Aberrant subcellular localization of BRCA1 in breast cancer.

The BRCA1 gene product was identified as a 220-kilodalton nuclear phosphoprotein in normal cells, including breast ductal epithelial cells, and in 18 of 20 tumor cell lines derived from tissues other than breast and ovary. In 16 of 17 breast and ovarian cancer lines and 17 of 17 samples of cells obtained from malignant effusions, however, BRCA1 localized mainly in cytoplasm. Absence of BRCA1 or aberrant subcellular location was also observed to a variable extent in histological sections of many breast cancer biopsies. These findings suggest that BRCA1 abnormalities may be involved in the pathogenesis of many breast cancers, sporadic as well as familial.

Blockade of the type I somatomedin receptor inhibits growth of human breast cancer cells in athymic mice.

Insulin and insulin-like growth factors (IGIs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR) expressed in these cells may mediate the growth effects of these peptides. We have examined the role of this receptor on human breast cancer growth with a monoclonal antibody (alpha-IR-3) that blocks the receptor binding domain and inhibits IGF-I-induced growth. alpha-IR-3 inhibited clonal growth in vitro and blocked the mitogenic effect of exogenous IGF-I in both MCF-7 and MDA-231 breast cancer cell lines. Antibody-induced blockade of the type I SR also inhibited the estrogen-independent MDA-231 cells growing in vivo in nude mice, but growth of the estrogen-dependent MCF-7 cells was unaffected. IGIs are important growth regulators of MDA-231 breast cancer cells. Blockade of this growth stimulatory pathway may provide a new treatment strategy.

Acquired tamoxifen resistance: correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen.

Acquired tamoxifen resistance represents a major cause of treatment failure in breast cancer. We implanted estrogen receptor-positive MCF-7 human breast cancer cells in athymic nude BALB/c mice as a model to study in vivo acquired tamoxifen resistance. After 4-6 months of tumor growth suppression by trans-tamoxifen, tumor progression was observed despite continued tamoxifen administration. Acquired resistance was not due to loss of estrogen receptors, to alterations in serum or tumor estrogen levels, or to changes in tamoxifen or its major metabolites in serum. Tamoxifen-resistant tumors remained estrogen dependent in vivo. However, resistance was also associated with the ability of tamoxifen to stimulate tumor growth. Resistant tumors were characterized by markedly lower intracellular tamoxifen levels and by isomerization of the potent antiestrogenic metabolite trans-4-hydroxy-tamoxifen to the less potent cis isomer. Metabolic tolerance, as manifested by alterations in cellular concentrations of tamoxifen and its metabolites, may thus be one mechanism for acquired tamoxifen resistance in breast cancer.

Analysis of loss of heterozygosity in 399 premalignant breast lesions at 15 genetic loci.

BACKGROUND: Usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors for invasive breast cancer (IBC), suggesting that these lesions may be direct precursors of IBC. To identify genetic changes that may be important in the early development of precursor lesions and their progression to malignant or invasive disease, we examined 399 putative precursors (211 UDH, 51 ADH, 81 non-comedo DCIS, and 56 comedo DCIS) for loss of heterozygosity (LOH) at 15 polymorphic genetic loci known to exhibit high rates of loss in IBC. We also assessed the sharing of LOH by putative precursors and synchronous cancers. METHODS: The polymerase chain reaction was used to analyze DNA from microdissected archival specimens. RESULTS AND CONCLUSIONS: In hyperplasias from noncancerous breasts (i.e., without DCIS and/or IBC in analyses of hyperplasias), LOH at any given locus was rare (range, 0%-15%), although 37% of UDH and 42% of ADH lesions showed loss for at least one locus, suggesting that the development of hyperplasias can involve many different tumor suppressor genes. In DCIS from noncancerous breasts (i.e., without IBC in analyses of DCIS), LOH was common, with 70% of noncomedo lesions and 79% of comedo lesions showing at least one loss. In DCIS, substantial rates of loss (up to 37%) were observed at loci on chromosomes 16q, 17p, and 17q, suggesting that inactivated tumor suppressor genes in these regions may be important in the development of noninvasive breast cancer. When DCIS lesions from cancerous and noncancerous breasts were compared, substantially more LOH was observed in the cancerous breasts at a few loci (on chromosomes 2p, 11p, and 17q), suggesting that genetic alterations in these regions may be important in the progression to invasive disease. Among specimens harvested from cancerous breasts, 37% of UDH, 45% of ADH, 77% of noncomedo DCIS, and 80% of comedo DCIS lesions shared LOH with synchronous cancers at one locus or more, supporting the idea that the putative precursors and the cancers are genetically related.

Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer.

BACKGROUND: The p53 (also known as TP53) tumor suppressor gene encodes for a nuclear phosphoprotein thought to regulate proliferation of normal cells. Most p53 mutations result in a nonfunctional protein that accumulates in tumor cell nuclei. These common mutations appear to be involved in the development and/or progression of several neoplastic diseases including human breast cancer. PURPOSE: Our purpose was to investigate the relationships between levels of mutant p53 protein expression, tumor cell proliferation rate, and clinical outcome in patients with node-negative breast cancer. METHODS: Expression of mutant p53 protein was evaluated by frozen-section immunohistochemistry (IHC) and light microscopy in 700 breast cancers from axillary lymph node-negative patients with long-term follow-up (median, 54 months). The immunostaining signal was expressed as the sum of scores representing the proportion and staining intensity of negative and positive tumor cell nuclei (ranges, 0 and 2-8, respectively). Statistical comparisons were made between levels of p53 protein expression and disease-free survival, overall survival, and tumor proliferation rate expressed as the percentage of cells in the S phase (%S phase) as determined by flow cytometry. RESULTS: Of the 700 tumors, 362 (52%) showed positive nuclear immunostaining (IHC score > 0). Proliferation rates were significantly higher (P = .0001) in positive tumors (median %S phase, 7.1%) than in negative tumors (4.1%). In a univariate cutpoint analysis, negative tumors (n = 388) versus low-positive tumors (IHC score = 2-6; n = 263) versus high-positive tumors (IHC score > 6; n = 99) showed progressively reduced disease-free survival (80% versus 72% versus 58% at 5 years, respectively; P < or = .05 for all pairwise comparisons). Analogous results for overall survival were 88% versus 84% versus 74%; only the result for negative versus high positive tumors was significant (P = .003). In a multivariate analysis, expression of p53 protein and high %S phase were independently associated with reduced disease-free survival (P = .008 and .01, respectively). CONCLUSIONS: Expression of mutant p53 protein was associated with high tumor proliferation rate, early disease recurrence, and early death in node-negative breast cancer. Despite the strong direct correlation between accumulation of p53 protein and tumor proliferation rate, both factors were independently associated with poor prognosis, suggesting that p53 may have other biological functions in addition to cell-cycle regulation. IMPLICATIONS: This test, when combined with other prognostic factors, may enhance our ability to identify node-negative breast cancer patients at high risk for early disease recurrence and/or death, for whom the use of adjuvant chemotherapy is unequivocally justified.

Loss of heterozygosity at D14S62 and metastatic potential of breast cancer.

BACKGROUND: In breast cancer progression, the prevalence of damage at specific genetic loci often increases with the stage of the lesion (i.e., from noninvasive to invasive to metastatic). By use of genetic markers and analysis of allelic imbalances (loss of heterozygosity [LOH]) to compare DNA samples from paired normal and breast tumor tissues, we examined whether specific genetic changes in primary breast cancers can serve as biomarkers of metastatic potential. METHODS: DNA samples from 76 patients with primary breast cancer (42 with axillary lymph node-negative disease and 34 with axillary lymph node-positive disease) were genotyped with four genetic markers spanning chromosome 14q31-q32. The intensity ratios of the two genetic alleles in normal-tumor DNA pairs were examined in genetically informative individuals. LOH was scored when the tumor allele intensity ratio (tumor allele 1/tumor allele 2) divided by the normal allele intensity ratio (normal allele 1/normal allele 2) was either less than 0.71 (tumor allele 1 LOH) or greater than 1. 4 (tumor allele 2 LOH). RESULTS/CONCLUSIONS: Contrary to our expectations, we found statistically significantly more LOH events at markers D14S62 (two-sided P =.001) and D14S51 (two-sided P =.02) in primary breast cancers from patients with lymph node-negative disease versus lymph node-positive disease, suggesting the presence of a gene in this region that affects metastatic potential. Analysis of small interstitial or terminal deletions in the tumors of six especially informative patients with lymph node-negative disease places the putative metastasis-related gene in a 1490-kilobase region near D14S62. IMPLICATIONS: LOH in the D14S62 region may impede the process of metastasis. Therefore, the D14S62 region LOH profile may have prognostic implications, and the isolation of the metastasis-related gene(s) in this region may lead to better diagnosis and treatment of breast cancer.

p53 is mutated in a subset of advanced-stage prostate cancers.

Inactivation of p53, a tumor suppressor gene, contributes to the genesis and/or progression of a substantial fraction of all human cancers, including > or = 50% of breast, lung, and colon carcinomas. Mutated p53 alleles typically contain missense single-base substitutions within exons 5-8 and encode abnormally stable p53 proteins that accumulate to high levels in tumor cell nuclei. To evaluate the frequency, type, and clinical significance of p53 mutation in human prostate cancer, archival tumor material from 150 prostate cancer patients was examined by immunohistochemistry (IHC) with anti-p53 antibodies. Abnormal nuclear p53 accumulation (IHC) was observed in 19 tumors (12.7%) and was strongly related to disease stage (23% of 69 stage III or IV tumors were IHC+ versus 4% of 74 stage 0-II tumors; P < 0.001, Fisher's exact test). The methods of polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing were used to identify mutations, predominantly missense single-base substitutions in exons 5, 7, or 8 in 9 of 14 IHC+ cases but in none of 20 IHC- cases; 5 of these mutations were G:C-->A:T transitions at CpG dinucleotides. These data indicate that mutated p53 alleles are quite uncommon in early prostate cancers but are found in 20-25% of advanced cancers, suggesting a role for p53 mutation in the progression of at least a subset of prostate cancers.

p53 protein accumulation detected by five different antibodies: relationship to prognosis and heat shock protein 70 in breast cancer.

Nuclear accumulation of p53 protein is associated with a poorer clinical outcome in breast cancer patients. Heat shock protein 70 (hsp70) is a chaperone that binds to mutant p53 and consequently could regulate its accumulation or localization. The aims of this study were to determine if the prognostic significance of p53 accumulation was dependent on the type of antibody used for detection and whether hsp70 was associated with this accumulation. Node-negative breast tumors (n = 169) were examined by immunohistochemistry for nuclear p53, cytoplasmic or nuclear hsp70, and for p53 gene alteration by single-strand conformation polymorphism analysis. Frozen sections of pulverized breast tumors were stained with five p53 antibodies (240, 1801, 421, BP53-12, and CM1), a cocktail of both 240 and 1801, and the hsp70 antibody C92. Protein level was expressed as the sum of a proportion and intensity score (total 0, 2-8) with > or = 2 defined as positive staining. The cocktail 240/1801 gave the highest rate of positive staining (45%), followed by BP53-12 (35%), 1801 (27%), 240 (25%), CM1 (24%), and 421 (18%), with a high correlation between antibodies. Positive staining with each individual antibody or the cocktail was significantly associated with estrogen receptor and progesterone receptor negativity, age < 50, and high S-phase fraction. Only staining detected by the 240/1801 cocktail was associated with significantly worse overall survival; 85 versus 70% at 5 years for p53-negative compared to p53-positive tumors, respectively (P = 0.02). There was no association between nuclear or cytoplasmic hsp70 staining and accumulation of p53. Patients that were p53-negative/cytoplasmic hsp70-positive had a better overall survival than those that were p53-negative/cytoplasmic hsp70-negative. No other combination of p53 and hsp70 status could further define subsets of patients with a significantly different prognosis compared to p53 status alone. Tumors without a detectable p53 gene alteration by single-strand conformation polymorphism but with accumulated p53 protein did not have relatively increased levels of hsp70. We conclude that in node-negative breast cancer, the cocktail of two antibodies, 240/1801, resulted in the highest rate of positive staining and was most strongly associated with overall survival compared with either antibody alone or with the other individual antibodies. By immunohistochemistry, nuclear accumulation of p53 was not associated with cytoplasmic or nuclear hsp70 levels.

Loss of heterozygosity events impeding breast cancer metastasis contain the MTA1 gene.

Breast cancer mortality is seldom attributable to the primary tumor, but rather to the presence of systemic (metastatic) disease. Axillary lymph node dissection can identify the presence of metastatic breast cancer cells and serves as a marker for systemic disease. Previous work in our laboratory determined that rates of loss of heterozygosity (LOH) of a 1.6-Mb region of chromosome 14q 31.2 is much higher in axillary lymph node-negative primary breast tumors than in axillary lymph node-positive primary breast tumors (P. O'Connell et al., J. NATL: Cancer INST:, 91: 1391-1397, 1999.). This unusual observation suggests that, whereas the LOH of this region promotes primary breast cancer formation, some gene(s) mapping to this 1.6-Mb region is rate-limiting for breast cancer metastasis. Thus, if primary breast cancers delete this region, their ability to metastasize decreases. To identify this gene(s), we have physically mapped this area of chromosome 14q, confirmed the position of two known genes and 13 other expressed sequence tags into this 1.6-Mb region. One of these, the metastasis-associated 1 (MTA1) gene, previously identified as a metastasis-promoting gene (Y. Toh et al., J. BIOL: CHEM:, 269: 22958-22963, 1994.), mapped to the center of our 1.6-Mb target region. Thus, MTA1 represents a strong candidate for this breast cancer metastasis-promoting gene.

Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors.

It is fairly well accepted that the presence of estrogen receptor (ER) and progesterone receptor (PgR) identifies breast cancer patients with a lower risk of relapse and better overall survival. But patients with discordant receptors, the ER+/PgR- phenotype, are often intermediate in clinical response. We focused upon this group of patients and have identified a truncated ER which is abundant in some ER+/PgR- breast tumors and which inhibits the binding of wild-type ER to its cognate response element. This variant interferes in a dominant negative manner with wild-type ER function and may represent a mechanism for modulation of estrogen responsiveness.

Mitosin (a new proliferation marker) correlates with clinical outcome in node-negative breast cancer.

Tumor proliferation rate is an important prognostic factor in breast cancer, and S-phase fraction (SPF), as measured by flow cytometry, is the most clinically validated of several methods for measuring it. However, flow cytometry is not well suited to evaluating the formalin-fixed, paraffin-embedded tumors that are routinely available or to the increasing number of small breast cancers. These and other limitations have motivated research into alternative methods for measuring proliferation, including immunohistochemistry (IHC) against cell cycle-related antigens, which are better suited for the evaluation of small archival tissue samples. Mitosin is a recently described 350 kD nuclear phosphoprotein that is expressed in the late G1, S, G2, and M phases of the cell cycle but not in G0. Using a new monoclonal antibody (14C10), this pilot study evaluated mitosin expression by IHC in a series of 386 node-negative, formalin-fixed, archival breast cancers and correlated the results with several prognostic factors and clinical outcome (median follow-up, 78 months; range 3-214 months). The median and range of mitosin positive cells were 7% and 1-47%, respectively. There was a strong positive correlation between mitosin and SPF (r = 0.57; P = 0.0001), and there were significant negative correlations with estrogen receptor, progesterone receptor, and patient age. Mitosin was not related to overall survival in this pilot study. However, in a univariate cutpoint analysis of disease-free survival (DFS), patients with high levels of mitosin (>9% positive cells) had significantly worse DFS than did patients with lower levels (68% versus 84% at 5 years, respectively). In a multivariate analysis of DFS, large tumor size (>2 cm) and high mitosin were the only independently significant predictors of recurrence (relative risks = 2.47 and 1.72, respectively) in a model containing the additional factors estrogen receptor, progesterone receptor, patient age, and SPF. These preliminary results suggest that mitosin as assessed by IHC may be superior to SPF as a prognostic factor in node-negative breast cancer, but additional studies are necessary to validate these promising findings.

A hypersensitive estrogen receptor-alpha mutation in premalignant breast lesions.

The best current model of breast cancer evolution suggests that most cancers arise from certain premalignant lesions. We have identified a common (34%) somatic mutation in the estrogen receptor (ER)-alpha gene in a series of 59 typical hyperplasias, a type of early premalignant breast lesion. The mutation, which affects the border of the hinge and hormone binding domains of ER-alpha, showed increased sensitivity to estrogen as compared with wild-type ER-alpha in stably transfected breast cancer cells, including markedly increased proliferation at subphysiological levels of estrogen. The mutated ER-alpha exhibits enhanced binding to the TIF-2 coactivator at low levels of hormone, which may partially explain its increased estrogen responsiveness. These data suggest that this mutation may promote or accelerate the development of cancer from premalignant breast lesions.

Growth factor signalling and response to endocrine therapy: the Royal Marsden Experience.

De novo resistance to endocrine therapy is a near-universal feature of oestrogen receptor (ER)- negative breast cancer. Although many ER-positive breast cancers also show no response to tamoxifen or aromatase inhibitors on objective clinical grounds the large majority show reduced proliferation indicating that some oestrogen dependence is present in almost all ER-positive breast cancer. In neoadjuvant studies HER2 positivity is associated with poor response rates to tamoxifen but not aromatase inhibitors, consistent with preclinical models. Acquired resistance to tamoxifen is associated with decreases in ER positivity but most recurrent lesions remain ER-positive. A small proportion of these show increased HER2 expression and in these patients increased phospho-p38 may contribute to the tamoxifen-resistant phenotype. There is an unfortunate paucity of clinical and biological data on acquired resistance to aromatase inhibitors.

Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer.

PURPOSE: Immunohistochemistry (IHC) is a newer technique for assessing the estrogen receptor (ER) status of breast cancers, with the potential to overcome many of the shortcomings associated with the traditional ligand-binding assay (LBA). The purpose of this study was to evaluate the ability of ER status determination by IHC, compared with LBA, to predict clinical outcome-especially response to adjuvant endocrine therapy-in a large number of patients with long-term clinical follow-up. PATIENTS AND METHODS: ER status was evaluated in 1,982 primary breast cancers by IHC on formalin-fixed paraffin-embedded tissue sections, using antibody 6F11 and standard methodology. Slides were scored on a scale representing the estimated proportion and intensity of positive-staining tumor cells (range, 0 to 8). Results were compared with ER values obtained by the LBA in the same tumors and to clinical outcome. RESULTS: An IHC score of greater than 2 (corresponding to as few as 1% to 10% weakly positive cells) was used to define ER positivity on the basis of a univariate cut-point analysis of all possible scores and disease-free survival (DFS) in patients receiving any adjuvant endocrine therapy. Using this definition, 71% of all tumors were determined to be ER-positive by IHC, and the level of agreement with the LBA was 86%. In multivariate analyses of patients receiving adjuvant endocrine therapy alone, ER status determined by IHC was better than that determined by the LBA at predicting improved DFS (hazard ratios/P = 0.474/.0008 and 0.707/.3214, respectively) and equivalent at predicting overall survival (0.379/.0001 and 0.381/.0003, respectively). CONCLUSION: IHC is superior to the LBA for assessing ER status in primary breast cancer because it is easier, safer, and less expensive, and has an equivalent or better ability to predict response to adjuvant endocrine therapy.

Tumor characteristics and clinical outcome of tubular and mucinous breast carcinomas.

PURPOSE: To comprehensively characterize the clinical and biologic features of tubular and mucinous carcinomas in a large cohort of patients and to relate this to clinical outcome and management. PATIENTS AND METHODS: The clinical and biologic features of 444 patients with tubular and 1,221 patients with mucinous carcinomas were compared with those of 43,587 patients with infiltrating ductal carcinoma, not otherwise specified (NOS). Disease-free survival (DFS) and overall survival (OS) for patients with tubular and mucinous carcinomas were compared with those of patients with NOS carcinomas and with age-matched sets from the general population. RESULTS: Tubular and mucinous carcinomas were more likely to occur in older patients, be smaller in size (tubular only), have substantially less nodal involvement, be estrogen receptor- and progesterone receptor-positive, have a lower S-phase fraction, be diploid, and be c-erbB-2- and epidermal growth factor receptor-negative compared with NOS carcinomas. Axillary node involvement was a poor prognostic feature in mucinous but not tubular carcinomas. Mucinous carcinomas < or = 1 cm had a < or = 5% incidence of node involvement. The 5-year DFS and OS were 94% and 88% for tubular, 90% and 80% for mucinous, and 80% and 77% for NOS carcinoma, respectively (P < .001 for differences among all three types for both DFS and OS). The 5-year OS of females from the general population age-matched to the patients with tubular and mucinous carcinomas was 89% and 82%, respectively, which is not different from the OS of patients with tubular or mucinous carcinomas. CONCLUSION: The biologic phenotype of tubular and mucinous carcinomas is quite favorable. Consistent with this observation, the survival of patients with tubular and mucinous carcinomas is similar to that of the general population. Systemic adjuvant therapy and node dissection may be avoided in many patients with these special types of carcinoma.

Cathepsin D by western blotting and immunohistochemistry: failure to confirm correlations with prognosis in node-negative breast cancer.

PURPOSE: We attempted to replicate and improve on our previous study (N Engl J Med 322:297-302, 1990) that showed that 34-kd cathepsin D levels as determined by Western blotting strongly correlated with disease-free survival (DFS) and overall survival (OS) of axillary node-negative (N-) breast cancer patients. We also examined the prognostic significance of cathepsin D measured by immunohistochemistry (IHC) in these patients. PATIENTS AND METHODS: Western blotting was performed on cytosols from frozen tumor specimens of 927 N- breast cancer patients in the San Antonio Breast Tumor Bank. The monoclonal antibody M1G8 was used to detect cathepsin D (in previous study, a polyclonal antiserum had been used). The same monoclonal antibody was also used for frozen-section IHC staining of tumor specimens from 562 N- patients from the same tumor bank. Levels of cathepsin D expression were then correlated with DFS and OS. RESULTS: Although the levels of cathepsin D expression as measured by Western blotting and IHC correlated with each other and with levels of cathepsin D measured in previous work using Western blotting with the polyclonal antiserum, in this present study, using the monoclonal antibody M1G8, we were unable to demonstrate that cathepsin D expression (measured by either Western blotting or by IHC) correlates with DFS or OS. CONCLUSION: In this study, cathepsin D expression as determined by either Western blotting or IHC using the monoclonal antibody M1G8 failed to improve the prognostic evaluation of N- breast cancer patients. The role of cathepsin D expression as a prognostic factor is still not precisely defined, raising questions about its use in the routine evaluation of N- breast cancer patients.

HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma.

PURPOSE: Amplification and/or overexpression of the HER-2/neu oncogene have been shown to correlate with poor clinical outcome in patients with axillary node-positive breast cancer. In contrast, the prognostic significance of HER-2/neu in node-negative disease is controversial. This study was undertaken to evaluate further the relationship between HER-2/neu and clinical outcome in node-negative disease. PATIENTS AND METHODS: Overexpression of HER-2/neu was evaluated by permanent-section immunohistochemistry in tumors from 613 patients with long-term clinical follow-up enrolled in the Intergroup Study 0011. Patients were stratified into low-risk (n = 307) and high-risk (n = 306) groups on the basis of tumor size and estrogen-receptor (ER) status. Low-risk patients were defined as having small (less than 3 cm), ER-positive tumors and were observed without additional treatment after initial surgery. High-risk patients had either ER-negative or large (greater than or equal to 3 cm), ER-positive tumors and were randomized to be observed (n = 146) or to receive adjuvant chemotherapy (n = 160) after surgery. RESULTS: The rate of HER-2/neu overexpression was 14.3% in all tumors combined and was higher in invasive carcinomas with (21.5%) than without (11.2%) a significant noninvasive or in situ histologic component (P less than .0001). There was no relationship between overexpression and clinical outcome in the natural history setting of combined low-risk and high-risk patients not receiving adjuvant therapy (n = 453). Based on the reasoning that the influence of HER-2/neu may have been obscured by high-risk features and/or the presence of noninvasive carcinoma, we also analyzed the subset of patients with low-risk lesions not containing a significant in situ component (n = 179). Patients of this group with HER-2/neu-positive tumors showed only 40% disease-free survival (DFS) at 5 years, compared with over 80% in patients with HER-2/neu-negative tumors (P less than .0001). A similar inverse correlation was observed between overexpression and overall survival in the same group of patients (P = .0001). In a separate analysis involving patients receiving adjuvant chemotherapy, those with HER-2/neu-negative tumors showed significantly improved DFS in response to therapy compared with patients with HER-2/neu-positive tumors. CONCLUSION: Overexpression of HER-2/neu is associated with poor clinical outcome in a subset of node-negative patients with small, ER-positive, predominantly invasive tumors and may play a role in resistance to adjuvant chemotherapy.

Prognostic value of p53 for local failure in mastectomy-treated breast cancer patients.

PURPOSE: The loss of p53 function is a recognized adverse prognostic factor in invasive breast cancer. Several studies have shown a relationship between the nuclear accumulation of p53 protein (a surrogate marker of p53 inactivation) and poor disease-free and overall survival. In general, however, these studies did not report the prognostic value of p53 for local failure, which we have therefore assessed retrospectively here. MATERIALS AND METHODS: Accumulation of p53 protein was evaluated by immunohistochemistry in 1,530 mastectomy-treated breast cancer patients (259 radiation therapy [RT]- and 1,271 mastectomy only [No RT]-treated patients). Statistical comparisons were made between p53 protein accumulation, estrogen/progesterone receptors, nodal status, tumor size, and local failure rate (LFR). Local failure was defined as tumor recurrence involving the chest wall and/or the ipsilateral supraclavicular/axillary lymph nodes. The median follow-up period was 62 months. RESULTS: In the No RT group, the LFR was 9.1% and 16. 5% in p53-negative and p53-positive patients, respectively (P<.001). Multivariate analysis revealed that p53 protein accumulation was significantly associated with an increased risk of local relapse (relative risk [RR], 1.7; 95% confidence interval [CI], 1.2 to 2.4). Nodal status and tumor size were also significant factors. In the RT group, the LFR was 9.3% and 21.5% in p53-negative and p53-positive patients, respectively (P = .009). Multivariate analysis revealed that p53 protein accumulation was significantly associated with an increased risk of local relapse (RR, 2.5; 95% CI, 1.1 to 5.7), as was nodal status. CONCLUSION: Nuclear accumulation of p53 protein is independently associated with a significantly increased local failure rate in breast cancer patients treated with mastectomy, with or without radiation.

Biologic markers as predictors of clinical outcome from systemic therapy for primary operable breast cancer.

PURPOSE: To determine whether pretreatment clinical features and molecular markers, together with changes in these factors, can predict treatment response and survival in patients with primary operable breast cancer who receive neoadjuvant therapy. PATIENTS AND METHODS: Mitoxantrone, methotrexate (with or without mitomycin), and tamoxifen chemoendocrine therapy was administered to 158 patients before surgery. Clinical response was assessed after four cycles of treatment. Fine-needle aspiration cytology for estrogen receptor (ER), progesterone receptor (PgR), c-erbB-2, p53, bcl-2, Ki67, S-phase fraction (SPF), and ploidy were performed pretreatment and repeated on day 10 or day 21 after the first cycle of treatment. RESULTS: Good clinical response (GCR, defined as complete response or minimal residual disease) was achieved in 31% of patients (49 of 158). Tumor size, nodal disease, response, ER, PgR, c-erbB-2, p53, bcl-2, Ki67, SPF, and ploidy were analyzed as predictors of survival. By univariate analysis, node-positive disease (P =.05), lack of ER (P <.05) and PgR (P <.05), and failure to attain GCR (P =.008) were associated with a significantly increased risk of relapse. A significantly increased risk of death was associated with node-positive disease (P =.02), lack of ER expression (P =.04), and failure to attain GCR. By multivariate analysis, GCR was an independent predictor for survival (P =.05). ER expression (P =.03), absence of c-erbB-2 (P =.03), and a decrease in Ki67 on day 10 or day 21 of the first cycle (P <.05) significantly predicted for subsequent GCR. CONCLUSION: Molecular markers may be used to predict the likelihood of achieving GCR, which seems to be a valid surrogate marker for survival.

bcl-2, p53, and response to tamoxifen in estrogen receptor-positive metastatic breast cancer: a Southwest Oncology Group study.

PURPOSE: To test the hypothesis that high bcl-2 expression and accumulation of p53 protein, both of which should inhibit apoptosis, are associated with a poorer tamoxifen response and a more aggressive clinical course in estrogen receptor (ER)-positive metastatic breast cancer. METHODS: A total of 205 paraffin-embedded tumor blocks were evaluated for nuclear p53 (a marker of p53 inactivation) and cytoplasmic bcl-2 by immunohistochemistry (IHC). All patients received tamoxifen as initial therapy for metastatic disease. The study began in 1982 and follow-up duration of the 24 patients last known alive is 8 years. RESULTS: Response to tamoxifen and time to treatment failure (TTF) were not significantly associated with p53 status, although patients with higher p53 had a worse survival (P = .008; median, 36 v 20 months). Higher bcl-2 expression was associated with higher levels of ER (P = .02), better response to tamoxifen (62% v 49%; P = .07), longer TTF (median, 9 v 5 months; P = .002), and better survival (median, 40 months v 25 months; P = .009). In multivariate analyses, including ER, progesterone receptor (PgR), and p53, high bcl-2 remained significantly associated with a longer TTF (P = .007) and survival (P = .07). p53 status was a significant factor for shorter survival (P = .05), but not for TTF (P = .61). CONCLUSION: p53 status, as determined by IHC is not significantly associated with response to tamoxifen, although tumors with altered p53 protein are inherently more aggressive. Contrary to expectation, high bcl-2 identifies a relatively indolent phenotype of ER-positive metastatic breast cancer, in which patients experience a better clinical response to tamoxifen and a longer survival.