bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Exclusive Radiotherapy an Option for Early Breast Cancers with Complete Clinical Response after Neoadjuvant Chemotherapy?
Authors: Daveau C. , Savignoni A. , Abrous-Anane S. , Pierga J. , Reyal F. , Gautier C. , Kirova Y. , Dendale R. , Campana F. , Fourquet A. , Bollet M.
Published: December 2010

Purpose: To determine whether exclusive radiotherapy (ERT) could be an option after complete clinical response (cCR) to neoadjuvant chemotherapy (NCT) for early breast cancers (EBC).Patients and methods: Between 1985 and 1999, 1477 patients received NCT for EBC considered to be too large for primary conservative surgery. Of 165 patients with cCR, 65 were treated by breast surgery (with radiotherapy) and 100 by ERT.Results: The two groups were comparable in terms of baseline characteristics, except for larger initial tumor sizes in the ERT group. There were no significant differences in overall, disease-free and metastasis-free survivals. Five-year and 10-year overall survivals were 91% and 77% in the no surgery group and 82% and 79% in the surgery group, respectively (p = 0.9). However, a non-significant trend towards higher locoregional recurrence rates (LRR) was observed in the no surgery group (31% vs. 17% at 10 years; p = 0.06). In patients with complete responses on mammography and/or ultrasound, LRR were not significantly different (p = 0.45, 10-year LRR: 21% in surgery vs. 26% in ERT). No significant differences were observed in terms of the rate of cutaneous, cardiac or pulmonary toxicities.Conclusion: Omission of breast surgery in patients who achieved cCR to NCT was not associated with an increased risk of distant metastases or death. However a trend towards an increased risk of locoregional recurrence was observed in the ERT group. Imaging modalities appear to be essential to monitor chemotherapy response and possibly select patients in whom ERT can be proposed.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4108.
Postmastectomy Radiotherapy in Intermediate-Risk Patients: Long Term Carcinologic and Quality of Life Results of the Montpellier Cancer Center.
Authors: Azria D. , Lemanski C. , Rat F. , Huguet H. , Llacer Moscardo C. , Gutowski M. , Dubois J.
Published: December 2010

Background:The impact of postmastectomy radiotherapy (PMRT) on survival has been controversial for decade in intermediate-risk patients.We present here our monocentric long-term carcinologic and quality of life results in the subset of patients classified at intermediate risk and treated with PMRT.Material and Methods: From 1990 to 2000, 1622 patients received adjuvant radiotherapy after mastectomy. Among them, 421 were defined at intermediate risk of local recurrence i.e. (i) one to 3 positive nodes (ii) negative node but younger than 35 years old and/or pT3-T4 and/or grade 3 and/or invasive multifocality and/or lymphovascular invasion. PMRT consisted in a 50 Gy-electron therapy to the chest wall with node irradiation if indicated. Adjuvant chemotherapy and/or hormonotherapy were administered according to our local guideline. Patients were visited every 6 months for at least 10 years. Quality of life questionnaires (EORTC QLQC30/BR23) were filled in at the last medical visit.Results: Median age was 57 years (range, 27-80). One-hundred thirty five patients (32%) and 286 (68%) were premenopausal and menopausal, respectively. Seventy-five percent of the patients presented a ductal invasive carcinoma and 55% one to three positive axillary nodes. The median number of nodes was 13 (range, 3-27). 77% of the patients presented an axillary dissection with more than 10 nodes. 32% of the nodes were associated with capsular effraction. In the node negative subgroups, grade 3, lymphovascular invasion, multifocality, pT3-4, and age < 35 were found in 57%, 19%, 33%, and 4%, respectively. Extensive DCIS was found in 227 patients (54%). Hormonal receptors (ER and/or PR) were positive in 321 patients (76%) and therefore treated with postoperative tamoxifen for 5 years. Adjuvant chemotherapy was delivered in 126 patients and mainly included anthracyclin. HER2 status was not available at that time in our center. Chest wall RT was delivered in all patients to a median total dose of 50 Gy (range, 43-62). Sus-clavicular and internal mammary chain were irradiated in 294 and 346 patients, respectively. With a 10-y median follow-up, 12 (2.8%) and 74 (17.6%) patients presented locoregional and metastatic recurrences, respectively. Locoregional-free survival (LRFS) was 98.3% and 97.1% at 5 and 10 years, respectively. None of the 12 patients presented metastasis before local recurrence. Multifactorial analysis showed a significant impact of LRFS on metastatic-free survival (p<0.001). Quality of life was good to excellent.Conclusion: In this study, only 2.8% recurrences were observed with a very long-term follow-up. Local recurrence is one of the main prognostic factors for metastatic progression. RT in this population seems to be clearly indicated.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4112.
In Vivo Tracking of Expression Levels of Human Growth Factors Secreted by Human ADRCs in a Murine Fat Graft Model.
Authors: Zhu M. , Eguchi M. , Xu S. , Eguchi C. , Fraser J. , Pinkernell K.
Published: December 2010

Background: Our previously published data show that Adipose-derived regenerative cells (ADRCs) enhance the long term retention of autologous fat grafting compared to non-cell-enhanced grafts. To further understand the time course of growth factor expression of ADRCs, we developed an in vivo xenogenic fat graft model.Materials and Methods: Male NCR nude mice were used as recipients to avoid immune rejection of human ADRCs. Inguinal fat pads from male C57Bl6 mice were minced and used as donor fat for transplantation. Human ADRCs from three healthy donors were isolated using the automated Celution® 800/CRS System (Cytori Therapeutics, San Diego, USA) and mixed with minced mouse fat at a concentration of 5x10E5/ml. The mixture was grafted into the dorsal subcutaneous space of nude mice. Grafted samples were harvested at day 0, day 1, day 7, day 14, day 28, day 56 and day 84, for a total of seven different time points (N=4 per time point). DNA and RNA was isolated for further human-specific detection of IGF-1 and VEGF-A and human-specific single copy DNA using real time RT-PCR. All data were normalized to time 0 samples as 100%.Results: Both of human IGF-1 and VEGF-A expression were detected in the first two weeks after fat grafting, and each of the human growth factor showed its unique pattern. For IGF-1, the highest expression level of 100±7.5% was detected at time 0 with a decline to 0.9±0.7% that was maintained for two weeks. By day 14 only 0.22% of the original signal was detected and no more signal could be detected after day 28. In contrast, on day 1 VEGF-A expression showed an increase to 212±82.6% of the time 0 value. It further declined to 30±23% on day 7 and 8.7±11.7% on day 14. After day 28 no more VEGF-A expression could be detected. The engraftment of ADRCs, measured by DNA content, declined to 12±10.7% on day 1 and remained at around 1% until day 14. By day 28, only 0.14% of human DNA signal could be detected and no more signal could be found after day 56.Conclusion: These findings demonstrate that this xenogenic approach allows the evaluation of single growth factor expression of human ADRCs in vivo using a mouse background when studying cell-enhanced fat grafting. It also allows quantitative assessment of cell survival. By using this model we find that human ADRCs secrete several growth factors within the grafts and that each growth factor has its own unique profile. Furthermore, it becomes evident that ADRCs are active for several days after implantation before growth factor expression subsides. This is an important safety feature of this adult cell therapy source since continued, high level expression of growth factors would not be desirable in cases of reconstruction after breast cancer removal, when concerns of residual tumor cells exist.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4124.
Magnetic Resonance Image Guided Focused Ultrasound Surgery of Early Breast Cancer: Efficacy and Safety in Excisionless Study.
Authors: Furusawa H. , Yasuda Y. , Shidooka J. , Nakahara H. , Hirabara E. , Inomata M. , Machida E. , Maeda Y. , Komaki K.
Published: December 2010

Background: The prognosis of the breast cancer patient doesn't depend on the local treatment, but on the systemic treatment. Breast conserving therapy, lumpectomy following radiotherapy, has been proved to be as good as mastectomy in several clinical trials. When focused ultrasound waves propagate in tissue, the energy is absorbed and converted to heat, resulting in tissue coagulation. MRI is the best modality in spatial resolution for the detection of breast cancer spread. Two phase II clinical studies (excision studies) to investigate pathological efficacy and clinical safety of MR guided focused ultrasound surgery (MRgFUS) were conducted and their outcome has been published.Purpose: The purpose of this study is to inspect the efficacy and safety of MRgFUS as a local treatment for early breast cancer followed by radiotherapy in a phase III study.Methods: The inclusion criteria include 1) tumor diagnosis by core needle biopsy under ultrasound 2) HER-2,HR status confirmation 3) tumor size is less than 15mm in diameter, 4) well demarcated mass in contrast enhanced MRI. The exclusion criteria include 1) pure type mucinous carcinoma, 2) invasive micropapillary carcinoma, 3)skin dimple or dimpling sign, previous surgical scar of the skin above the tumor, 4) location of the tumor requires a high sonication angle. Core needle biopsy was performed again within three weeks after ablation and no residual viable cancer cells were identified by HE stain and single strand DNA immunohistochemical stein. The following radiotherapy is whole breast(50Gy) and boost(10Gy). Results: Fifty patients entered this study and forty-one lesions were completely treated from April 2005 to June 2009. The median age was 57 years old (37 - 72). The average tumor size was 11.0mm (6 - 15). The median follow up period was 29 months (1 - 48). Thirty five cases were followed up for more than 12 months. There were no severe adverse events and no local recurrence cases.Conclusions: Although the number of cases is still small and the follow-up period is short, MRgFUS has the potential of replacing usual breast conserving surgery. Strict selection of cases is essential.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4126.
Targeting Pleural Carcinoma Cells with the Anti-EpCAM BiTE Antibody MT110 by Stimulating Microenvironment T Cells.
Authors: Mayer B. , Witthauer J. , Winter H. , Funke I. , Baeuerle P. , Jauch K.
Published: December 2010

Rationale: Despite therapy, most patients with advanced breast cancer develop malignant pleural effusion (MPE) associated with short survival. Strikingly, the metastatic cancer cells are frequently hormon receptor negative and show a reduced Her2/neu expression compared to the corresponding primary tumors. Thus, new treatment strategies are urgently needed.Objectives: In the present study, the efficacy of the bispecific single-chain antibody MT110 targeting both the epithelial antigen EpCAM (CD326) and the T cell antigen CD3 was investigated ex vivo with MPE samples of breast cancer patients.Methods: Target antigen expression of MPE cells was analyzed by immunohistochemistry. Percentage of redirected target cell lysis by MT110 was determined by double staining of cells with 7-amino actinomycin and an anti-EpCAM antibody using FACS analysis. Activation of autologous CD4+ and CD8+ cells in response to MT110 was studied by expression of CD25 and granzyme B using FACS staining and different cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) using ELISA analysis.Results: EpCAM+ cells were found in 14 out of 18 (78%) MPE samples from metastatic breast cancer patients. The fraction of EpCAM+ pleural carcinoma cells varied between 30% and 100% (mean 78%). CD3-positive cells were detected in all MPE samples ranging from 60% to 93% (mean 80%). Seven effusion samples were analyzed for MT110 treatment and revealed a dose-dependent and specific redirected lysis of EpCAM+ cells after 48h and 72h with 10 ng/ml (48h, 72h: p=0.03, respectively) and 1000 ng/ml (48h, p=0.03; 72h, p=0.016). After 72 h, 57% ± 29.5% (mean ± SD) of EpCAM+ cells were killed using 1000 ng/ml MT110. The ratio between effector (E) and target (T) cells revealed no influence on the extent of the redircted specific lysis. Antibody treatment increased the fraction of CD25/CD4 cells after 48h (p=0.03) and 72h (p=0.016). Similar, CD25 expression on CD8 cells was stimulated after 48h (p=0.03) and 72h (p=0.016) using 1000 ng/ml MT110. Cytokine analysis revealed a strong TH1 immune response detecting an increased TNFα (p=0.016) and IFNγ (p=0.03) secretion. Conclusion: Single-agent therapy with MT110 is capable of activating unstimulated autologous T cells for efficient and specific redirected lysis of EpCAM+ tumor cells in MPE from breast cancer patients. MPE revealed interindividual differences regarding the extent of specific lysis and the stimulation of the microenvironmental T cells. Thus, treatment with the bispecific antibody MT110 might by an option in the management of a subcohort of metastatic breast cancer patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4133.
Local Radiotherapy Rescues Responsiveness to Anti-CD137 Immunotherapy in a Mouse Model of Advanced Breast Cancer.
Authors: Pilones K. , Kawashima N. , Formenti S. , Demaria S.
Published: December 2010

BACKGROUND: The benefits of immunotherapy are often hampered by immunosuppressive networks in the tumor microenvironment. Accumulating evidence indicates that local ionizing radiation (IR) can synergize with immunotherapy and improve tumor response.CD137/4-1BB is a co-stimulatory molecule expressed on activated T-cells that provides a survival signal to promote the expansion and differentiation of antigen-specific T-cells. Pre-clinical studies have shown the ability of agonistic anti-CD137 antibodies to enhance anti-tumor immunity. 4T1 is a poorly immunogenic experimental mouse model of metastatic breast cancer. 4T1 cells injected s.c. form a tumor and rapidly spread systemically giving rise to lung metastases that kill the animals. We have previously shown that IR as single modality can delay growth of the irradiated 4T1 s.c. tumors but not extend mice survival.METHODS: BALB/c mice were inoculated s.c. with 4T1 cells on day 0. Treatment with anti-CD137 mAb (200 mg, BMS-469492 from Bristol Myers Squibb) was started on day 7 or 15 and repeated every 3 days for two or three times. For radiotherapy mice were randomized into 4 groups receiving (1) no treatment, (2) IR alone, (3) anti-CD137 mAb and (4) IR+ anti-CD137 mAb. IR was delivered exclusively to the tumor in 2 fractions of 12 Gy at 24 hours interval starting on day 13. Mice were followed for tumor growth and survival.RESULTS: Administration of anti-CD137 mAb to 4T1 tumor-bearing mice starting at day 7 resulted in significant tumor growth delay which correlated with an 8-fold increase in the ratio of intratumural effector CD8 T cells (Teff) to regulatory T cells (Treg) at day 15. In contrast, when administration of anti-CD137 mAb was delayed until day 15 no anti-tumor effect was observed.The median survival of mice (39 days) was not increased by treatment with anti-CD137 mAb or IR used as single modalities (39 days). On the other hand, the combination of IR and anti-CD137 mAb increased survival to 49 days, although no complete tumor remission was observed.CONCLUSIONS:Overall, our studies indicate that with tumor progression mice became resistant to immunotherapy with single modality anti-CD137 mAb. However, a therapeutic effect was seen when anti-CD137 mAb was combined with local radiotherapy. We are currently investigating the mechanisms of resistance to anti-CD137 mAb treatment that develop with 4T1 tumor progression. Accumulation of Treg and myeloid-derived suppressor cells (MDSC) within the tumors likely contribute to resistance to immunotherapy by creating a highly immunosuppressive microenvironment. Identification of these mechanisms will provide new therapeutic targets to improve the response to treatment. Importantly, data support the combination of local radiotherapy with immunotherapy in advanced breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4134.
Elevated Circulating Myeloid Derived Suppressor Cells (MDSC) Are Associated with Inferior Overall Survival (OS) and Correlate with Circulating Tumor Cells (CTC) in Patients with Metastatic Breast Cancer.
Authors: Cole S. , Montero A. , Garret-Mayer E. , Onicescu G. , Vandenberg T. , Hutchens S. , Diaz-Montero C.
Published: December 2010

Background: MDSC acumulation is an important mechanism of tumor escape from immune surveillance and cancer progression. Pre-clinical studies have shown a correlation between tumor progression and MDSC. While we and others have shown that circulating MDSC correlate with clinical stage, to date there are few clinical studies that have explored the correlation of MDSC with metastatic tumor burden or as an adverse prognostic factor. Consequently, the goal of this study was to determine whether levels of MDSC in patients with stage IV breast cancer correlate with tumor burden using CTC and Swenerton Scores (SS) as surrogates for metastatic disease burden, and if circulating MDSC levels were predictive of overall survival.Methods: Flow cytometric analysis was performed on peripheral whole blood from 25 patients with stage IV breast cancer prior to initiation of therapy and at defined intervals during therapy. The MDSC population was defined as lineage-, CD33+, HLA-DR-, CD11b+. CTC (CellSearch®) and MDSC were drawn simultaneously and SS were calculated using a standardized scoring system previously defined and published in the literature. A generalized estimating equation regression model was created and fitted individually for each predictor. OS was defined as time of study enrollment to date of death.Results: Using a generalized estimating equation regression model the percentage of MDSC in peripheral blood was found to have a close correlation with CTC (Pearson correlation 0.62, p=0.0001) and a coefficient of 0.29 (0.14, 0.43 95%CI). The correlation between percent MDSC and SS was not significant (Pearson 0.31, p=0.12). Interestingly, no significant correlation between CTC and SS was noted either. MDSC levels were also found to predict OS in patients with the highest percentages at the first visit having significantly decreased median overall survival [6.8 vs. 19.6 months; p=0.05]Similarly, high MDSC levels at the final blood draw was also found to correlate with significantly shorter median OS [p=0.023].Conclusions. MDSC correlated well with CTC but not SS. High circulating MDSC levels were associated with significantly decreased overall survival in patients with metastatic breast cancer. MDSC appear to be a promising prognostic immunologic biomarker in metastatic breast cancer. Further studies are needed to better define the role of MDSCs in breast cancer progression and may be a useful therapeutic target to enhance the effectiveness of immune based therapies in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4135.
Estrogen Receptor-Mediated Repression of Target Genes: Mechanism of Action, and Biological Significance.
Authors: Oesterreich S. , Jiang S. , Verdin E. , Lee A. , Malik S.
Published: December 2010

Estrogen-bound estrogen receptor (ER) can regulate the transcription of a large number of genes. Although estrogen represses as many genes as it induces, induction has been studied in detail while the repression mechanisms remain largely unknown. In this study we characterized several estrogen-repressed ERα target genes that are directly repressed by estrogen in breast cancer cells. Interestingly, many repressed genes are tumor suppressor genes, playing critical roles in cell cycle inhibition and/or apoptosis. One gene that particularly piqued our interest is Reprimo (RPRM) because of its robust repression in a number of cell lines, and its role as a cell cycle inhibitor. Additionally, it is highly methylated in a variety of cancers including breast cancer and maps to a locus that displays loss of heterozygosity, suggesting that it may be a tumor suppressor gene. As a result, E2-mediated repression of this gene may be a crucial step in the progression of breast cancer. We did show that RPRM decrease (by siRNA) enhances estrogen-mediated S-phase entry, strongly suggesting a biological role of its repression. RPRM was actively and strongly repressed by estrogen and this repression was not due to a quicker turnover of the RPRM mRNA by estrogen. E2-mediated repression of RPRM levels did not require new protein synthesis since it is also repressed in the presence of the translation inhibitor, cycloheximide. We find that estrogen repression of RPRM requires a tripartite interplay between ERα, FoxA1, and HDAC7 as knockdown of these proteins abrogates its repression. Remarkably, silencing of HDAC7 significantly relieved repression of the majority of eleven estrogen-repressed genes tested. Further examination of the interplay between HDAC7 and ERα revealed that HDAC7 can interact with ERα and repress its transcriptional activity in a deacetylase-independent manner. Chromatin immunoprecipitation assays demonstrate that ERα, FoxA1, and HDAC7 are all recruited to a cis-regulatory enhancer (-4.8 kb) in the RPRM gene in the presence of estrogen, which was associated with a release of RNA Pol II from the proximal promoter. Repression requires FoxA1 to be recruited to an epigenetic signature characterized by H3K4 mono- and di-methylation. In summary, we have uncovered a unique requirement for a deacetylase-independent function of HDAC7 in estrogen repression of genes such as RPRM.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4136.
NM23-H2, an ERβ Associated Protein, as a Prognostic Marker in Breast Cancer.
Authors: Hamilton-Burke W. , Speirs V. , Cummings M. , Horgan K. , Shaaban A.
Published: December 2010

Purpose: Endocrine therapy is a well established therapy for estrogen receptor (ER) positive breast cancer. Two ER have been identified but currently only ERα is used in clinical practice. The role of ERβ in breast cancer is less well understood. Two human NM23 genes exist, NM23-H1 and NM23-H2. Several studies have shown an inverse relationship between overexpression of NM23-H1 in breast cancer and disease aggressiveness. NM23-H2, a metastasis suppressor protein, was recently identified as ERβ associated protein in vascular wall of atherosclerotic lesions, but the prognostic significance of this protein and its association with ERβ has not being studied in breast carcinoma. The purpose of this study was to investigate whether NM23-H2 is expressed in breast cancer and its potential clinical implications.Material and Methods: A cohort of 427 patients with diagnosis of primary invasive breast carcinoma between the period of 1994 to 1997 was used for this study. They all were patients diagnosed and treated at the Leeds Teaching Hospitals, UK. Immunohistochemistry was used primarily to identify the expression of NM23-H2 in this study. Regulation of NM23-H2 in vitro in MCF-7 cells was assessed by Western blotting. p<0.05 was considered statistically significant.Results: Patients ages were 27-92 years (median = 58 years, IQ range 47-69 years) with median follow up of 110 months. Tumour size varied from 1 and 130 mm (median=22.7mm, IQ range 14-26mm). Seventy eight percent were invasive ductal carcinoma NST and 11.5% were invasive lobular; 3.9% were special type (tubular, mucinous); and 6.6% were mixed type. Twenty-three percent of the tumours were grade 1, 43.3% were grade 2 and 33.7% were grade 3. Axillary metastases were present in 51% of the cases. NM23-H2 expression was variable between tumours and demonstrated only in the cytoplasm of breast carcinomas. The percentage of staining ranged from 0% to 100% (median 80%).NM23-H2 was strongly correlated with ERβ1 and ERα (p <0.001; correlation coefficient 0.252 and 0.176 respectively). No correlation was found between NM23-H2 with age, size, grade or lymph node status. NM23-H2 when expressed at levels higher than 30% was associated with improved overall survival (p= 0.030). In a multivariate Cox hazard analysis, NM23-H2 overexpression was a significant predictor of better survival independent of tumor grade, lymph node status, size or ERα. MCF-7 cells treated with 17β-estradiol (E2) expressed higher levels of NM23-H2(17.5kDa) compared with serum treated cells.Conclusion: This is the first time to our knowledge that the expression of NM23- H2 in breast cancer and its association with ERβ has been investigated. Our data provide evidence that immunohistochemical overexpression of NM23 H2 is associated with ERβ and ERα in breast cancer and with improved survival. NM23-H2 might be a good prognostic marker in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4148.
QuantiGene 2.0 Assay for ER, PR and HER2 RNA Levels Is a Useful Adjunctive for the Evaluation of ER, PR and HER2 in Breast Cancer.
Authors: Lee A. , Chae B. , Song B. , Jung S. , Yim H.
Published: December 2010

Immunohistochemistry (IHC) for the estrogen receptor (ER) and progesterone receptor (PR) is an established procedure in the routine treatment of patients with breast cancer, primarily to predict responses to hormonal therapy. HER2 IHC and FISH are also recommended to select patients for anti-HER2 based therapy. The IHC method for ER and PR is semi-quantitative and needs standardization. Furthermore, ER and PR RNA levels are continuous variables and quantitative measurement of ER levels can help predict the tamoxifen benefit breast cancer. In this study, the RNA levels of ER, PR and HER2 were measured using the QuantiGene 2.0 assay in formalin-fixed, paraffin- embedded (FFPE) tissue samples from breast cancer (n=40) and compared with conventional ER/PR IHC results and HER2 FISH results. There were no cases of discrepancy in ER status between staining with the SP1 antibody and staining with the 6F11 antibody; however, the ER score was statistically different between them (5.3 ± 3.3 vs. 4.9±3.2, P=0.02). One out of 40 cases (2.5%) showed a false negative result when performed using a 2mm representative tissue sample (TMA), and 1 out of 40 cases (2.5%) showed a discrepant ER status between the results using the Allred scoring system and the fractional scoring system. These results reconfirmed that various factors such as the primary antibodies used, interpretation methods and TMA vs. whole section, influence the results of IHC. When the cut-off values for ER, PR and HER2 RNA levels were 5.0, 7.2 and 50, respectively, the sensitivity, specificity, positive predictive value and negative predictive value of the QuantiGene 2.0 Assay were 96.6%, 90%, 96.7% and 90%, respectively, for ER; 89.7%, 81.8%, 92.9% and 75% for PR, and 83.3%, 96.4%, 90.9% and 93.1% for HER2. The Allred scores for ER and PR were correlated with RNA levels (P=0.046, R=0.32; P=0.00, R=0.61, respectively), and the HER2 FISH ratio was correlated with RNA levels (P=0.00, R=0.76). In conclusion, measuring ER, PR and HER2 RNA levels, which is known to have additional benefits in patient treatment, from FFPE tissue may be a helpful adjunctive in determining the status of ER, PR and HER2 in breast cancer. And the QuantiGene 2.0 assay, which does not require an RNA extraction step, uses FFPE samples, and expensive modern equipment, is easy to perform even in a small scale laboratory.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4153.
Effect of ER/PR Weak Expression on Neoadjuvant Chemotherapy.
Authors: Min S. , Park C. , Lee S. , Jung S. , Lee S. , Kim S. , Kang H. , Kwon Y.
Published: December 2010

Background: Neoadjuvant chemotherapy(NAC) is now a relatively standard treatment for advanced breast cancer. Many reporters suggest that the response rate of NAC on ER/PR positive breast cancer is lower than on ER/PR negative cancer. However, there are few reports regarding the effectiveness of NAC according to expression of ER/PR. The aim of this study is to assess the effect of NAC on ER/PR weak positive breast cancer compared with ER/PR negative and strong positive breast cancer.Patients and Methods: 495 patients who underwent NAC between 2001 and 2008 were included. The patients were treated with variable chemotherapy regimens. We analyzed the response rate to NAC by revewing the preoperative breast imaging study including breast US and postoperative pathologic report. ER/PR expression by IHC were scored as negative, weak positive, intermediate and strong positive. We compared weak positive group with negative and strong positive group. We assessed clinicopathologic characteristics, response rate, disease progression according to ER/PR expression and chemotherapy regimens.Results: There were 165(33.3%) of ER/PR negative, 47(9.5%) of weak positive and 207(41.8%) of strong positive, respectively. Most of preoperative clinical stage was stage IIIA, 166(39.6%). Mean age of patients was 46.7±10.1 year. The pathologic complete response(pCR) was 47(11.2%). In weak positive group, pCR, partial response, stable disease, progressive disease was 9(19.1%), 23(48.9%), 9(19.1%) and 6(12.8%), respectively. There was no significant difference between weak positive group and negative group. However, pCR was less(11(5.3%)) in strong positive group compared with weak positive group. (p=0.004). In patients treated with preoperative anthracycline-based NAC and taxen-based NAC weak positive group was similar to negative group. However, pCR rate of strong positive group was lower than other groups.Conclusion: The clinicopathologic behaviors and effect of NAC on weak positive breast cancer were similar to ER/PR negative breast cancer. Therefore, neoadjuvant chemotherapy might be more effective in ER/PR weak positive breast cancer rather than strong positive breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4157.
DCIS-Associated Myoepithelial Cells Expressing the αvβ6 Integrin Modify Tumour Cell Behaviour: A Predictor of Invasive Progression?
Authors: Jones J. , Allen M. , Thomas G. , Dawoud M. , Vallath S. , Payne S. , Edwards D. , Pennington C. , Marshall J. , Hart I.
Published: December 2010

Ductal carcinoma in-situ (DCIS) now accounts for up to 25% of breast cancer presentations. DCIS will progress to invasive breast cancer in 25-40% of cases, though the mechanisms involved in this progression are poorly understood. Understanding the factors that promote progression to invasive disease is of importance both in terms of developing better prognostic and predictive markers, and also in the development of novel therapies.One of the key features in the progression of DCIS to invasive disease is penetration of the myoepithelial-basement membrane barrier. As well as being a physical barrier myoepithelial cells (MEC) also have been described as tumour suppressors exerting their effects through autocrine and paracrine pathways, but there is evidence that these cells change in DCIS.The integrin αvβ6 is an epithelial-restricted protein, not usually expressed in normal adult tissue, which has been demonstrated to be up-regulated in invasive cancer where it activates TGF-β. Here we show that αvβ6 is up-regulated in the myoepithelial cell compartment of high grade DCIS (∼ 100% in ∼200 cases) especially where invasion also is present. To understand the relevance of this observation we have created a model for DCIS-associated MEC by introducing β6 cDNA into an immortalised MEC line (1089) to create the αvβ6 expressing derivative, β6-1089. Using Transwell assays we demonstrate increased invasive activity of a panel of breast cancer cell lines exposed to conditioned media from β6-1089 cells relative to the control cell line. Using our organotypic assay, to examine invasion in a more physiological manner, we also found that the invasive capacity of MDA-MB-231 cells is enhanced after they are co-cultured with β6-1089 cells. Furthermore, in-vivo experiments demonstrate increased growth of tumour cells injected with β6-1089 MEC compared to control 1089 cells. Expression of αvβ6 de novo in β6-1089 cells caused an increase in MMP9 expression while a chemical inhibitor of MMP9 was able to block the observed increased invasion. Tissue studies revealed a highly significant correlation between MEC expression of αvβ6 and of MMP-9 (p<0.001), supporting these in vitro findings.These results show that up-regulation of αvβ6 expression in MEC from DCIS with associated invasion is a common event and promotes invasion of breast cancer cells through increased elaboration of MMP-9. This suggests expression of αvβ6 by myoepithelial cells may be an important prognostic marker for the progression of DCIS.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4159.
Overexpression or Lack of beta 2-Adrenergic Receptor Correlate with Progression of Breast Cancer.
Authors: Ou Z. , Wang L. , Hong X. , Shen Z. , Shao Z.
Published: December 2010

Background: Recent studies showed that stress can modulate biological behavior of ovarian carcinoma through adrenergic receptors. Since both nonselective beta-adrenergic receptor antagonist propranolol and selective beta 2-adrenergic receptor (b2AR) antagonist ICI 118,551 instead of b1AR antagonist atenolol could eliminate most of the effects, b2AR is accordingly regarded as the key molecule. In this study, we investigated the role of b2AR in breast cancer. Material and Methods: Gene expression of b2AR was analyzed by RT-PCR in human breast cancer cell lines MDA-MB-231, MDA-MB-435, MDA-MB-468, MCF-7, T47D, BT-549, HCC1937, BCaP-37 and normal mammary epithelial-like cell line HBL-100. b2AR cDNA was transfected into MDA-MB-435 cells. Protein level of b2AR was analyzed by immunohistochemistry in clinical samples, including 25 normal human breast or benign disease breast tissues and 202 breast cancer tissues. Results: b2AR was detected in majority of breast cancer cell lines, except for MDA-MB-435, MCF-7 and T47D, and all of normal or benign disease breast tissues. Both nonselective beta-adrenergic receptor agonist norepinephrine (NE) and selective b2AR agonist terbutaline promoted proliferation and invasion of spontaneous b2AR-positive MDA-MB-231 cells and transfectant MDA-MB-435-b2AR cells, these effects were completely inhibited by selective b2AR antagonist ICI 118,551. In the breast cancer patients, 125 samples (61.9%) expressed b2AR weakly or moderately just as normal breast or benign diseases tissue, and 43 (21.3%) presented significantly stronger b2AR expression, while no b2AR staining was found in other 34 samples (16.8%). Higher expression or lack of b2AR were associated with axillary lymph nodes metastasis and poor disease free survival (DFS). Discussion: Based on our preliminary results, the breast cancer patients could be classified into three subgroups: b2AR “normal”, high expression and negative groups. Overexpression or lack of b2AR associated with metastasis and clinical prognosis in breast cancer. Obviously, abnormal expression of b2AR may influence the outcome of breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4165.
Gene Targets of Insulin-Like Growth Factor-I Receptor (IGF-IR) Signaling and Their Potential Role in Breast Cancer.
Authors: Potter A. , Potter A. , Casa A. , Casa A. , Lee A. , Lee A.
Published: December 2010

BACKGROUND: Insulin-like Growth Factor-I Receptor (IGF-IR) has been implicated in a number of human tumors including breast cancer. IGF-IR is over-expressed and potentially hyper-phosphorylated in breast cancer. The specific gene targets of the IGF-IR signaling pathway, and the role of these genes in the development and progression breast cancer is not known.METHODS: MCF7B breast caner cells, and MCF10A immortalized mammary epithelial cells were treated with either serum-free medium or serum-free medium plus 100 ng/ml IGF-I for 3 hours and 24 hours. RNA was isolated and hybridized to U133A Affymetrix microarrays. Candidate genes were selected based on fold change and p-value. Q-RT-PCR was used to validate expression of potential candidate genes. 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) was used (50µM) to block transcription. Cycloheximide (CHX) (20µg/mL) was used to block translation.RESULTS: Data from the microarray revealed 3673 and 3727 genes that were regulated by IGF-I treatment in MCF7B and MCF10A cells, respectively, at 3 hours. Various genes have been validated by Q-RT-PCR, and of these, Suppressor of Cytokine Signaling 2 (SOCS2) SOCS2 was repressed by IGF-I in MCF7B cells, but induced by IGF-I in MCF10A cells. The repression of SOCS2 by IGF-I was inhibited by pretreatment of MCF7B cells with a small molecule inhibitor of IGF-IR, but not by knockdown of IGF-IR by siRNA. This suggests that the repression of SOCS2 is specific to IGF-I signaling, but may be occurring through IGF-I stimulation of an alternate pathway other than IGF-IR. IGF-I doesn't alter levels of SOCS2 mRNA following blockade of transcription, suggesting that IGF-I does not have an effect on SOCS2 mRNA stability. Furthermore, IGF-I-mediated repression of SOCS2 does not require new protein synthesis, and is thus likely an immediate and direct response.CONCLUSION: SOCS2 is a candidate tumor suppressor gene in breast cancer, with loss of SOCS2 being related to cell proliferation and tumor growth. Here we show that SOCS2 is one of the gene targets of IGF-I signaling and is repressed by IGF-I stimulation in MCF7B breast cancer cells. Furthermore, the repression of SOCS2 by IGF-I appears to be via a direct inhibition of transcription. Together, this suggests that SOCS2 is a direct gene target of IGF-I signaling and that IGF-I repression of SOCS2 may be a crucial event that plays a role in driving the development or progression of breast cancer. Further studies are planned to determine how IGF-I affects SOCS2 function and the role of SOCS2 in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4167.
The Growth Factor GP88 (Progranulin) Confers Aromatase Inhibitor Resistance to Breast Cancer Cells.
Authors: Serrero G. , Serrero G. , Brodie A. , Brodie A. , Sabnis G. , Macedo L. , Abrhale T.
Published: December 2010

The development of aromatase inhibitors has provided novel therapeutic strategies for post-menopausal patients with estrogen receptor positive (ER+) breast tumors. Aromatase inhibitors, such as letrozole, inhibit ER+ breast cancer cell growth by blocking the synthesis of estrogen by the enzyme aromatase in the tumor stroma. However, ER+ breast cancer cases can sometimes fail or cease to benefit from such therapy. Several mechanisms have been proposed for aromatase inhibitor resistance. Here, we investigate the role of the 88 kDa autocrine growth factor PC-Cell Derived Growth Factor, also known as GP88 or progranulin on the acquisition of aromatase inhibitors resistance by ER+ breast cancer cells. GP88 is the largest member of the epithelin-granulin family characterized by a unique cysteine rich motif. GP88 has been shown to play a role in breast tumorigenesis. GP88 expression increased in breast cancer cells in a positive correlation with tumorigenesis whereas inhibition of GP88 expression lead to a 98% reduction in tumor incidence and growth rate in vivo. Pathological studies of paraffin embedded breast cancer biopsies have shown that GP88 is expressed in infiltrating ductal carcinoma (IDC) in correlation with expression of parameters of poor prognosis but was independent from Her-2 expression. Most importantly, increased GP88 expression in ER+ IDC was associated with increased recurrence and decreased overall survival. The present study investigated the effect of GP88 on the proliferation and letrozole responsiveness of ER+ breast cancer cells that express high aromatase activity (MCF-7 CA and MCF-7 AC1 cells). For both cell types, GP88 added exogenously conferred letrozole resistance in a time and dose-dependent fashion. GP88 also stimulated survival and soft agar colony formation of MCF-7-CA and AC1 cells in the presence of letrozole. GP88 overexpressing cells displayed higher basal levels of phosphorylated ERK1/2 and AKT, both known to be activated in GP88 mitogenic signaling pathways. In addition, naturally letrozole resistant breast tumors displayed a 10-fold increase in GP88 expression when compared to letrozole sensitive cells. Treatment of these letrozole resistant cells with GP88 SiRNA lead to a dose dependent inhibition of proliferation and restoration of letrozole sensitivity indicating that GP88 is regulating the ability of the cells to respond to letrozole.These data show the importance of GP88 as a target for the development of diagnostic and therapeutic products for aromatase inhibitor resistant breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4171.
SOLTI-0701: A Multinational Double-Blind, Randomized Phase 2b Study Evaluating the Efficacy and Safety of Sorafenib Compared to Placebo When Administered in Combination with Capecitabine in Patients with Locally Advanced or Metastatic Breast Cancer (BC).
Authors: Baselga J. , Roché H. , Costa F. , Getúlio Martins Segalla J. , Pinczowski H. , Ma Ciruelos E. , Cabral Filho S. , Gómez P. , Van Eyll B. ,   Grupo Español de Estudio Tratamiento y Otras Estrategias Experimentales en Tumores Sólidos
Published: December 2010

INTRODUCTION: Sorafenib (SOR) is a potent multikinase inhibitor with anti-angiogenic and anti-proliferation activity. It is indicated for the treatment of advanced renal cell carcinoma and unresectable hepatocellular carcinoma. As a single agent, SOR has shown modest activity in patients with advanced BC. Here, we report results from one (SOLTI-0701) of four Phase 2b trials of sorafenib in combination with chemotherapy for advanced breast cancer.METHODS: SOLTI-0701 is a multinational, double-blind, randomized, placebo-controlled phase 2b study in patients with locally advanced or metastatic BC. Eligibility criteria included HER-2 negative tumors and <2 prior chemo regimens for advanced/metastatic BC. Patients with active brain metastasis were excluded. Patients were randomized (1:1) to receive CAP (1000 mg/m2, orally, twice daily [BID], for 14 of every 21 days) with placebo (pl) or SOR (400 mg, orally, BID, continuously). Randomization is stratified by metastatic site (visceral vs nonvisceral). The primary endpoint is progression-free survival (PFS), and secondary endpoints include overall survival, time to progression, response rate, response duration, safety, and QoL. Disease assessments occur every 6 weeks for the first 24 weeks of the study and then every 9 weeks thereafter until disease progression. Based on projected event and accrual rates, a sample size of 220 patients has been calculated. We estimate 120 PFS events within 17 months of study initiation. The study is registered at EudraCT (ID 2007-000290-32).RESULTS: Enrollment began in August 2007 and was completed in December 2008 with 229 patients randomized. Study findings demonstrated that the median PFS was extended in patients treated with SOR and CAP compared to patients receiving CAP and PL. These results were statistically significant (p=0.0006). The safety and tolerability of the combination did not show any new or unexpected toxicities. A subset analysis will be presented.CONCLUSION: The SOLTI-0701 study will provide important efficacy and safety data for the use of sorafenib in combination with oral chemotherapy for advanced breast cancer. These data will help us better understand the potential role for sorafenib in BC and if phase III trials are warranted.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 45.
Targeting Intrinsically-Resistant Breast Cancer Stem Cells with Gamma-Secretase Inhibitors.
Authors: Chang J. , Landis M. , Schott A. , Pavlick A. , Dobrolecki L. , Korkaya H. , Zhang X. , Froehlich A. , Rodriguez A. , Rimawi M. , Wicha M. , Lewis M. , Hayes D.
Published: December 2010

Background: We showed previously that tumorigenic, mammosphere-forming human breast cancer cells characterized by high CD44 and low or undetectable CD24 levels (CD44+/CD24-/low) are intrinsically resistant to conventional chemotherapy, and therefore may be responsible for cancer relapse. Our goal is identify novel drugs that selectively target these chemotherapy-resistant, tumor-initiating cells. Gene expression analysis of CD44+/CD24-/low cells vs. non-tumorigenic cells implicated the Notch, PI3K, and Hedgehog signaling pathways in regulating CD44+/CD24-/low cells. Thus, Notch, PI3K-AKT, and/or Hedgehog inhibitors may eliminate this unique subpopulation of cancer cells, either alone or in combination with chemotherapy, and could improve patient outcome. To test this hypothesis, we are carrying out a series of preclinical and clinical studies using a gamma-secretase inhibitor (GSI) to target the Notch pathway. Methods: For preclinical studies, stable xenograft lines were generated by transplantation of human tumor biopsy fragments into immunocompromised mice. Mice with tumors (n = 32, 150-300 mm3) were randomized to four treatment groups: 1) vehicle control, 2) chemotherapy: docetaxel 3) drug: GSI (MRK-003, Merck) or 4) combination: docetaxel + MRK-003. During treatment, mice were monitored for tumor volume and body weight. At the end of the treatment cycle, residual tumors were characterized by FACS for the percentage of CD44+/CD24-/low cells, as well as for mammosphere-forming efficiency (MSFE) and tumor-initiating capacity. In a complementary clinical trial, breast cancer biopsies taken before and after treatment with GSI (MK-0752, Merck) were characterized for expression of CD44, CD24, and ALDH by FACS and for MSFE. Results and Conclusions: In preclinical studies using two independent triple negative xenograft lines, Notch pathway inhibition reduced mammosphere formation but did not affect tumor volume, with no consistent change in marker expression by FACS. In patient samples, MSFE also declined after the first cycle of GSI/chemotherapy and remained low after subsequent cycles. This response corresponded with a stasis of metastatic growth during five cycles of treatment, but metastatic burden began to increase coincident with the sixth cycle of treatment. Marker analysis suggests that GSI treatment chemo-sensitizes a significant proportion of the otherwise chemo-resistant CD44+/CD24-/low cell population indicating that they are dependent on the Notch pathway for survival. The decrease of MSFE in both preclinical and clinical studies suggests that inhibition of the Notch pathway by GSI may reduce the number of tumorigenic cancer cells that would otherwise remain after chemotherapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 48.