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.

Stress response protein (srp-27) determination in primary human breast carcinomas: clinical, histologic, and prognostic correlations.

Expression of an estrogen-regulated protein known as the 27,000-d heat-shock or stress-response protein (srp-27) was evaluated in human breast carcinomas and established breast cancer cell lines. Results obtained by Northern and Western blot analyses and immunohistochemical methods were concordant. Immunohistochemical assessment of srp-27 expression in 300 breast carcinomas (with median patient follow-up of 8 years) was performed. Twenty-six percent of lymph node-negative and 45% of lymph node-positive tumors were overexpressors. Univariate analysis demonstrated significant correlations between srp-27 overexpression and estrogen receptor (ER) content, pS2 protein expression, nodal metastases, advanced T stage, lymphatic/vascular invasion, and a shorter disease-free survival period (but not a shorter overall survival) for the study population as a whole. Regression tree analysis showed that srp-27 expression was an independent prognostic indicator for disease-free survival only in patients with one to three positive lymph nodes. The Cox proportional hazards model confirmed the independent prognostic significance of nodal involvement, T stage, and ER content but failed to recognize srp-27 overexpression as a significant independent parameter predictive of patient outcome in the patient population as a whole. The observed associations between srp-27 overexpression and more aggressive tumors suggest a biologic role for srp-27 in human breast carcinomas.

Tumor angiogenesis as a prognostic assay for invasive ductal breast carcinoma.

BACKGROUND: Tumor angiogenesis, as assayed by microvessel density, has been proposed as an independent prognostic marker for clinical outcome in breast cancer patients. PURPOSE: The present study evaluated the microvessel density assay and assessed its utility, alone and together with the evaluation of other tumor characteristics, in predicting outcome in patients with invasive ductal carcinomas. METHODS: In a blinded design, cases of invasive ductal carcinoma were selected from a registry containing the records of and tumor specimens from 386 breast cancer patients treated at the Massachusetts General Hospital from 1977 through 1982. After the exclusion of ineligible patients and inadequate specimens, 220 patients were included in the study; their median time of follow-up was 11.5 years. Half of these patients (n = 110) were positive for axillary lymph node metastases. Histologic sections of the tumors were stained immunocytochemically for factor VIII, a coagulation protein expressed by blood vessel endothelium, and for p53 protein. Independently, two analysts counted microvessels in three microscope fields selected from separate vascular regions of the tumor. Variability in microvessel scores between analysts and among different fields of the same tumor was summarized by the coefficient of variation. The kappa statistic tested for agreement between the analysts while correcting for chance agreement. The effects of tumor characteristics on metastasis-free survival and overall survival were tested univariately by the Harrington-Fleming rank test procedure. The effect of multiple factors on survival was tested under a Cox multivariate proportional hazards model. RESULTS: Microvessel count showed considerable variability between the two analysts and among regions within each tumor, with an overall concordance for tumor classification of 73%. Univariate analysis revealed no association between microvessel count and any other tumor or patient characteristic. Multivariate analysis indicated, for these patients, that nodal status and p53 staining predicted metastasis-free survival and that nodal status, estrogen receptor (ER) status and tumor grade predicted overall survival. CONCLUSIONS: Microvessel count showed much variation among different regions of each tumor. It did not predict metastasis-free survival or overall survival. Nodal status was the most powerful criterion to stratify these patients with invasive ductal carcinoma of the breast into different survival groups. Only ER status, tumor grade, and p53 staining had additional prognostic utility for these patients after they had been stratified by nodal status.

Accumulation of p53 tumor suppressor gene protein: an independent marker of prognosis in breast cancers.

BACKGROUND: Mutations of the tumor suppressor gene p53 have been identified in breast cancer cell lines, and some breast carcinomas are detectable by immunohistochemical assay because of p53 protein accumulation. PURPOSE: This study was designed to determine whether p53 protein accumulation in breast cancers correlates with p53 gene mutation, with survival, and with five pathobiologic factors associated with prognosis. METHODS: IgG1 monoclonal antibody to human p53 protein (PAb 1801) and immunohistochemical methods were used to detect p53 protein accumulation in archival formalin-fixed, paraffin-embedded, randomly selected carcinomas. We studied 295 invasive ductal carcinomas from the Massachusetts General Hospital; 151 were determined to be sporadic (not hereditary). We also studied 97 invasive ductal carcinomas--21 sporadic and 76 familial (hereditary)--from Creighton University. In addition, we examined 31 archival in situ carcinomas, 15 snap-frozen invasive ductal carcinomas, primary cell cultures from three benign breast tissue samples, and breast carcinoma cell lines MDA-MB-231 and MDA-MB-468. RESULTS: Nuclear p53 protein was observed in 16% of the 31 in situ carcinomas, 22% of the 172 sporadic carcinomas, 34% of the 50 tumors from patients with familial breast cancer, 52% of the 23 tumors from patients with the familial breast and ovarian cancer syndrome, and all three tumors from two patients with the Li-Fraumeni syndrome. There was complete concordance between p53 gene mutation and p53 protein accumulation in the 15 snap-frozen carcinomas and in both breast carcinoma cell lines. Statistically significant associations of p53 protein accumulation with estrogen receptor negativity and with high nuclear grade were found. There were statistically significant associations, independent of other prognostic factors, between p53 protein accumulation and metastasis-free and overall survival, for randomly accrued and for both sporadic and familial tumors. CONCLUSIONS: Immunohistochemically detected p53 protein accumulation was an independent marker of shortened survival and was seen more often in familial than in sporadic carcinomas. Our findings also suggest a correlation between p53 protein accumulation and p53 gene mutation.

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.

pS2 expression and response to hormonal therapy in patients with advanced breast cancer.

Seventy-two patients with advanced breast carcinoma (42% bone, 25% visceral, 5.5% soft tissue, and 27.5% multiple site metastases) were evaluated to determine the relationship between tumor expression of the estrogen-regulated protein pS2, estrogen receptor (ER) or progesterone receptor (PgR) content, and response to hormonal therapy. Twenty-nine % of tumors were pS2 positive, 64% were ER positive, and 29% were PgR positive. Of the ER-positive patients (n = 43), 15 (35%) had greater than 10% of the invasive carcinoma which immunostained for pS2 (these were considered pS2 positive). Only 3 of 24 ER-negative tumors were pS2 positive. A weak association between pS2 expression and ER content (P = 0.08) but not PgR content was observed. Of pS2-positive patients, 52% had a partial or complete response to hormonal therapy. In 24% of pS2-positive patients the disease stabilized with treatment. In contrast, 27% of pS2-negative patients had a partial or complete response. In 10% of these patients the disease stabilized. Similar associations between therapeutic response and ER or PgR were not observed. The odds of having a clinical response to hormonal therapy was greater for pS2-positive than for ER- or PgR-positive tumors. pS2 expression may define a subset of ER-positive tumors that are more likely to respond to hormonal treatment.

Analysis of c-erbB-2 expression in breast carcinomas with clinical follow-up.

Various monoclonal antibodies reactive with protooncogene products or tumor-associated antigens have been utilized to investigate breast carcinoma biology or antigen expression with potential prognostic relevance. Murine monoclonal antibody TA1, generated by immunization of BALB/c mice with whole c-erbB-2 (neu) transformed NIH/3T3 cells, recognizes the extracellular domain of the c-erbB-2 protein and binds a Mr 185,000 protein by immunoprecipitation. Using avidin-biotin-peroxidase techniques and monoclonal antibody TA1, 313 archival primary adenocarcinomas of the breast were evaluated for c-erbB-2 overexpression; 290 of these were used for multiparametric statistical analysis. Historical, clinical (age, laterality), histological (nuclear grade, tumor size, lymph node status, lymphatic or blood invasion), and hormone receptor data as well as clinical outcome (minimal follow-up, 6 years; median follow-up, 8.5 years) were compared to TA1 staining. For these 290 patients Cox regression multivariate analysis showed the strongest correlation between lymph node status or estrogen receptor status and overall survival (P = 0.0001 and 0.049, respectively). TA1 staining did not significantly correlate with survival (P = 0.395). However, univariate analysis of certain patient subpopulations showed a significant correlation if the examined tumors were subdivided into negative or focally reactive and those with greater than or equal to 40% cellular reactivity. For T3, T4 patients, strong TA1 immunoreactivity correlated with a shortened disease-free survival (log rank P = 0.0018; Wilcoxon p = 0.0078) and overall survival (log rank P = 0.0002; Wilcoxon P = 0.0013). For these patients the overall survival at 6 years was markedly different between the strongly reactive tumors (0%) and the negative to weakly reactive tumors (55%). In lymph node-positive patients a trend between high TA1 reactivity and a worse overall survival was also noted (log rank P = 0.128; Wilcoxon P = 0.054), with a 6-year survival of 42% in the strongly reactive tumors (n = 16) and 65% in the negative to weakly reactive carcinomas (n = 105). No correlation between TA1 immunoreactivity and other historical, clinical, and histological features were noted. c-erbB-2 overexpression as measured by immunohistochemical techniques, therefore, may have clinical significance in certain patient subpopulations.

HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer.

HER2/Neu is overexpressed in 25-30% of all human breast cancers as a result of both gene amplification and enhanced transcription. Transcriptional upregulation of HER2/neu leads to a 6-8-fold increased abundance of its mRNA per gene copy and likely results from the elevated activity of transcription factors acting on the HER2/neu promoter. Here we report that transcripts of PEA3, an ETS transcription factor implicated in oncogenesis, were increased in 93% of HER2/Neu-overexpressing human breast tumor samples. Analyses to uncover the molecular basis for elevated PEA3 transcripts in HER2/Neu-positive breast tumors revealed that the HER2/Neu receptor tyrosine kinase initiated an intracellular signaling cascade resulting in increased PEA3 transcriptional activity; transcriptionally-activated PEA3 stimulated HER2/neu and PEA3 gene transcription by binding to sites in the promoters of these genes. PEA3 also activates transcription of genes encoding matrix-degrading proteinases, enzymes required for tumor cell migration and invasion. These findings implicate PEA3 in the initiation and progression of HER2/Neu positive breast cancer, and suggest that PEA3 and signaling proteins affecting its regulation are appropriate therapeutic targets.

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.

Oncologists' recognition of depression in their patients with cancer.

PURPOSE: This study was performed as part of a large depression screening project in cancer patients to determine the degree of physician recognition of levels of depressive symptoms in cancer patients and to describe patient characteristics that influence the accuracy of physician perception of depressive symptoms. METHODS: Twenty-five ambulatory oncology clinics affiliated with Community Cancer Care, Inc of Indiana enrolled and surveyed 1,109 subjects treated by 12 oncologists. Subjects completed the Zung Self-Rating Depression Scale (ZSDS) and physicians were asked to rate their patients' level of depressive symptoms, anxiety, and pain using numerical rating scales. Subjects' sex, age, primary tumor type, medications, primary caregiver, and disease stage at diagnosis were also recorded. RESULTS: Physician ratings of depression were significantly associated with their patients' levels of endorsement of depressive symptoms on the ZSDS. However, agreement between physicians and patients is most frequently clustered when patients report little or no depressive symptoms. While physician ratings are concordant with patient endorsement of no significant depressive symptomatology 79% of the time, they are only concordant 33% and 13% of the time in the mild-to-moderate/severe ranges, respectively. Physician ratings were most influenced by patient endorsement of frequent and obvious mood symptoms, ie, sadness, crying, and irritability. Physician ratings also appeared to be influenced by medical correlates of patients' level of depressive symptoms (functional status, stage of disease, and site of tumor). Additionally, patients whose depression was inaccurately classified reported significantly higher levels of pain and had higher levels of disability. Physicians' ratings of depression were most highly correlated with physicians' ratings of patients' anxiety and pain. CONCLUSION: Physicians' perceptions of depressive symptoms in their patients are correlated with patient's ratings, but there is a marked tendency to underestimate the level of depressive symptoms in patients who are more depressed. They are most influenced by symptoms such as crying and depressed mood, and medical factors that are useful, but not the most reliable, indicators of depression in this population. Physicians' ratings of their patients' distress symptoms seem to be global in nature--they are highly correlated with anxiety, pain, and global dysfunction. Physician assessment might be improved if they were instructed to assess and probe for the more reliable cognitive symptoms such as anhedonia, guilt, suicidal thinking, and hopelessness. Screening instruments and the use of brief follow-up interviews would help to identify patients who are depressed.

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.

p53 mutations in benign breast tissue.

PURPOSE: Patients with benign breast biopsies that exhibit atypical epithelial proliferation or fibroadenoma may be at increased risk for invasive breast cancer. We hypothesized that molecular markers might also be useful to evaluate the malignant potential of nonneoplastic breast tissue. PATIENTS AND METHODS: Study subjects belonged to a cohort of 6,805 women who underwent biopsy for nonmalignant breast disease at the Mayo Clinic and Rochester-affiliated hospitals between 1967 and 1981. As part of a nested case-control study that compared subjects who developed invasive breast cancer with those who did not, we analyzed a sample of 60 benign breast biopsies for the following markers: HER-2/neu and p53 over-expression by immunohistochemistry, HER-2/neu and PRAD-1 amplification using differential polymerase chain reaction (PCR), and p53 mutation using single-strand conformation analysis (SSCA) and direct DNA sequencing by asymmetric PCR. RESULTS: None of 60 biopsies showed amplification of HER-2/neu or PRAD-1. Five samples exhibited low-level immunoreactivity to the HER-2/neu protein product. Fourteen samples exhibited focal or diffuse immunoreactivity to the p53 protein. Point mutations in the p53 gene were found in five samples: three of these samples exhibited mutations that altered the amino acid sequence. Only two of five samples with p53 mutation exhibited p53 overexpression. Histologic diagnoses on three samples with nonconservative p53 mutation were, respectively, nonproliferative fibrocystic change, papillomatous hyperplasia, and fibroadenoma. CONCLUSION: The clinical significance of p53 mutation, p53 overexpression, and low-level HER-2/neu expression in benign breast tissue remains to be determined. Further research will be necessary to evaluate whether these markers could serve as useful adjuncts to histology in evaluation of the malignant potential of benign breast tissue.

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.

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.

Analysis of Bax and Bcl-2 expression in p53-immunopositive breast cancers.

Bax and Bcl-2 are proteins that regulate programmed cell death and apoptosis. The expression of these proteins can be regulated, at least in part, by the tumor suppressor p53, but the effects of p53 are highly tissue specific. In an effort to better understand the relation between p53 and the in vivo control of the expression of Bax and Bcl-2 in adenocarcinomas of the breast, we evaluated by immunohistochemistry the expression of Bcl-2 and Bax in 149 invasive ductal carcinomas, 135 of which were chosen because of their p53 immunopositivity. The percentages of Bcl-2-immunopositive tumor cells were significantly lower in the p53-positive (median 20%) subsets as compared to the p53-negative (median 85%) subsets (P = 0. 004). Comparisons of the percentages of p53-immunopositive tumor cells with the percentages of Bcl-2- and Bax-positive cells (as continuous variables) revealed a significant inverse correlation between Bcl-2 and p53 (r = -0.41, P < 0.001) but not between Bax and p53. In the p53-positive subset, the percentages of Bax- and Bcl-2-immunopositive tumor cells were correlated positively (r = 0. 27, P = 0.002), suggesting that the expression of these genes may be co-regulated to some extent in these breast cancers. Higher percentages of Bcl-2-positive tumor cells were also associated with estrogen receptor positivity (P = 0.03), low histological tumor grade (P = 0.03), and low T stage (P = 0.02), whereas Bax immunostaining was associated only with c-erbB-2 immunopositivity (P = 0.02). Although the number of cases was small and treatment was non-uniform, preliminary correlations with clinical outcome data suggest that the prognostic significance of Bcl-2 may be enhanced by inclusion of Bax data in patients with p53-immunopositive adenocarcinoma of the breast, at least for patients with node-negative disease. Taken together, these data suggest that, despite the ability of p53 to bind directly to the Bax gene promoter, the regulation of Bax in human breast cancers does not necessarily correlate with p53 status, implying that regulation of this pro-apoptotic gene in these tumors is complex and probably influenced by several factors.

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.

Monoclonal antibodies reactive with human breast or ovarian carcinoma: in vivo applications.

Monoclonal antibodies (MoAbs) are unique and useful bioprobes that allow in vivo targeting of membrane-associated or circulating antigens. Most of the clinical trials to date have used low dosages of radiolabeled MoAb given in a single dose. Newer studies have included antibody fragments, repeated injections, intraperitoneal (IP) administration, and other labels such as 90Y. Clinical MoAb trials are often arduous, expensive, and time-consuming to perform. Before human use, animal studies and extensive MoAb characterization are required. The production of pharmaceutical grade, radiolabeled MoAb is technically difficult and costly. Clinical trials require administrative and patient consent as well as extensive written protocols. These studies necessitate interdepartmental and intradepartmental cooperation and coordination. Furthermore, the use of in vivo radiolabeled probes impacts many levels of health care providers from janitorial, nursing, and technical staff to laboratories and physicians. Simple blood tests or disposal of body excretions may concern nursing or technical staff with the possibility of radiation exposure. The responsibility for study design, personnel involvement, and prospective use in patients without a definitive cancer diagnosis ultimately rests with the physician. While many issues have been addressed, additional clinical trials, consideration of safety issues, and standardization between institutions will be necessary before the use of radiolabeled MoAb for diagnosis, management, or therapy of human tumors becomes routine. Continued cooperation and funding should ensure its achievement.

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.

Evaluation of in vitro bromodeoxyuridine labeling of breast carcinomas with the use of a commercial kit.

The prognostic significance of S-phase fraction analyses of breast carcinomas has been reported by several investigators. The Cell Proliferation Kit (Amersham Corporation, Arlington Heights, IL), which uses in vitro bromodeoxyuridine (BRDU) labeling to evaluate cell cycle kinetics without a flow cytometer or radioisotopes, simplifies this assay for the clinical-based laboratory by providing standardized reagents and recommended methods. This study was performed to determine whether data derived from its use were comparable to published S-phase data from the use of thymidine labeling, BRDU, or other methods on breast carcinomas. Primary infiltrating ductal carcinomas (n = 142) and primary lobular carcinomas (n = 6) showed mean and median BRDU labeling of 4.63% and 3%, 1.3% and 1%, respectively, with a range of 0-28%. Benign lesions always had less than 3% BRDU uptake (n = 21). Estrogen receptor (ER) and progesterone receptor (PR) status correlated with BRDU labeling (P less than 0.05), with the highest S-phase fractions in ER- and PR-negative tumors. Correlations between BRDU uptake and histologic tumor type or size were observed. Significant correlations between BRDU uptake and lymph node status, patient age, or histologic tumor grade were not observed. S-phase studies of breast carcinomas using other techniques have shown similar data, therefore, the Cell Proliferation Kit appears to be a practical and useful method for in vitro S-phase analysis that allows concomitant histologic examination of the same tumor tissue sample.

erbB-2 (HER-2) and breast cancer progression.

The purpose of this study was to explore differences in erbB-2 alterations in recurrent or metastatic disease, as compared with the primary tumor. Primary invasive breast cancers and subsequent local, regional, or distant metastases from 113 patients were examined. Primary and all metastatic or recurrent tumors were evaluated for erbB-2 expression using immunohistochemistry (with CB11, TAB 250, DAKO anti-erbB-2, HercepTest) and fluorescence in situ hybridization. Immunohistochemical data derived from the 4 reagents on the same tumor sample were highly correlated (pair-wise correlation range, 78-90%). Immunohistochemical and fluorescence in situ hybridization erbB-2 data were also generally concordant (average agreement, 82%; range, 75-87%). Approximately 80% of the primary and recurrent or metastatic tissues from the same patient had similar patterns of erbB-2 protein expression and gene copy number, although in approximately 20%, disagreement was observed. Discordant cases were mostly erbB-2 normal primary tumors with altered metastases, although the opposite pattern was also observed. Interestingly, erbB-2 discordance, based on CB11 data, was associated with subsequent survival, whereas there were no similar associations with other erbB-2 stains. In approximately 80% of patients, erbB-2 protein expression and gene copy number were similar in the primary tumor and locally recurrent or distant metastases. The lack of complete concordance suggests clonal selection or genetic drift in some cases.