bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipomodelling of the breast.
Authors: ELFadl D , Mahapatra T , McManus P , Drew P
Published: December 2010

Abstract #4149

Background: Lipomodelling is the autologous transfer of fat to correct congenital and post-surgical deformities. Recent improvements in technique have provided the opportunity for a greater volume of fat transfer thus increasing its potential application following surgery for benign and malignant breast disease.  Materials & methods: After injecting a solution of local anaesthetic, normal saline and 1:106 adrenaline into donor sites in the abdomen and thighs, fat was aspirated with 3mm cannulae and centrifuged for 5 minutes. The fat was then re-injected into pre-marked areas in the breast using a 3mm injection cannula. Patients were followed up in a specialist breast clinic.  Results: 23 patients (22 female, 1 male) were treated between November 2007 and May 2008. The mean age was 50.6 years (36-61).    Prior to lipomodelling, volume deficit was observed in all patients. A visible depression was identified in 43.5% (n=10), significant flap atrophy in 21.7% (n=5), skin tethering in 21.7% (n=5) and implant rippling in 13% (n=3). Ten patients (43.5%) required lipomodelling to the upper pole, while in 7 patients (30.4%), the location was closely associated with the surgical scar.  The average volume of fat harvested was 210.7 ml (85-510), and that injected per breast was 107.1 ml (45-206). Injections were stopped when slight overfilling was observed. Excessive tissue tension was avoided. Operative time varied between 45-100 minutes.  During the first follow up visit, satisfactory results were recorded. 3 patients were booked for their second operation.  Nine patients were seen at 3-4 months (second follow up visit). Two patients had clinical and radiological evidence of fat necrosis. One patient had marked volume resorption and numbness at the donor site. Eight patients had good volume retention. Two patients were booked for further lipomodelling at this stage.  One patient had further lipomodelling five months after the first session, with satisfactory results, and is awaiting a third session.    Discussion: Early results have shown satisfactory cosmetic outcome and patient satisfaction. The technical simplicity and autologous nature make lipomodelling an attractive option to both surgeon and patient. However it is not universally successful and further follow-up is required before the long term effects of the technique in cancer patients can be confirmed.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4149.
A new enrichment model for high sensitivity detection and downstream analyses of circulating tumor cells in breast cancer patients.
Authors: Deng G , Mishaeli M , Miller M , Zayed AA , Huntsman D , Gelmon K , Yerushalmi R , Manna E , Krag D , Habib I , Williamson J , Burke J
Published: December 2010

Abstract #4162

The detection of circulating tumor cells (CTCs) in breast cancer patients have the potential to improve prognostication and the monitoring of response to treatment. Most CTC enrichment technologies are based on binding to anti-EpCAM antibodies. The sensitivity of such assays is limited by tumors that express no or undetectable levels of EpCAM. Improvements in CTC detection coupled with the development of systems to interrogate CTCs for therapeutic target expression could lead to novel applications for patient monitoring, clinical diagnosis and treatment. In this study, we describe a sensitive and reproducible enrichment method for CTCs. We defined cells as circulating tumor cells with three criteria: Positive for cytokeratin (CK+) and DAPI (nuclear) (DAPI+) and negative staining for CD45 (CD45-). We have previously reported that this system has a higher sensitivity for circulating tumor cell detection and provides a better platform for CTC downstream analyses compare to the methods currently available in the market. Herein, we describe the use of this platform for the evaluation of breast cancer biomarkers in CTCs. Blood samples from patients with metastatic breast cancer were used for CellSearch™ assay (Veridex , LLC ) and our CTC assay (A1000 CTC enrichment and detection kit, Genetix). We performed the CTC enrichment assay using the combination of anti-CK and anti-EpCAM antibodies. CTCs were identified with brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images. The Ariol® system (Applied Imaging Corporation) was employed for automated cell image capture and analysis of CTCs on glass slides. CTCs enriched on the glass slides were used for CTC downstream analysis. Our CTC enrichment model is designed to have the capability to enrich all the three types of CTCs including CK+ & EpCAM+, CK+ & EpCAM-/low and CK-/low & EpCAM+ cells. Compared to the enrichment methods using anti-EpCAM or anti-cytokeratin antibody alone, a higher CTC detection rate and a larger dynamic CTC detected range were obtained with our new enrichment model. Interestingly there were clear CTC number differences with enrichment methods in the higher CTC count patient samples which indicate that the different enrichment methods may enrich different types of CTCs from patient blood samples.  Results of DNA and RNA FISH analyses on enriched CTCs indicate that the CTCs on glass slides can be used for its downstream analyses directly or indirectly. Our method may have better performance on enrichment of heterogeneous CTCs and provide a better platform for CTCs profiling for biomarker evaluations and CTC downstream analyses.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4162.
MM-111: a novel bispecific antibody targeting ErbB3 with potent anti-tumor activity in ErbB2 over-expressing malignancies.
Authors: Nielsen U , Huhalov A , Harms B , Paragas V , Adams S , Gu J , Nguyen S , Luus L , Oyama S , Razlog M , Overland R , Wallace M , Kohli N , Feldhaus M , Schoeberl B , McDonagh C
Published: December 2010

Abstract #4166

ErbB3 has been identified as a preferred dimerization partner of ErbB2, critical for driving the proliferation of ErbB2 over-expressing breast tumors. We have designed a bispecific antibody, MM-111, which inhibits ligand-induced phosphorylation of ErbB3 with sub-nanomolar potency by exploiting the abundant expression of its dimerization partner, ErbB2, for specific targeting to cancer cells that express both receptors. We employed computational physicochemical modeling to guide the kinetic optimization of the monovalent binding affinities to the ErbB2 and ErbB3 receptors to increase the potency and specificity of MM-111 for tumor cells. We have demonstrated that MM-111 inhibits activation of the phosphatidylinositol 3-kinase pathway in vitro and in vivo, resulting in attenuation of tumor proliferation. Inhibition of growth by MM-111 has been observed in several murine xenograft models including BT474 and MDA-MB-361 breast tumors. While the antitumor activity of MM-111 is positively correlated with ErbB2 expression levels, MM-111's potent inhibition of ErbB3 phosphorylation and signaling downstream from this receptor differs markedly from currently available therapies targeting ErbB2 over-expressing breast tumors and thus provides a novel approach to treatment for these malignancies. In conclusion, our data demonstrate that the combination of computational biology with antibody engineering has resulted in the development of a promising, novel therapeutic, MM-111, that has potent antitumor activity in malignancies driven by the ErbB2/3 oncogenic unit.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4166.
First clinical experience of contrast enhanced digital mammography (CEDM) for the detection of breast carcinomas.
Authors: Dromain C , Balleyguier C , Tardivon A , Rimareix F , Delaloge S
Published: December 2010

Abstract #5001

Purpose: CEDM is a recent development of digital mammography using the intra-venous injection of an iodinated contrast agent in conjunction with a mammography examination. The objective of this paper is to present two techniques developed to perform CEDM examinations, the preliminary clinical experiences and potential clinical applications of this new breast imaging procedure.  Material and methods: Between 2005 and 2008, 140 consenting women with suspicious findings on mammography and or ultrasonography underwent CEDM using modified full-field digital mammography systems (GE Healthcare, Chalfont St-Giles, UK). A temporal subtraction sequence with acquisition of high-energy images before and after contrast medium injection was used for the first 20 patients. A dual energy technique with acquisition of a pair of low and high-energy images only after contrast medium injection was used for the other 120 patients.  Results: CEDM, either using temporal or dual energy technique, improved the probability of malignancy and the BIRADS assessment compared to conventional mammography alone. The temporal subtraction technique offered the possibility to analyze the kinetic curve of enhancement of breast lesions, similar to breast MRI. Dual energy technique did not provide information about the kinetic of tumor enhancement but allowed the acquisition of multiples views of the same breast or bilateral examination and was less sensitive to patient motion than temporal CEDM. The potential clinical applications are the determination of the extent of disease, the assessment of recurrent disease, the clarification of mammographically equivocal lesions, the detection of occult lesions on standard mammography, particularly in dense breast, and the monitoring of the response to chemotherapy.        Conclusion: Initial clinical experience has shown the ability of CEDM to map the distribution of neovasculature induced by cancer using mammography. CEDM should result in a simple way to enhance the detection and the characterization of breast lesions and to provide some prognostic factors of breast carcinomas.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5001.
A potential serum panel for breast cancer diagnosis: ongoing clinical validation for its clinical implication.
Authors: Lee J , Kim B , Han W , Ko E , Kim E , Hwang K , Kim S , Park P , Kim C , Noh D
Published: December 2010

Abstract #5006

Although every effort has been made to discover single potent biomarkers in oncology, the promising single biomarker often loses its clinical potency in the course of clinical validations. We aimed to construct and verify a biomarker panel with multiple proteins for breast cancer diagnosis. Using Surface-Enhanced Laser Desorption/ Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS) and microbead array system, epidermal growth factor (EGF), vascular cell adhesion molecule-1 (VCAM-1), CD40L, vitronectin, plasminogen activation inhibitor-1 (PAI-1), vitamin-D binding protein (VDBP), C-reactive protein (CRP), kininogen, apolipoprotein A1 and proapolipoprotein A1 were identified and validated as useful biomarkers for detection of breast cancer. The multiplexing panel (MP) which was constructed by combining algorithm (random forest, support vector machine) with above biomarkers showed that the diagnostic accuracy approached 91% in 216 test samples. A following double blind test verified that the diagnostic accuracy of the MP was 68% with an additional validation set including sera from 49 patients with breast cancer and 51 healthy women (sensitivity=74%; 95% confidence interval (CI)=58.9–85.0%, specificity=62.7%; 95% CI=48.1–75.9%, positive predictive value=65.5%; 95% CI=55.7-75.3%, negative predictive value=71.7%; 95% CI=61.7-80.35%). We suggest that these results support the potential use of the MP as an early detector of breast cancer. In order to validate the role and potency of the MP in routine clinical practice, a large scale clinical validation in a cohort consisting of 500 patients with breast cancer and the same number of healthy women are underway and the results will be released in the near future.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5006.
Detecting BRCA2 protein truncation in breast tissue biopsies to identify hereditary cancer.
Authors: Lieberman R , Snyder C , Clark V , Lynch H , Holt J
Published: December 2010

Abstract #501

Background: Mutations in the BRCA2 gene are dominantly inherited but cause cancers when the wildtype allele has LOH (loss of heterozygosity) within the cancer. It is important to identify breast cancer patients with BRCA mutations so they can be appropriately treated and affected family members can implement cancer prevention strategies. Because most disease-associated BRCA2 mutations are truncating mutations, a test for truncated BRCA2 proteins should identify most BRCA2 hereditary cancers. Methods: We have developed a tissue truncation test to identify truncated BRCA2 proteins in breast cancer tissue biopsies by direct immunohistochemistry, without amplification or genetic manipulations. N-terminal and C-terminal antibodies are used to visualize protein truncation by demonstrating that the beginning of the protein is present but the end (terminus) is absent. Results: A quantitative C-terminal immunostaining score or a C/N terminal truncation ratio correctly classified 20/21 breast cancers from BRCA2 mutation carriers and 57/58 sporadic breast cancers. This represents a sensitivity of 95% and specificity of 98%. Figure 1 shows a loss of C-terminal staining (except for a lymphocyte) in the upper panel but strong N-terminal staining in the lower panel sections of the same hereditary breast cancer.    Due to the presence of C-terminal BRCA2 protein and atypical clinical features of the misclassifed cancer in a BRCA2 mutation carrier,we performed PCR and sequence analyses on this cancer. The results showed continued presence of the BRCA2 wildtype allele in the cancer, indicating that this represents a sporadic cancer which occurred in a mutation carrier. Conclusions: This immunohistochemistry based test (which takes only 4 hours) appears to identify BRCA2 hereditary cancer with high accuracy. The test also appears to diagnose the biochemical loss of BRCA2 protein in cancers (BRCA2 mutant genotype) which will usually but not always agree with the presence of a germline BRCA2 mutation found by susceptibility testing by DNA sequencing of blood samples. This rapid but accurate tissue test can diagnose hereditary BRCA2 breast cancer at time of biopsy, allowing medical oncologists to implement targeted therapies and surgeons to perform timely prophylactic surgery without the usual 4 week wait for DNA sequencing results.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 501.
Evolution of breast cancer screening in France from 2005 to 2008: results of the EDIFICE survey.
Authors: Pivot X , Coscas Y , Morere J , Namer M , Blay J , Roussel C , Eisinger F
Published: December 2010

Abstract #5010

Background: In 2005, a first EDIFICE survey was aimed to provide a better understanding of population s adherence to the screening tests available in France and results pertaining to breast cancer are reported (Int. J. Med. Sci. 2008, 5: 106-112). In 2008, EDIFICE survey is performed again to assess the evolution 3 years later. Taking into account that the Implementation of breast cancer screening has been generalized in France since 2003: All women aged between 50 and 74 years are systematically invited to perform a mammography every two years.  Methods: This second nationwide observational survey was carried out from December 12, 2007 to January 07, 2008 among a representative samples of 1802 French people aged between 40 and 85 years and 600 general practitioners (GPs). Information collected included socio-demographic characteristics, attitude towards cancer screening and actual experience of cancer screening, as well as GPs practice regarding screening. The precision of the results is 4.3% for a 95% confidence interval.  Results: In EDIFICE–1 and -2, 501 and 488 participating women aged between 50 and 74 years are assessed, respectively.    In EDIFICE-2, there was a significant relationship (p<0.05) between the existence of a first mammography screening on women s own initiative and age (odd ratio 0.35; 95%CI 0.13 – 0.95). Main factors associated with performing the screening test every two years were: knowledge of screening process (odd ratio 3.59 ; 95%CI 1.54 – 8.38), Own motivation (odd ratio 5.03; 95%CI 1.6 – 15.8), First mammography performed in the organized screening (odd ratio 6.53 ; 95%CI 1.15 – 37.3), and gynecologist consultation (odd ratio 8.68; 95%CI 2.21 – 34.11).  Of the 600 participating GPs, 68% systematically recommended a mammography to their patients in the screening national plan. GPs perceptions of the reasons for women s avoidance of the screening test were unwillingness to be aware of mammography results (46%) and négligence (24%).  Conclusion: The main result of this second EDIFICE Survey is to stress the impact of the breast cancer screening in France. Between 2005 and 2008 the rate of women s attendance at mammography screening has increased and the proportion of women who repeat the test every two years has been dramaticaly improve.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5010.
Improving community access for breast cancer screening.
Authors: Bell JL , Gray KD , Huggler PZ
Published: December 2010

Abstract #5016

Background: The Breast Health Outreach Program (BHOP) at the University of Tennessee Medical Center's Cancer Institute began in 1996 delivery breast health education and mobile mammography to underserved areas in East Tennessee. Through grant funding, dedicated personnel and community support, the program is able to provide breast health services to 25 East Tennessee counties.  Study Design: The BHOP is designed to deliver culturally and educationally sensitive breast health education classes prior to onsite mobile mammography screening. Thirty-minute classes inform women of appropriate, best breast health practices, eliminate fears associated with mammography and encourage women to obtain annual screening. Once relationships are established, the BHOP is offered annually in churches, industrial work sites, and community centers.    Results: Since 1996, the BHOP has educated over 13,500 women, delivered approximately 8,800 screening mammograms and diagnosed 20 cancers. As delivery processes improved, 6,851 screening mammograms were provided between 2002 and 2006 with 1,080 women receiving their first-ever mammogram. Fourteen breast cancers were diagnosed in this period. Further data analysis from this time period reveals increases in education, screenings, first-ever mammograms and diagnosed cancers.  Conclusions: Mobile mammography is becoming "standard" in rural communities of East Tennessee. Most of the women now participating in the program have had previous mammograms through the BHOP. Each year the BHOP has increased the number of women obtaining their first-ever mammogram by targeting new sites. Companies and communities are moving to support healthy living practices and providing healthier environments for employees and their families. As the BHOP has matured and become widely recognized, measures of program effectiveness have improved. Future areas for research will address longitudinal data tracking to assess mortality impact and early diagnosis trends.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5016.
Characterization of the HER2 status on circulating breast cancer cells.
Authors: Alix-Panabières C , Riethdorf S , Fehm T , Vendrell J , Pantel K
Published: December 2010

Abstract #5018

Background: Detection and characterization of circulating tumor cells (CTC) in the peripheral blood of breast cancer patients could help to guide treatment decisions before the onset of overt metastasis as well as in the setting of advanced disease. The HER2 oncogene is the most prominent therapeutic target in breast cancer and shedding of the extracellular domain of this receptor seems to contribute to constitutive activation of HER2. Here we developed a novel assay to detect shedding of the extracellular domain (ECD) of the HER2 oncogene on single CTC and applied this assay to the analysis of blood from metastatic breast cancer patients.  Material and Methods: Using a new dual fluorescent CK19/HER2-EPISPOT assay, the shedding of HER2 ECD by CTC from 49 metastatic breast cancer patients was assessed. In parallel, using the CellSearch system, the number of CTC and their HER2 expression were determined on 42 samples from metastatic breast cancer patients. A subgroup (n=34) of these patients was analyzed with both methods. This work was performed in the context of the activities of the DETECT study group.  Results: In the CK19/HER2-EPISPOT assay 24 of 49 (48.9%) patients had CTC (mean, 12.4; median, 5; range 1-141) and among these positive patients, 7 of 24 (29.2%) showed also HER2-secreting CTC (mean, 3.3; median, 2; range, 1-14). Using the CellSearch™ system, in 23/42 (54.8%) patients CTC were found (mean, 82; median, 13; range, 1-656). HER2 staining intensity was heterogeneous even among CTC from the same patient. Regarding the samples analyzed with both methods, we found that 23.5%, 50.0%, 50.0% and 76.5% of the patients were positive with both detection methods, the EPISPOT assay only, the CellSearch™ system only and one or the other method, respectively.  Discussion: These findings demonstrate, for the first time, that (i) CTC can secrete HER2 and (ii) HER2 secretion is not closely linked to HER2 CellSearch™ status. Both assays (i.e., EPISPOT and CellSearch™) have a comparative sensitivity and appear to be complementary for the detection and characterization of the HER2-status of CTC.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5018.
HER-2 expression on circulating tumor cells in metastatic breast cancer.
Authors: Hayashi N , Yagata H , Hamaoka T , Sarumaru S , Tsugawa K , Yoshida A , Tsunoda-Shimizu H , Suzuki K , Shimada M , Shimoda Y , Nakamura S
Published: December 2010

Abstract #5020

Background: HER-2 Overexpression in breast cancer is associated with poor prognosis. The circulating tumor cells (CTCs) in peripheral blood can be identified in patients with breast cancer. They are recognized as a predictor of the effect of systemic therapy, and also as a prognostic marker recently. Cristofanilli et al reported that progression-free Survival (PFS) and overall survival(OS) in metastatic breast cancer patients with <5CTCs per7.5ml are significantly better than those with >5CTCs before initiation of a new line of therapy and at the 1st follow-up visit. However, the detection rate and value of HER-2 Overexpression on CTCs has been not determined well.Materials and Methods:CTCs were obtained prospectively for 22 patients with metastatic breast cancer to start a new line of treatment at single institution. Blood specimens were collected at monthly intervals for a period of up to 12 weeks. CT scans were also performed before start of the treatment and 12 weeks after. HER-2 expression on primary lesions and CTCs were determined by both immunohistochemical methods and fluorescence in situ hybridization (FISH) analysis. Mean age was 54.4 years (range, 32-75 years). 8 patients received first line therapy, one patient received second line therapy, and 13 patients received over third line therapy. Of the 22 primary cancer, 7(31.8%) were HER-2 positive(score,+3 or +2/FISH positive) and 15 (68.2%) were HER-2 negative. CTCs were determined using Cell Search™ System.Results:14(63.6%) patients had clinical benefit (partial response and stable disease) during the course. CTCs were detected in 16 patients (72.7%). OS for patients with <5CTCs were significantly longer than those with 5< at any period. In 15 patients with HER-2 negative primary cancer, 5 patients (33.3%) were with HER-2 positive CTCs. However, in 7 patients with HER-2 positive primary cancer, 6 patients (85.7%) who had been treated with trastuzumab was with HER-2 negative CTCs. But only one patient (14.3%) treated without trastuzumab was with HER-2 positive CTCs. The patient had been treated with trastuzumab and failed in past. And the median OS time for those patients with HER2 Positive CTCs were significantly shorter (130.5 days) than for those patients with HER2 negative CTCs (283.3 days). Surprisingly, 5 (83.3%) of 6 patients with HER-2 positive CTCs were dead within 7 months.Conclusion:The number of CTCs is a prognostic marker for the patients with metastatic breast cancer. Furthermore, HER-2 positive CTCs is much related to poor prognosis. And these results indicated that the possibility of negative conversion of HER2 expression on CTCs for patients with HER-2 positive primary cancer due to trastuzumab. Trastuzumab may be effective for the patients with HER2 negative primary cancer and HER-2 positive CTCs.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5020.
Multicenter study on the detection of disseminated tumor cells in primary breast cancer.
Authors: Effenberger KE , Borgen E , Christine EE , Andrea G , Marit S , Kaaresen R , Jahn NM , Pantel K , Bjorn N
Published: December 2010

Abstract #5021

Background: Detection of disseminated tumor cells (DTC) in bone marrow (BM) has prognostic value in primary breast cancer (BC) and is currently under investigation for therapy stratification and monitoring. Anti-cytokeratin (CK) antibodies (Abs) have been established as markers for detection of such DTC in patients with BC. Here we present a direct comparison of the two Abs most frequently used for DTC detection over the past 10 years.  Material and Methods: We screened BM samples from 391 BC patients of which 64 were node-positive (N+). All patients were free of any signs of overt metastases (stage M0, pT1-3, pN0-3). Tumor cells in BM were enriched by Ficoll-Hypaque gradient and the mononuclear cell (MNC) fraction was spun on slides. For each patient 2*106 MNC were analyzed per Ab, using A45-B/B3 (A45) and AE1/AE3 (AE) Abs in parallel. A45 staining was, however, performed a couple of years later than AE staining, on cytospins stored at -80°C. All samples were screened automatically for the presence of CK-positive cells. By morphological evaluation only TC-compatible cells were scored positive. Median follow-up times ranged from 5 to 118 months.  Results: For both Abs, the number of DTC revealed was 5 or less. Of the 391 patients, 24 patients (6.1%) were positive for A45, and 41 patients (10.5%) for AE, respectively. Concordance obtained by cross-analysis for the two Abs was 84.4%, whereas the overlap in positive cases was only 3.2%. Regarding immunohistopathological characteristics AE- and A45-positive BM were found significantly more frequent in tumors of higher nodal status (p=0.003 and p=0.026), and for AE also in hormone receptor negative (HR-) compared to HR+ patients (p=0.006). Positivity for AE and/or A45 staining in BM correlated with high nodal status and large tumor size (p=0.001 and p= 0.016, respectively). Survival analyses revealed shorter relapse-free survival for A45-positive patients (p=0.026). In node-positive patients, a significantly worse prognosis regarding overall and BC specific survival was observed for AE-positive (p=0.0245 and p=0.0125) and for AE- and/or A45-positive patients (p=0.035 and p=0.044, respectively). In node-negative patients, A45-positivity was correlated to a significantly shorter relapse-free survival (p=0.032). Immunocytochemical double staining of BM samples from 26 patients (M+ and M0) revealed subpopulations of DTC labeled with both Abs (in 8/26 cases) and other DTC exclusively stained for either A45 or AE (4/26).  Discussion: Our results indicate that the two anti-cytokeratin Abs most frequently used for DTC detection in BM do not give identical results. They appear to identify at least partially different subpopulations of DTC in BC patients. This implies a possible advantage of the combined use of these Abs in future trials using DTC diagnostics.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5021.
Circulating tumour cell (CTC) detection: a direct comparison between the CellSearch system, the AdnaTest, CK19/MAM RT-PCR and unmethylated/methylated free DNA in patients with metastatic breast cancer.
Authors: Van Der Auwera I , Elst H , Benoy I , Prové A , Wuyts H , Maes H , Van Dam P , Huget P , Vermeulen P , Dirix L
Published: December 2010

Abstract #5025

Introduction: The detection, enumeration and isolation of CTC has considerable potential to influence the clinical management of patients with breast cancer. There is however a substantial variability in the rates of positive samples using existing detection techniques. This study was designed to compare the sensitivities and specificities of three techniques for detecting CTC in blood of patients with metastatic breast cancer (MBC). Furthermore, correlations between CTC and circulating total and tumor-related methylated DNA were investigated.  Material and Methods: The presence of CTC in blood samples of 80 patients with MBC and 20 healthy controls was assessed by two commercial systems: i) the CellSearch System (Veridex LLC, Warren, NJ), in which epithelial cells are immunomagnetically separated and fluorescently labeled, and nucleated (DAPI+) cells with the EpCAM+, cytokeratin (CK) 8/18/19+, and CD45- phenotype are counted as CTC and ii) the AdnaTest and a real-time qRT-PCR assay for the detection of CK-19 and mammaglobin (MAM) transcripts. Plasma total DNA levels were measured by a qPCR method. Sera were analyzed by methylation-specific qPCR for three methylated markers: APC, RASSF1A and ESR1. The results obtained with AdnaTest are being analyzed and will be presented at the meeting.  Results: Numbers of CTC identified by the CellSearch test were significantly higher in blood samples of patients with MBC than in healthy controls: the median number of CTC detected in 7.5 ml of blood was 1 (range 0-2617) in patients with MBC and 0 (range 0-1) in controls (P<0.001). Using ≥2 cells as a threshold for positivity, which corresponds to 100% specificity in the control population, 34% of samples from patients with MBC were positive with the CellSearch test. Using a cut-off to ensure 100% specificity in the control population, 31% of patients were positive for CK-19, 49% for MAM and 61% for CK-19 and/or MAM by qRT-PCR. When the CellSearch test and the qRT-PCR for CK-19 and MAM were compared, the concordance was 57%. Positive samples with the CellSearch test, but not with the qRT-PCR assay, also showed significantly higher levels of circulating total DNA and were more likely to be positive for one of the methylated DNA markers in serum.  Conclusion : We observed a substantial variation in the detection rates of CTC in blood from breast cancer patients using different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and MAM, which suggests that this is currently the most sensitive technique for detecting CTC.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5025.
Circulating tumor cells in operable breast cancer: a pilot study.
Authors: Sandri MT , Zorzino L , Munzone E , Cassatella C , Luini A , Bassi F , Salvatici M , Botteri E , Rotmensz N , Esposito A , Adamoli L , Goldhirsch A , Nolè F
Published: December 2010

Abstract #5028

Background. Circulating tumor cells (CTCs) in the peripheral blood are an ideal source for the detection of disseminated tumor cells because of an easy sampling procedure. Their prognostic significance has been demonstrated in metastatic breast cancer, while little data exist in operable patients. Aims of this study are the evaluation of the prevalence and kinetics of CTCs before and after surgical treatment and the possible correlation between CTCs and clinico-pathological characteristics and biological features. Methods. We analyzed 30 ml of peripheral blood from 60 T1-T3, any N, M0 breast cancer pts, before surgery and after 5 days. In case of positivity of one of the perioperative samples, a further sample was taken after 30 days. The presence of CTCs was assessed with the CellSearch System (Veridex, USA). Samples were subjected to immunomagnetic enrichment with an anti-Epcam-antibody: CTCs were defined as nucleated cells expressing cytokeratin 8, 18 and 19 but lacking CD45. A sample was considered positive when 1 or more cells were detected. Results. Data are available for 50 pts. We found ≥1 CTC in 28% of the pts (n=14/50) before surgery, and in 29% of the patients (n= 12/42 ) at 5 days. The median number of CTCs was 1, with a range of 1-3. We found a borderline significant association between the presence of CTCs at baseline and the presence of vascular invasion (p=0.09). The presence of CTCs at baseline did not correlate with tumor size (p=0.66), grading (p=0.88), presence of estrogens receptor (p=0.30), presence of progesteron receptor (p=0.30), HER2/neu status of the tumor (p=1.0), or with the presence of lymph node metastases (p=0.71). In 42 pts we have the data of CTCs determined at baseline and at day 5: 27 patients showed concordant results (23 negative and 4 positive). Among the 15 patients with different results, 8 were negative at baseline and positive at day 5: 6 out of the 8 patients had positive nodes. At day 30 we analyzed samples from 18 subjects: persistence of at least 1 CTC was found in 18 % of patients (n=5). Conclusions. The results of this explorative study are very preliminary and a larger number of patients and a long-term follow-up will be required. However, it is of interest the borderline correlation between presence of CTCs at baseline and vascular invasion, and between appearance of CTCs and nodal involvement in postoperative samples. The study is ongoing and the patients will be followed during and after adjuvant treatment to explore the prognostic significance of persistency of CTCs. Complete data will be presented.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5028.
Behaviour and metastatic potential of her2/neu positive and her2/neu negative circulating epithelial tumor cells (CETC) during adjuvant systemic therapy in breast cancer.
Authors: Camara O , Hammer U , Gajda M , Kroll T , Krauspe S , Gellner A , Joerke C , Runnebaum IB , Hoeffken K , Pachmann K
Published: December 2010

Abstract #5030

Background: Most tumor patients do not die from the primary tumor but from metastases in vital organs. Here we report on the influence of adjuvant chemotherapy and trastuzumab on the remnant CETC patients with her2/ positive tumors after surgery and the impact on metastasis formation.  Material and Methods: Tumor cells were quantified with an automated microscope (Laser Scanning Cytometer®, Icis or ScanR) from anticoagulated blood drawn before each new therapy cycle from patients treated with systemic chemotherapy after surgery and subsequent maintenance therapy with trastuzumab. After red blood cell lysis leucocytes were stained with PE-anti-CD45 and tumor cells with FITC-anti-EpCAM. FISH analysis was performed for determination of the proportion of her2/neu positive cells and changes registered as % increase or decrease. The response to therapy was correlated to outcome.  Results: Almost all her2/neu positive breast cancer patients scheduled for systemic treatment had CETC before therapy. A decrease in cell numbers highly correlated with relapse free survival. Her2/neu positive patients responding to therapy with an increase in CETC were at increased risk to develop further increasing cell numbers also during subsequent trastuzumab treatment. Her2/neu positive patients who did never receive trastuzumab all had increasing CETC numbers during the postoperative observation time and all have suffered relapse. Patients who received trastuzumab only after adjuvant treatment all had decreasing CETC numbers and all are in sustained complete remission after up to 4 years. Of the patients who received trastuzumab together with adjuvant treatment those who showed no change or an increase in CETC in spite of trastuzumab had increasing proportions of FISH high amplified cells and a higher risk of relapse than patients with decreasing CETC during trastuzumab.  Discussion: Monitoring CETC will not only provide the earliest and most reliable indicator of successful adjuvant treatment and spare patients unnecessary treatment but also patients with the highly aggressive her2/neu positive tumors can be discerned into such with high and low risk of relapse even during trastuzumab therapy. This warrants further therapy studies to control what patients may benefit from addition of e.g. small molecules already during therapy before development of metastases.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5030.
Abundance and distribution of polychlorinated biphenyls (PCBs) in breast tissue.
Authors: Ellsworth DL , Gillard D , Love B , Ellsworth RE , Deyarmin B , Hooke JA , Kostyniak PJ , Shriver CD
Published: December 2010

Abstract #5039

Background: Breast cancer is a complex disease whose causative factors remain ambiguous and poorly defined. Many environmental chemicals accumulate in human tissues and may contribute to breast cancer risk. Polychlorinated biphenyls (PCBs), which constitute a class of chlorinated compounds that are ubiquitous in the environment, are believed to be associated with adverse health effects. Because the rising incidence of breast cancer may result from exposure to increasing levels of exogenous chemicals, there is keen interest in determining relationships between exposure to environmental chemicals and breast cancer risk.  Material and Methods: Frozen sections of breast tissue were collected from each quadrant and the central region of the breast from 47 patients enrolled in the Clinical Breast Care Project at Walter Reed Army Medical Center. Pathological diagnoses ranged from disease free prophylactic mastectomy to metastatic breast cancer. From a portion of each sample, 98 PCB congeners were assayed by pressurized liquid extraction followed by high resolution gas chromatography.  Results: PCBs were abundant in breast tissues. Only one congener was not observed in any patient and four (153, 138+163, 180, 206) were seen in all patients in at least one quadrant. Mean total PCB concentration in 202 breast quadrants was 602 ng/g lipid (range 0-4030), while for individual congeners, PCB 153 had an average level of 107 ng/g lipid (range 8-773 ng/g). Total PCB concentration (P<<0.001) and presence of 26 congeners (P<0.05) were highly correlated with patient age, particularly in the UIQ and LOQ, but not in the central region, suggesting that chemicals may accumulate at different rates in different regions of the breast. Similarly, no significant differences were observed for PCB concentrations among the four traditional quadrants, but 23 PCBs were present at significantly lower levels (P<0.05) in the central region. When examining PCB levels by disease status, seven individual congeners and median overall levels were significantly higher (P<0.05) in patients with invasive cancer compared to disease free patients.  Discussion: Population monitoring is beginning to assess exposures of the general public to environmental chemicals to evaluate long-term health effects. This study showed that many PCB residues are present in human breast tissue, these chemicals are not distributed evenly, and several are correlated with age and presence of invasive disease. Improved understanding of the true carcinogenic potential of environmental chemicals may be useful in efforts to reduce individual risk for breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5039.
Promoter hypermethylation is an early event in breast carcinogenesis.
Authors: Park M , Yoon J , Lee J , Choi M , Yoon H , Jung S , Lee B , Fackler M , Sukumar S
Published: December 2010

Abstract #5041

Background. Aberrant methylation of CpG islands in the promoter region of tumor suppressor genes has become established as an important epigenetic mechanism for gene silencing. Promoter hypermethyaltion in precursor lesions at risk for progression to invasive cancer may be biomarkers of cancer risk and targets for cancer chemoprevention. In breast cancer promoter hypermethylation has been described for several genes covering all aspects of cellular function. However, the methylation studies of precursor lesion of the breast are rather sparse and mostly done in a purely qualitative manner.  Design. To evaluate the significance of alterations in promoter hypermethylation during multistage carcinogenesis of the breast, quantitative multiplex methylation-specific PCR of six genes (APC1, Cyclin D2, HIN-1, RAR-b, RASSF1A, and Twist) was performed on DNA from 15 normal breast tissues, 21 usual ductal hyperplasia (UDH), 48 ductal carcinoma in situ (DCIS), and 35 stage I invasive ductal carcinoma (IDC), not otherwise specified.  Results. We found no to very low levels of promoter methylation in normal samples. In general UDH, DCIS, and IDC samples revealed varying levels of methylation ranging from 0 to 100%. One-way analysis of variance showed that the methylation levels of all six genes increased significantly with the progression of breast neoplasia from normal epithelium, through hyperplasia, to DCIS. However, methylation levels were not significantly different between DCIS and IDC.  Conclusion. Our results suggest that promoter hypermethylation is an early event in breast carcinogenesis. Promoter hypermethylation in the precursor lesions of the breast cancer may be used as a target for cancer chemoprevention.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5041.
GSTP1 promoter hypermethylation during breast cancer development.
Authors: Park M , Yoon J , Lee J , Choi M , Youn H , Jung S
Published: December 2010

Abstract #5042

Background. Promoter hypermethylation in precursor lesions of the breast cancer may be biomarkers of cancer risk and targets for cancer chemoprevention. GSTP1 is inactivated by promotor hypermethylation in invasive breast cancers. However, little is known about epigenetic silencing of GSTP1 gene by promoter hypermethylation in precursor lesions.  Method. To determine the significance of GSTP1 promoter hypermethylation in breast carcinogenesis, methylation status of GSTP1 gene was studied by nested methylation-specific PCR and GSTP1 expression was studied by immunohistochemistry in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), usual ductal hyperplasia (UDH) and normal breast tissue.  Results. GSTP1 promoter hypermethylation was detected in 4/24 (16.7%) of UDH, 18/49 (36.7%) of DCIS, and 14/36 (38.9%) of IDC. No hypermethylation was detected in normal breast tissues. GSTP1 promoter hypermethylation was found to be progressively elevated during breast carcinogenesis (P < 0.01). GSTP1 promoter hypermethylation was associated with loss of GSTP1 expression (P < 0.01 for UDH, P < 0.001 for DCIS and IDC).  Conclusion. Our results suggest that GSTP1 promoter hypermethylation is an early event in breast carcinogenesis and appears to functionally silence GSTP1 expression. GSTP1 promoter hypermethylation in the precursor lesions of breast cancer may be used as a target for cancer chemoprevention.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5042.
Physical activity and the risk of breast cancer in BRCA1/2 mutation carriers in the Netherlands.
Authors: Pijpe A , Manders P ,   the Netherlands Collaborative Group on Hereditary Breast Cancer (HEBON), van Leeuwen FE , Rookus MA
Published: December 2010

Abstract #505

Background: Physical activity reduces the risk of breast cancer in the general population by 20-40%. Suggested biological mechanisms include a reduction of adipose tissue and decreased lifetime exposure to estrogens. This is one of the first studies that investigates the association between physical activity and breast cancer risk in BRCA1/2 mutation carriers (carriers).  Patients and methods: We analyzed data from a nationwide retrospective cohort study (GEO-HEBON), including 1026 carriers, of which 465 had a history of breast cancer. We examined the association between breast cancer risk and average (METhr/wk) sports, walking/cycling, occupational, household, and total physical activity, lifetime and for age-periods below and above age 20, separately. All analyses were stratified for birth cohort and gene, clustered on family, and adjusted for confounders and other physical activity variables (when investigating separate components).  Preliminary results: For adulthood sports activity we found a risk reduction for active carriers (low: Hazard Ratio (HR) = 0.72; 95% CI = 0.54-0.96; medium: HR = 0.64; 95% CI = 0.46-0.90; high: HR = 0.71; 95% CI = 0.48-1.06) when compared to carriers who had never engaged in sports. No risk change was found for sports activity before age 20. For lifetime (mainly adulthood) household activity we found a risk reduction of 0.74 (95% CI = 0.57-0.96) for the second versus the lowest quartile but for higher quartiles HRs were not significantly decreased, and a similar pattern for occupational activity (0.53 (0.35-0.82)). We found neither an effect for walking/cycling activity nor for total physical activity.  Discussion: Our physical activity information was relatively extensive, and we used proxy-questionnaires to examine survival bias. Limitations include the retrospective character of the cohort and potential misclassification bias. In conclusion, these preliminary results suggest that physical activity may reduce the risk of breast cancer in carriers. The inverse associations were clearest for physical activity during adulthood. Time-dependent, weighted (for testing bias), and stratified (for example by menopausal status and Body Mass Index) analyses will be conducted.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 505.
Do stem cell markers have significant implication in breast cancer? Immunohistochemical study for CD44 and CD24.
Authors: Kim H , Kim M , Ahn S , Son B , Kim S , Jung K , Ahn J , Kim H , Gong G
Published: December 2010

Abstract #5058

Background: We reported that breast cancer expressing CD44+CD24-/low showed a favorable prognosis in contrary to the in vitro/in vivo studies. We further followed this data up to 99 months and analyzed it according to CD44 expression, CD24 expression and hormone expression.  Design: immunohistochemical stainings for CD44s and CD24 as well as prognostic markers including estrogen receptor, progesterone receptor, p53, and Her2/neu were done using tissue microarray blocks containing 645 consecutive cases of invasive breast carcinomas from 1993 to 1998. Mean follow up periods were 99.5 months. Cases were categorized into four subgroups (CD44-/CD24+, CD44+/CD24+, CD44-/CD24-, CD44+/CD24-) based on the immunohistochemical staining results and compared them with clinicopathologic parameters. Immunostainings for CD44s and CD24 interpreted as positive if at least 1% of tumor cells show distinct membranous and/or cytoplasmic stainings. In the positive group of CD24, we categorized it as three subgroups according to the degree of positivity.  Results: CD44+CD24-/low group showed inversely associated with lymph node metastasis and the tumor stage than other subgroups (p=0.001 and p=0.018, respectively). And CD44+CD24-/low group was showed an increase in disease free survival and overall survival (p=0.003, p=0.002) In the subgroup analysis of CD24 expression (0, grade 1, grade 2, grade 3), the incidence of metastasis and death was more frequently observed in the cases with the higher expression of CD24. (DFS: p=0.03, OS: p=0.001). With respect to the CD44, CD44- group showed frequent metastasis and death (p=0.01, both) however, for the receptor positive groups, not CD44 but CD24 expression resulted negatively to the overall survival significantly(p=0.01, Relative risk=1.90) on multivariate analysis. For the receptor negative groups, especially triple negative group, lack of CD44 expression made overall decreased to 50%(p=0.03, hazard ratio=0.5)  Conclusion: In contrast to cell line studies, CD44+CD24-/low phenotype is considered a favorable prognostic subgroup of breast cancer associated with less frequent nodal metastasis, lower tumor stage and infrequent distant metastasis. For receptor positive breast caner, CD24 expression effect DFS, OS significantly, and For receptor negative group, especially triple negative breast cancer, Lack of CD44 expression made and effect OS inversely.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5058.