Breast Cancer Patient Specimens Research Articles

Modeling ductal carcinoma in situ: a HER2–Notch3 collaboration enables luminal filling

A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as 3- dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional up-regulation of multiple components of the Notch survival pathway. Importantly, luminal filling required up-regulation of a signaling pathway comprising Notch3, its cleaved intracellular domain (NICD) and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and Cyclin D1. In line with HER2- Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report up-regulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.

P118 Clinical usefulness of liquid-based cytology in hormone receptor analysis for fine needle aspiration specimens of breast cancer patients

P118 Clinical usefulness of liquid-based cytology in hormone receptor analysis for fine needle aspiration specimens of breast cancer patients

Strategies for H-score normalization of preanalytical technical variables with potential utility to immunohistochemical-based biomarker quantitation in therapeutic response diagnostics.

Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.

Strategies for H-score Normalization of Preanalytical Technical Variables with Potential Utility to Immunohistochemical-Based Biomarker Quantitation in Therapeutic Reponse Diagnostics

Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.

DNA repair biomarker alterations in breast cancer relevant to adjuvant chemotherapy treatment decisions.

10634 Background: It is increasingly evident that DNA Repair pathways are important determinants, prognostically and predictively, in breast cancer outcomes. Although anthracycline-based therapies are standard-of-care in breast cancer, some patients may benefit from other available therapies. Based on the DNA-damaging mechanism of action of anthracyclines and radiation, we sought to discriminate the phenotypic tumor changes to DNA repair by investigating multiple DNA repair biomarkers. We utilized a digital pathology platform to explore the association of these biomarkers with chemotherapy outcome. Methods: Tumor from 1,491 breast cancer patient specimens collected at Helsinki University Central Hospital between 1997 and 2000 were formatted into Tissue Microarrays (TMAs). The FFPE samples included 6,392 cores with four 0.6mm diameter cores per patient. IHC was performed with antibodies against 6 DNA repair proteins: BRCA1, FANCD2, RAD51, PAR, XPF, and phosphoMapKapKinase2 (pMK2). Fifty or greater tumor cells per core were analyzed using a pattern recognition and image analysis platform. Nuclear plus/minus cytoplasmic intensity and quantity measurements were acquired for the markers. Results: Digital pathology platforms are a robust means to determine levels of protein biomarkers as evidenced by DNA repair analysis. Pattern recognition and manual pathologist selection of tumor zones for scoring achieved high similarity with R2 correlations of >0.96, allowing increased throughput of biomarker analysis. RAD51 and FANCD2 biomarkers had the greatest fraction of breast cancer patients with low score values with mean scores of 44 and 30 respectively (0-300 scale). For BRCA1, pMK2, and PAR, the scores were variable amongst breast cancer patients with mean scores of 119 (0-300 scale), 166 (0-300 scale), and 50 (0-100 scale). Statistical analysis with univariate and multimarker models for subtype definition and treatment associations to determine the prognostic and predictive significance of DNA repair biomarkers are to be presented. Conclusions: Significant DNA repair differences are evident in breast cancer, and may be important determinants of therapy selection. No significant financial relationships to disclose.

Prediction of Response to Paclitaxel by ChemoFx assay Correlates with Estrogen Receptor Status and Changes in Apoptosis Pathway in Human Breast Cancers.

Abstract Background: Paclitaxel belongs to the taxane family of therapeutics, which have emerged as critically important drugs for breast cancer treatment. In addition to inhibiting cell growth by interfering with microtubule disassembly, its mechanism of action also includes induction of apoptosis. Recent studies suggest that besides being a key predictor for endocrine therapy response, Estrogen Receptor (ER) status also influences sensitivity of breast cancer to paclitaxel, with ER negative tumors being more responsive to the drug.Methods: The ChemoFx live cell chemoresponse assay was performed on 25 breast cancer cell lines (10 ER+ and 15 ER-). These cells were treated with a range of 10 doses of paclitaxel for 72 hours before DAPI staining of nuclei and counting. AUC (Area Under Curve) values were calculated and additional statistical analysis was performed on the resulting dose-response curves. Differential gene expression analysis was conducted to compare ER+ (n=82) and ER- (n=51) breast cancer patients using a public Microarray database. In addition, 2 of the 25 breast cancer cell lines, T47D (ER+) and SKBR3 (ER-), were treated with paclitaxel, lysed, and analyzed with Western blotting to detect cleaved caspase-3 and cleaved PARP expression, with beta-actin employed as a normal control.Results: The ChemoFx assay results revealed that none of the ER+ cells were categorized as R (responsive) to paclitaxel, with seven NR (non-responsive) and one IR (intermediate responsive). On the contrary, of the 15 ER- cell lines, three were categorized as R, only four were categorized as NR, and eight were categorized as IR. Statistical analysis suggested that paclitaxel responsiveness based on ChemoFx assay correlates with ER status (Chi-square test, p<0.05), with ER- breast tumors being more responsive to paclitaxel. Microarray analysis revealed differential expressions of genes implicated in the apoptosis pathway (q< 0.05) in ER+ and ER- breast cancers. Western blot analysis showed that paclitaxel induced cleaved caspase-3 and cleaved PARP expressions, both of which are indicators of activation of apoptosis, in SKBR3 cells (ER-), but not in T47D cells (ER+).Conclusions: ER status appears to predict in part, the response of breast cancer cells to paclitaxel as determined by the ChemoFx assay. ER-negative breast cancer cells are more likely to be responsive, which is consistent with established clinical findings. Our assay also distinguishes between NR/IR and R to paclitaxel within the ER- population. Similar ChemoFx assays are being performed on primary cultures from ER+ and ER- breast cancer patient specimens. Results from RNA microarray and Western blot analyses indicate that differences in gene expression in the apoptosis pathway, and in activation of apoptosis pathway, namely changes in expressions of cleaved PARP and cleaved caspase-3 in response to paclitaxel, may explain differences in the responsiveness of ER+ and ER- breast cancers to paclitaxel. This also suggests a potential role of cleaved PARP and cleaved caspase-3 as biomarkers in addition to ER for prediction of paclitaxel responsiveness in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2028.

A study on the relationship between CD44v6 expression and biological characteristics in breast cancer

Objective To investigate the correlative relationship between the expression of adhesion molecule CD44v6 and biological characteristics of breast cancer.Methods Using immunhistochemical techniques,we examined the expression of CD44v6 in 65 specimens of breast cancer patients,who were diagnosed in our hospital in 2006,and assessed about biological characteristics of the tumor.Results Fifty-three of sixty-five (81.5%) women with breast carcinoma were CD44v6 positive.The positive rate of CD44v6 in cases with metastases of axillary nodes was significantly higher than that without metastases of axillary nodes (90.9% vs 61.9%,P＜0.01).The positive rate of CD44v6 in cases with Ki-67 expression was 76.9%,and there is no expression of CD44v6 in cases of Ki-67 negative (P＜0.01).In multivariate analysis,using the Logistic model,the main factors on CD44v6 expression were metastases of axillary nodes (OR=1.604) and Ki-67 expression (OR =2.800).Conclusion CD44v6 expression was closely correlated with metastases of axillary nodes and Ki-67 expression.It is an important indicator of detecting patient's condition and predicting patient's prognosis in breast cancer. Key words: Breast cancer; CD44v6; Biological characteristics

Use of imprint cytology for assessment of surgical margins in lumpectomy specimens of breast cancer patients

In the past several years, breast-conservation therapy has provided an alternative to mastectomy. In order to reduce the subsequent local tumor recurrence, it is critical that all the measures are in place to find the residual foci of occult microscopic tumor at the time of the initial lumpectomy procedure. An accepted method to evaluate the lumpectomy margins for presence of residual tumor is the use of imprint cytology (also called touch-prep), which is assessment of the presence or absence of the tumor cells by cytological preparation. This is a rapid, cost effective, and easy to use procedure with added advantage of saving tissue for permanent sectioning and rendering a definitive diagnosis. In this report, we present our experience using intraoperative imprint cytology for evaluation of the status of lumpectomy specimens in breast cancer patients. The objective of this study was to evaluate the diagnostic accuracy of intraoperative imprint cytology for assessment of surgical resection margins in lumpectomy margins of patients with breast carcinoma. This is a retrospective study of 100 cases of breast lumpectomy specimens, which had undergone intraoperative imprint cytology. The cases were retrieved from the archived files of the University of Florida, Department of Pathology at Shands Jacksonville. The results of intraoperative imprint cytology were compared with the histological findings of the corresponding permanent sections of the same cases as the gold standard. Overall, we reviewed 510 cytology imprint slides, which were obtained from 100 lumpectomy specimens. Among these cases, 37 slides from 22 cases were reported positive and the remaining were negative. Only eight slides from six cases of lumpectomy showed discrepancy between the result of intraoperative imprint cytology and the permanent sections of the same cases. In our study, intraoperative imprint cytology showed a sensitivity of 97%, specificity of 99%, with positive predictive value of 84%, and negative predictive value of 99%. This study demonstrates that intraoperative imprint cytology can be used as a reliable diagnostic procedure for the evaluation of the status of lumpectomy margins in breast cancer patients.

Detection of Circulating Tumor Cells in Peripheral Blood of Breast Cancer Patients During or After Therapy Using a Multigene Real-Time RT-PCR Assay

To evaluate the utility of a multigene real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay to detect circulating tumor cells in peripheral blood specimens of breast cancer patients during or after treatment. Using this assay, peripheral blood samples were analyzed for expression levels of mammaglobin and three complementary transcribed breast cancer-specific genes: B305D, gamma-aminobutyrate type A receptor pi subunit (GABA pi; GABRP), and B726P. We examined 172 blood specimens from 82 breast cancer patients during or after therapy for the presence of circulating tumor cells using the multigene real-time RT-PCR assay. In 63.4% of the blood samples, a positive signal for mammaglobin and/or three breast cancer-associated gene transcripts was detected. Of breast cancer patients, 75.6% had at least one positive blood sample. Blood specimens from 51 of 53 healthy female volunteers tested negative in the assay whereas two samples had a low expression signal. In addition, three patients were monitored for more than a year during their adjuvant therapy treatment. This assay could be a valuable tool for monitoring breast cancer patients during and after therapy.

Opportunities of use of biochemical markers for early diagnostics of breast cancer

Mammaglobin, known for its mammary tissue specificity, has been discussed as a promising diagnostic marker in breast cancer for almost 10 years. In particular, the application of mammaglobin RT-PCR to detect disseminated breast cancer cells has been reported. More than 25 publications evaluate the detection of mammaglobin mRNA in lymph node, blood, and bone marrow specimens of breast cancer patients. Recently, structural details about the mammaglobin complex have been discovered, and these findings can be implemented to optimize detection of the secreted protein. This review summarizes the findings of almost 50 published studies and the current knowledge about the diagnostic utility of mammaglobin.

Organochlorines, Breast Cancer, and GATT

To the Editor. —Breast cancer, which now affects one in eight American women, must truly be regarded as a modern epidemic. This year 180 000 American women will be diagnosed as having this disease and a third of them will die of it. In the past two decades, breast cancer has claimed the lives of more American women than the total fatalities of the Korean War, the Vietnam War, World War I, and World War II combined. Recent evidence strongly supports the relationship between tissue levels of organochlorines like dichlorodiphenyltrichloroethane (DDT) and the incidence of breast cancer, with some studies showing as much as a fourfold increase in the relative risk of breast cancer in women with high organochlorine compound concentrations. 1,2,3 Metabolites of DDT have been noted to be 50% to 60% higher in breast cancer patient specimens compared with controls ( P 4 The US government, hoping to

Prognostic value of the combined assessment of proliferating cell nuclear antigen immunostaining and nuclear DNA content in invasive human mammary carcinomas.

The expression of the S-phase associated, nuclear protein proliferating cell nuclear antigen (PCNA) was investigated in routinely paraffin-embedded surgical specimens from 209 breast cancer patients. Cytometric DNA assessments were performed on fine-needle aspirates, upon which the primary diagnosis of breast cancer had been based. The mean clinical follow-up was 16 years (range 13-20 years). The percentage of PCNA immunoreactive tumour cells ranged between less than 5 to 60% (mean value 13.34%). There was a direct association between PCNA expression, high histological tumour grade (p < 0.01), and DNA aneuploidy (p = 0.009). In a subgroup of 22 patients with near-diploid DNA distribution patterns the PCNA expression yielded additional prognostic information. Patients with tumours of near-diploid DNA histograms and more than 20% of PCNA immunoreactive neoplastic cells had a significantly worse clinical course, than patients with near-diploid tumours containing less than 20% PCNA immunoreactive cells (p = 0.0001). In contrast, the PCNA immunoreactivity did not yield additional prognostic information for patients with distinctly diploid or highly aneuploid tumour variants. In a multivariate analysis comprising all 209 patients, nodal status (p < 0.01), tumour size (p < 0.01), and DNA ploidy (p < 0.01) were found to have significant prognostic effect. The findings indicate that carcinomas characterised by high proliferative activity and near-diploid DNA distribution patterns can show rapid tumour progression. The combined assessment of the PCNA immunoreactivity and of the nuclear DNA content in routinely processed surgical specimens of breast cancer patients appears to be of prognostic value.

Use of monoclonal antibody MBr1 to detect micrometastases in bone marrow specimens of breast cancer patients

Bone marrow specimens obtained from 121 breast cancer patients immediately after surgery were examined by an immunofluorescence method with monoclonal antibody MBr1 to detect tumour cells undetectable by other diagnostic procedures. 80 women were node-negative and 41 node-positive. In no case could conventional histology demonstrate tumour cells, whereas MBr1 was positive in 20 (16.5%) of the 121 cases. No difference was observed in MBr1 positivity between node-negative and node-positive cases (17% vs. 15%). With regard to clinical outcome (median follow-up 48 months) 27 women relapsed, including 6 to 20 MBr1-positive and 24 of 101 MBr1-negative patients. First distant metastases or death from progression of disease were taken as end-points. Multivariate analysis showed that the additional contribution of MBr1 positivity, after making allowance for other prognostic factors, was negligible.

Analysis of c‐erbB‐2 protein expression in conjunction with DNA content using multiparameter flow cytometry

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.