Atul K. Tandon

HER-2/neu oncogene protein and prognosis in breast cancer.

Amplification of the HER-2/neu oncogene was recently reported to predict poor clinical outcome in node-positive breast cancer patients. Since expression of the oncogene as its protein product might be even more closely related than gene amplification to disease progression, we have now examined levels of the HER-2/neu oncogene protein for its prognostic potential in both node-positive and node-negative breast cancer. Using Western blot analysis, levels of this protein were determined in 728 primary human breast tumor specimens. We examined relationships between this protein and other established markers of prognosis, as well as clinical outcome. In node-negative patients (n = 378), the HER-2/neu protein failed to predict disease outcome. However, in node-positive patients (n = 350), those patients with higher HER-2/neu protein had statistically shorter disease-free (P = .0014) and overall survival (P less than .0001) than patients with lower levels of the protein. Higher HER-2/neu protein was found in tumors without estrogen receptor (ER) (P = .02) or progesterone receptor (PgR) (P = .0003), and in patients with more than three positive lymph nodes (P = .04). A significant correlation between levels of the HER-2/neu gene protein and amplification of the gene itself was also found (n = 48, P less than .001). Multivariate analyses in these patients showed that the HER-2/neu protein is a significant independent predictor of both the disease-free and the overall survival in node-positive breast cancer, even when other prognostic factors are considered.

Cathepsin D and Prognosis in Breast Cancer

We investigated the possibility that cathepsin D, an estrogen-induced lysosomal protease, might have value as a prognostic factor in breast cancer by studying frozen tissue specimens from 397 patients. We measured the 34-kd mature form of the enzyme by Western blot assay and densitometry. Among 199 patients with node-negative disease, but not among 198 with node-positive disease, high levels of cathepsin D proved to be a significant predictor of reduced disease-free survival (median follow-up, 64 months), either as a continuous variable (log cathepsin D; P = 0.018) or as a dichotomous variable with an optimized cutoff point (P = 0.0001). Results were similar for overall survival (P = 0.009 and 0.0001, respectively). Relating the level of cathepsin D to other prognostic factors in the patients with node-negative disease, we found an association with aneuploidy but none with estrogen or progesterone receptors, tumor size, or the age of the patient. In multivariate analyses, a high level of cathepsin D was the most important independent factor in predicting shorter disease-free and overall survival in patients with node-negative disease. As compared with the risk in women with low levels of cathepsin D, the relative risk of tumor recurrence was 2.6 (95 percent confidence interval, 1.6 to 4.4) and the relative risk of death was 3.9 (95 percent confidence interval, 2.1 to 7.3) among those with high levels of cathepsin D. For disease-free survival, cathepsin D status was predictive of outcome primarily among those with aneuploid tumors; the actuarial five-year recurrence rates of aneuploid tumors were 60 percent among women with high levels of cathepsin D and 29 percent among those with low levels, as compared with 22 percent for all diploid tumors. We conclude that cathepsin D may be an independent predictor of early recurrence and death in node-negative breast cancer.

Electrophoretic analysis of 248 clinical breast cancer specimens for P-glycoprotein overexpression or gene amplification.

Multiple drug resistance (MDR), consisting of acquired cross resistance to anthracyclines, vinca alkyloids, and other antineoplastic antibiotics, has been described in a variety of cell lines. This MDR phenotype is associated with overexpression and sometimes amplification of a gene coding for a 170 kDa glycoprotein, termed P-glycoprotein. To understand the role of this mechanism in clinical breast cancer, 248 breast cancer specimens representing both untreated primary and refractory relapsing disease were probed for evidence of P-glycoprotein gene amplification or overexpression using Southern, Northern, or Western blot techniques. In no case was an increase in P-glycoprotein gene copy number or expression detected. Though these findings do not necessarily rule out a role for P-glycoprotein in mediating drug resistance in breast cancer, electrophoretic analysis of clinical specimens is unlikely to provide useful predictive information. More sensitive assays must be developed to overcome the difficulties inherent in analyzing heterogenous tissue samples.

Glutathione transferase GSTπ in breast tumors evaluated by three techniques

The glutathione transferases are involved in intracellular detoxification reactions. One of these, GST pi, is elevated in some breast cancer cells, particularly cells selected for resistance to anticancer agents. We evaluated GST pi expression in 60 human breast tumors by three techniques, immunohistochemistry. Northern hybridization, and Western blot analysis. There was a significant positive correlation between the three methods, with complete concordance seen in 64% of the tumors. There was strong, inverse relationship between GST pi expression and steroid receptor status with all of the techniques utilized. In addition, there was a trend toward higher GST pi expression in poorly differentiated tumors, but no correlation was found between tumor GST pi content and DNA ploidy or %S-phase. GST pi expression was also detected in adjacent benign breast tissue as well as infiltrating lymphocytes; this expression may contribute to GST pi measurements using either Northern hybridization or Western blot analysis. These results suggest that immunohistochemistry is the method of choice for measuring GST pi in breast tumors.

Progesterone receptor assays in low-protein cytosols: a modified charcoal-gelatin procedure.

Quantitative measurement of steroid receptors including progesterone receptor (PgR) is usually accomplished by the dextran-coated charcoal (DCC) assay. At protein concentrations below about 1 mg/ml, however, serious underestimation of receptor content by DCC occurs, presumably because of adsorption of receptor to the charcoal and possibly to assay tubes, etc. We have therefore developed a modified charcoal-gelatin (MCG) procedure which largely avoids receptor losses even in samples with extremely low protein concentrations. In this MCG procedure, 0.1% gelatin is added to both sample and charcoal suspension, the charcoal content is increased to 1%, and dextran is no longer necessary. Comparison of the MCG procedure with the standard DCC and several other methods at decreasing protein concentrations shows that MCG retains acceptable efficiency for PgR at much lower protein than the others, even as low as 10 micrograms/ml. This MCG procedure will be useful in determining receptors for prognosis in very small human breast cancer biopsies, as shown here, but also for receptor determination in very small tissues such as specific brain regions, and for receptor assay during purification.

HER-2/neu Oncogene Amplification and Expression in Human Mammary Carcinoma

Publisher Summary This chapter provides an overview of HER-2/neu oncogene amplification and expression in human mammary carcinoma. HER-2/neu is a proto-oncogene highly homologous to the epidermal growth factor-receptor (EGFR) gene that resides on human chromosome 17. It codes for a 185-kDa membrane protein with intracellular tyrosine-kinase activity, and is thought to function as a growth factor receptor. Amplification and overexpression of the HER-2/neu oncogene is present in up to 30% of invasive human breast cancers. The incidence of expression is nearly twice as high in pure in situ carcinomas of the breast, suggesting that HER-2/neu plays an important role in the early stages of tumor development. The observation that expression dramatically declines, as tumors progress from noninvasive to pure invasive lesions, suggests that individual tumors lose expression over time, and that many invasive carcinomas arise de novo by mechanisms not involving HER-2/neu. Amplification and expression of HER-2/neu are strongly associated with poor prognosis in breast cancer patients with positive axillary lymph nodes. In contrast, the prognostic significance of HER-2/neu in patients with node-negative disease appears to be restricted to small subsets of these patients.

Expression and Prognostic Significance of the HER-2/neu Oncogene During the Evolutionary Progression of Human Breast Cancer

About half of all breast cancer patients have metastatic disease in axillary lymph nodes when they are first seen by a physician. The untreated prognosis for these patients is very poor, and the decision to use adjuvant therapy (i.e., radiation, endocrine or chemotherapy) has become almost routine. The other half of patients, however, present without clinical-pathological evidence of metastatic disease, and appear to be cured by initial surgery. Unfortunately, the disease will recur in 20–30% of these patients within 5 years. The choice for adjuvant therapy in this setting is difficult and controversial. On the one hand, evidence from recent studies has shown that adjuvant endocrine and/or chemotherapy can significantly improve disease-free survival (DFS) in some patients with apparently localized breast cancer (i.e., axillary node-negative tumors) (1–3). This has resulted in an official recommendation by the National Cancer Institute that all patients with node-negative breast cancer should be considered for some form of adjuvant therapy (4). On the other hand, the disease does not recur in the majority (70–80%) of node-negative patients, supporting an alternative point of view that all of them should not receive potentially harmful adjuvant therapy (5). Proponents of both views would agree that patients at high-risk for recurrence should receive adjuvant therapy if there were reliable methods of identifying them.

Direct counting of tritium by dissolving MicroelisaR wells in scintillation fluid

Radioassays with low-energy beta-emitting nuclides (e.g., 3H, 14C, 35S) in 96-well plastic plates are tedious and frequently inaccurate because of the necessity of quantitatively removing and transferring the contents of each well to scintillation fluid. We therefore investigated the possibility of counting these nuclides by directly placing the entire break-apart well (Microelisa) along with the sample into one of four scintillation counting fluids: toluene-PPO-POPOP with or without Protosol, ACSR, and EcoLite. Although some of these scintillation fluids fully dissolved the plastic wells and other did not, we found that the presence of the wells did not appreciably interfere with the efficiency of tritium counting. This technique saves considerable time and reduces possible errors in liquid scintillation counting of samples from plastic microtitration plates.