Medical Device Link.

Originally Published IVD Technology January/February 2003

Molecular Diagnostics

Differential proteomic analysis of nipple aspirate fluid is a sensitive approach to personalized diagnosis of breast cancer

Ira L. Goldknopf, Helen R. Park, and Henry M. Kuerer

Fluorescent two-dimensional gel electrophoresis images can be used to screen patients for breast cancer.

Despite the many breakthroughs in diagnosing and treating cancer, the life expectancy of breast cancer patients has not substantially improved for several generations. Breast cancer is the second-largest cancer-related cause of death for women in the United States, with an average of 40,000 deaths and 200,000 new cases per year. What is especially troubling about breast cancer is that once the disease has progressed to the lymph nodes, the survival rate decreases rapidly.

With increased concerns about the effectiveness of mammography as a screening tool for breast cancer, genetic testing using individual genes or proteins as markers, such as HER-2/neu, has improved screening for potential sensitivity to some treatments such as Herceptin.¹ However, a low percentage of breast cancer cases are found positive for such cancer-related genes, which is underscored by the fact that these genomic tests are the primary screening method in pre-menopausal patients.² Moreover, standard estrogen and progesterone receptor tests, which require a biopsy of the tumor, and other similar combinations of diagnostics have improved the predictability of breast cancer survival by only a small percentage.³

New tests with greater scope and improved sensitivity and specificity are needed for screening, prognosis, determining specific treatment protocols, and predicting complications to provide a higher quality of life for breast cancer patients. A noninvasive method of early risk stratification and diagnosis, and the ability to combine that method with treatment selection and monitoring from the same noninvasive procedure could benefit breast cancer patients.

Molecular diagnostic testing, especially proteomic testing, could provide the source of such new tests for breast cancer screening. For example, surface enhanced laser desorption and ionization time of flight (SELDI-TOF) mass spectrometry has recently been used for testing ovarian cancer and has sparked a considerable amount of interest.⁴ This article examines a comprehensive and noninvasive approach that involves a large number of proteins with high sensitivity and specificity and uses two-dimensional gel electrophoresis with image analysis.

Obtaining the Sample

Figure 1. The Criterion Dodecacell, a 12-gel second-dimension simultaneous electrophoresis apparatus of the ProteomeWorks system by Bio-Rad Laboratories (Hercules, CA).

Analyzing the biochemical and cellular contents of breast ductal fluid has gained notice as a potential noninvasive method for studying the development and progression of breast carcinoma.^5–7 In most breast cancer cases, the disease originates in the ductal or lobular epithelial cells of the breast, which secrete into the ducts.⁷ Only a small amount of the breast secretion, as little as 1–2 µl, is needed to study its protein concentration. This fluid is obtained through nipple aspiration by placing a handheld suction cup on the nipple to extract the fluid quickly and noninvasively.

A recent study demonstrated a promising new application for nipple aspiration. It involves obtaining ductal fluid samples from a patient's breast containing a known carcinoma and from the other healthy contralateral breast as an internal control.⁷ This retrospective preliminary study conducted at ProteEx Inc. (The Woodlands, TX) concluded that comparing the protein expression profiles of both samples from the same individual appears to be a practical method for identifying clinically relevant tumor markers and drug targets.⁷ The patterns of these differentially expressed proteins may become useful in screening, risk stratification, diagnosis, drug selection, treatment monitoring, and detection of cancer recurrence. This approach may also have additional value in discovering new diagnosable tumor biomarkers and drug targets for early detection and more-effective therapy.

Research has shown that cytological analysis of breast nipple aspirate fluid and fluid obtained by ductal lavage is effective as a predictor of cancer risk.^5,6 In fact, FDA and the Blue Cross Blue Shield Association (Chicago) have approved cytological tests using either ductal lavage or nipple aspiration.^5,6

Figure 2. A two-dimensional gel image analysis using PDQUEST software by Bio-Rad Laboratories (Hercules, CA). In this analysis, a seven-gel match set of images has been enlarged for comparison.

Proteomic testing may be the next step in ductal fluid analysis by providing more information of a broader nature. Investigators at the National Cancer Institute (Bethesda, MD) have generated proteomic spectra from serum samples in ovarian cancer patients and from nipple aspirate fluid samples using SELDI-TOF.^4,8 While these results have great promise, this technique is limited in that the final data output is restricted to protein molecular weight. However, the 2-D gel approach first separates the proteins on the basis of their isoelectric points and then by molecular weight. Many important changes that are observed with the 2-D gels of nipple aspirate fluid are differences resolved by the first dimension separation through isoelectric focusing, which would likely not be detected by SELDI-TOF.

Using Proteomic Analysis

Since the 1970s, 2-D gel electrophoresis has been considered a highly specialized, labor intensive, and non-reproducible method that has been restricted primarily to research purposes.⁹ However, with the advent of integrated supplies, robotics, and software combined with bioinformatics, using this proteomics technique in diagnostics has become feasible. Now, 2-D gel electrophoresis is well suited for detecting changes in protein expression and discriminating protein isoforms due to amino acid sequence variations and protein postsynthetic modifications such as phosphorylation, ubiquitination, conjugation with ubiquitin-like proteins, acetylation, and glycosylation.¹⁰ These factors are important variables in cell regulatory processes involved in cancer and other diseases.

Figure 3. Matched pair nipple aspirate protein comparisons in a unilateral breast cancer patient: normal breast aspirate (a) and cancerous breast aspirate (b). The enlarged images show where cancerous aspirate exhibits additional proteins (d) compared with the normal aspirate (c). (click to enlarge)

There are few comparable alternatives to 2-D gels for tracking these types of processes. The introduction of high sensitivity fluorescent staining, digital image processing, and computerized image analysis has greatly simplified the detection of unique species and quantification of proteins. For example, by utilizing known protein standards as landmarks within each gel run, computerized analysis can detect unique differences in protein expression and modifications between the breasts of an individual or among several individuals. The proteins of interest can then be excised from the gels, and the exact proteins can be identified by in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry–based peptide mass fingerprinting and database searching, or by liquid chromatography with tandem mass spectrometry (LCMS/ MS) partial sequencing of individual peptides. Studies using 2-D gels, MS, and MS/MS of breast cancer tissues have also been used to catalog and characterize many potential drug targets and biomarkers.¹¹

In addition, these studies could lead to the discovery and development of an integrated solution for breast cancer treatment, including early detection, prognosis, determination of appropriate drug treatments, and monitoring for treatment and recurrence of the disease. The preliminary 2-D gel electrophoresis studies characterized differences in protein patterns in cancerous and noncancerous breast nipple aspirates from the same patients.⁷ This ability to characterize these samples underscores the potential for developing differential protein pattern tests for biomarker-based risk stratification, early detection, diagnosis, treatment, and monitoring of diseases such as breast cancer.

The Breast Cancer Example

At the University of Texas M. D. Anderson Cancer Center (Houston), nipple aspirate protein samples were taken from a group of patients who had been diagnosed with unilateral primary invasive ductal breast carcinoma and also had an apparently normal contralateral breast. Corresponding samples were also taken from healthy volunteers whose mammograms were negative.

The protein samples were aspirated in liquid form from the nipples of each breast through a noninvasive suction device similar to a manual breast pump. The proteins were prepared and separated by 2-D polyacrylamide gel electrophoresis using a fully integrated proteomics platform, the ProteomeWorks system by Bio-Rad Laboratories (Hercules, CA; see Figure 1). The gels were then stained and subjected to fluorescent digital image analysis in which the protein patterns of each patient were compared using an image analysis software, PDQuest by Bio-Rad (see Figure 2).

Figure 4. Matched pair nipple aspirate protein comparisons in a volunteer with normal breasts: left-breast aspirate (a) and right-breast aspirate (b). Enlarged images of the left-side (c) and right-side (d) nipple aspirates. (click to enlarge)

In the samples from the breast cancer patients, qualitative differences were found between the protein expression patterns of the fluid from the cancerous breasts compared with the fluid from the noncancerous breasts (see Figure 3). For example, the number of protein spots detected in the protein patterns of the breasts with cancer not only differed from the breasts without cancer in the same patients, but also varied from patient to patient. In the cancer patients, proteins that were detected in one breast and not in the other varied in molecular weight (vertical position), charge (isoelectric point, horizontal position), and abundance (spot intensity). Some of these factors also differ from patient to patient. With this approach, the patient is acting as her own control, and the differences between the ductal fluid protein patterns of her two breasts are the important variables.

In contrast, the protein expression patterns of the two breasts in normal individuals are almost qualitatively identical (see Figure 4). While the protein pattern differences between the breast cancer patients and the healthy volunteers are evident on initial visual inspection, the image analysis software enables further detailed comparisons of quantitative and qualitative differential expression. Enlarging the images delineates the extent of the differences between the breasts of the cancer patients versus the remarkable similarities between the breasts of normal individuals. Among the patients studied, many of the differences involved proteins that are found in relatively low abundance and detected by the high-sensitivity fluorescent stain. The results of the comparisons between matched cancerous and noncancerous breasts from eight unilateral breast cancer patients have been summarized (see Table I).

Reproducibility and Sensitivity

Figure 5. Reproducibility of protein quantitation using 75 ng of bovine serum albumin on nine separate two-dimensional gels. (click to enlarge)

The reproducibility of the 2-D gel electrophoresis procedure has been an issue in the past. However, with the use of preformed first-dimension immobilized pH gradient strips and second-dimension gels, as well as the ability to run concurrently up to 12 2-D gels in the same apparatus, the past problems with reproducibility has been greatly diminished. This is particularly evident in the enlargements of the nipple aspirate gels from normal individuals in which the patterns from both breasts are mirror images (see Figure 4).

To assess reproducibility, 75 ng of bovine serum albumin (BSA) were run on nine separate 2D gels. The gels were stained with a highly sensitive and linear responsive stain, and the five spots in the BSA region of the gel were subjected to quantitative analysis with an image analysis software using the Gaussian peak value method. The electrophoretic patterns were reproducible, and the reproducibility of the quantitation was independent of spot amount over the range tested, with the percentage of coefficient of variation less than 20% (see Figures 5 and 6).

The 2-D gel electrophoresis analysis involves the use of highly sensitive staining techniques that can detect proteins in the picogram range. Subtle differences emerge, which may lead to the detection of breast cancer at very early stages and may be used in the future to augment breast cancer detection by other methods.

Figure 6. A plot showing the reproducibility of two-dimensional gel quantitation using 75 ng of bovine serum albumin. (click to enlarge)

In addition, though not yet proven, the sensitivity of this protein detection may indicate that with further proof, detecting precancerous conditions prior to mammograms may be possible, especially with protein information that can also be used for treatment decisions and monitoring. In fact, using enzyme-linked immunosorbent assays, researchers have demonstrated the presence of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor in nipple aspirates in small amounts.¹²

Conclusion

The 2-D gel electrophoresis analysis of matched-pair breast nipple aspirate fluid proteins could potentially be used as a tool for noninvasive early detection, prognosis, risk stratification, drug treatment, and monitoring of breast cancer. This method requires a complete clinical trial compared with the gold standards (i.e., mammograms, ultrasound, biopsy, nipple lavage and aspirate cytology, and serum markers).

Presently, 2-D gel electrophoresis has limitations. For example, performing the test takes time, as much as three days for sample preparation, staining, and image comparisons. Some of this labor intensiveness could be addressed with more robotics, automation, and miniaturization. And while the subjectivity of this technique has been largely removed with the use of software, using 2-D gels combined with MS to catalog the markers found in the fluids makes it feasible to transport the test to other platforms. Thus, with time and technological advances, this highly sensitive approach may become very useful, such as to help determine whether to perform a needle biopsy of a suspicious mass seen in a mammogram.

Among the differential expression patterns of ductal fluid proteins, some evidence of known and possibly new biomarkers and drug targets for breast cancer has been observed. The patient-to-patient variability of these differences may reflect variables in the disease structure and may prove to be of clinical diagnostic and therapeutic significance to individual patients. For example, the presence or absence of known biomarkers detected in the differences in the fluids can be used to determine the aggressiveness of the cancer (e.g., the presence or level of Cyclin E) or signal the appearance of a cancer-related genetic instability or hereditary component (e.g., the absence or level of BRCA1).¹³ The presence of known drug targets detected in the differences in the fluids may also be used in the future to indicate what drugs to use (e.g., HER-2/neu indicating sensitivity to Herceptin).

References

1. DF Hayes et al., "Circulating HER-2/erbB-2/c-neu (HER-2) Extracellular Domain as Prognostic Factor in Patients with Metastatic Breast Cancer: Cancer and Leukemia Group B Study 8662," Clinical Cancer Research 7, no. 9 (2001): 2601–2604.

2. J Bradbury, "Low Susceptibility Genes for Breast Cancer: The Way Forward?" Lancet Oncology 3, no. 1 (2002): 2.

3. A Molino et al., "Prognostic Significance of Estrogen Receptors in 405 Primary Breast Cancers: A Comparison of Immunohistochemical and Biochemical Methods," Breast Caner Research Treatment 45, no. 3 (1997): 241–249.

4. EF Petricoin et al., "Use of Proteomic Methods in Serum to Identify Ovarian Cancer," Lancet 359 (2002): 572–577.

5. W Doley et al., "Ductal Lavage for Detection of Cellular Atypia in Women at High Risk for Breast Cancer," Journal of the National Cancer Institute 93 (2001): 1624–1632.

6. MR Wrensch et al., "Breast Cancer Risk in Women with Abnormal Cytology in Nipple Aspirates of Breast Fluid," Journal of the National Cancer Institute 93 (2001): 1791–1798.

7. HM Kuerer et al., "Identification of Distinct Protein Expression Patterns in Bi-Lateral Matched Pair Ductal Fluid Specimens from Women with Unilateral Invasive Breast Cancer: A Unique Opportunity for High-Throughput Biomarker Discovery," Cancer 95 (2002): 2276–2282.

8. CP Paweletz et al., "Proteomic Patterns of Nipple Aspirate Fluids Obtained by SELDI-TOF; Potential for New Biomarkers to Aid in the Diagnosis of Breast Cancer," Disease Markers 17 (2001): 301–307.

9. IL Goldknopf et al., "Isolation and Characterization of Protein A24, a Histone-Like Non-Histone Chromosomal Protein," Journal of Biological Chemistry 250 (1975): 7182–7187.

10. IL Goldknopf and H Busch, "Modification of Nuclear Proteins: The Ubiquitin-Histone 2A Conjugate," Cell Nucleus 6 (1978): 149–179.

11. H Hondermarck et al., "Proteomics of Breast Cancer for Marker Discovery and Signal Pathway Profiling," Proteomics 1 (2001): 1216–1232.

12. Y Liu et al., "Breast Cancer Diagnosis with Nipple Fluid bFGF," Lancet 356, (2000): 567.

13. K Keyomarsi et al., "Cyclin E and Survival in Patients with Breast Cancer," New England Journal of Medicine 347 (2002): 1566–1575.

Ira L. Goldknopf, PhD, is chief scientific officer, and Helen R. Park is chief executive officer at ProteEx Inc. (The Woodlands, TX). The authors can be reached via irag@proteex.com and hpark@proteex.com, respectively. Henry M. Kuerer, MD, PhD, is assistant professor of surgical oncology at the University of Texas MD Anderson Cancer Center (Houston). He can be reached via hkuerer@mdanderson.org.

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