bluuebunny/crossref_metadata_2025 huggingface dataset:
P1-07-11: Consistency and Control in Clinical Assay Technology over Time: The Oncotype DX Recurrence Score and Assessment of Single Gene Expression Levels.
Authors: Baehner FL , Butler SM , Anderson JM , Ballard J , Tan V , Shak S
Published: June 2012

Background: ASCO® and CAP have recently highlighted the importance of consistency and control in clinical assay technology. To date the Oncotype DX® Recurrence Score® (RS) has been ordered to assist in individualized treatment decision making in over 200,000 estrogen receptor positive, early stage breast cancer patients. The assay quantifies gene expression using RT-PCR from fixed paraffin embedded tissue (FPET) and employs a large number of controls and calibrators to enhance precision and reproducibility. Over 6 years of data on the assay presents an opportunity to explore consistency over time in the RS and quantitative single gene levels (ER, PR, HER2).

Methods: All tumors successfully analyzed in the Genomic Health Laboratory from 1/1/05-3/31/11 were included. Descriptive statistics for the RS, the average reference gene expression level, and expression levels for quantitative single genes were obtained for each calendar year. This was done for the entire data set as well as for subgroups defined by histological tumor type. The associations by year between HER2 and GRB7, ER and HER2, and ER and PR expression levels were explored including scatterplots and summary statistics.

Results: There were a total of 207,691 breast cancer cases, and the number in each calendar year increased over time as shown in the Table. In general the median age increased slightly over time, as did the proportion of patients in the low RS Risk Group. There was no systematic change over time in the average reference gene expression level, or in the expression levels for the individual genes ER, PR or HER2 during the period from 2008 to 2011 when individual gene testing was provided as part of the Oncotype DX assay report. The relationships between HER2 and GRB7, ER and HER2, and ER and PR remained consistent over time.

Conclusions: Active monitoring of the Oncotype DX assay as mandated by ASCO/CAP shows a high degree of consistency in results for both the multigene Recurrence Score and the quantitative single gene results. These results and the approaches used for monitoring consistency are relevant to other institutions in their efforts to maintain and improve assay quality control.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-11.
P1-07-14: Quantum Dot-Labelled Antibodies To Assess HER2 Expression in Breast Cancer.
Authors: Zona S , Blackburn E , Hojjatoleslami AS , Brown IR , Gullick WJ
Published: June 2012

Background: The Human Epidermal Growth Factor Receptor 2 (HER2) is overexpressed in 18–20% of breast cancers. Herceptin is an effective drug for the treatment of breast cancers expressing high levels of HER2. It is well known that there is a correlation between levels of HER2 expression and response to Herceptin. However a proportion of patients selected for Herceptin treatment do not respond to the drug. The accuracy of the assessment of HER2 levels in breast cancer is therefore important to predict patients’ response to Herceptin therapy. The current techniques in clinical use for the assessment of HER2 are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH); however both IHC and FISH have several limitations which may result in patient misclassification. A more accurate technique, which can quantify HER2 levels more precisely, could help to improve clinical diagnosis, prediction of prognosis and allow more accurate select individual patients for particular drug therapies.

Materials and Methods: We employed QDs labelled antibodies, laser scanning confocal microscopy and image segmentation techniques to quantify HER2 expression in cases of formalin fixed paraffin embedded breast cancers. Quantum dots (QDs) are a new class of fluorochromes made of semiconductor nanocrystal which have several useful properties for quantitative image analysis. In order to validate the system we used a breast cancer HER2−IHC control tissue array with negative, low, moderate and strong expressing cases, and a tissue microarray containing 60 samples of formalin fixed paraffin embedded breast cancer sections, previously examined by IHC and scored semiquantitatively (0-3+). We then applied this system to quantify HER2 expression a tissue microarray containing 150 primary breast cancers scored as 3+ by IHC from patients treated with Herceptin.

Results: We first demonstrated that the QD system could reliably detect HER2 expression in IHC 3+ cases. A comparison of immunofluorescent staining with conventional immunohistochemistry showed that QDs give more linear and scalable measurements of receptor levels. We also quantified HER2 within a set of 150 breast cancers scored as 3+ by IHC, and we found that HER2 is expressed at very different levels, ranging over fifty fold between individual (IHC3+) cases. We currently are evaluating the correlation between HER2 receptor levels, measured by QDs, and patient's response to Herceptin.

Conclusion: QDs and image analysis can produce a more precise measurement of HER2 levels of expression than IHC. This should help to improve clinical diagnosis, prognosis, identify specific treatments for individual patients and reduce costs associated with Herceptin treatment. This technology may be applied to study other members of the EGF family in breast cancers or more widely as a quantitative measurement of biomarkers in tumours.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-14.
P1-07-19: Analysis of HER2−Status in Breast Cancer by Mass Spectrometry in Archival, Formalin-Fixed Tissues.
Authors: Sanders M , Sprung R , Ham A , Sanchez V , Manning S , Arteaga C , Liebler D
Published: June 2012

HER2 (ERBB2) is overexpressed in about 25% of breast cancers and predicts clinical benefit from trastuzumab, as well as response to anthracycline-based chemotherapy. Fluorescence in situ hybridization (FISH) to detect HER2 gene copy number and immunohistochemistry (IHC) to detect HER2 protein levels are approved by the FDA to identify HER2−positive (H2) tumors. However, the 2007 ASCO/CAP report concluded that approximately 20% of HER2 testing may be inaccurate. Further, the available data did not clearly demonstrate clear superiority of either IHC or FISH as a predictor of benefit from anti-HER2 therapy. Discordance between these methods is as high as 5%. Thus, novel complementary quantitative methods for interrogating HER2 expression in tumors are needed.

Targeted protein analysis by multiple reaction monitoring mass spectrometry (MRM-MS) offers a powerful approach to configure assays for specific proteins without using antibodies. Our studies using this platform have demonstrated applicability to formalin-fixed, paraffin-embedded (FFPE) specimens. In the current studies, we used this approach to measure signals from two tryptic peptides specific to HER2, one each from the extracellular and intracellular domains, selected from among 28 candidates based on their signal intensity and sharpness of their chromatographic profiles. Preliminary studies with a HER2−overexpressing BT474 xenograft in mice demonstrated quantitation and detected previously reported HER2 ectodomain shedding. Subsequent analysis of FFPE tissue from five H2 and five triple-negative (TN) tumors yielded measurement of at least 1 femptomole of receptor for H2 tumors and less than 0.2 femptomole of receptor for TN tumors per microgram of digest analyzed. If we assume 200 picograms of protein per cell, the results suggest 110,00 to 468,000 receptors per cell in the H2 tumors and only 2,000 to 14,000 receptors per cell in the TN tumors. Despite significant biological variability in receptor levels measured among the specimens of each type, a clear separation of the H2 and TN tumors was achieved based on the peptide quantitation. This preliminary study demonstrates the potential of MRM-MS in FFPE tissue to provide an alternate approach to IHC-based protein analysis. MRM-MS offers the potential for more, accurate and robust HER2 quantification in clinical breast cancer tissues. The next phase of this work will encompass a larger sample set, including tumors with equivocal and negative FISH and/or IHC test results. Correlation with response to anti-Her2 therapy will be performed in samples with available follow-up data.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-19.
P1-08-07: Survival in US Women Following an Indication of Metastatic Breast Cancer Diagnosis and Chemotherapy Initiation: A SEER-Medicare Analysis.
Authors: Rugo H , Taylor D , Sanon M , Clements K , Balu S , Faria C , Teitelbaum A
Published: June 2012

Objective: Use SEER-Medicare data to identify US women with metastatic breast cancer (MBC) treated (tx) with chemotherapy (CTX) and evaluate survival.

Methods: Key inclusion criteria were women diagnosed (dx) with breast cancer (BC) in 2001–2005 with 1) Medicare enrollment 12 mo prior to dx through follow-up (2008) or death; 2) an indication of tx with CTX at any time after initial BC diagnosis (any stage). Dx of MBC between 2001–2006. MBC dx date was defined as the initial BC dx date if the patient had “distant” disease at entry into SEER or the date of first appearance of secondary malignancy in claims data (ICD9 codes 197.0−198.1, 198.3−198.7, 198.82−198.89) during follow up, at least 2 mo after initial dx. CTX date was defined as the date of first receipt of a CTX agent commonly used to treat BC after the MBC date. Data on oral medications (e.g., most hormone therapies, capecitabine) were not available. Kaplan-Meier survival analyses were performed to describe survival after MBC dx (patients were censored at end of follow-up). Survival was assessed both from the date of MBC and receipt of first intravenous (IV) chemo. Subgroup analyses were performed based on age, type of MBC dx and estrogen receptor (ER) status.

Results: Patient demographics and survival are shown in the table.

Conclusions: Despite recent advances in treatment options, median survival following a dx of MBC is less than two years, and is approximately 16 months after start of IV CTX. Survival is shorter for patients who develop MBC after an initial breast cancer dx compared to those with MBC at diagnosis, and for those with ERnegative tumors. In order to improve clinical outcomes, it is critical to pursue a better understanding of tumor biology and appropriate use of new agents.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-07.
P1-08-09: Increased Mortality in Swedish Women Diagnosed with Breast Cancer during and Shortly after Pregnancy.
Authors: Johansson ALV , Andersson TM-L , Cnattingius S , Hsieh C-C , Lambe M
Published: June 2012

Background: The incidence of pregnancy-associated breast cancer (PABC), i.e. breast cancer diagnosed during pregnancy or within two years after delivery, is increasing in Sweden. Results from epidemiological studies suggest that women with PABC have worse prognosis than women of same age and no recent birth. We studied the mortality in women with PABC, compared to women with a diagnosis of breast cancer not close to pregnancy, and in relation to when the PABC diagnosis occurred (during pregnancy, different postpartum periods). We also studied whether mortality varied over time since diagnosis.

Materials and Methods: We used data from the Swedish National Cancer Register to identify all women diagnosed with breast cancer between ages 15–44 years during 1963–2002 (n=15,729). The study cohort was linked to the Multi-Generation Register to obtain information on childbirths and when they occurred. We estimated mortality rates among women with and without PABC, and subdivided by time of detection among PABC patients (PABC during pregnancy, or during months 1–3, 4–6, 7–12, 13–18, 19–24 after delivery). Hazard ratios (HR) with 95% confidence intervals (CI) were estimated using the novel method of flexible parametric survival models. These models use splines to model the baseline hazard rate, and are able to pick up more subtle time-dependent effects than the more commonly used piecewise splitting of the timescale (for example in Poisson regression).

Results: Women with PABC had higher mortality than women diagnosed at the same age and calendar period and with no recent birth. In women with PABC (n=1,110), 46% died during follow-up, compared to 34% of the non-PABC patients (n=14,611). The mortality in both groups peaked at around two years after diagnosis, with the highest peak occurring in women diagnosed 4–6 months after delivery (HR=3.8, 95% CI 2.4−5.9). Also, among women diagnosed during pregnancy the mortality was higher compared to women diagnosed further away from delivery. An increased mortality among women with PABC remained up to 10 years after diagnosis.

Discussion: Women with PABC had a poorer prognosis compared to women with breast cancer and no recent birth. The worse prognosis may be due to diagnostic delays or tumour promoting hormonal boosts or changes in the tumour micro-environment post-partum. Taken together, our findings indicate that women with PABC represent a high-risk group of young breast cancer patients that may require special management. Also, by applying novel statistical methods we were able to detect time dependent effects in that the elevated mortality associated with PABC varied by timing of diagnosis and time since diagnosis.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-09.
P1-08-21: Demographic and Clinical Characteristics of Metastatic Breast Cancer Patients and Biomarker-Based Prevalence in the UK, Germany, France, Spain and Italy (EU-5).
Authors: Ganguli A , DeKoven M , Bonthapally V , Lee WC , Ray S
Published: June 2012

Background: Much peer-reviewed literature focuses on metastatic breast cancer (mBC) treatment regimens. However, research around mBC patients’ demographic/clinical characteristics across Europe is limited. This study compared such mBC characteristics as well as biomarker-based prevalence across the EU-5.

Methods: IMS LifeLink™ Oncology Analyzer (OA) database, based upon practicing oncologist surveys, was used to identify mBC patients aged ≥18 between 01/2005-06/2010. The study investigated the distribution of mBC population based on age, biomarker, co-morbidities and stage at diagnosis. This study also estimated the proportion of patients, and sites of metastasis, by lines of drug therapies (LOT).

Results: A total of 186,640 mBC patients were identified — Germany (30.2%), France (22.4%), UK (21.2%), Italy (17.7%) and Spain (8.4%). The majority of patients were aged 61–70yrs (24%-32%), except in Spain (22.3% aged 71–80yrs). Proportion of mBC patients with ≥1 co-morbid condition were highest in Germany (36.3%), followed by Spain (32.8%), UK (31.5%), Italy (27.6%) and France (20.1%), with diabetes (12.9−23.9%) and cardiac dysfunction (5.2−21.7%) being most prevalent. The distribution of mBC patients by biomarker status was 53.9% HER-/HR+, 17.8% HER+/HR+, 11.1% HER+/HR- and 17.2% triple negative, and was similar amongst the EU5 countries. The top three metastatic sites were bone (54.4%), lung (36.0%) and liver (32.7%), with proportion of bone metastasis increasing from 1st LOT (38.1%) to 4th LOT (69.6%). Almost 50% of the mBC patients were diagnosed at Stage IV, 14% at Stage III, 26% at Stage II and 6% at Stage I. Of all mBC patients, 93% received 1st LOT, 31% received 2nd LOT, 27% received 3rd LOT and 6.8% received 4thLOT.

Conclusion: In the EU-5, mBC patients were primarily elderly and HER-/HR+. The burden of bone metastasis was higher in later LOTs. Following the 1st LOT, fewer patients moved to subsequent treatments. Additional research on EU-5 clinical practice patterns is warranted.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-21.
P1-08-22: Treatment Patterns and Clinical Outcomes in Elderly Patients with HER2−Positive Metastatic Breast Cancer from the registHER Observational Study.
Authors: Kaufman PA , Brufsky AM , Mayer M , Rugo HS , Tripathy D , Ulcickas Yood M , Feng S , Wang LI , Brammer MG , Yardley DA
Published: June 2012

Background: Data are lacking regarding treatment patterns and outcomes in elderly patients (pts) with HER2−positive (HER2+) metastatic breast cancer (MBC).

Methods: registHER is a large, observational cohort of pts with HER2+ MBC diagnosed within 6 months of enrollment. Pts (N=1,001) were followed until death, disenrollment, or June 2009 (median follow-up 27 months). In these analyses, pts were stratified into three groups based on age at MBC diagnosis: younger (<65 years), older (65-74 years), elderly (≥75 years). For Progression Free Survival (PFS) and Overall Survival (OS) analyses of 1st-line trastuzumab (T) vs. no T, older and elderly pts were combined due to small number of events in elderly. Hierarchical multivariate analyses were adjusted for baseline characteristics and treatments.

Results: ER/PR status was similar across age groups (Table 1). Elderly pts with HER2+ MBC had higher rates of underlying cardiovascular disease (CVD) than younger or older pts. In pts receiving T-based 1st-line treatment, elderly pts were less likely to receive chemotherapy (C), and more likely to receive T alone or combined with hormone therapy (HT). Central nervous system (CNS) events decreased with increasing age. In T-treated pts, incidence of left ventricular dysfunction (grade ≥3) was higher in elderly pts (3/63 [4.8%]) than in younger (21/746 [2.8%]) or older pts (2/134 [1.5%]). Across age groups, unadjusted median PFS (months) was significantly higher for pts treated with T in 1st-line than those who were not (<65 years T: 11.0; <65 years no T: 3.4; ≥65 years T: 11.7; ≥65 years no T: 4.8). In pts <65 years, unadjusted median OS (months) was significantly higher in T-treated pts; in pts ≥65 years, median OS was similar (<65 years T: 40.4, <65 years no T: 25.9; ≥65 years T: 31.2, ≥65 years no T: 28.5). In multivariate analyses, T in 1st-line was associated with significant improvement in PFS across age (Table 2). In OS, significant improvement was observed for pts <65 years; results were suggestive for pts ≥65 years.

Conclusions: Elderly pts (≥75 years) with HER2+ MBC in registHER had higher rates of underlying CVD than younger counterparts and received less aggressive treatment, including less 1st-line T. These population-based, real-world data suggest improved PFS with T as 1st-line therapy across all age groups.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-22.
P1-09-09: Fetal Microchimerism and In Situ Breast Cancer.
Authors: Eun JK , Gadi VK
Published: June 2012

Background: Haploidentical fetal cells acquired and maintained by a woman in her blood and tissues long after pregnancy completion, also known as fetal microchimerism, is inversely associated with invasive breast cancer. Furthermore, in healthy women, absence of fetal microchimerism serves as a risk factor for development of future breast cancer (J Clin Oncol 29: 2011 suppl; abstr 1574). Here, we test the hypothesis that failure to maintain fetal microchimerism following pregnancy is also associated with in situ breast cancer.

Methods: Buffy coat specimens were obtained from the Roswell Park Cancer Institute Data Bank and BioRepository from parous patients with in situ breast cancer and parous control volunteers with no prior history of cancer. Using quantitative PCR, DNA extracts from peripheral blood buffy coat specimens were tested by a single female operator blinded to case:control status for the presence (and concentrations) of Y chromosome sequence DYS14, a marker of male fetal microchimerism.

Results: In this interim analysis, 83% (34/41) of healthy control women and 55% (29/53) in situ breast cancer patients tested positively for the presence of DYS14. The unadjusted odds ratio was 0.25 (95%-CI of 0.08−0.72; p=0.008). Median concentrations were also greater in healthy women compared to women with in situ cancer, 0.15 versus 0.02 fetal cell equivalents per 10^5 proband genomes, respectively (p=0.004).

Summary: Preliminary results suggest that the associations of fetal microchimerism and in situ breast cancer are similar in magnitude and direction to that observed for invasive disease. The natural history of in situ breast cancer is for a large portion of patients to develop invasive disease in 8–10 years absent of comprehensive locoregional control; results here point to a failure to maintain or loss of haploidentical fetal cells well before development of invasive disease. Final results from the entire cohort planned (n=200) with adjustment for anthropometric, reproductive, and cancer specific factors will be presented at the meeting.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-09-09.
P1-12-08: Induction of Adhesion Molecules on Target Cells Maximizes Antibody-Dependent Cell-Mediated Cytotoxicity of Anti-Her2 Therapeutic Antibody with High Fucose Glycan.
Authors: Jia X , Wong P-Y , Gazzano-Santoro H
Published: June 2012

Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one pivotal effector function of certain therapeutic antibodies. ADCC is the lysis of antibody-coated target cells by effector cells expressing Fc receptors (FcR) that recognize the fragment crystallizable (Fc) region of the bound antibody. Higher fucosylated terminal glycan on Fc results in significantly lower ADCC activity because core fucose sterically hinders the binding of Fc region to FcR on effector cells. The role of adhesion molecules in ADCC is unclear but it has been reported that adhesion molecules help better orientation of effector cells (natural killer cells) toward adhesion molecule-coated beads. We hypothesize that a preferable association of effector cells and target cells mediated by adhesion molecules may compensate ADCC loss due to the steric hindrance of high core fucose on Fc. Recombinant humanized monoclonal anti-Her2 antibodies (anti-Her2 rhuMAb) at various fucosylation levels have been used in this study to evaluate the role of adhesion molecules in ADCC of antibodies with high fucose glycan.

Materials and Methods: Natural killer (NK) cell line transfected with FcR were used as effector cells. Her2-expressing breast cancer cell lines BT-474 and SK-BR3 were used as target cells. Adhesion molecules were induced in target cells by incubation with 50ng/ml of recombinant human tumor necrosis factor (TNFa) for 1–2 days. Anti-Her2 rhuMAbs with different Fc fucosylation (G0-F from <1% to >2%) were added in the co-culture of effector cells and target cells. ADCC activities were quantitated by target cells lysis assessment using Europium-TDA release assay.

Results: Untreated BT-474 or SK-BR3 target cells had very little adhesion molecule expression. A high fucosylated anti-Her2 rhuMAb induced 52% of target cell lysis, which represented two-thirds of cell lysis by a low fucosylated Ab. Increment of E:T ratio could not maximize ADCC activities. With TNFa treatment, target cell lysis was increased from 52% to 100% by high fucose rhuMAb. This enhancement of ADCC activities is probably due to the expression of adhesion molecules such as ICAM-1 and VCAM-1, which was shown to be up-regulated by 100-fold on target cells in the presence of TNFa. Our data suggested that ADCC activities in Her2-expressing breast cancer cell lines were HER2−specific, anti-HER2 rhuMAb-dependent and adhesion molecules were indispensible for the enhanced ADCC activities.

Discussion: rhuMAbs produced in Chinese hamster ovary (CHO) cell lines may contain various Fc fucosylation, hence various ADCC activities. Although up-regulation of adhesion molecules alone cannot lyse target cells directly, our data suggested maximized ADCC activities by rhuMAbs with high Fc fucosylation level could be achieved by induction of adhesion molecule expression on target cells.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-08.
P1-12-16: HER-2 Testing and Treatment – Is Age a Factor?
Authors: Singh JK , Ewing K , Howell S , Howe M , Cramer A , Bundred NJ
Published: June 2012

Aims: Elderly breast cancer patients have a poorer prognosis due to late diagnosis and sub-optimal treatment. In the UK, HER-2 testing was introduced in 2005 and we sought to determine whether HER-2 testing was performed on patients of all ages and whether HER-2 positive patients of all ages subsequently received Herceptin, from implementation in 2005 until 2008.

Methods: Review of all newly diagnosed breast cancers in women over 50 in our Unit between January 2005 and December 2008 was performed. Cases were identified from the hospital pathology electronic database. Patients were considered HER-2 positive if tissue sections scored 3+ on immunohistochemical analysis (intense membranous staining) or if gene amplification was detected using fluorescence in-situ hybridisation (FISH). Patients were eligible for treatment with Herceptin if the tumour exceeded 1cm in size or if they had lymph-node positive disease, per UK guidelines. Herceptin treatment was determined for all patients.

Results: In total 703 patients with a median age of 68 years (range 56 to 98) were identified and 628 (89.3%) underwent definitive surgery (mastectomies: 246; wide local excisions: 382) and axillary surgery. In total 371 patients (52.8%) underwent HER-2 testing by immunohistochemistry or FISH. HER-2 tests performed by year: 2005 (51/131; 38.9%); 2006 (59/187; 31.6%); 2007 (71/189; 37.6%) and 2008 (190/196; 96.9%). Fifty six patients (15.1%) were HER-2 positive by immunohistochemistry or FISH. Median age of HER2 positive patients was 66 years (range 57 to 98); median age of HER2 negative patients was 68 years (range 56 to 96). Forty four HER-2 positive patients (78.6%) were eligible for treatment with Herceptin; of these 28 patients (63.6%) were treated with Herceptin. HER2 testing, treatment with Herceptin and overall 5-year mortality rate for patients between 50 to 69 years and patients aged 70 years or older is summarised in Table1.

Twenty percent of patients aged between 50 to 69 years and 71.4% of patients aged 70 years or older eligible for Herceptin were not treated. Reasons for non-treatment included: multiple co-morbidities (4); treatment declined by oncologists (1); no referral to oncologists (1); death prior to treatment (2) and unknown (8). Median follow up time was 43.3 months (range 0.2 to 73.8). All cause mortality was higher in the over 70 year old breast cancer group (Log Rank Test p=0.001) regardless of HER-2 status.

Conclusions: National introduction of routine HER-2 testing for all newly diagnosed breast cancers led to a testing rate increase to almost 97%. Despite this only 63.6% of HER-2 positive patients eligible for treatment with Herceptin received it. Patients aged 70 years or older were less likely to receive Herceptin due to multiple factors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-16.
P2-01-13: Impact of Erlotinib on MSC-Mediated TIC Expansion and EMT.
Authors: Lacerda L , Solley T , Debeb B , Xu W , Krisnamurthy S , Ueno N , Reuben J , Klopp A , Woodward W
Published: June 2012

Background: Recently, we have demonstrated that mesenchymal stem cells (MSC) and MSC secreted factors (MSC-CM) have a profound effect on tumor initiating cells (TIC) enriched mammosphere formation and latency of tumor xenografts formation from breast cancer cell lines. Furthermore these interactions increased the expression of epithelial mesenchymal transition (EMT)-associated proteins which are associated with tumor cell invasion and metastasis as well as the TIC phenotype. (Klopp, A. H. et al., 2010, PLoS One. 5, e12180). Our data suggest that the presence of MSC in the tumor microenvironment may increase metastases by conferring stem progenitor cell biology on more differentiated non-metastatic cells. In addition, preliminary data suggested MSC-CM upregulated EGFR signaling in breast cancer cells. Therefore, we hypothesized that inhibiting EGFR signaling with erlotinib (tyrosin kinase inhibitor) can suppress MSC-mediated TIC expansion and EMT.

Methods & Results: In order to demonstrate that erlotinib inhibits MSC-CM promoted expansion of TIC, we cultured breast cancer cells lines (SUM149, SUM159, SUM190, MDA-IBC3 and MCF-7) in anchorage independent conditions with MSC-CM and treated them with increasing concentrations of erlotinib. The efficiency of mammosphere formation was examined after 5 days. We found that erlotinib inhibited MSC mediated increase in mammosphere formation in triple negative cell lines SUM149 and SUM159, and HER2−positive cell lines SUM190 and MDA-IBC3, but not in ER-positive, erlotinib resistant MCF-7 cells. Furthermore, we evaluated the impact of erlotinib on cell cultures grown with breast cancer patient-derived fluids, such as seroma and malignant pleural effusions. We observed that the effect of erlotinib on mammospheres formation was attenuated by both types of patient fluids.

Discussion: Patients with triple negative breast cancer have the highest rates of metastases and no available targeted therapies for treatment. EGFR is expressed in a significant proportion of triple negative breast cancers, and recent clinical and preclinical studies suggest that EGFR may contribute to the metastasis or aggressiveness of triple negative breast cancer. Here we demonstrate that host and environmentally-derived factors are critical for determining resistance to erlotinib. In vivo studies regarding the ability of erlotininb to prevent MSC-enhanced TIC survival and metastases are underway.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-13.
P2-01-14: High Level of CD44 Expression and Soluble CD44 Secretion Mediate Trastuzumab Resistance in HER2 Positive Breast Cancer.
Authors: Jin Q , Baek JM , Boulbes DR , Esteva FJ
Published: June 2012

Background: Overexpression of HER2 occurs in 15–25% of invasive breast cancer. Trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of HER2, markedly improves disease-free and overall survival in patients with HER2−positive breast cancer. However, resistance to trastuzumab develops in most patients with HER2−overexpressing metastatic breast cancer. Breast cancer stem cells have been shown to express HER2, although its role in the development or maintenance of stem cells is not well characterized. CD44 is one of the proposed markers for breast cancer stem cells. We investigated the role of CD44 in acquired trastuzumab resistance in HER2 positive breast cancer.

Methods: CD44 mRNA expression was measured by quantitative real-time PCR. The proportion of CD44 positive cells was measured by fluorescence-activated cell sorting (FACS). Soluble CD44 level was detected by enzyme-linked immunosorbent assay (ELISA). In vitro tumorigenicity was evaluated by soft agar colony formation assay. Cell invasion was tested by using a matrigel invasion assay. In vivo tumor growth was evaluated using a mammary fat pad xenograft mouse model.

Results: CD44 mRNA expression, CD44 positive cell population by FACS, and CD44 secretion were significantly increased in both SKBR3 derived trastuzumab resistant SKBR3 clone 3 and BT474 derived trastuzumab resistant HR20 cells compared to parental, trastuzumab sensitive cells. Consistent with the increase in CD44, trastuzumab resistant cells showed an increase in soft agar colony formation and cell invasion, which were blocked by siRNA-induced downregulation of CD44 expression. Tumor growth and incidence were enhanced in mice injected with trastuzumab resistant HR20 cells compared to the mice injected with trastuzumab sensitive parental BT474 cells. Knock-down of CD44 expression by the transfection with CD44-specific siRNA suppressed in vivo tumor growth in the mice injected with trastuzumab resistant HR20 cells. Finally, we found high serum CD44 levels correlated with advanced tumor progression in HER2 positive breast cancer.

Conclusion: Our findings indicate that high levels of CD44 mediate trastuzumab resistance in HER2 positive breast cancer cells as well as tumor progression in mice. Circulating CD44 is prognostic in patients with HER2 positive breast cancer. CD44 may be a therapeutic target for HER2 positive breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-14.
P2-01-17: L1-CAM Promotes Adhesion of Breast Cancer Cells to the Endothelium.
Authors: Milde-Langosch K , Dippel V , Schröder C , Wicklein D , Hein S , Jänicke F , Müller V , Schumacher U
Published: June 2012

Background: Overexpression of the adhesion molecule L1 -CAM (L1) in breast cancer is correlated with nodal involvement, high grading, and a shorter recurrence-free and overall survival, but the mechanism leading to this effect is poorly understood. L1 is also expressed in endothelial cells promoting the interaction between lymphatic cells and endothelia via homophilic L1-L1 interaction. This mechanism might also mediate adhesion of breast cancer cells to endothelia and thus promote metastasis. To examine this likely role of L1, the impact of L1 expression in breast cancer cells on their adherence to human pulmonary microvascular endothelial cells (HPMEC) was investigated and potential L1 ligands on these cells were identified. Methods: MDA-MB231-Fra2 breast cancer cells expressing high levels of L1 were stably transfected with shRNA vectors to generate two clones with strong L1 downregulation (L1low clones). Adherence of these clones to endothelial cells was studied in dynamic cell-flow adhesion assays in channels seeded with HPMEC simulating in vivo-conditions. Rolling or adherent cells per minute were counted using CapImage software. For the cell-flow assay HPMEC were activated with TNFα. In order to identify the L1 ligand, endothelial cells were partly pre-incubated with blocking antibodies directed to the potential binding partners L1, ALCAM, ICAM-1 and E-selectin before interaction with tumor cells. In additional experiments, flow chambers were coated with recombinant L1-CAM, ICAM1, ALCAM or E-selectin, and the adherence of MDA-MB231 cells with high or low L1 expression to these proteins was investigated.

Results: Adhesion of MDA-MB231 cells to activated HPMEC was significantly higher in L1high cells compared to L1low clones, where the number of adherent cells was only 40–50% of the L1high control (p=0.025; p=0.035). Blocking experiments showed that the adherence of L1high cells could be reduced by antibodies directed to ALCAM (p=0.0007), but not to ICAM1. Anti-L1 antibodies had a significant effect only in passages of endothelial cells which showed L1 expression, whereas E-selectin blocking strongly diminished adherence of breast cancer cells irrespective of their L1 expression. In addition, L1low clones showed significantly lower adhesion to recombinant L1, ALCAM and E-selectin proteins compared to the parental cells.

Conclusion: Our experiments indicate that L1 expression in breast cancer cells leads to an increased adherence to activated endothelial cells via homophilic (L1-L1) or heterophilic (L1-ALCAM) interactions. This mechanism is a possible explanation for the increased metastatic potential and poor prognosis in L1-positive carcinomas observed in vivo. Our results suggest that this adhesion molecule might be a suitable target for therapeutic interventions.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-17.
P2-01-23: Seprase Promotes the Growth and Impairs the Migratory Ability of Breast Cancer Cells.
Authors: Jia J , Martin TA , Jiang WG
Published: June 2012

Background: Seprase or Fibroblast Activation Protein alpha (FAP-a) is an integral membrane serine peptidase. It has been shown that it plays an important role in tumour proliferation, migration, invasion and angiogenesis. However, previous work has not satisfactorily explained both the suppression and promotion effects that have been observed. Furthermore, the interaction of Seprase with other membrane molecules or pathway had not been well investigated. This study aimed to investigate the role of Seprase in human breast cancer. Methods: The full length of Seprase cDNA was isolated from normal human prostate tissue using RT-PCR and then cloned into an expression vector (pEF6-V5 His, TOPO). Following confirmation of successful cloning, the plasmid was transfected into the to human breast cancer cell lines, MCF-7 and MDA-MB-231. The function of Separase in these two cell lines was then analysed using cell function assays and ECIS, and the potential interaction with other signalling pathways explored.

Results: Compared with the MCF-7 wild type cells (MCF-7wt) and the MCF-7 cells transfected with pEF6/V5 empty vector (MCF-7pef), the MCF7 cells transfected with Seprase (MCF-7exp) had increased growth ability and impaired migratory ability. The growth rate of MCF-7exp cells was 1132.72% on day 5 while those of MCF-7wt and MCF-7pef control cells were 897.24% and 990.99% respectively (p=0.011 and 0.032). The resistance increase of MCF-7exp cells at 2 hours after wounding in ECIS assays was 478.22ohms compare with 1063.75 ohms and 1563.09 ohms in MCF-7wt and MCF-7pef control cells (p<0.05). In MDA-MB-231 cells, over-expression of Seprase resulted in impaired motility. The resistance increase of the MDA-MB-231 wild type cells (MDA-MB-231wt), MDA-MB-231 cells tranfected with pEF6/V5 vector (MDA-MB-231pef), and the MDA-MB-231 cells transfected with Seprase (MDA-MB-231exp) at 3 hours after wounding in ECIS assay were 216.75ohms, 216.00ohms and 120.16ohms respectively. The growth of MDA-MB-231 cells and the adhesion and invasion ability of both MCF-7 cells and MDA-MB-231 cells were not dramatically influenced with Seprase expression. An inhibitor to focal adhesion kinase (FAK) restored the reduced motility ability of both MCF-7exp cells and MDA-MB-231 exp cells. However, inhibitors to PI3K, ERK and ROCK had no influence on it.

Conclusion: These results suggest that Seprase promotes proliferation but inhibits the migration of breast cancer cells by potentially regulating the FAK pathway.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-23.
P2-02-09: TP53 Mutation Patterns in Breast Cancer Subgroups.
Authors: Dumay A , Feugeas J-P , Wittmer E , Lehmann-Che J , Bertheau P , Espié M , de Cremoux P , André F , Sotiriou C , Pusztai L , de Thé H
Published: June 2012

Tumor protein 53 (TP53) is the most commonly altered gene in human cancers. In breast cancers, TP53 is mutated in approximately 30% of all cases, but this frequency fluctuates widely within the different molecular subclasses. Different types of mutations may be observed, such as substitutions (replacement of a nucleotide pair by another one), or complex mutations (deletions or insertions of one or more nucleotides). Mutation types may reflect mechanism of DNA lesion or DNA repair deficiencies. Furthermore, mutations can give rise to different effects such as truncating mutations leading to loss of function, or missense mutations often leading to dominant negative activity. Those mutation effects can be advantageous in tumorigenesis and thus can be subject to selective pressure.

Here we classified 572 breast tumors in three groups, according to microarray data: luminal, basal and molecular apocrine. TP53 status was assessed by a yeast-based functional assay (FASAY) and cDNA sequencing. We then assessed whether any feature of TP53 mutations would be preferentially associated to a specific subtype of breast cancer.

- In term of TP53 mutation frequencies, as expected, lowest frequency was observed in luminal subgroup (26%) and highest in basal (90%) and molecular apocrine (70%). Notably, much higher rate of TP53 mutations occurred in luminal B subgroup (41%) than in luminal A one (17%), suggesting that TP53 may be an important feature in progression from luminal A to B.

- In term of mutations types, luminal tumors showed high frequency of substitutions, while molecular apocrine and basal presented increased rate of deletions and insertions, reflecting probably increased rate of DNA breaks. This suggests that same mutational events may occur in basal and molecular apocrine tumors.

- In term of mutation effects, we found high frequency of missense mutations in luminal tumors (notably AT to GC) and much higher rate of truncating mutations in basal tumors. These observations point to an existence of different selection pressure in each of them, such as a strong pressure for P53 mutations with potential dominant negative inhibition of P73/P63 (recently shown to favor invasion), in luminal tumors. Collectively, these results point not only to different mechanisms of P53 gene inactivation, but also different functional consequences among the different breast cancer subclasses.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-09.
P2-03-05: Attenuation of TGF-beta Signaling Suppresses Premature Senescence in a p21-Dependent Manner and Promotes Oncogenic ras-Mediated Metastatic Transformation in hTERT-Immortalized Basal-Like Human Mammary Epithelial Cells.
Authors: Lin S , Yang J , Elkahloun AG , Bandyopadhyay A , Wang L , Cornell JE , Yeh I-T , Agyin JK , Sun L-Z
Published: June 2012

Triple negative, basal-like breast cancer is highly aggressive and has a poor prognosis. The molecular mechanisms that drive its progression are elusive and no molecular target has been identified for its prevention or treatment. Here, we demonstrate that triple negative human breast cancer cells and clinical samples show an attenuated transforming growth factor-beta (TGF-β) signaling. Therefore, we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered TGF-β sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative RII (DNRII) receptor of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, co-expression of DNRII and H-ras-V12 rendered HMECs to become highly tumorigenic and metastatic in vivo in comparison with H-ras-V12-transformed HMECs that spontaneously escaped H-ras-V12-induced SLGA. By using microarray analysis, we found that p21 is the major player mediating Ras-induce SLGA and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling is blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-ras-V12-induced SLGA. Our results identifies that autocrine TGF-β signaling is an integral part of cellular antitransformation network by suppressing the expression of a host of genes including p21-regulated genes that mediate oncogene-induced transformation in basal-like breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-05.
P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.
Authors: Liu H , Luo AZ , Mu Z , Chu K , Ye Z , Alpaugh RK , Fernandez S , Boley KM , Jin J , Wright MC , Cristofanilli M , Robertson FM
Published: June 2012

Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC.

Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis.

Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC.

Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.
P2-06-01: Genomic Instability in Breast Cancers from Atomic Bomb Survivors: An Analysis of Microarray-Comparative Genomic Hybridization with Old Archival Tissues.
Authors: Oikawa M , Yoshiura K-I , Kondo H , Miura S , Kurashige T , Nagayasu T , Yano H , Nakashima M
Published: June 2012

Background: Sixty-six years after two atomic bombs (A-bombs) explosion on Hiroshima and Nagasaki, the incidence of breast cancer in A-bomb survivors still higher than that in controlled populations. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors, which suggested genomic instability (GIN) induced by A-bomb radiation exposure might promote breast carcinogenesis in A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on GIN in breast cancer using microarray-comparative genomic hybridization (aCGH).

Materials and Methods: DNA was extracted from the paraffin-embedded tissues of invasive ductal cancers from 11 survivors exposed at 1.5km from the hypocenter and 9 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray (Agilent® SurePrint G3 8×60k microarray). The total length of chromosomal aberrant region was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested.

Results: The mean of the derivative log ratio spread (DLRSpread), which estimates the log ratio noise by calculating the spread of log ratio differences between consecutive probes along all chromosomes, was 0.60 (range, 0.30 to 1.05). The concordance of aCGH results with FISH results for HER2 gene amplification was 90%. Samples from A-bomb survivors had significantly more copy number aberrations (CNA) than samples from control patients (P= 0.040). Samples with C-MYC gene amplification determined by aCGH analysis tended to harbor more CNA (P= 0.20), and age at the time of diagnosis tended to be inversely associated with the total length of chromosomal aberrant regions (P= 0.13). The multivariate analysis with covariance revealed that the status of A-bomb exposure was the only independent factor which was significantly associated the total length of chromosomal aberrant regions (P= 0.0324).

Conclusions: This study suggested that A-bomb radiation may affect the development of chromosomal aberrant regions including oncogene amplification by inducing GIN and may be associated with a higher histological grade in breast cancer found in A-bomb survivors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-06-01.