demonstrate the utility of complex patterns without knowledge of their underlying biology, and to reliably reproduce profiles on a large scale.52 There are several methods of proteomic profiling and, for now, there are controversies about which methods are best. Indeed, many findings of very high accuracy are not confirmed by subsequent studies using other methods.

“This may be one of the greatest challenges this field currently faces,” according to researcher Emmanuel Petricoin.52 “Scientific consensus and standards are needed to develop, to evaluate, and to accept new statistical models for establishing the significance of linking gene and protein pattern analyses to more conventional diagnostic endpoints or outcomes.” It may be necessary, he concludes, to agree on different degrees of validation, depending on an individual product’s intended purpose, its stage of development, and the role of profile data in the evaluation of the product’s performance.

Finally, all molecular profiles have an important limitation: Although they may detect signals indicating the presence of cancer or its precursors

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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somewhere in the body, they cannot locate the source of those signals. Thus a molecular profile, like any individual cancer biomarker, must be paired with an imaging technique such as mammography, histochemistry, or—in the future—molecular imaging, a biologically based technology described in the next section of this chapter.

PICTURES OF BREAST CANCER: MOLECULAR IMAGING

Along with the promise of bioassays and therapies directed at the molecular roots of breast cancer comes a need to locate incipient disease, determine its extent, and monitor response to therapy, all at the molecular level.68 Molecular imaging, the in vivo measurement, characterization, and quantification of biological processes at the cellular and subcellular level, completes the picture of molecular medicine sketched in this chapter.41,68,77 The ability to “see” the molecular signatures of breast cancer is critical to fulfilling biologically based technologies’ promise of earlier detection and better disease management.

Today, the vast majority of breast cancers are detected with mammography and other imaging methods that measure nonspecific physical, physiological, anatomic, or metabolic phenomena such as electron density, acoustic interfaces, or temperature.41,53 While conventional images can sometimes differentiate pathological from normal tissue, molecular images can identify specific events—such as altered gene expression or changes in the proteome—that cause disease. In the future, molecular images will also play a key role in monitoring therapeutic response to biologically or molecularly based cancer treatment. Targeted molecular therapies are likely to inhibit cell proliferation rather than kill tumor cells, so their impact cannot be judged by radiological measures of tumor size.9 Gene therapy will necessitate tracking the transgene’s location, level of expression, and duration of effect.41 To gauge the success of anti-angiogenesis drugs, clinicians will need to measure changes in the number or viability of the blood vessels that feed tumors.68

Molecular imaging could one day be used throughout the cancer care pathway, to detect early stage alterations in gene expression, to guide therapeutic choices, and to evaluate and adjust treatment protocols.53 Ultimately, researchers envision molecular image-guided therapy systems to treat cancer as soon as it is found.68 However, the development of molecular imaging for breast cancer faces many of the same hurdles described for biomarkers and molecular profiles, particularly the need for deeper knowledge of cancer biology and the ability to evaluate new technologies in large-scale epidemiological trials. Most of the technologies described in this chapter are currently being tested in animal models. Despite their distance from

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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clinical use, their promise is immense: to revolutionize cancer medicine at every stage of patient care.

Functional Images Reveal Biological Processes

Molecular imaging is a high-resolution form of functional imaging, which reveals physiological, cellular, or molecular processes in living tissue.41,77 Existing functional imaging technologies depict cancer-associated physiological processes—such as glucose metabolism, oxygen consumption, and blood flow—in vivo and in real time. A few such technologies have been clinically approved for cancer imaging, including the detection of radiolabeled fluorodeoxyglucose by positron emission tomography, which provides a somewhat nonspecific, but interpretable, indicator of tumor metabolism. Similarly, optical imaging of near-infrared absorption by hemoglobin appears to correlate with malignancy.45 This technique, called diffuse optical tomography, is currently being tested in clinical trials.

In scintimammography, tracers such as 99mTc-Sestamibi are injected intravenously and then visualized using gamma camera/single-emission photon emisison computed tomography (SPECT) imaging of the breasts. Although it has not been evaluated as a viable screen for breast cancer, this technology has been found to detect the expression of a key multidrug resistance gene in tumors. Therefore scintimammography could potentially be used for prognosis (because multidrug resistance is an indicator of poor prognosis) and to guide therapeutic choices.53

Ultrasound can be used to assess blood flow in tumors, but in its present form, cannot reliably distinguish benign from malignant lesions.20 However, targeted ultrasonic agents are being developed to provide high-contrast images for specific cell surface receptors.34,41 In this guise, ultrasound would become a true molecular imaging technology—a technology cultivated much as other molecular imaging methods to be described, through parallel advances in probe and imaging design.

Molecular Probes Amplify Biological Signals

The biological processes targeted by molecular imaging are also key to cancer therapy: signal transduction, cell cycle regulation, multidrug resistance, angiogenesis, apoptosis, and telomerase expression (Table 5-3).

To visualize these processes in action, molecular probes may need to penetrate vascular, tissue, and cell membrane barriers, and they must be biocompatible.9 The smallest possible probes, at low concentration, stand the greatest chance of success against these odds. However, with target molecules at extremely low (picomolar) concentrations or even lower, molecular imaging probes must also bind specifically; thus for some applica-

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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TABLE 5-3 Molecular Imaging in Breast Cancer Targets and Agents53

Target

Imaging Agent

Imaging Modality

Current Status

Glucose transporter-1 (Glut 1)

Fluorodeoxyglucose (FDG)

PET

Clinically approved

Hexokinase 1

FDG

PET

Clinically approved

Multidrug resistance 1 P-glycoprotein (MDR1 Pgp)

99mTechnetium

SPECT

Clinically verified

Estrogen receptors (ERs)

Fluoroestradiol

PET

Clinically verified

Vasoactive intestinal peptide receptors

Labeled peptides

SPECT/PET

Preclinical/ early clinical

Met tyrosine kinase

HGF/SF60

Blood oxygenation level dependent MRI

Preclinical/ early clinical

Sigma-2 receptors

Iodobenzamide

SPECT/PET

Preclinical/ early clinical

Na+/I symporter (NIS)

131/125Iodine

SPECT

Preclinical/ early clinical

Mucin-1 glycoprotein (MUC1)

Pre-targeting antibody fragments

PET

Preclinical/ early clinical

Cell proliferation activities

Fluorothymidine (FLT)

PET

Preclinical/ early clinical

Cathepsin D

Cy-CDF-PGC72c

NIR Optical

Preclinical/ early clinical

MMP2

C-PGC79d

NIR Optical

Preclinical/ early clinical

cCy5.5 Cathepsin D Sensitive Peptide Protected Graft Copolymer.

dCy5.5 Poly L-Lysine Methoxypolyethylene Glycol.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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tions, larger molecules with high affinities for their targets, such as antibodies or recombinant proteins, make superior probes.9,41,53

Low target concentrations also mean that molecular affinity probes must produce a strong signal, a challenge that has been met by attaching the target-binding affinity component to a signaling component.41 The signal may be produced by a radioisotope, as detected by PET or another tomographic method; by a paramagnetic atom, as revealed by molecular resonance imaging; or by a fluorochrome, as visualized by imaging. (See Appendix A for description of these technologies.) Some nonradiolabeled probes send a signal only after being biochemically “turned on” by enzymatic activity that occurs upon target binding.41,78 Such activatable imaging probes reduce background noise caused by nonspecific probe binding.

Molecular probes have already revealed a variety of cancer-related processes—so far, mostly in experimental animals—in unprecedented detail. These include events and features occurring in the extracellular milieu, such as the activity of cathepsins B and H in breast and other cancers; at the cell surface, such as tumor receptors, multidrug resistance transporters, and membrane phospholipids associated with apoptosis; within the cell, such as DNA replication; and even oncogene activity within the nucleus.7,8,10,16,39,41,61,75,83

Imaging Technologies Bring Probes into Focus

In addition to probes that bind specifically to their targets and produce clear signals, noninvasive molecular imaging techniques are being developed that can distinguish between probe signals and non-specific “noise” from other biological activity within the body. Molecular imaging technologies include radiological methods, such as positron emission tomography (PET) and single-emission photon emission computed tomography (SPECT); optical imaging approaches for fluorescent or bioluminescent probes; and MRI and magnetic resonance spectroscopy (MRS) (see Table 5-4).

Radiological Imaging

PET-based molecular imaging could eventually be used to diagnose a variety of molecular or genetic diseases, to predict a patient’s response to a particular molecular therapy, and, if a molecular therapy is chosen, to determine whether it reaches its target and is effective.12 Researchers have used PET with molecular probes to track gene expression in living animals; in addition to taking the technology one step closer to monitoring gene therapy in humans, mouse models such as these could be used to assess new drugs.12,41 Like PET, SPECT, a similar technology that visualizes a different

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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TABLE 5-4 Characteristics of Various Molecular Imaging Modalities in Animal Studies

Imaging Technique

Portion of EM Spectrum Used

Spatial Resolution*

Tissue Penetration Depth

Type of Probe

Amount of Probe Used

Cost Rankinge

Positron emission tomography (PET)

High-energy gamma rays

1-2 mm

No limit

Radiolabeled

Nanograms

$$$$

Single photon emission computed tomography (SPECT)

Low-energy gamma rays

1-2 mm

No limit

Radiolabeled

Nanograms

$$$

Optical bioluminescence imaging

Visible light

3-5 mmf

1-2 cm

Activatable

Micrograms to milligrams

$$

Optical fluorescence

Visible light or near-infrared

2-3 mmg

<1 cmh

Activatable

Micrograms to milligrams

$-$$

Magnetic resonance imaging (MRI)

Radiowaves

25-100 µm

No limit

Activatable

Micrograms to milligrams

$$$$

Computed tomography (CT)

X-rays

50-200 µm

No limit

Under study

Not applicable

$$

Sonography

High-frequency sound waves

50-500 µm

Millimeters to centimeters

Limited activatable

Micrograms to milligrams

$$

*These values are for microPET and microSPECT which are used in animal studies. Most PET results in humans have spatial resolution of about 6-8 mm, with newer models being 5-6 mm. SPECT resolution for humans is about 10 mm.

eIncludes cost of equipment and cost per study.

fResolution is tissue depth dependent.

gFluorescence tomography may have better resolution.

hFluorescence tomography can likely image at greater depths (2-6 cm).

SOURCE: Adapted from Massoud and Gambhir, 2003.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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range of isotopes, is currently used in functional imaging.42 PET is more adaptable to molecular imaging, however, because the positron-emitting isotopes it employs are more easily incorporated into probes than the gamma-emitting isotopes visualized by SPECT.41

“It is clear that the first gene imaging to obtain FDA approval will be with PET, because the imaging probes are used in such low amounts that they will not produce pharmacologic or physiologic effects,” according to Michael Phelps, chair of molecular and medical pharmacology at the University of California at Lost Angeles.9 PET imaging probes are also relatively easy to construct, because drugs or existing molecules known to interact with a specific target can be modified with a radiolabel with minimal perturbation.41 But even if a PET-based molecular imaging technology gains clinical approval in the near future, it is not likely to be widely adopted without the introduction of less-expensive PET scanners with better resolution and sensitivity.12

Another development likely to boost the clinical value of molecular imaging with PET is the advent of “multimodality” imaging systems combining PET scans—which do not clearly reveal the anatomy of regions of probe uptake—with high-resolution x-ray computed tomography (CT) imaging.12 PET/CT scanners are already in clinical use for functional imaging.12,41,71 Work is also under way to develop combined PET and MRI; however, although MRI provides better soft-tissue contrast than CT, it will be more technically challenging to integrate with PET. Researchers are exploring additional combinations of optical, radiological, MRI, and CT techniques capable of producing truly multimodal images.6,29,41

MRI

Functional MRI was introduced as an imaging technique in the 1970s, but was not widely used to detect breast cancer until the late 1990s.19 Although orders of magnitude less sensitive than PET or optical techniques, molecular resonance has attracted the attention of molecular imaging researchers because of its higher spatial resolution and simultaneous depiction of molecular and anatomical information.9,41 Antibody- and protein-based MRI probes have been used to visualize cell-surface molecules including cancer antigens and a protein associated with apoptosis.23,31,41,59,83 Novel cancer therapies containing gadolinium, a paramagnetic species commonly used in magnetic resonance applications, could be tracked with MRI to image tumors and monitor their uptake of the labeled drugs over the course of treatment.19 Activatable MRI agents for visualizing intracellular processes are possible, but only if the large target-binding molecules used in current probes can be replaced by smaller ones or made penetrable to cell membranes.9 This constraint, for example,

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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challenges work in progress on an MRI-based reporter system employing the intracellular enzyme beta-galactosidase.53

Optical Imaging

Optical imaging will be widely adopted because its capabilities exceed those of other imaging technologies, according to Ralph Weissleder, director of the Center for Molecular Imaging Research at Massachusetts General Hospital.9 By identifying cancer-related alterations in gene expression, optical imaging will permit early diagnosis, “perhaps before morphological or clinical signs of disease can be seen,” he says. Yet, as abnormalities are detected earlier, confirming the presence of cancer for a definitive diagnosis will become more difficult. Optical technology presents the possibility of using multiple probes, each with a distinct spectrum, to monitor several events or molecular species simultaneously.41,77 Promising optical techniques for molecular imaging feature targeted bioluminescent probes, near-infrared (IR) fluorochromes (including activatable probes), and red-shifted fluorescent proteins.41,80

Bioluminescent probes emit light that is essentially free of background, and are therefore attractive because they can be detected at very low concentrations.41 However, viable technology has yet to be developed for bioluminescent imaging in the human body, and this strategy would still require injecting mass levels of substrates, such as D-Luciferin, into the body.41 Fluorescent probes have higher background, but offer two advantages: they can be used as reporters in both live and fixed tissues, and they can often be visualized without the addition of a substrate.64

Fluorescent probes that emit in the near IR have maximal tissue penetration and minimal background fluorescence.41 An activatable near-IR probe has been used in vivo to monitor activity of cathepsin D, an extracellular protease that is overexpressed in many tumors.41,53,68 Fluorescence-mediated tomography, an approach that is still in its infancy, is being developed to penetrate further than is possible with existing near-IR methods.41,46,47 Multimodality probes that are capable of fluorescence and bioluminescence are also under active investigation.

Multidisciplinary Research Is Key to Bringing Molecular Imaging to the Clinic

A review article by Massoud and Gambhir (2003) identifies the following goals for molecular imaging, leading from the research laboratory to the clinic:

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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  • To develop noninvasive in vivo imaging methods that reflect specific cellular and molecular processes, such as gene expression and protein-protein interactions

  • To monitor multiple molecular events in concert

  • To follow trafficking and targeting of cells

  • To optimize drug and gene therapy

  • To image drug effects at the molecular and cellular level

  • To assess the molecular pathology of disease progression

Meeting these goals and translating that achievement into rapid, reproducible and quantitative clinical technologies will be a critical step toward the molecular management of cancer.

Many basic questions remain to be answered in the course of developing and refining molecular imaging technologies. Overcoming the theoretical and practical challenges of biocompatibility, barriers to probe delivery, and signal amplification will require continued research.40,41 Investigators are concentrating their efforts on selecting appropriate cellular and subcellular imaging targets, probing the development and delivery, amplification strategies for targets at nanomolar to picomolar concentrations, and the development of high-resolution, real-time imaging systems that can ultimately be used in humans.41,77,80

If the potential of molecular imaging is fulfilled, imaging will influence all aspects of cancer care, from diagnosis to treatment evaluation, and will play an increased role in the development of new molecular therapies. Researchers are already looking beyond the previously described imaging technologies to the design of molecular biosensors that can be injected into the bloodstream to find and destroy cancer cells. Advances such as these can only be achieved through collaborative, multidisciplinary research that brings together molecular and cellular biologists, imaging scientists, nanotechnologists, and cancer clinicians. Consequently, a key online resource, Molecular Imaging Central, has been created to provide links among the various areas of research in molecular imaging, background information on different types molecular imaging, as well as highlighting the latest research findings. Supporting agencies for such research include the NCI, which funds a variety of molecular imaging initiatives (see Box 5-4). Bridging these disparate fields is perhaps the greatest challenge to the development of molecular imaging, but one which, if met, could establish a new research paradigm for the advancement of molecular medicine.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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BOX 5-4
National Cancer Institute Support for Molecular Imaging Research43

The following NCI initiatives foster advances in functional and molecular imaging:

In Vivo Cellular and Molecular Imaging Centers: Bring together experts from diverse scientific and technological backgrounds to conduct multidisciplinary research on cellular and molecular imaging in cancer. Five Centers established as of 2002; support provided for 14 potential sites, including a site for researching functional imaging of low-activity genes.

Novel Imaging Technologies Program: Supports collaboration of academic scientists with industry and foreign institutes to create unique imaging technology, including the next generation of PET/CT scanner for improved localization and evaluation of difficult-to-locate cancers and therapeutic monitoring.

Clinical Imaging Drugs and Enhancers Program: Fosters the development of new imaging contrast agents and molecular probes to improve cancer diagnosis and treatment. Several agents or probes currently in development for measuring blood vessel formation and cell death, evaluating cell growth, and enhancing visualization of various cancers.

Molecular Imaging Database (MOLI): A publicly available imaging database intended to help researchers develop new imaging agents and to help clinicians find existing agents for imaging specific cancers. The database is expected to be released in mid-2004.

Clinical Trial Cooperative Groups: Networks of healthcare professionals affiliated with medical schools, teaching hospitals, and community-based cancer treatment centers who encourage movement of promising imaging advances from discovery and development to clinical use (e.g., American College of Radiology Imaging Network).

Cancer Therapy Evaluation Program: Exploring the use of imaging as a biomarker or surrogate marker for cancer, instead of biopsy, to monitor treatment effectiveness.

Mouse Models of Human Cancer Consortium: Includes researchers who are developing novel imaging modalities for use in preclinical studies.

Small Animal Imaging Resource Program: Resource to allow scientists from different disciplines to use small animal imaging technology, including molecular imaging.

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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NOVEL IN VITRO DIAGNOSTIC TESTS POSE UNIQUE REGULATORY CHALLENGES

Innovative in vitro diagnostics, such as tests for genetic susceptibility to various diseases, pose a new spectrum of regulatory challenges. These include overcoming both the inexperience of the FDA with such cutting-edge technology and the inexperience of the budding companies that are developing it, as well as narrowing down the complex genetic data being generated, and providing uniformity in analysis and testing.

The FDA is currently in a learning mode about genomics and proteomics. These methods use patterns in the activation of specific genes or production of specific proteins to help determine the diagnosis, prognosis, or risk of developing various diseases. New products from these endeavors have emerged in significant numbers only in the past decade.

In the past 3 years, the agency has had about 50 presentations about this technology from industry, academia, or the government. The FDA’s in vitro diagnostics office has an internal “Omics” working group that has met periodically over the last 3 years to discuss new developments in the field and to interact with their counterparts in the FDA’s Center for Drug Evaluation and Research, and the agency released a guidance document for pharmacogenetics and pharmacogenomics in November 2003.

Hundreds or thousands of results are generated by genomic and proteomic tests for each specimen as opposed to one result per sample in the conventional diagnostic tests that the FDA is used to seeing. Regulation of these array tests will be much easier if manufacturers of genomic or proteomic tests can reduce the amount of data required for a given diagnostic determination. Regulatory submissions for such diagnostics include imprecise measurements of every analyte and a lack of standardization in the analysis approach. These technologies are so new that clear standards have not yet emerged, which means that the FDA must not only conduct its usual evaluation for adherence to established methodological and analytic standards, but it must evaluate the validity of new methods as they are evolving.

SUMMARY

The biological revolution in breast cancer detection and management is under way, but it is likely to proceed slowly and by degrees. Significant progress has been made toward the identification of key breast cancer biomarkers, as well as aggregate profiles of breast cancer in the genome, transcriptome, and proteome; the theoretical promise of molecular imaging is beginning to be realized in animal models. When molecular medicine for breast cancer first enters the clinic, it will most likely come in the form of techniques to monitor therapeutic response and recurrence. The use of

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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molecular screening technologies, such as blood tests for routine screening of normal-risk, symptom-free women, likely lies in the more distant future. Measures of recurrence or response to therapy are intrinsically easier to develop than screening tests, because each woman can serve as her own reference point for changes that can be measured over days or months. In contrast, a screening test needs to provide interpretable results based on a single time point.

Even further in the future, researchers envision individualized management of each case of breast cancer, based on its specific molecular characteristics. “We’ll have a roadmap, a wiring diagram of the deranged cellular circuitry of each patient’s cancer, not just a named diagnosis, but a molecular profile,” according to Lance Liotta of the NCI. “Instead of choosing therapy by a category of disease, we’ll use combination therapy tailored to the individual molecular profile of the tissue, the tumor microenvironment, and the cancer. Instead of single targets and single therapeutic agents, we’ll have multiple targets all along the length of key signal transduction pathways, both intracellular and extracellular, at the tumor-host interface. And finally, instead of determining efficacy by waiting for a change in tumor size or recurrence, we’ll have direct monitoring of cellular targets before, during, and after therapy by biopsy—or ideally by molecular imaging or serum proteomics—to monitor changes that are going on in the tissue microenvironment following treatment.” However, the nonimaging biological techniques must be linked to additional procedures that can localize the cancer and examine its pathology. In addition, the problem of a test being too sensitive—detecting cancer before it could be physically located with current imaging technology—could be traumatic for patients. True positives would thus be indistinguishable from false positives and create a high level of anxiety among women with a positive test.

Fulfilling the potential of molecular medicine for breast and other cancers will require collaboration between molecular biologists and scientists from a broad spectrum of disciplines. It will fall to epidemiologists and biostatisticians to guide the rational design of biologically based cancer diagnostics, to establish their significance and reproducibility, and, in the case of clinical epidemiologists, to adapt them for routine clinical use.56 Ultimately, they will have to develop standardized statistical methods for analysis of large genomic and proteomic data sets. Once these new biologically based detection and diagnostic tools have been developed, they will need to be tested for safety and effectiveness beyond the research setting in multicenter clinical trials. Yet, a lack of regulatory standards for the validation of novel diagnostic tests may hinder clinical trials by making them more difficult to design and the results more challenging to interpret. Finally, these tools will not be used in isolation but will become part of an arsenal of tools—each with distinctive capacities and caveats. Developing

Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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evidence-based systems for integrating this new technology will require attention at all levels of the health care system—physicians, payers, purchasers, and patients.

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Suggested Citation:"5 Biologically-Based Technologies." Institute of Medicine and National Research Council. 2005. Saving Women's Lives: Strategies for Improving Breast Cancer Detection and Diagnosis. Washington, DC: The National Academies Press. doi: 10.17226/11016.
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The outlook for women with breast cancer has improved in recent years. Due to the combination of improved treatments and the benefits of mammography screening, breast cancer mortality has decreased steadily since 1989. Yet breast cancer remains a major problem, second only to lung cancer as a leading cause of death from cancer for women. To date, no means to prevent breast cancer has been discovered and experience has shown that treatments are most effective when a cancer is detected early, before it has spread to other tissues. These two facts suggest that the most effective way to continue reducing the death toll from breast cancer is improved early detection and diagnosis.

Building on the 2001 report Mammography and Beyond, this new book not only examines ways to improve implementation and use of new and current breast cancer detection technologies but also evaluates the need to develop tools that identify women who would benefit most from early detection screening. Saving Women’s Lives: Strategies for Improving Breast Cancer Detection and Diagnosis encourages more research that integrates the development, validation, and analysis of the types of technologies in clinical practice that promote improved risk identification techniques. In this way, methods and technologies that improve detection and diagnosis can be more effectively developed and implemented.

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