Abrogated response to cellular stress identifies DCIS associated with subsequent tumor events and defines basal-like breast tumors.

Approximately 15%-30% of women diagnosed with ductal carcinoma in situ (DCIS) develop a subsequent tumor event within 10 years after surgical lumpectomy. To date, little is known about the molecular pathways that confer this differential risk for developing subsequent disease. In this study, we demonstrate that expression of biomarkers indicative of an abrogated response to cellular stress predicts DCIS with worse outcome and is a defining characteristic of basal-like invasive tumors. Mechanistic studies identify the Rb pathway as a key regulator of this response. Conversely, biomarkers indicative of an intact response to cellular stress are strongly associated with a disease-free prognosis. Assessment of these biomarkers in DCIS begins to allow prediction of tumor formation years before it actually occurs.

Genomic and transcriptional aberrations linked to breast cancer pathophysiologies.

This study explores the roles of genome copy number abnormalities (CNAs) in breast cancer pathophysiology by identifying associations between recurrent CNAs, gene expression, and clinical outcome in a set of aggressively treated early-stage breast tumors. It shows that the recurrent CNAs differ between tumor subtypes defined by expression pattern and that stratification of patients according to outcome can be improved by measuring both expression and copy number, especially high-level amplification. Sixty-six genes deregulated by the high-level amplifications are potential therapeutic targets. Nine of these (FGFR1, IKBKB, ERBB2, PROCC, ADAM9, FNTA, ACACA, PNMT, and NR1D1) are considered druggable. Low-level CNAs appear to contribute to cancer progression by altering RNA and cellular metabolism.

In situ analyses of genome instability in breast cancer.

Transition through telomere crisis is thought to be a crucial event in the development of most breast carcinomas. Our goal in this study was to determine where this occurs in the context of histologically defined breast cancer progression. To this end, we assessed genome instability (using fluorescence in situ hybridization) and other features associated with telomere crisis in normal ductal epithelium, usual ductal hyperplasia, ductal carcinoma in situ and invasive cancer. We modeled this process in vitro by measuring these same features in human mammary epithelial cell cultures during ZNF217-mediated transition through telomere crisis and immortalization. Taken together, the data suggest that transition through telomere crisis and immortalization in breast cancer occurs during progression from usual ductal hyperplasia to ductal carcinoma in situ.

Increased beta1 integrin is associated with decreased survival in invasive breast cancer.

Aberrant microenvironments and loss of balance in cell-extracellular matrix signaling are associated with breast cancer invasion, metastasis, and resistance to therapy. We have recently shown that increased beta1 integrin signaling is involved in malignant progression and that inhibitory antibody to beta1 integrin leads to selective apoptosis and decreased proliferation in three-dimensional cultures and in xenograft models of breast cancer in vivo. To investigate the clinical importance of these findings, in the present study we examined the expression of beta1 integrin and extracellular beta1 integrin ligands fibronectin and laminin-1 in a cohort of 249 breast cancer patients who had a median follow-up of 8.4 years. Among the 149 scorable cases, the highest beta1 integrin intensity score (3+ versus 0-2+) was associated with significantly decreased 10-year overall survival of 48% versus 71% (P<0.03) and decreased disease-free survival of 50% versus 80% (P<0.05). Importantly, high fibronectin expression was associated with decreased overall and disease-free survival on univariate analysis (P<0.04) and beta1 integrin intensity score was significantly correlated with fibronectin expression (Kendall's tau-b=0.19; P=0.03). In a multivariate Cox proportional hazards model, beta1 integrin intensity score remained a significant independent predictor of overall survival [hazard ratio (HR), 1.69; 95% confidence interval (95% CI), 1.19-2.38; P<0.003] and disease-free survival (HR, 1.87; 95% CI, 1.21-2.88; P<0.005). These findings show that beta1 integrin expression has potential prognostic value in invasive breast cancer and that coexpression of fibronectin may help identify patients with more aggressive tumors who may benefit from targeted therapy.

Risk estimation of distant metastasis in node-negative, estrogen receptor-positive breast cancer patients using an RT-PCR based prognostic expression signature.

BACKGROUND: Given the large number of genes purported to be prognostic for breast cancer, it would be optimal if the genes identified are not confounded by the continuously changing systemic therapies. The aim of this study was to discover and validate a breast cancer prognostic expression signature for distant metastasis in untreated, early stage, lymph node-negative (N-) estrogen receptor-positive (ER+) patients with extensive follow-up times. METHODS: 197 genes previously associated with metastasis and ER status were profiled from 142 untreated breast cancer subjects. A "metastasis score" (MS) representing fourteen differentially expressed genes was developed and evaluated for its association with distant-metastasis-free survival (DMFS). Categorical risk classification was established from the continuous MS and further evaluated on an independent set of 279 untreated subjects. A third set of 45 subjects was tested to determine the prognostic performance of the MS in tamoxifen-treated women. RESULTS: A 14-gene signature was found to be significantly associated (p < 0.05) with distant metastasis in a training set and subsequently in an independent validation set. In the validation set, the hazard ratios (HR) of the high risk compared to low risk groups were 4.02 (95% CI 1.91-8.44) for the endpoint of DMFS and 1.97 (95% CI 1.28 to 3.04) for overall survival after adjustment for age, tumor size and grade. The low and high MS risk groups had 10-year estimates (95% CI) of 96% (90-99%) and 72% (64-78%) respectively, for DMFS and 91% (84-95%) and 68% (61-75%), respectively for overall survival. Performance characteristics of the signature in the two sets were similar. Ki-67 labeling index (LI) was predictive for recurrent disease in the training set, but lost significance after adjustment for the expression signature. In a study of tamoxifen-treated patients, the HR for DMFS in high compared to low risk groups was 3.61 (95% CI 0.86-15.14). CONCLUSION: The 14-gene signature is significantly associated with risk of distant metastasis. The signature has a predominance of proliferation genes which have prognostic significance above that of Ki-67 LI and may aid in prioritizing future mechanistic studies and therapeutic interventions.

Aging impacts transcriptomes but not genomes of hormone-dependent breast cancers.

INTRODUCTION: Age is one of the most important risk factors for human malignancies, including breast cancer; in addition, age at diagnosis has been shown to be an independent indicator of breast cancer prognosis. Except for inherited forms of breast cancer, however, there is little genetic or epigenetic understanding of the biological basis linking aging with sporadic breast cancer incidence and its clinical behavior. METHODS: DNA and RNA samples from matched estrogen receptor (ER)-positive sporadic breast cancers diagnosed in either younger (age or= 70 years) Caucasian women were analyzed by array comparative genomic hybridization and by expression microarrays. Array comparative genomic hybridization data were analyzed using hierarchical clustering and supervised age cohort comparisons. Expression microarray data were analyzed using hierarchical clustering and gene set enrichment analysis; differential gene expression was also determined by conditional permutation, and an age signature was derived using prediction analysis of microarrays. RESULTS: Hierarchical clustering of genome-wide copy-number changes in 71 ER-positive DNA samples (27 younger women, 44 older women) demonstrated two age-independent genotypes; one with few genomic changes other than 1q gain/16q loss, and another with amplifications and low-level gains/losses. Age cohort comparisons showed no significant differences in total or site-specific genomic breaks and amplicon frequencies. Hierarchical clustering of 5.1 K genes variably expressed in 101 ER-positive RNA samples (53 younger women, 48 older women) identified six transcriptome subtypes with an apparent age bias (P < 0.05). Samples with higher expression of a poor outcome-associated proliferation signature were predominantly (65%) younger cases. Supervised analysis identified cancer-associated genes differentially expressed between the cohorts; with younger cases expressing more cell cycle genes and more than threefold higher levels of the growth factor amphiregulin (AREG), and with older cases expressing higher levels of four different homeobox (HOX) genes in addition to ER (ESR1). An age signature validated against two other independent breast cancer datasets proved to have >80% accuracy in discerning younger from older ER-positive breast cancer cases with characteristic differences in AREG and ESR1 expression. CONCLUSION: These findings suggest that epigenetic transcriptome changes, more than genotypic variation, account for age-associated differences in sporadic breast cancer incidence and prognosis.

Enhanced NF kappa B and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer.

BACKGROUND: Signaling pathways that converge on two different transcription factor complexes, NFkappaB and AP-1, have been identified in estrogen receptor (ER)-positive breast cancers resistant to the antiestrogen, tamoxifen. METHODS: Two cell line models of tamoxifen-resistant ER-positive breast cancer, MCF7/HER2 and BT474, showing increased AP-1 and NFkappaB DNA-binding and transcriptional activities, were studied to compare tamoxifen effects on NFkappaB and AP-1 regulated reporter genes relative to tamoxifen-sensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA) or the proteasome inhibitor bortezomib (PS341), alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF node-negative ER-positive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFkappaB and AP-1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using node-negative ER-positive breast cancer cases identified from three other published data sets (Rotterdam, n = 209; Amsterdam, n = 68; Basel, n = 108), each having different patient age and adjuvant tamoxifen treatment characteristics. RESULTS: Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFkappaB and AP-1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional co-repressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFkappaB and AP-1 upregulated genes--cyclin D1, uPA and VEGF--capable of dichotomizing node-negative ER-positive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of node-negative ER-positive breast cancer cases (UCSF, Rotterdam, Amsterdam, Basel), high expression of all three NFkappaB and AP-1 upregulated genes was associated with earliest metastatic relapse. CONCLUSION: Altogether, these findings implicate increased NFkappaB and AP-1 transcriptional responses with tamoxifen resistant breast cancer and early metastatic relapse, especially in younger patients. These findings also suggest that agents capable of preventing NFkappaB and AP-1 gene activation may prove useful in restoring the endocrine responsiveness of such high-risk ER-positive breast cancers.

Identification of a robust gene signature that predicts breast cancer outcome in independent data sets.

BACKGROUND: Breast cancer is a heterogeneous disease, presenting with a wide range of histologic, clinical, and genetic features. Microarray technology has shown promise in predicting outcome in these patients. METHODS: We profiled 162 breast tumors using expression microarrays to stratify tumors based on gene expression. A subset of 55 tumors with extensive follow-up was used to identify gene sets that predicted outcome. The predictive gene set was further tested in previously published data sets. RESULTS: We used different statistical methods to identify three gene sets associated with disease free survival. A fourth gene set, consisting of 21 genes in common to all three sets, also had the ability to predict patient outcome. To validate the predictive utility of this derived gene set, it was tested in two published data sets from other groups. This gene set resulted in significant separation of patients on the basis of survival in these data sets, correctly predicting outcome in 62-65% of patients. By comparing outcome prediction within subgroups based on ER status, grade, and nodal status, we found that our gene set was most effective in predicting outcome in ER positive and node negative tumors. CONCLUSION: This robust gene selection with extensive validation has identified a predictive gene set that may have clinical utility for outcome prediction in breast cancer patients.

p38 regulates cyclooxygenase-2 in human mammary epithelial cells and is activated in premalignant tissue.

The immediate-early gene, cyclooxygenase-2 (COX-2), is induced in a variety of inflammatory and neoplastic processes and is believed to play an important role in tumorigenesis. In this study, we identify an important upstream regulatory pathway of COX-2 expression in variant human mammary epithelial cells (vHMEC), which has been shown to exhibit phenotypes important for malignancy. We find that the stress-activated kinase, p38, is phosphorylated and activated in vHMEC compared with HMEC and is responsible for the expression of COX-2 in vHMEC as cells grow in culture. Furthermore in this capacity, p38 acts to stabilize the COX-2 transcript rather than activate COX-2 transcription. Inhibition of p38 kinase, using a chemical inhibitor, down-regulates COX-2 and decreases cell survival. Examination of archived tissue from women with ductal carcinoma in situ reveals epithelial cells that not only overexpress COX-2 but also have an abundance of activated phospho-p38 in the nucleus and cytoplasm, mirroring the expression observed in vitro. These epithelial cells are found within premalignant lesions as well as in fields of morphologically normal tissue that surround the lesions. In contrast, low phospho-p38 staining was observed in the majority of normal tissue obtained from reduction mammoplasty. These data help define the regulation of COX-2 expression in early carcinogenesis and provide alternative candidates for targeted prevention of COX-2-induced phenotypes and breast cancer.

Arsenic-related chromosomal alterations in bladder cancer.

BACKGROUND: Previous studies have demonstrated that ingestion of arsenic in drinking water is a strong risk factor for several forms of cancer, including bladder cancer. It is not known whether arsenic-related cancers are genetically similar to cancers in unexposed individuals or what mechanisms of carcinogenesis may underlie their formation. This study was designed to compare chromosomal alterations in bladder cancers of arsenic-exposed individuals to provide insight into the mechanism of how arsenic may induce or promote cancer. METHODS: A case-case study was conducted in Argentina and Chile examining chromosomal alterations in bladder tumor DNA in 123 patients who had been exposed to arsenic in their drinking water. Patients were placed into one of four arsenic exposure categories according to their average 5-year peak arsenic exposure. Patients were also classified as ever smokers or never smokers. Comparative genomic hybridization was used to identify chromosomal alterations throughout the genome. All statistical tests were two-sided. RESULTS: The total number of chromosomal alterations was higher in individuals exposed to higher arsenic levels (5.7 +/- 5.1, 5.6 +/- 5.1, 7.3 +/- 7.4, and 9.1 +/- 6.5 [mean +/- standard deviation] chromosomal alterations per tumor with increasing arsenic exposure; P(trend) =.02, adjusted for stage and grade). The trend was stronger in high-grade (G2-G3) tumors (6.3 +/- 5.5, 8.3 +/- 4.7, 10.3 +/- 7.8, and 10.5 +/- 6.4 alterations per tumor; P(trend) =.01) than it was in low-grade (G1) tumors (3.5 +/- 3.1, 1.1 +/- 1.1, 2.5 +/- 2.5, and 3.6 +/- 3.2 alterations per tumor; P(trend) =.79). The mean number of chromosomal alterations also increased with tumor stage and grade (P(trend)<.001) independently of arsenic exposure but was not associated with smoking history. Deletion of part or all of chromosome 17p (P(trend)<.001) showed the strongest association with arsenic exposure. CONCLUSIONS: Bladder tumors in patients with higher levels of arsenic exposure showed higher levels of chromosomal instability. Most of the chromosomal alterations associated with arsenic exposure were also associated with tumor stage and grade, raising the possibility that bladder tumors from arsenic-exposed patients may behave more aggressively than tumors from unexposed patients.

Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia.

Cultures of human mammary epithelial cells (HMECs) contain a subpopulation of variant cells with the capacity to propagate beyond an in vitro proliferation barrier. These variant HMECs, which contain hypermethylated and silenced p16(INK4a) (p16) promoters, eventually accumulate multiple chromosomal changes, many of which are similar to those detected in premalignant and malignant lesions of breast cancer. To determine the origin of these variant HMECs in culture, we used Luria-Delbrück fluctuation analysis and found that variant HMECs exist within the population before the proliferation barrier, thereby raising the possibility that variant HMECs exist in vivo before cultivation. To test this hypothesis, we examined mammary tissue from normal women for evidence of p16 promoter hypermethylation. Here we show that epithelial cells with methylation of p16 promoter sequences occur in focal patches of histologically normal mammary tissue of a substantial fraction of healthy, cancer-free women.

Cyclooxygenase-2 expression is related to nuclear grade in ductal carcinoma in situ and is increased in its normal adjacent epithelium.

Cyclooxygenase-2 (COX-2) is emerging as an important cancer biomarker and is now an experimental target for solid tumor treatment.However, no study has exclusively focused on COX-2 expression in early lesions such as ductal carcinoma in situ (DCIS). We examined COX-2 expression by immunohistochemistry in 46 cases of women undergoing surgical resection for DCIS. We found that COX-2 expression was detected in 85% of all DCIS specimens, with increased COX-2 staining correlating with higher nuclear grade. Strikingly, COX-2 staining intensity in the normal adjacent epithelium was stronger than in the DCIS lesion itself. Our observations demonstrate that COX-2 is up-regulated in the normal adjacent epithelium and supports the hypothesis that the surrounding epithelial tissue is part of the disease process in DCIS.

Expression of the coxsackie adenovirus receptor in normal prostate and in primary and metastatic prostate carcinoma: potential relevance to gene therapy.

Adenovirus-based gene therapy may provide an alternative mode of treatment for prostate cancer, especially for late-stage and androgen-independent disease for which there is currently no effective treatment. Efficient adenovirus infection of target cells depends upon the presence of the coxsackie adenovirus cell surface receptor, CAR, which is the primary receptor for group C adenoviruses and is important for the attachment of adenovirus to the cell membrane. To evaluate the potential efficacy of adenoviral therapy for prostate cancer, we evaluated CAR expression in normal prostate tissue and in prostate carcinoma of increasing Gleason grades in paraffin-embedded, archival tissues using a polyclonal antibody raised against human CAR. Immunohistochemical analysis of benign prostate epithelia demonstrated intense luminal and lateral cell membrane staining. There was a statistically significant difference in CAR membrane expression with respect to Gleason score. In addition, metastatic prostate specimens demonstrated strong membrane staining for CAR. Adenovirus therapy may, therefore, provide an alternate modality in the treatment of prostate cancer and may be especially efficacious in the treatment of metastatic disease.

Differentiation of lobular versus ductal breast carcinomas by expression microarray analysis.

Invasive lobular and ductal breast tumors have distinct histologies and clinical presentation. Other than altered expression of E-cadherin, little is known about the underlying biology that distinguishes the tumor subtypes. We used cDNA microarrays to identify genes differentially expressed between lobular and ductal tumors. Unsupervised clustering of tumors failed to distinguish between the two subtypes. Prediction analysis for microarrays (PAM) was able to predict tumor type with an accuracy of 93.7%. Genes that were significantly differentially expressed between the two groups were identified by MaxT permutation analysis using t tests (20 cDNA clones and 10 unique genes), significance analysis for microarrays (33 cDNA clones and 15 genes, at an estimated false discovery rate of 2%), and PAM (31 cDNAs and 15 genes). There were 8 genes identified by all three of these related methods (E-cadherin, survivin, cathepsin B, TPI1, SPRY1, SCYA14, TFAP2B, and thrombospondin 4), and an additional 3 that were identified by significance analysis for microarrays and PAM (osteopontin, HLA-G, and CHC1). To validate the differential expression of these genes, 7 of them were tested by real-time quantitative PCR, which verified that they were differentially expressed in lobular versus ductal tumors. In conclusion, specific changes in gene expression distinguish lobular from ductal breast carcinomas. These genes may be important in understanding the basis of phenotypic differences among breast cancers.

Patterns of chromosomal alterations in breast ductal carcinoma in situ.

PURPOSE: Ductal carcinoma in situ (DCIS) is thought to be a nonobligate precursor of invasive cancer. Genomic changes specific to pure DCIS versus invasive cancer, as well as alterations unique to individual DCIS subtypes, have not been fully defined. EXPERIMENTAL DESIGN: Chromosomal copy number alterations were examined by comparative genomic hybridization in 34 cases of pure DCIS and compared with 12 cases of paired synchronous DCIS and invasive ductal cancer, as well as to 146 additional cases of invasive breast cancer of ductal or lobular histology. Genomic differences between high-grade and low/intermediate-grade DCIS, as well as between pure DCIS and invasive cancer, were identified. RESULTS: Pure DCIS showed almost the same degree of chromosomal instability as invasive ductal cancers. A higher proportion of low/intermediate-grade versus high-grade DCIS had loss of 16q (65 versus 12%, respectively; P = 0.002). When compared with lower grade DCIS, high-grade DCIS exhibited more frequent gain of 17q (65 versus 41%; P = 0.15) and higher frequency loss of 8p (77 versus 41%; P = 0.04). Chromosomal alterations in those cases with synchronous DCIS and invasive ductal cancer showed a high degree of shared changes within the two components. CONCLUSIONS: DCIS is genetically advanced, showing a similar degree of chromosomal alterations as invasive ductal cancer. The pattern of alterations differed between high- and low/intermediate-grade DCIS, supporting a model in which different histological grades of DCIS are associated with distinct genomic changes. These regions of chromosomal alterations may be potential targets for treatment and/or markers of prognosis.

Cytology of ductal lavage fluid of the breast.

The cytologic evaluation of nipple aspirate fluids has been shown to identify women at increased risk for developing breast cancer. One limitation of this assay is the often scant cellularity of the specimen. An improved technique, ductal lavage, utilizes a microcatheter inserted into individual breast ducts to collect large numbers of cells for cytologic evaluation. Epithelial cells in ductal lavage fluids can be categorized as benign, malignant, or showing mildly or markedly atypical changes. The cell characteristics which were most helpful in identifying abnormal cells were related to cell arrangement, cell size, nuclear size, and size variation, nuclear membrane irregularity, chromatin granularity, and the presence of large nucleoli. Cell size, nuclear size variation, and large nucleoli were the most robust features, as determined by agreement between two pathologists. Moderate cell enlargement and the presence of large nucleoli were the features selected by structured tree analysis for classifying the specimens into the diagnostic groups. The similarity of the cytology of ductal lavage fluid to nipple aspirate fluid strongly suggests that these specimens will also be useful for predicting breast cancer risk.

Multiple sampling for increasing the diagnostic sensitivity of nipple aspirate fluid for atypical cytology.

OBJECTIVE: To determine if repeated collection of nipple aspirate fluid (NAF) can improve the diagnostic sensitivity for cytologic atypia, a marker of increased risk of breast cancer. STUDY DESIGN: Two hundred sixty-seven women without known breast disease volunteered for NAF cytology at 5 6-month intervals over 2 years. NAF samples were prepared on Millipore filters (Millipore Filter Corp., Bedford, Massachusetts, U.S.A.) and stained with a modified Papanicolaou method. Fluid availability and cellular abnormalities were evaluated for each collection attempt. Cellular findings were classified as benign, hyperplasia or atypia. RESULTS: NAF was obtained from 178 women (66.6%) at the first visit and from an additional 15, 10, 2 and 4 women at visits 2, 3, 4 and 5, respectively, for a cumulative total of 78.2% by visit 5. The number of women yielding NAF containing hyperplastic or atypical epithelial cells was determined at each visit. Hyperplastic cells were found in 34 (19.1%) at visit 1 and in an additional 20, 10, 5 and 4 women at visits 2, 3, 4 and 5, respectively. Atypical epithelial cells were present in 12 (6.7%) women at the initial visit and in an additional 11, 7, 5 and 1 women at visits 2, 3, 4 and 5, respectively, for a cumulative percent of 18.2 at visit 5. NAF could not be obtained from 58 women at any visit. CONCLUSION: These findings suggest that an optimum collection method for NAF cytology should consist of at least 3 or 4 separate fluid aspiration attempts. Reviewing repeated multiple samples instead of 1 increases the number of women who can be evaluated and the likelihood of detecting cytologic atypia.

Biomarker expression and risk of subsequent tumors after initial ductal carcinoma in situ diagnosis.

BACKGROUND: Studies have failed to identify characteristics of women who have been diagnosed with ductal carcinoma in situ (DCIS) and have a high or low risk of subsequent invasive cancer. METHODS: We conducted a nested case-control study in a population-based cohort of 1162 women who were diagnosed with DCIS and treated by lumpectomy alone from 1983 to 1994. We collected clinical characteristics and information on subsequent tumors, defined as invasive breast cancer or DCIS diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a regional or distant site greater than 6 months after initial treatment of DCIS (N = 324). We also conducted standardized pathology reviews and immunohistochemical staining for the estrogen receptor (ER), progesterone receptor, Ki67 antigen, p53, p16, epidermal growth factor receptor-2 (ERBB2, HER2/neu oncoprotein), and cyclooxygenase-2 (COX-2) on the initial paraffin-embedded DCIS tissue. Competing risk models were used to determine factors associated with risk of subsequent invasive cancer vs DCIS, and cumulative incidence survival functions were used to estimate 8-year risk. RESULTS: Factors associated with subsequent invasive cancer differed from those associated with subsequent DCIS. Eight-year risk of subsequent invasive cancer was statistically significantly (P = .018) higher for women with initial DCIS lesions that were detected by palpation or that were p16, COX-2, and Ki67 triple positive (p16(+)COX-2(+)Ki67(+)) (19.6%, 95% confidence interval [CI] = 18.0% to 21.3%) than for women with initial lesions that were detected by mammography and were p16, COX-2, and Ki67 triple negative (p16(-)COX-2(-)Ki67(-)) (4.1%, 95% CI = 3.4% to 5.0%). In a multivariable model, DCIS lesions that were p16(+)COX-2(+)Ki67(+) or those detected by palpation were statistically significantly associated with subsequent invasive cancer, but nuclear grade was not. Eight-year risk of subsequent DCIS was highest for women with DCIS lesions that had disease-free margins of 1 mm or greater combined with either ER(-)ERBB2(+)Ki67(+) or p16(+)COX-2(-)Ki67(+) status (23.6%, 95% CI = 18.1% to 34.0%). CONCLUSION: Biomarkers can identify which women who were initially diagnosed with DCIS are at high or low risk of subsequent invasive cancer, whereas histopathology information cannot.

High mammographic breast density is independent predictor of local but not distant recurrence after lumpectomy and radiotherapy for invasive breast cancer.

PURPOSE: Biologically meaningful predictors for locoregional recurrence (LRR) in patients undergoing breast-conserving surgery (BCS) and radiotherapy (RT) are lacking. Tissue components, including extracellular matrix, could confer resistance to ionizing radiation. Fibroglandular and extracellular matrix components of breast tissue relative to adipose tissue can be quantified by the mammographic breast density (MBD), the proportion of dense area relative to the total breast area on mammography. We hypothesized that the MBD might be a predictor of LRR after BCS and RT for invasive breast cancer. METHODS AND MATERIALS: We conducted a nested case-control study of 136 women with invasive breast cancer who had undergone BCS and RT and had had the MBD ascertained before, or at, diagnosis. Women with known recurrence were matched to women without recurrence by year of diagnosis. The median follow-up was 7.7 years. The percentage of MBD was measured using a computer-based threshold method. RESULTS: Patients with a high MBD (>/=75% density) vs. low (/=30 kg/m(2)). In a multivariate Cox proportional hazards model, patients with MBD in the greatest quartile were at significantly greater risk of LRR (hazard ratio, 6.6; 95% confidence interval, 1.6-27.7; p = 0.01). Obesity without a high MBD also independently predicted for LRR (hazard ratio, 19.3; 95% confidence interval, 4.5-81.7; p < 0.001). CONCLUSION: The results of our study have shown that a high MBD and obesity are significant independent predictors of LRR after BCS and RT for invasive breast cancer. Additional studies are warranted to validate these findings.

Fluorescence in situ hybridization of ductal lavage samples identifies malignant phenotypes from cytologically normal cells in women with breast cancer.

BACKGROUND: Ductal lavage (DL) does not routinely identify cytologically malignant cells. For this study, the authors asked whether molecular analyses of DL specimens from women with cancer would identify abnormal cells, even if they appeared cytologically normal. METHODS: DL was performed and yielded fluid in 29 of 45 consenting women who were undergoing breast cancer surgery. Array comparative genomic hybridization (CGH) was performed on the corresponding tumor tissue from 14 women. There was no single, common alteration; thus, bacterial artificial chromosome-specific fluorescence in situ hybridization (FISH) probes were selected based on CGH alterations. RESULTS: FISH copy number changes were detected in tumor sections in 9 women. In the corresponding 9 DL samples, 1 sample was clearly malignant on cytology, 1 showed marked atypia, 1 showed mild atypia, and the rest were benign. Five of the 9 DL samples had epithelial cells that showed genetic changes identical to those observed in the tumor by FISH. The remaining 4 of 9 DL samples that did not show molecular changes were probably (N = 1) or possibly (N = 3) from the same duct as the tumor. CONCLUSIONS: Although only 11% of the DL samples were identified as malignant cytologically, 55% showed molecular changes that were identical to those observed in the tumor. FISH was more sensitive for finding tumor in DL specimens than cytology. However, the ductal system in which the tumor was located did not always yield fluid, limiting the sensitivity of DL. The results from this study showed that genetic changes can be detected in the absence of morphologic changes in cytologically benign cells, but the application will be limited without a better approach for acquiring cells and a common set of probes for detecting molecular abnormalities that are found in breast malignancies.