Metformin Targets Glucose Metabolism in Triple Negative Breast Cancer.

Metformin is the most widely administered anti-diabetic agent worldwide. In patients receiving metformin for metabolic syndrome or diabetes, it reduces the incidence and improves the survival of breast cancer (BC) patients. We have previously shown that metformin is particularly potent against triple negative breast cancer (TNBC), with a reduction of proliferation, oncogenicity and motility, inhibition of pro-oncogenic signaling pathways and induction of apoptosis. These BCs are well recognized to be highly dependent on glucose/glucosamine (metabolized through anaerobic glycolysis) and lipids, which are metabolized for the production of energy and cellular building blocks to sustain a high rate of proliferation. We have previously demonstrated that metformin inhibits lipid metabolism, specifically targeting fatty acid synthase (FASN), cholesterol biosynthesis and GM1 lipid rafts in TNBC. We also reported that glucose promotes phenotypic aggression and reduces metformin efficacy. We now show that metformin inhibits several key enzymes requisite to glucose metabolism in TNBC, providing additional insight into why metformin is especially toxic to this subtype of BC. Our data suggests that the use of metformin to target key metabolic defects in lipid and carbohydrate metabolism in cancer may be broadly applicable, especially against highly aggressive malignant cells.

Reactivation of epigenetically silenced HER4/ERBB4 results in apoptosis of breast tumor cells.

Experimental and clinical data support a growth inhibitory role for HER4 in breast cancer. Clinically HER4 expression is extinguished during breast tumorigenesis supporting a tumor suppressor function for HER4, however, a molecular mechanism to explain the selective loss of HER4 expression has remained elusive. Epigenetic mechanisms, for example, aberrant gene promoter hypermethylation, have been shown to ablate tumor suppressor gene expression in breast carcinomas. We identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite-treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Treatment of the HER4-negative BT20 cell line with the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC)-enhanced HER4 expression, confirming a role for DNA methylation in suppressed HER4 expression. DAC treatment to reactive HER4 expression in combination with the HER4 ligand heregulin-ß1 (HRG) resulted in apoptosis of BT20 cells providing a novel therapeutic strategy for triple-negative tumors. The BT20 cells were rescued from apoptosis when preincubated with HER4 small interfering RNA, thereby confirming a role for HER4 in DAC/HRG-induced apoptosis. We verified HER4 promoter methylation in primary breast carcinomas and detected a significant increase in HER4 promoter methylation in HER4-negative breast tumors (P<0.001). Furthermore, increased levels of HER4 promoter methylation were significantly associated with worse patient prognosis (P=0.0234). Taken together, our data support a tumor suppressor function for HER4, which is epigenetically suppressed in breast tumors through promoter hypermethylation.

Docetaxel combined with trastuzumab is an active regimen in HER-2 3+ overexpressing and fluorescent in situ hybridization-positive metastatic breast cancer: a multi-institutional phase II trial.

PURPOSE: To determine the efficacy and safety of weekly docetaxel and trastuzumab as first- or second-line therapy in women with HER-2-overexpressing metastatic breast cancer and to correlate the efficacy of trastuzumab with HER-2 status as determined by immunohistochemistry assay and fluorescent in situ hybridization (FISH). PATIENTS AND METHODS: Twenty-six women with HER-2-positive (HercepTest [Dako Corp, Carpenteria, CA]2 to 3+) metastatic breast cancer were enrolled onto this study of trastuzumab (4 mg/kg load; 2 mg/kg/wk administered intravenously) and docetaxel (35 mg/m2/wk for 6 weeks). RESULTS: Using an intent-to-treat analysis, the overall response rate was 50% (13 of 26 patients). Eight patients (31%) had a period of stable disease posttherapy. Among HER-2 3+ patients, the overall response rate was 63% (12 of 19 patients) compared with a 14% response rate (one of seven patients) for HER-2 2+ patients (P=.07). Patients with FISH-positive tumors experienced an overall response rate of 64%. Median time to progression was 12.4 months for the entire cohort (HER-2 3+ tumors, 12.3 months; HER-2 2+ lesions, 9.5 months) and median survival was 22.1 months. All HER-2 3+ patients were FISH-positive; the only HER-2 2+ patient responding to treatment was also FISH-positive. Grade 4 toxicities occurred in four patients; most toxicities were mild. CONCLUSION: Trastuzumab plus docetaxel is an active and well-tolerated regimen in women with HER-2 3+ overexpressing or FISH-positive metastatic breast cancer.

Understanding the age dependency of breast cancer biomarkers.

This review and study of nearly 4,000 primary breast cancers evaluates the hypothesis that human aging not only increases breast cancer incidence but also alters breast cancer biology. Clinically validated biomarkers were chosen as surrogate measures of genetic instability and tumor growth, invasiveness and metastatic potential. Our results support the premise that breast cancer clinical behavior and biology are significantly affected by patient age, but call into question key aspects of the current cancer-aging paradigm.

Gelsolin as a negative prognostic factor and effector of motility in erbB-2-positive epidermal growth factor receptor-positive breast cancers.

PURPOSE: erbB-2 and epidermal growth factor receptor (EGFR) may mediate motility via signaling that enables changes in the actin cytoskeleton. A physical basis for this motility may depend on the coexpression of gelsolin, a M(r) 80,000 actin-binding protein. EXPERIMENTAL DESIGN: The expression of erbB-2, EGFR, and gelsolin was analyzed in 790 archival invasive breast cancers. These data were compared with histological, clinical, and outcome data (median follow-up, 16.3 years). RESULTS: Protein overexpression was observed in overlapping subsets of breast cancers (38% of cases were erbB-2+; 15% of cases were EGFR+; and 56% of cases were gelsolin+). Tumor gelsolin was associated with overexpression of erbB-2 and EGFR, as well as with an aggressive tumor phenotype. By univariate and multivariate analyses, tumor gelsolin alone was not a prognostic factor. Overexpression of all three factors significantly predicted poor clinical outcome by univariate and multivariate analyses. For example, in node-positive patients, coexpression of all three markers was associated with a 3-year disease-specific survival (as compared with erbB-2+, EGFR+, gelsolin- patients, who had a median survival of 6 years). CONCLUSIONS: These data suggest that gelsolin coexpression may be an important additional prognostic factor in erbB-2+, EGFR+ breast cancer patients. We hypothesize that this is due to the role of gelsolin in mediating motility and invasion.

A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation.

The ribonucleoprotein telomerase synthesizes telomeric DNA by copying an intrinsic RNA template. In most cancer cells, telomerase is highly activated. Here we report a telomerase-based antitumor strategy: expression of mutant-template telomerase RNAs in human cancer cells. We expressed mutant-template human telomerase RNAs in prostate (LNCaP) and breast (MCF-7) cancer cell lines. Even a low threshold level of expression of telomerase RNA gene constructs containing various mutant templates, but not the control wild-type template, decreased cellular viability and increased apoptosis. This occurred despite the retention of normal levels of the endogenous wild-type telomerase RNA and endogenous wild-type telomerase activity and unaltered stable telomere lengths. In vivo tumor xenografts of a breast cancer cell line expressing a mutant-template telomerase RNA also had decreased growth rates. Therefore, mutant-template telomerase RNAs exert a strongly dominant-negative effect on cell proliferation and tumor growth. These results support the potential use of mutant-template telomerase RNA expression as an antineoplastic strategy.

Measures of cell turnover (proliferation and apoptosis) and their association with survival in breast cancer.

Our objective was to investigate the prognostic significance of cell turnover (apoptosis and proliferation) in breast cancer patients. Apoptosis was microscopically quantitated on histological sections from 791 breast cancer patients with long-term follow-up (median, 16.3 years). Apoptotic counts were also compared with proliferation data (mitotic counts and MIB-1 labeling); apoptosis data derived from terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay; and pathobiological variables, including p53, erbB-2, and estrogen receptor (ER). High apoptotic counts were associated with increased cellular proliferation, ER negativity, immunopositivity of erbB-2 and p53 (P < 0.0001), and shortened disease-specific survival (DSS; P = 0.0009) and disease-free survival (DFS; P = 0.0006). Other factors associated with shortened DFS and DSS by univariate analysis were high tumor grade, nodal metastases, and large tumor size (P < 0.0001 for each). Multivariate analysis of data for all of the patients demonstrated that tumor size, nodal status, ER, histological grade, and erbB-2 showed independent prognostic value. In node-negative patients, tumor size and mitotic rate per 1000 cells independently predicted DFS (P = 0.0055). Tumor grade was the only independent predictor of DSS. For node-positive patients, tumor size, nodal status, ER, and erbB-2 were independent prognostic factors. The number of mitoses per 1000 was independently associated with DFS (P = 0.043) but not with DSS. Apoptosis data did not provide independent prognostic value in any, node-positive or node-negative, breast cancer patients.

Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis.

MCF-7, a breast cancer-derived cell line, is deficient of caspase 3 and relatively insensitive to many chemotherapeutic agents. To study the association of caspase 3 deficiency and chemotherapeutic resistance, we reconstituted caspase 3 in MCF-7 cells and characterized their apoptotic response to doxorubicin and etoposide. Western blots demonstrated that caspase 3 was constitutively expressed in the reconstituted MCF-7 cells. Both morphological observation and survival assays showed that caspase 3 reconstitution significantly sensitized MCF-7 cells to both drugs. Remarkably increased activation of caspases 3, 6, and 7, cleavage of cellular death substrates, and DNA fragmentation were detected in the reconstituted MCF-7 cells after drug treatment. Together, these data demonstrated a specific role for caspase 3 in chemotherapy-induced apoptosis and in activation of caspases 6 and 7. Our results also suggest that caspase 3 deficiency may contribute to chemotherapeutic resistance in breast cancer.

HER-2/neu and p53 expression versus tamoxifen resistance in estrogen receptor-positive, node-positive breast cancer.

PURPOSE: An association between the overexpression of proto-oncogene HER-2/neu and resistance to tamoxifen in estrogen receptor (ER)-positive primary and metastatic breast cancer has been suggested. We examine a possible interaction between HER-2/neu or p53 expression and tamoxifen effectiveness in patients with ER-positive, node-positive disease treated with cyclophosphamide, doxorubicin, and fluorouracil in a large adjuvant chemotherapy trial (Cancer and Leukemia Group B [CALGB] 8541). Tamoxifen assignment was not randomized-physician discretion was used for premenopausal and postmenopausal women. Trial protocol then specified assignment to postmenopausal women with ER-positive tumors, although not all took tamoxifen. PATIENTS AND METHODS: CALGB 8541 assessed HER-2/neu expression in patients with ER-positive disease by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) and amplification by differential polymerase chain reaction (PCR). IHC assessed expression of p53. Univariate and multivariate proportional hazards models assessed tamoxifen-HER-2/neu status interactions and tamoxifen-p53 status interactions. RESULTS: HER-2/neu status was available for 651 patients with ER-positive disease; 650, 608, and 353 patients were assessed by IHC, PCR, and FISH, respectively. Approximately one half received tamoxifen. Reduction in risk of disease recurrence or death resulting from tamoxifen was approximately 37% (32% with overexpression and 39% with normal expression of HER-2/neu; n = 155 by IHC). The tamoxifen-HER-2/neu status interaction was not significant in multivariate analysis of all three HER-2/neu assessment methods. Tamoxifen-p53 interaction did not significantly predict outcome. CONCLUSION: Disease-free and overall survival benefit of tamoxifen in patients with ER-positive, node-positive breast cancer does not depend on HER-2/neu or p53 status. Our data suggest that neither HER-2/neu nor p53 expression should be used to determine assignment of tamoxifen.

Activation (tyrosine phosphorylation) of ErbB-2 (HER-2/neu): a study of incidence and correlation with outcome in breast cancer.

PURPOSE: We hypothesize that phosphorylated ErbB-2 (P-ErbB-2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB-2 (HER-2/neu) and P-ErbB-2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS: Eight hundred sixteen invasive breast cancers with a median of 16.3 years of follow-up were immunostained for ErbB-2 (using antibody CB11) and P-ErbB-2 (using antibody PN2A). ErbB-2 and P-ErbB-2 data were compared with clinical, histologic, immunohistochemical, and outcome variables. RESULTS: Of 816 primary breast cancers, 307 (38%) were positive for ErbB-2 and 37 (12% of ErbB-2 positive and 5% of the study population) expressed P-ErbB-2. P-ErbB-2 was not detected in ErbB-2-negative cases (n = 509). ErbB-2 immunohistochemical data were bimodal; patients with > or = 80% cellular expression had the shortest disease-free and disease-specific survival. P-ErbB-2 was associated with a higher percentage of ErbB-2-positive cells, a higher number of positive lymph nodes, and cellular proliferation. ErbB-2 and P-ErbB-2 were indicators of poor prognosis in node-positive patients in both univariate and multivariate analyses. We found that either P-ErbB-2 expression or high (> or = 80%) ErbB-2 expression provided the most significant prognostic value in node-positive cases by multivariate analyses. There were too few P-ErbB-2-positive cases and events in the node-negative patient group to allow statistical analysis of P-ErbB-2 in that subgroup. CONCLUSION: PN2A immunostaining identified a subset (approximately 12% of ErbB-2-positive breast cancers) with activation (phosphorylation) of the receptor ErbB-2. P-ErbB-2 expression was strongly associated with higher levels of ErbB-2 expression (> or = 80%), although it was not a surrogate. Identification of cases with a high percentage of invasive breast cancer cells expressing ErbB-2 or determination of receptor activation via P-ErbB-2 may provide additional prognostic value in node-positive breast cancers.

p(21WAF1/CIP1) expression in breast cancers: associations with p53 and outcome.

p21(WAF1/CIP1) is transcriptionally activated by wt p53 and inhibits G1 associated cyclins, a major mechanism by which p53 inhibits cellular proliferation. Archival breast cancers (798) with a median follow-up of 16.3 years were used to explore the prognostic value of p21 immunohistochemical analyses. p21 immunostaining was detected in the majority (726/798: 91%) of breast cancers as well as adjacent in situ carcinomas (125/170: 74%), hyperplastic lesions (140/349: 40%) and normal breast epithelium adjacent to carcinoma (3/89: 3%). Complete immunonegativity was observed in only 9% of invasive cancers and was associated with p53 immunopositivity (p < 0.05). Univariate analysis of all patients showed that p21 negativity was associated with a longer disease specific survival (relative risk (RR) 1.5). Node positive p21- patients also showed a longer disease free and disease specific survival as compared to tumor p21+ patients. In node negative patients, p53 positivity but not p21 alone, was significantly associated with a shortened disease free survival (RR = 1.6). Node negative patients who were p53+ p21-, in particular had the shortest disease free survival compared to other p53, p21 subgroups (i.e., p21 negativity was associated with a worse outcome). Multivariate analysis of lymph node negative patients (n > 300) demonstrated that tumor size and tumor grade were independently predictive of outcome, whereas neither p53 nor p21 were significant. For node positive patients, p21 positivity (p = 0.05), p53 positivity (p = 0.03), a higher number of positive nodes, larger tumor size, steroid receptor negativity, high proliferation rate, and erbB-2 expression were each independently associated with poor outcome. In summary, p21 negativity was inversely correlated with p53 immunopositivity in the majority of cases. p21 negative tumor patients had an improved outcome if they were node positive, whereas p21 status was not significantly associated with survival in node negative patients. This observation may be due to the reported 'uncoupling of S phase and mitosis' associated with a loss of p21 expression which may result in enhanced sensitivity to chemotherapy.

Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999.

BACKGROUND: Under the auspices of the College of American Pathologists, a multidisciplinary group of clinicians, pathologists, and statisticians considered prognostic and predictive factors in breast cancer and stratified them into categories reflecting the strength of published evidence. MATERIALS AND METHODS: Factors were ranked according to previously established College of American Pathologists categorical rankings: category I, factors proven to be of prognostic import and useful in clinical patient management; category II, factors that had been extensively studied biologically and clinically, but whose import remains to be validated in statistically robust studies; and category III, all other factors not sufficiently studied to demonstrate their prognostic value. Factors in categories I and II were considered with respect to variations in methods of analysis, interpretation of findings, reporting of data, and statistical evaluation. For each factor, detailed recommendations for improvement were made. Recommendations were based on the following aims: (1) increasing uniformity and completeness of pathologic evaluation of tumor specimens, (2) enhancing the quality of data collected about existing prognostic factors, and (3) improving patient care. RESULTS AND CONCLUSIONS: Factors ranked in category I included TNM staging information, histologic grade, histologic type, mitotic figure counts, and hormone receptor status. Category II factors included c-erbB-2 (Her2-neu), proliferation markers, lymphatic and vascular channel invasion, and p53. Factors in category III included DNA ploidy analysis, microvessel density, epidermal growth factor receptor, transforming growth factor-alpha, bcl-2, pS2, and cathepsin D. This report constitutes a detailed outline of the findings and recommendations of the consensus conference group, organized according to structural guidelines as defined.

HER-2/neu amplification in benign breast disease and the risk of subsequent breast cancer.

PURPOSE: The purpose of this study was to determine whether the presence of HER-2/neu gene amplification and/or overexpression in benign breast disease was associated with an increased risk of subsequent breast cancer. PATIENTS AND METHODS: We conducted a nested case-control study of a cohort of women who were diagnosed with benign breast disease at the Mayo Clinic and who were subsequently observed for the development of breast cancer. Patients who developed breast cancer formed the case group, and a matched sample from the remaining cohort served as controls. Benign tissue samples from 137 cases and 156 controls and malignant tissues from 99 cases provided DNA or tissue for evaluation of HER-2/neu amplification and protein overexpression. RESULTS: Among the controls, seven benign tissues (4.5%) demonstrated low-level HER-2/neu amplification, whereas 13 benign (9.5%) and 18 malignant (18%) tissue specimens from cases exhibited amplification. HER-2/neu amplification in benign breast biopsies was associated with an increased risk of breast cancer (odds ratio ¿OR = 2.2; 95% confidence interval ¿CI, 0.9 to 5.8); this association approached statistical significance. The risks for breast cancer associated with benign breast histopathologic diagnoses were OR = 1.1 (95% CI, 0.6 to 1.9) for lesions exhibiting proliferation without atypia and OR = 1.5 (95% CI, 0.4 to 5.6) for the diagnosis of atypical ductal hyperplasia. For women having both HER-2/neu amplification and a proliferative histopathologic diagnosis (either typical or atypical), the risk of breast cancer was more than seven-fold (OR = 7.2; 95% CI, 0.9 to 60.8). Overexpression of the HER-2/neu protein product, defined as membrane staining in 10% or more of epithelial cells, was found in 30% of the breast tumors but was not detected in any of the benign breast tissues. Case patients who had HER-2/neu gene amplification in their malignant tumor were more likely to have had HER-2/neu amplification in their prior benign biopsy (P =.06, Fisher's exact test). CONCLUSION: Women with benign breast biopsies demonstrating both HER-2/neu amplification and a proliferative histopathologic diagnosis may be at substantially increased risk for subsequent breast cancer.

Ets regulation of the erbB2 promoter.

Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nuclease-sensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)-polypyrimidine(TCC) mirror-repeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (Hr-DNA) at the mirror-repeat element. Mutations preventing Hr-DNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that Ets-erbB2 promoter complexes cannot form when the mirror-repeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2-regulating breast cancer Ets factors (PEA3, ESX/Elf-3), Elf-1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promoter-targeted therapeutics, an EBS-targeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity.

HER2 testing: laboratory, technical and clinical considerations.

HER2 measurement holds great promise in predicting response to a variety of systemic therapies an exciting step forward in the management of breast cancer patients. The enthusiasm surrounding the clinical importance of HER2, however, is tempered by the uncertainty regarding the clinical usefulness and accuracy of the different methodologies currently available to assess HER2 status. In this paper the authors address laboratory and technical issues associated with methods which measure HER2 overexpression or amplification. We also discuss how these issues can influence the clinical utility and routine application of this marker. While a tumor marker may be considered clinically relevant, it must be proven to be clinically useful. Optimally, this should occur in the setting of a randomized clinical trial, using methods that are reliable, reproducible, and biologically accurate. For HER2 testing to be a useful, routine clinical marker several critical issues need to addressed. These include the determination and validation of the clinical utility of each method to predict response to the different systemic therapies currently associated with HER2. In addition, there is need to set standards for assay performance and interpretation of assay results, based on criteria that are clinically validated.

Comparison of mitotic index, in vitro bromodeoxyuridine labeling, and MIB-1 assays to quantitate proliferation in breast cancer.

PURPOSE: To investigate the hypothesis that in vitro bromodeoxyuridine (BrDu) labeling might be superior to MIB-1 immunostaining for prognostic value, because it more selectively labels cells during the S phase. METHODS: Four hundred eighty-six patients with breast cancers (59% lymph node-negative, 41% lymph node-positive) surgically excised between 1988 and 1993 (median follow-up, 62 months) were evaluated for cellular proliferation using prospective in vitro BrDu uptake assays, retrospective mitotic indices, and MIB-1 labeling. RESULTS: MIB-1, BrDu labeling, and mitotic index-derived proliferation data were highly correlated. Each was similarly associated with most other markers of prognosis, although these relationships were not identical. By univariate analysis, nodal status was the most significant prognostic variable for all patients. Higher BrDu labeling index, MIB-1 immunolabeling, and mitotic index were also associated with shortened disease-free survival (DFS) and disease-specific survival for the entire patient group, as well as for node-negative patients. The association between cellular proliferation and survival was much weaker for node-positive patients. Multivariate models confirmed that nodal status, tumor size, and proliferation data predicted survival in all patients as well as those with node-negative disease, although MIB-1 was somewhat more closely associated with outcome than mitotic index or in vitro BrDu data. For patients with T1NOMO disease (n = 172), the only significant predictors of DFS were proliferation rate (mitotic index or MIB-1) and tumor grade. CONCLUSIONS: Proliferation rate predicts recurrence and survival in breast cancer. This effect is more pronounced in node-negative patients. In vitro BrDu data are not superior to MIB-1 and mitotic counting.

Invasive micropapillary carcinoma of the breast: a prognostic study.

Invasive micropapillary carcinoma (IMC) of the breast is a rare variant of infiltrating ductal carcinoma that has been associated with an extremely high incidence of lymph node metastases. Follow-up studies on patients with pure IMC breast cancer histology have been limited by low patient numbers, short duration of follow-up, and a lack of multivariate analyses. Using invasive breast cancers from 1,287 patients (median follow-up, 13.8 years), histological review showed 21 cases (1.7%) with pure IMC histology. Pure IMC histology was associated with high-grade histology (P = .04), metastases to regional lymph nodes (P < .001), a high mitotic index (P = .02), and erbB-2 immunopositivity (P = .007). Univariate analyses showed a strong association between IMC histology and shortened survival (disease-free survival [DFS], P = .0052; median, 44 months for IMC and 63 months for non-IMC; disease-specific survival [DSS], P = .014; medians, 71 and 78 for IMC and non-IMC, respectively) only in an analysis of all patients. Because only 1 case of node-negative IMC histology was available, univariate analysis of IMC histology was performed only on node-positive patients without significance. Multivariate analyses comparing IMC histology with either node-positive or all other breast cancers failed to show independent prognostic significance. In summary, breast cancer patients with pure IMC histology showed survival rates similar to those of other patients with equivalent numbers of lymph node metastases.

erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer.

BACKGROUND: We have previously reported that high expression of the erbB-2 gene (also known as HER-2/neu and ERBB2) in breast cancer is associated with patient response to dose-intensive treatment with cyclophosphamide, doxorubicin (Adriamycin), and 5-flurouracil (CAF) on the basis of short-term follow-up of 397 patients (set A) with axillary lymph node-positive tumors who were enrolled in Cancer and Leukemia Group B (CALGB) protocol 8541. METHODS: To validate those findings, we conducted immunohistochemical analyses of erbB-2 and p53 protein expression in an additional cohort of 595 patients (set B) from CALGB 8541, as well as a molecular analysis of erbB-2 gene amplification in tumors from all patients (sets A and B). Marker data were compared with clinical, histologic, treatment, and outcome data. RESULTS: Updated analyses of data from set A (median follow-up, 10.4 years) showed an even stronger interaction between erbB-2 expression and CAF dose, by use of either immunohistochemical or molecular data. A similar interaction between erbB-2 expression and CAF dose was observed in all 992 patients, analyzed as a single group. However, for set B alone (median follow-up, 8.2 years), results varied with the method of statistical analysis. By use of a proportional hazards model, the erbB-2 expression-CAF dose interaction was not significant for all patients. However, in the subgroups of patients randomly assigned to the high- or the moderate-dose arms, significance was achieved. When patient data were adjusted for differences by use of a prognostic index (to balance an apparent failure of randomization in the low-dose arm), the erbB-2 expression-CAF dose interaction was significant in all patients from the validation set B as well. An interaction was also observed between p53 immunopositivity and CAF dose. CONCLUSIONS: The hypothesis that patients whose breast tumors exhibit high erbB-2 expression benefit from dose-intensive CAF should be further validated before clinical implementation. Interactions between erbB-2 expression, p53 expression, and CAF dose underscore the complexities of predictive markers where multiple interactions may confound the outcome.

Altered expression of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG) in breast cancer.

We examined expression of N-methylpurine-DNA glycosylase (MPG), a DNA repair enzyme that removes N-alkylpurine damage, in normal, malignant, and immortalized breast epithelial cells, and breast cancer cell lines (MDA-MB-231, MCF7, T47D). Northern analysis showed increased expression in cancer versus normal breast epithelial cells (2-24-fold). Southern blots revealed no gene amplification or polymorphisms. Immunofluorescence, immunohistochemistry, and Western blot analysis demonstrated increased MPG protein expression in the tumor cells that correlated with elevated glycosylase activity. Since MPG overexpression has been shown to be paradoxically associated with increased susceptibility to DNA damage, up-regulation of this gene may suggest a functional role in breast carcinogenesis.

The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression.

RHAMM is an oncogene that regulates signaling through ras and controls mitogen-activated protein kinase [extracellular signal-regulated protein kinase (ERK)] expression in embryonic murine fibroblasts. ERK is a dual-specificity kinase that controls expression of proteins relevant to tumorigenesis, proliferation, and motility. To assess whether RHAMM and ERK are involved in human breast tumor progression, we examined RHAMM, ras, and ERK expression in two cohorts of breast cancer patients using reverse transcription-PCR and immunocytochemistry. We show that overexpression of RHAMM in primary tumors of two patient cohorts was significantly prognostic of poor outcome in breast cancer progression. Furthermore, RHAMM overexpression occurred within subsets of tumor cells in the primary tumor, and this staining pattern was associated with lymph node metastases. The metastases exhibited a significantly higher level of staining for RHAMM than did the primary tumor. RHAMM expression strongly correlated with overexpression of both ras and ERK, although overexpression of either of these two signaling molecules was not by itself a prognostic indicator. These results identify a new parameter that is involved in lymph node metastasis of primary breast cancers and suggest that quantification of RHAMM overexpression may be a useful prognostic indicator for breast carcinoma progression.