bluuebunny/crossref_metadata_2025 huggingface dataset:
P4-16-08: Pilot Randomized Trial of De-Escalated (q12 Weekly) Versus Standard (q3-4 Weekly) Intravenous Bisphosphonates in Women with Low-Risk Bone Metastases from Breast Cancer.
Authors: Amir E , Freedman O , Carlsson L , Usmani T , Lee E , Dranitsaris G , Clemons M
Published: June 2012

Background: Bone-targeted agent such as bisphosphonates (BPs) can reduce skeletal complications from bone metastases but have no effect on either disease progression or survival. Despite substantial variability in the frequency and severity of skeletal complications, patients are empirically treated with BPs at the same dose and schedule irrespective of their individual risk.

Materials and Methods: A pilot, randomized, non-inferiority trial was conducted. Patients with low-risk bone metastases (serum C-telopeptide [CTx] <600ng/L after at least 3 cycles of monthly IV BP) were assigned to pamidronate 90mg IV either every 3–4 weeks (control) or every 12 weeks (de-escalated). Data on serum CTx and bone alkaline phosphatase (BAP), pain scores (brief pain inventory [BPI] and functional assessment of cancer therapy-bone pain [FACT-BP]) were collected at 12 weekly intervals for 48 weeks.

Results: Fifty-four patients were approached, 44 provided consent and 38 were eligible for randomization. Median age was 55 (range 29–77) and mean baseline CTx was 319ng/L (range 10–526). Thirty-five participants (92%) completed the trial, 2 withdrew consent and one participant died. Fourteen control group participants (73.7%) and 13 experimental group participants (68.4%) maintained CTx in the low risk range (test for two proportions p=0.64). All patients not maintaining CTx in the low risk range showed evidence of both visceral and bone progression. Compared to the control group, there was a trend towards increasing CTx with time in the experimental group (p=0.10). There was no significant difference in BAP (p=0.37), BPI (p=0.21) or FACT-BP scores (p=0.59) between the two groups. Over the 48 week follow-up, two skeletal events were observed in each group.

Conclusions: Randomized trials of de-escalated BP therapy in women with low-risk bone metastases are feasible. Twelve-weekly pamidronate appears non-inferior to 3–4 weekly treatment. Larger trials are required to assess whether; 1) increasing CTx levels with de-escalated therapy will lead to higher rates of skeletal complications and 2) whether BP should be given every 3–4 weeks in patients with progressive visceral and bone disease.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-16-08.
P4-17-08: Is Cranial Magnetic Resonance Imaging (MRI) Necessary for Staging of Asymptomatic HER2−Positive Breast Cancer Patients?
Authors: Kaplan MA , Inal A , Kucukoner M , Urakci Z , Isikdogan A
Published: June 2012

Background

Breast cancer one of the most common tumors of the adults. Approximately 20% to 25% of all breast cancers are HER2−positive. It can be assumed that up to 30% of HER2−positive metastatic breast cancer (MBC) patients may experience brain metastasis during the course of their disease. The aim of this study to investigate that whether cranial MR is necessary for staging in asymptomatic HER2−positive breast cancer patients.

Material and metods: HER2−positive breast cancer patients without symptoms of brain metastasis included in the study. Cranial MRI was added to the staging procedures. Subgroup analysis was performed to determine risk factors for developing brain metastasis.

Results: Seventy five patients included in the study. Of whom, 71 were women (94.7%). Median age was 42.5 (22-76). Ten of the 75 patients (13.3%) had brain metastasis. The distribution rate of brain metastasis according to breast cancer stage at diagnosis was: 0% for stage I, 9.1% for stage 2, 4.3% for stage 3 and 28.6% for stage 4 (p=0.027). There was no association between presence of brain metastasis and the site of metastasis, estrogen and progesterone receptor positivity, grade, histological subgroup, menopausal status and gender.

Discussion: Brain metastasis is an important problem for breast cancer patients. Treatment strategies may vary in the presence of brain metastasis. In our study we found brain metastasis nearly one quarter of patients with metastatic HER2−positive breast cancer. Cranial MRI imaging can be performed routinely in patient with metastatic HER2−positive breast cancer patients.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-17-08.
P4-18-04: Hypofractionated Radiotherapy for Breast Ductal Carcinoma In Situ.
Authors: Hathout L , Fortin B , Vulpe H , Hijal T , Bahig H , Dubé P , Yassa M
Published: June 2012

Background

Conventional radiotherapy at a dose of 50 Gy in 25 fractions after breast conservative surgery is the standard treatment for ductal carcinoma in situ of the breast (DCIS). Hypofractionation is an interesting alternative for the treatment of breast cancer, providing a less intense treatment scheme for the patient. While the hypofractionated regimen of 42.5 Gy in 16 fractions has been shown to be equivalent to 50 Gy in 25 in infiltrating ductal carcinoma, few studies and no prospective study have reported results using hypofractionation in DCIS.

Materials and Methods: In this multicenter collaborative effort, we retrospectively reviewed the records of women with a diagnosis of DCIS at two Canadian institutions, treated with conservative surgery followed by hypofractionated radiotherapy (42.5 Gy in 16 fractions) to the whole breast. Tumor, patient and treatment factors were collected. Local control was evaluated using the Kaplan Meier method. Curves were compared using log rank.

Results: Between 2003 to 2008, 292 patients with DCIS underwent conservative surgery followed by hypofractionated radiation in 16 fractions for a total dose of 42.5 Gy. Treatment delivery was similar at both institutions. Mean age at diagnosis was 59 years, with 70 % of women being postmenopausal. Nuclear grade was 1–2 in 65 % of patients while 28 % had grade 3 tumors. Complete surgical excision with negative margins was achieved in 92 % of patients. Radiotherapy boost was given to 92 patients (31.5 %) at the discretion of the radiation oncologist. After a mean follow up of 3.35 years (range 1–8 years), 10 patients (3.4 %) had ipsilateral local relapse resulting in a disease-free survival (DFS) of 96 % at 4 years. The histology at recurrence was DCIS for 8 patients; infiltrative disease for one patient and one patient had an unknown histology at recurrence. Age was statistically associated with local relapse (DFS 89 % for patients under 50 years old vs 97 % for those older than 50, p=0.027). Grade was also a significant prognostic factor (DFS 98% for grade 1–2 vs 91 % for grade 3, p = 0.015). Finally, administration of boost did not have an impact on local control (DFS boost 98 % vs 95 % for no boost, p=0.44).

Discussion: Hypofractionated radiotherapy (42.5 Gy in 16 fractions) provides excellent local control for patients with DCIS undergoing breast conservative surgery. This regimen is more convenient to both patient and physician since it is less time consuming. Further work needs to be done to determine if this regimen is as effective as conventional radiotherapy in younger women (age less than 50) and those with grade 3 tumors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-18-04.
P4-19-06: Ghrelin Expression Is Associated with Favorable Outcome in Male Breast Cancer.
Authors: Grönberg M , Nilsson C , Johansson I , Hedenfalk I , Janson ET , Fjällskog M-L
Published: June 2012

Background: Expression of the peptide hormones ghrelin and obestatin has previously been demonstrated in normal breast as well as in breast cancer. Interestingly, ghrelin expression has been shown to be associated with longer survival in female patients suffering from invasive breast cancer. However, the clinical implications of the expression of these peptides in male breast cancer are unclear. The aim of this study was to further investigate the role and potential clinical value of these peptides in male breast cancer.

Methods: A tissue microarray containing male breast cancer specimens from 197 patients was immunostained with antibodies directed towards the peptides. The expression of the peptides was evaluated and correlated to previously known prognostic factors in breast cancer and to the outcome. Cox regression analysis was used to assess whether these markers may predict survival of male breast cancer patients.

Results: Immunoreactivity for ghrelin and obestatin was observed in 81% and 87% of the cases, respectively. No strong correlations were found between ghrelin or obestatin expression and other known prognostic factors. Only ghrelin expression was significantly correlated to breast cancer-specific survival (HR=0.37−0.39, p=0.01−0.03) in both uni- and multivariate analyses. Reproducibility between the two readers was good to very good for both stainings with kappa values of 0.65−0.89.

Conclusions: Male breast cancer patients with tumors expressing ghrelin had 3 times lower risk for breast cancer death than those lacking ghrelin expression. Ghrelin expression is easily assessable with good reproducibility using immunohistochemistry. Further studies are needed to establish the clinical significance of ghrelin as a biomarker in breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-19-06.
P4-20-01: Multicentric Phase II PACS 09/Beverly1 Trial: First Efficacy and Safety Results of Neoadjuvant Chemotherapy Combined with Bevacizumab in HER2−Negative Patients with Non-Metastatic Inflammatory Breast Cancer.
Authors: Viens P , Petit T , Dalenc F , Pierga JY , Delozier T , Romieu G , Bonneterre J , Ferrero J-M , Kerbrat P , Mouret-Reynier M-A , Bachelot T , Soulie P , Lerebours F , Eymard J-C , Deblock M , Lortholary A , Hardy-Bessard A-C , Boher J-M , Asselain B , Charafe-Jauffret E , Lemonnier J , Martin A-L , Andre F
Published: June 2012

Background: Inflammatory breast cancer (IBC) is a relatively rare form of breast cancer. Because this disease exhibits angiogenic properties, bevacizumab (BEV) may improves the efficacy of neoadjuvant chemotherapy (CT) in IBC.

Methods: Pts with HER2−negative IBC (IHC 0 or 1+ or FISH/CISH-, T4d, any N) were included to receive 4 cycles of FEC100 + BEV 15 mg/kg, d1 q3w, followed by 4 cycles of Docetaxel 100 mg/m2 + BEV 15 mg/kg q3w. Complete surgery was performed 4–6 weeks after the last dose of CT, followed by radiotherapy and endocrine therapy for hormone receptor positive (HR+) pts. BEV was re-introduced as adjuvant therapy for 10 cycles, giving a total of 18 cycles of BEV. The primary endpoint of the PACS09/Beverly1 trial was to evaluate the pathologic complete response (pCR; Sataloff classification) rate in HER2−negative non-metastatic IBC patients(pts).

Results: Between Jan 2009 and Sep 2010, 101 pts were included (100 pts were evaluable). Main baseline characteristics were: median age 49 years; postmenopausal status, 39 pts; SBR 2, 32 pts SBR3 61 pts, both HR-negative, 55 pts. 85 pts completed neoadjuvant therapy according to the protocol, ***.90 pts underwent complete mastectomy, 4 had a breast-conservative surgery. After neoadjuvant treatment, investigator-assessed pCR rate was 27% (18%-36%, CI 95%). Interestingly, a significantly greater proportion of pCR rate was observed in the HR-negative group compared to HR-positive group (38% vs 13% respectively; p=0.007). Results from central review of pCR are currently in progress. Main Grade 3,4 toxicities were: neutropenia (61 pts),febrile neutropenia (36 pts),mucitis (23 pts) Neither gr3/4 HTA nor cardiac failure or proteinuria was reported. There were no treatment-related deaths.

Conclusions: These data suggest that the use of BEV with neoadjuvant CT is active in HER2−negative IBC (pCR rate 27%), with an acceptable safety profile. Interestingly, the HR-negative subgroup is significantly more responsive than the HR-positive subgroup.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-20-01.
P4-20-05: Inflammatory Breast Cancer: Comparison of Epidemiology, Biology, and Prognosis between Japan and the United States, a Hospital-Based Study.
Authors: Yamauchi H , Natori A , Hayashi N , Soejima K , Takahashi O , Fukui T , Nakamura S , Cristofanilli M , Ueno N
Published: June 2012

Background: Inflammatory breast cancer (IBC) is a challenging disease characterized by low incidence, rapid progression, and poor survival. The epidemiology of IBC has been slow to emerge, given that the rarity of IBC makes large prospective clinical trials difficult. Compared to non-IBC, well-established features of IBC include a younger age of onset and lower frequency of hormone receptor positivity. Higher body mass index (BMI) is a purported risk factor. Previous reports also suggest that IBC is associated with more racial disparities, including incidence and age at diagnosis. The epidemiology of IBC in East Asia, however, has not been investigated. We performed a comparative study of IBC in Japan versus the United States to determine its epidemiologic and clinical features and to evaluate the differences in epidemiological factors between the two countries.

Patients and Method: Patients who visited St. Luke's International hospital (SLIH) in Tokyo, Japan and The University of Texas MD Anderson Cancer Center (MDA) in Texas, USA between 2003 and 2009 were identified. Epidemiological and biological data were collected from electronic medical records. Patient and tumor characteristics were tabulated and stratified by hospital. Kaplan-Meier curves were created for survival estimates and log-rank test was used for cross-group comparisons. Cox proportional-hazard analysis was used to identify a subset of significant prognostic variables that related to overall survival.

Result: 34 patients at SLIH and 531 patients at MDA were identified. Mean age at diagnosis was 52 years old (range, 32–81, SD, 10.8) and 50 years old (range, 22–87, SD, 11.6), respectively (P=0.476). Mean BMI was 22.9 kg/m2 (range, 17.3−30.5, SD 3.3 ) and 31.0 kg/m2 (range, 13.6−88.9, SD, 7.8) respectively (P<0.01). Clinical Staging was not significantly different; Stage IIIB 38.2%, Stage IIIC 26.5%, and Stage IV 32.4% at SLIH versus 48.6%, 23.7%, and 27.3% at MDA (P= 0.167). Estrogen receptor (ER) and progesterone receptor (PR) negative cases were, respectively, 50.0% and 64.7% at SLIH and 50.5% and 64.2% at MDA (ER, P= 0.935; PR, P=0.908). Her-2 over-expression cases were 38.2% at SLIH and 28.6% at MDA (P=0.174). A significant difference in nuclear grade was seen between SLIH and MDA: 20.6% at SLIH were Grade 3 versus 68.7% at MDA (P<0.01). Median overall survival at SLIH was 3.6 years versus 2.3 years at MDA (P=0.570). No prognostic factors were associated with overall survival.

Conclusion: Though IBC at SLIH differed significantly from IBC at MDA by several epidemiologic and biologic factors, there was no significant difference in survival. To define the epidemiological, prognostic, and risk factors of IBC in Japan, as well as in the world, further studies are needed.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-20-05.
P5-01-01: Identification, Validation and Assessment of Transcriptional Relevance of a PDGFR-Activation Signature in (Inflammatory) Breast Cancer.
Authors: Van Laere SJ , Van Golen KL , Joglekar M , Ueno NT , Finetti P , Van Dam PA , Viens P , Birnbaum D , Bertucci F , Vermeulen PB , Dirix LY
Published: June 2012

Introduction. Breast cancer can be divided into several subgroups characterized by unique patterns of pathway activation. Platelet-derived growth factor receptor (PDGFR) signalling has not yet been included in this classification scheme, although it has been reported to be a potential target for therapy. In this study, we have constructed a PDGFR-activation signature and investigated its relevance in breast cancer.

Materials and Methods. Sixteen PDGFR-modulated genes were identified by intersecting two published PDGFR-modulated gene lists. The resulting gene signature was applied onto a publicly available gene expression data set of GIST (GSE17743) using principle component analysis. The segregation of PDGFR- and KIT-mutated GIST samples was investigated using permutation analysis and classification sensitivity and specificity were assessed. Using the regression coefficients from the first principal component, a PDGFR-activation score was constructed and applied onto a second data set in order to validate the score (GSE1923). Finally, the score was applied onto a gene expression data set of 389 breast cancer ***samples, including 137 samples from patients with IBC.

Results. Sixteen PDGFR-modulated genes (NR4A1, EGR3, JUNB, IER3, TIEG, JUN, BCL3, MYC, NR4A3, PLAU, MCL1, DUSP1, DUSP5, DUSP6, SGK, GADD45A) were able to discriminate PDGFR-mutated GIST samples from KIT-mutated GIST samples with a sensitivity of 75% and a specificity of 85%. Application of the PDGFR-activation score onto a data set of control and PDGF-treated glioblastoma cells showed a significant increase in the PDGFR-activation score in the treated condition (P=0.0302). Application of the PDGFR-signature onto our series of IBC and nIBC samples demonstrated a significant and molecular subtype-independent increase in PDGFR-activation in IBC (P=0.0015; FDR=3%). In addition, in our series of nIBC samples only, PDGFR-activation was associated with decreased DMFS and RFS (P=0.0038 and P=0.0137 respectively). In fact, PDGFR-activation was an independent prognosticator in a multivariate model incorporating the molecular subtypes.

Discussion. We identified a gene signature composed of 16 genes able to predict PDGFR-activation in tissue samples by gene expression analysis. PDGFR-activation is significantly increased in samples from patients with IBC, an aggressive form of locally advanced breast cancer. In addition, in nIBC, PDGFR-activation is associated with DMFS and RFS, independently of the molecular subtypes suggesting that PDGFR-activation might add another level of clinically relevant heterogeneity in breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-01.
P5-01-13: High Levels of Nuclear Heat Shock Factor 1 (HSF1) Are Associated with Poor Prognosis in Breast Cancer: Results from the Nurses' Health Study.
Authors: Santagata S , Hu R , Lin NU , Mendillo ML , Collins LC , Hankinson SE , Schnitt SJ , Whitesell L , Lindquist S , Tamimi RM , Ince TA
Published: June 2012

Purpose: Heat shock factor 1 (HSF1) is the master transcriptional regulator of the cellular response to heat and a wide variety of other stressors. We previously reported that HSF1 promotes the survival and proliferation of malignant cells. At this time, however, the clinical and prognostic significance of HSF1 in cancer is unknown.

Patients and methods: Breast cancer samples from 1,841 participants in the Nurses’ Health Study (NHS) were scored for levels of nuclear HSF1. Associations of HSF1 status with clinical parameters and survival outcomes were investigated by Kaplan-Meier analysis and Cox proportional hazard models. The associations were further delineated by Kaplan-Meier analysis using publicly available mRNA expression data.

Results: Nuclear HSF1 levels were elevated in ∼80% of in situ and invasive breast carcinomas. In invasive carcinomas, HSF1 expression was associated with high histologic grade, larger tumor size, and nodal involvement at diagnosis (P<0.0001). Overall, in multivariate analysis, high-HSF1 levels were associated with increased breast cancer-specific mortality (HR, 1.62; 95% CI, 1.21−2.17). This association was seen in the ER-positive population (HR, 2.10; 95% CI, 1.25−2.47), even in early-stage lymph node negative cases (HR, 1.98; 95% CI, 1.17−3.33). In public expression profiling data, high-HSF1 mRNA levels were also associated with an increase in ER-positive breast cancer-specific mortality.

Conclusions: Increased HSF1 is associated with reduced survival in breast cancer. The findings indicate that HSF1 should be evaluated prospectively as an independent prognostic indicator in ER-positive breast cancer and that HSF1 may provide a useful therapeutic target.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-13.
P5-02-03: Lipid Raft Disruption by Docosahexaenoic Acid Induces Apoptosis in Transformed Human Mammary Luminal Epithelial Cells Harboring HER-2 Overexpression.
Authors: Ravacci GR , Tharcisio  , Junior T , Logullo AF , Torrinhas RS , Brentani MM , Waitzberg DL
Published: June 2012

Background, In HER-2 overexpressing breast cells, HER-2 receptors exist on the cell surface as monomers, homodimers and heterodimers with EGFR and HER-3. For signal activation and transduction to occur, the HER-2 receptors must be located in lipid rafts. Therefore, HER-2 overexpression must be accompanied by increased lipid rafts on the cell membrane to ensure constant signal transmission.

Methods, To test this hypothesis, we used HB4a — untransformed human mammary epithelial cell strain — and HB4aC5.2 cells — HB4a cells co-transfected with ErbB-pJ5E.c-2 (normal full length human HER-2 cDNA). We analyzed the lipid rafts, cholesterol and fatty acids from cell membrane by confocal microscopy and gas chromatography. The proteins associated to HER-2 activity and lipid rafts formation, such as Akt, ERK ½ and FASN were analyzed by western blotting. To evaluate the lipid rafts importance for HER-2 signaling, we treated cells with docosahexaenoic acid (DHA) to disrupt the lipid rafts. Thereby, all analysis was repeated and cells death and apoptosis were analyzed by flow cytometry. Values were obtained by the mean and standard deviation of five independent experiments and developed in triplicate, by using one-way analysis of a variance (ANOVA) test, followed by Bonferroni post-test, to identify significant (p<0.05) differences between cell lines and results with different concentrations of DHA treatment. Results, HB4aC5.2 cells have five copies of pJ5E.c-ErbB-2 inserted into their genome and express approximately 900 times more HER-2 than HB4a cells. HER-2 overexpression in HB4aC5.2 cells was accompanied by increased lipid rafts in cell membranes, hyperactivation of downstream Akt and ERK1/2 proteins, and rate of cell growth increase as compared to the HB4a strain. In addition, HER-2 overexpression was associated to increased activation of FASN, a key enzyme involved in cellular lipogenesis. The final product of FASN activation, palmitate, is used to synthesize saturated phospholipids, necessary to form lipid rafts. Treatment of HB4aC5.2 cells with DHA, an omega-3 fatty acid, disrupted the lipid rafts by its increased incorporation into the cell membrane; inhibited HER-2 activation by decreasing Akt, ERK1/2, and FASN proteins; and induced apoptosis. The same treatment maintained the structure of HB4a cell lipid rafts, accompanied by a low percentage of DHA-induced apoptosis.

Conclusion, Although little is yet known about lipid rafts, our data support the theory that disturbances in these micro-domains may represent a useful tool in controlling cell signaling triggered by HER-2 receptors and contribute to a better understanding of the role of this receptor in HER-2 positive breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-02-03.
P5-02-04: Disruption of Endothelial Cells Barrier Integrity by Invasive Breast Cancer Cells.
Authors: Haidari M , Zhang W , Chen Z , Mortazavi A , Dixon R
Published: June 2012

Despite its critical role in cancer metastasis the molecular mechanisms regulating breast cancer cells transendothelial migration are poorly understood, but clearly depend on the invasive capacity of tumor cells and their ability to breach the endothelial cell barrier. Vascular endothelial-cadherin (VE-cadherin) is found specifically in the endothelial cell adherens junction and has been implicated in playing a fundamental role in controlling the transport across the endothelial barrier. Tyrosine phosphorylation of VE-cadherin has been implicated in the disruption of endothelial cells adherens junctions and diapedesis of metastatic cancer cells. We tested this hypothesis that interaction of breast cancer cells with endothelial cells initiates the signal transductions that disrupt the endothelium barrier integrity. Our studies demonstrated that the attachment of MDA-MB-231 human breast cancer cells to Human Umbilical Vein Endothelial Cells (HUVECs) leads to tyrosine phosphorylation of VE-cadherin and the formation of gaps between endothelial cells. These were accompanied by activation of two tyrosine kinases, Src and proline rich tyrosine kinase (Pyk-2). In addition, immunoprecipitation studies indicated that the endothelial cells adherens junction structure was disrupted through MDA-MB-231-induced dissociation of VE-cadherin and β-catenin complex. Activation of RhoA and HRas by over expression of constitutively active forms of the genes leads to tyrosine phosphorylation of VE-cadherin and Pyk-2 in HUVECs. Over expression of dominant negative forms of RhoA, HRas, Raf and ERK2 but not Rac1 and Cdc42 attenuated breast cancer cell-induced tyrosine phosphorylation of VE-cadherin and Pyk-2 in HUVECs. Indicating that breast cancer cell-induced VE-cadherin tyrosine phosphorylation and disruption of adherens junction in endothelial cells is mediated by RhoA and HRas\Raf\MEK\ERK signaling cascade. Understanding the precise molecular mechanisms that facilitate breast cancer cells transendothelial migration could develop novel therapeutic strategies targeting cancer cell metastasis by improving the protective role of endothelial cells.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-02-04.
P5-05-01: Vitamin D Status in Newly Diagnosed Breast Cancer Patients Inversely Correlates with Tumor Size and Moderately Correlates with Outcome.
Authors: Hatse S , Lambrechts D , Verstuyf M , Smeets A , Brouwers B , Vandorpe T , Brouckaert O , Peuteman G , Laenen A , Kriebitzsch C , Dieudonné A-S , Paridaens R , Neven P , Christiaens M-R , Bouillon R , Wildiers H
Published: June 2012

Aims: To study the impact of Vitamin D (VitD) status and genetic variability in key VitD regulatory genes on patient and breast tumor characteristics, and on breast cancer related outcome.

Methods: We examined serum 25-hydroxyvitamin D3 (25OHD) levels in a cohort of 1800 early breast cancer patients treated in Leuven between 2003 and 2010. Serum was collected at diagnosis for all patients; germline DNA from peripheral blood was also available for the majority of them. Serum 25OHD was measured by radioimmunoassay, and single nucleotide polymorphisms (SNP's) were assessed by Sequenom. Statistical analysis was done by multivariable regression models including age, BMI and season and by a Cox proportional hazard model for analysis of disease-specific survival and disease-free interval.

Results: Lower 25OHD serum levels were significantly correlated with larger tumor size (0,4 ng/ml decrease in 25OHD per 1 cm increment in tumor size, p=0.0063) but not with lymph node invasion, estrogen receptor and HER2 status, or tumor grade. Serum 25ODH level was significantly affected by genotypes of rs10741657 and rs1993116 (25-hydroxylase; CYP2R1) and of rs222040, rs7041 and rs4588 (D-binding protein; DBP), yet its observed association with tumor size did not differ between distinct genotypes. Although not statistically significant, patients with higher 25OHD levels tended to have slightly improved survival. Also, a significant increase in relapse risk became apparent after 3 years for patients with 25OHD<30 ng/mL.

Conclusion: Vitamin D Level in breast cancer patients inversely correlates with tumor size and moderately correlates with outcome.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-01.
P5-05-07: The Msp1 Polymorphism of Cytochrome P-450 CYP1A1 in Asymptomatic Women with Breast Cysts.
Authors: Fenile R , Nazário A , Facina G
Published: June 2012

The objective of this study was to determine the prevalence of the cystic form of fibrocystic breast changes among women from different age groups correlated with the presence of the Msp1 polymorphism of cytochrome P-450 CYP1A1. Methods: A retrospective, case-control study (was carried out with ultrasound examination) was performed on 197 women divided into two groups: 44 women with cystic breast disease (cases) and 153 women without breast disease (controls), the majority Caucasian, aged 41–50 years, having regular menstrual cycles, pregnancies and complaining of mastalgia. Genetic analyses were carried out by performing polymerase chain reaction to detect the Msp1 polymorphism in CYP1A1. Results: Cystic breast disease as diagnosed by ultrasound was present in 22% of the population studied. In the genetic analysis of CYP1A1, we observed the homozygous wild-type genotype in 68.2% of cases and 66% of controls. The heterozygous genotype occurred in 31.28% of cases and 26.8% of controls. The homozygous mutant genotype was seen in zero cases and in 7.2% of controls. There were no statistically significant differences in the genotype frequencies observed between the case and control groups (p = 0.42). Conclusion: The prevalence and the general epidemiological profile of cystic breast disease in this group were consistent with the values previously reported the literature. There was a higher frequency of heterozygotes in the case than in the control group, but this was not true for the homozygote mutant genotype. There was no statistically significant association detected between the CYP1A1 Msp1 polymorphism and fibrocystic breast disease.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-07.
P5-06-02: Response and Acquired Resistance of BRCA1-Deficient Triple-Negative Breast Cancer Xenografts to Alkylators or PARP Inhibitors.
Authors: ter Brugge P , van der Burg E , Kristel P , Boon U , Moutinho Freixas C , Esteller M , Hogervorst F , Gevensleben H , Turner N , Wesseling J , Jonkers J
Published: June 2012

Background. BRCA1 mutated breast tumor cells are defective in DNA repair by homologous recombination and therefore especially sensitive to treatment with DNA double-strand break (DSB) inducing agents, such as alkylators or PARP inhibitors. However, such tumors can eventually develop therapy resistance. Understanding therapy resistance mechanisms may help in designing treatment strategies to overcome therapy resistance.

Methods. We have developed BRCA1-deficient triple-negative breast cancer (TNBC) xenograft models by implantating fresh human breast tumor pieces and subsequent serial passaging. These models show epigenetic loss of BRCA1 due to promoter hypermethylation or genetic inactivation of BRCA1 due to a frameshift mutation (c.2210delC) resulting in a premature stop codon (p.Thr737LeufsX15). We have used these BRCA1-deficient TNBC models to study response and acquisition of resistance to alkylating therapy (cisplatin, melphalan) and the clinical PARP inhibitor olaparib.

Results. Treated tumors were sensitive to the alkylators cisplatin or melphalan or the PARP inhibitor olaparib, in some cases resulting in complete remission with no palpable tumor left. However, relapses did occur in most cases and repeated treatment of recurrent tumors eventually led to aquired resistance.

Since restoration of BRCA1 function has been suggested as a mechanism of therapy resistance (Swisher et al., Cancer Res 2008; 68: 2581), we determined BRCA1 expression in therapy-sensitive and -resistant tumors by Western blot analysis. While no full length BRCA1 protein could be detected in the therapy-sensitive tumors, expression of full length BRCA1 protein was found in the majority of cisplatin resistant and olaparib resistant tumors. BRCA1 reexpression in the therapy-resistant BRCA1-c.2210delC tumors was caused by genetic restoration of the reading frame due to reversion to the wildtype sequence or additional deletions near the c.2210delC mutation. BRCA1-re-expression in the therapy-resistant TNBC xenografts with epigenetic loss of BRCA1 was caused either by loss of BRCA1 promoter methylation or by complex rearrangements at the BRCA1 locus.

Conclusion. Although BRCA1-deficient TNBC xenografts are initially very sensitive to alkylating agents and olaparib, resistance to treatment develops in almost all treated tumors. This acquired resistance is frequently associated with re-expression of BRCA1 due to secondary mutations or loss of promoter methylation.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-02.
P5-06-09: Acquired Sensitivity to TRAIL Mediated Apoptosis in Lapatinib Resistant SKBR3 Cells.
Authors: Eustace AJ , Browne BC , McDermott M , Gallagher C , Watson W , Crown J , O'Donovan N
Published: June 2012

Introduction Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor is approved for the treatment of trastuzumab-refractory HER2 positive metastatic breast cancer. However, not all HER2 positive tumors respond to lapatinib and patients who initially respond frequently relapse. Understanding the molecular alterations associated with acquisition of lapatinib resistance may lead to the identification of targets to overcome resistance. Thus, the aim of this study was to examine changes in apoptosis in a model of acquired lapatinib resistance.

Methods SKBR3 cells were treated with 250 nM lapatinib twice weekly for 6 months to establish the lapatinib resistant cell line SKBR3-L. The effects of TNF-related apoptosis-inducing ligand (TRAIL) and tumour necrosis factor-α (TNF-α) on cell survival and apoptosis induction were examined in SKBR3 and SKBR3-L cells, using the acid phosphatase proliferation assay and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Cleavage of poly ADP ribose polymerase (PARP) was used to confirm apoptosis. TRAIL-1 and TRAIL-2 receptor expression were measured by flow cytometry (FACS).

Results In SKBR3-L cells, TRAIL treatment (25 ng/ml) significantly inhibited cell survival (94.0 ± 12.0 %) compared to the SKBR3 cells (11.0 ± 2.2%). Treatment with TNF-α (0.5 ng/ml) also resulted in significant inhibition of cell survival (68.2 ± 10.1 %) in SKBR3-L cells compared to SKBR3 cells (9.4 ± 9.0 %). TRAIL (25 ng/ml) induced a low level of apoptosis in SKBR3 cells (7.0 ± 4.0%), whereas in SKBR3-L cells, TRAIL (25 ng/ml) induced significant apoptosis (54.8 ± 2.6%, p < 0.001), as determined by the TUNEL assay. We confirmed by western blotting that TRAIL treatment for 72 hours resulted in higher levels of PARP cleavage in SKBR3-L cells compared to SKBR3 cells. Both western blotting and FACS analysis of TRAIL receptor expression showed that neither TRAIL-1 nor TRAIL-2 receptor expression was significantly elevated in SKBR3-L cells compared to SKBR3 cells.

Conclusions SKBR3-L cells, a model of acquired lapatinib resistance, display significant sensitivity to TRAIL induced apoptosis compared to the parental cell line SKBR3. Thus, targeting TRAIL may represent a novel therapeutic strategy to overcome acquired lapatinib resistance.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-09.
P5-07-05: Hypoxic Mammary Epithelial Cells in Three-Dimensional Culture Develop a Cancer-Like Phenotype.
Authors: Vaapil M , Helczynska K , Påhlman S , Jögi A
Published: June 2012

Due to inadequate vascularization and tumor cell proliferation solid tumors are hypoxic compared to corresponding normal tissues. Tumor hypoxia has been associated with increased metastatic potential and worse patient outcome. The hypoxic response is mainly driven by the hypoxia inducible transcription factors; HIF −1α and HIF-2α. We have reported that hypoxia leads to a less differentiated phenotype in breast cancer and neuroblastoma, and in the clinic low tumor cell differentiation is related to poor prognosis in these diseases. We have shown that high levels of HIF-2α protein correlate to distant metastasis and poor survival in invasive breast cancer. The aim of the present study was to investigate the effect of hypoxia on mammary epithelial cell differentiation and epithelial organization. We employed the well-characterized model of acinar morphogenesis on extra cellular matrix substrate. In this model mammary epithelial cells that are cultured in three-dimensional culture on a laminin-rich matrix differentiate and form acini-like structures with evacuated lumen surrounded by polarized palisade cells. We found that hypoxia (1% oxygen) impaired the process of acinar morphogenesis and the hypoxic cells failed to obtain polarity and that markers of differentiation were affected at expression levels as well as cellular localization. The hypoxic cells remained proliferative whereas normoxic cells lost Ki-67 expression within a week. We found that hypoxia impaired the global histone deacetylation that has been shown by others to take place in the process of acini formation in mammary epithelial cells. Three days post-seeding, during the proliferation stage, both normoxic and hypoxic cells stained positive for acetylated histone 4. When the normoxic cells went through morphological changes and acinar formation, the acetylation of histone 4 was reduced whereas it was maintained in the hypoxic cell-clusters at day 10 post-seeding. Thus, the hypoxic conditions maintained the epithelial cells in an acetylated state, which is generally associated with an open chromatin structure, high level of transcription and an undifferentiated stage. These data indicate that hypoxia promotes loss of differentiation also at a non-malignant stage and render the cells a cancer-like phenotype. We are currently extending this study in primary cells.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-05.
P5-08-06: Comparison of Mammographic Density between Ductal Carcinoma In Situ and Benign Breast Disease.
Authors: Lee JW , Shin EJ , Yi OV , Lee JW , Shin HJ
Published: June 2012

Background: High breast density is an independent risk factor for breast cancer as well as benign breast diseases. However there are few studies about breast density of ductal carcinoma in situ (DCIS) and benign breast disease(BBD). In this study, we investigated patterns of breast density of DCIS and BBD, and comparatively analyzed breast densities of DCIS and BBD.

Material and Methods From 2008 to 2009, 345 patients underwent surgery for DCIS at Asan Medical Center, and 295 patients for BBD at 2010. We retrospectively reviewed each groups and estimated cranio-caudal view of digital mammogram of unaffected breast by computer-assisted thresholding methods, using Cumulus 4, version 4.0. For BBD we included atypical ductal hyperplasia, fibroadenoma, papilloma(atypical/intraductal), phyllodes tumor(benign/borderline), and excluded unavailable mammogram(mostly, because of scanned films from outside hospitals and previously diagnosed breast cancer).

Results Mean ages of each group were 46.51 year old in DCIS group and 41.43 year old in BBD group, and the mean percentage density(PD) were 44.89% and 45.27%, respectively.

There was no significant difference between two groups in total population(p=0.79). We categorized into two groups according to the age, ≥50 and <50, for each DCIS and BBD group. Mean percentage density of below 50 year old was 52.92% in DCIS gruop and 49.58% in BBD group(p=0.031). Mean percentage density above 50 year old was 33.67% in DCIS group and 33.29% In BBD group(p=0.867).

Conclusion The mean values and distribution patterns of PD were similar between BBD group and DCIS group. However, in the subgroup aged under 50, the DCIS group has significantly higher breast density than the BBD group. Although the causal association of breast density with breast cancer has been relatively well documented, this kind of cross-sectional study has an inherent limitation. The absolute difference between the DCIS group and the BBD group under age 50 (3.34% in table2) needs more investigation for its clinical implications.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-08-06.
P5-10-02: Relevance of Magnetic Resonance Imaging To Predict Disease-Free Survival after Neoadjuvant Chemotherapy of Breast Cancer.
Authors: Loo CE , Vrancken Peeters M-JTFD , Wesseling J , Gilhuijs KGA
Published: June 2012

Purpose: To explore the relevance of magnetic resonance imaging (MRI) to predict disease free survival (DFS) after neoadjuvant chemotherapy (NAC) of breast cancer.

Patients and Methods

Between July 2000 and December 2007, MRI examinations were performed in 188 women before, during and after NAC in the context of a single-institution review board approved study. Patients were consecutively included after informed consent. Interpretation of MRI was revised by an experienced breast MR radiologists and included assessment of lesion morphology, changes in morphology, size, and contrast uptake kinetics (initial and late enhancement).

MRI features and known prognostic markers of breast cancer (age, tumor response at pathology, tumor size at baseline, immunohistochemistry-derived breast cancer subtype (triple-negative; HER2−positive or ER-positive/HER2−negative)) were correlated with DFS. Multivariate Cox regression analysis was used with forward LR feature selection. Kaplan Meier curves were obtained to explore the impact of relevant associations.

Results: Follow-up data were available of 184 women. The median follow-up time was 57,6 months (range 32,4 — 125). In 30 women events were found (26 breast cancer related deaths; 29 distant metastasis and 12 local/regional recurrences).

At multivariate analysis, only breast cancer subtype (p=0.004) and largest diameter of late enhancement at MRI after NAC (p=0.006) retained prognostic value in favour of response at final pathology, age and tumor size at baseline. Cumulative survival at 60 months (kaplanmeier curves) was 70%, 86.7%, and 89.7% for triple-negatives, HER2−positive and ER-positive/HER2−negative subtypes [figure 1], respectively, and 88.5%, 81.4 % and 55.1% for absence of washout/plateau after NAC, between 0 and 30 mm washout/plateau, and ≥ 30 mm washout/plateau, respectively [figure 2].

Conclusions MRI after NAC in conjunction with breast cancer subtype has potential as complementary predictor for DFS. Larger diameters of residual late enhancement at MRI after NAC are associated with inferior DFS at 60 months.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-10-02.
P5-11-04: A New Immunohistochemistry-Based Assay SBrC5 Classifies Invasive Breast Cancer Subtypes – Profiling Five Biomarkers in One Single Test.
Authors: Liu H , Muralitharan S
Published: June 2012

Background: Obtaining expression status of estrogen receptor (ER), progesterone receptor (PR) and Her2 is essential for guiding treatment and predicting outcome for breast cancers. By adding cytokeratin 5/6 and EGFR to the above mentioned three common biomarkers, a five-biomarker breast cancer panel has been validated in previous studies for identifying the basal-like subtype with superior prognostic values than the triple negative subtype1. The current study aimed to evaluate a new immunohistochemical assay, named Subtyping Breast Cancers 5-in-1 Assay (abbreviated as SBrC5, or “the assay” in this poster), for classifying breast cancer subtypes.

Material and Methods: A cocktail of ER, PR, Her2, cytokeratin 5/6 and EGFR antibodies (SBrC5 Cocktail) and the MultiVision Detection Kit (TFS Cat. #: TL-012-MARH) were used in generating the data for this poster. The Multivision Detection Kit is a double staining system with two visualizing colors, red and blue. The assay was performed on a breast cancer tissue microarray containing 75 cases in duplicate. Both color and cellular localization of the staining generated from the assay was used for the result interpretation. There were three staining clusters: 1) Red Nuclei indicated ER or PR positive; 2) Red Membrane indicated Her2 expression; and 3) Blue Membrane or Cytoplasm indicated EGFR or cytokeratine 5/6 positivity respectively. The same breast cancer tissue microarray was also subjected to individual IHC tests of ER, PR, Her2, cytokeratin 5/6 and EGFR. The concordance between the assay and the individual tests was analyzed.

Results: The assay SBrC5 was found to have excellent effectiveness in detecting ER or PR positivity, Her2 over expression, and cytokeratine 5/6 or EGFR positivity. The concordance for the assay when compared to the individual tests for each biomarker were 94.1%, 93.8% and 92.4% for Her2, combined results of ER and PR, and combined results of cytokeratine 5/6 and EGFR respectively. When a similar comparison was made, the sensitivity was 87.9%, 94.3% and 73.7%; the specificity was 100%, 93.1% and 100%.

Discussion: This study has demonstrated that a single test of the assay SBrC5 was able to accomplish the collective task of the individual IHC tests of the five-biomarker breast cancer panel. The assay is able to provide sufficient information for classifying breast cancer subtypes with only a single IHC test. Thus it makes it possible to obtain subtypes information in biopsy settings where the tumor tissue obtained is often insufficient for multiple tests required to accomplish such task by existing methods.

Reference: Maggie C.U. Cheang, David Voduc, Chris Bajdik, et al. Clin Cancer Res March 1, 2008 14; 1368.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-04.
P5-13-16: Reduction in PET Uptake-Value Is an Early Predictor for Response to Neoadjuvant Therapy Including Anthracycline and Taxane in Stage II-IIIa Breast.
Authors: Lao Romera J , Puértolas Hernandez T , Godoy Molias A , Alvarez Alejandro M , Madani Perez J , Garcia Mur C , Ubieto MA , Cebollero de Miguel A , Artal Cortes A , Anton Torres A
Published: June 2012

Background and Aim: Reduction in FDG-PET uptake as response predictor after induction chemotherapy for breast cancer has been described. Our aim was to determine whether a SUV reduction ≥45% in the breast lesion could accurately predict pathological Complete Response (pCR).

Material and Method: Stage II-IIIa breast cancer patients (p) with primary tumor ≥3cm or positive axilla were included provided they were amenable to neoadjuvant chemotherapy with Docetaxel-Doxorubicin-Cyclophosphamide (TAC). A FDG-PET scan was performed on day −1 of the first course and was repeated on day+8 of the same course. SUV-uptake differences (day 8 minus day −1) were correlated to the eventual pathological response according to Miller-Payne criteria.

Results: 42p were included. Median age was 45 years (30-66). 11p (26.2%) were triple-negative tumors. 2p (4.8%) were Her-2 +. By stage, 13p (30.9%) were IIA, 17p (40.4%) IIB and 12p (28.2%) IIIA. 23p (54.7%) were N+ (FNAB positive). Median breast-SUV on first PET scan was 5.51 (1.3−18.50). 40p (95.2 %) completed the scheduled 6 courses. 41p (97.6%) were submitted to surgery. 11p (26.2%) achieved a pCR. 40p (95.2%) were evaluable for the primary objective. 1p (0.02%) died of disease progression before surgery and 1p (0.02%) with pCR could not be assessed by PET. 7 out of 14 p (50%) with uptake reduction ≥45% with respect to the basal value, and 3/26p (11.5%) whose reduction was <45% achieved a pCR (p=0.007). SUV decrease >45% accurately predicted 7/10 pCR (70%) that could be assessed.

Conclusions: Our study confirmed the value of PET-SUV reduction on day 8th as an early predictor of pCR with neoadjuvant TAC chemotherapy.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-16.