bluuebunny/crossref_metadata_2025 huggingface dataset:
T47D/PKCα Cells Are Nonmigratory and Noninvasive In Vitro Despite Tamoxifen Resistance In Vivo.
Authors: Perez White B. , Tonetti D.
Published: December 2010

Background: We have previously reported that overexpression of PKCα in T47D (T47D/PKCα) breast cancer cells results in hormone independence, growth inhibition by estradiol, and tamoxifen resistance (Chisamore, 2001). This has clinical relevance because we have shown that PKCα overexpression may predict disease recurrence and tamoxifen treatment failure suggesting consideration of other treatments (Tonetti, 2003). Further characterization reveals that T47D/PKCα cells are noninvasive in vitro compared to T47D/neo control cells in Matrigel invasion assays and that knockdown of PKCα reverses the phenotype (Perez White, AACR 2009). We have further characterized the in vitro phenotype of T47D/PKCα cells using migration assay and profiling of proteins associated with epithelial-mesenchymal transition (EMT). Adherens junctions are made of a complex of proteins including E-cadherin whose dissolution is thought to play a role in EMT. E-cadherin can be transcriptionally repressed by the embryonic transcription factor Snail1. Generally, cells that lose expression of E-cadherin gain expression of N-cadherin and/or vimentin which is correlated to increased migration, invasion, and metastasis.Methods: To determine migration, cells were plated in serum-free media into Boyden chambers for 24 h with media containing 10% fetal bovine serum as chemoattractant. Protein expression levels were determined by western blot of whole cell extracts and transcript levels by RT-qPCR. Statistical analyses were performed by t-test with GraphPad Prism 5.Results: Migration assay results show that T47D/PKCα cells are nonmigratory compared to T47D/neo cells (P < 0.0001). Western blots show that there is significantly lower expression of E-cadherin in T47D/PKCα cells compared to T47D/neo cells (P < 0.005). However, transcripts of E-cadherin are expressed at 3-fold higher levels in T47D/PKCα cells compared to T47D/neo cells. Neither cell line expresses vimentin, N-cadherin, or Snail1 proteins.Conclusions: T47D/PKCα cells have reduced invasive and migratory potential even though they have decreased expression of E-cadherin. Results indicate that regulation of E-cadherin may be due to translational repression or increased degradation due to increased transcripts and absence of a transcriptional repressor. In summary, despite the fact that PKCα overexpression correlates with tamoxifen resistance, these tumors may have a better prognosis due to the less invasive and migratory nature of these cells. Future mechanistic and clinical studies are needed to determine the full prognostic impact of PKCα tumor overexpression in patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6173.
Phase II Clinical Study of Protection of Nail Change and Skin Toxicity by Using a Frozen Glove in Japanese Patients with Early Breast Cancer Treated by Docetaxel and Cyclophosphamide (TC) [TBCRG-03 Study].
Authors: Hayashi T. , Fujita T. , Mase T. , Nakano S. , Wada M. , Kashizuka T. , Sugiura H. , Mizuno T. , Iwata H. , Ohashi Y.
Published: December 2010

Background: The combination docetaxel with cyclophosphamide (TC) regimen is one of standard treatment for early breast cancer patients. TC regimen has been widely used in Japan. However, a management for adverse event such as especially edema and nail/skin toxicity by docetaxel is very important due to give a completely full dosage. This multicenter phase II study was designed to evaluate the protection of nail and skin toxicity by using a frozen glove for early breast cancer patients treated by TC.Methods: Four cycles of docetaxel (75mg/m2) and cyclophosphamide (600mg/m2) administered intravenously (i.v.) every 21 days after surgery for primary breast cancer. All patients were attached frozen glove at right or left hand (protected hand) from 15 minutes before to 30 minutes after administration of docetaxel. The other hand (control hand) of same patient is not attached frozen glove between a administration of TC. Safety was assessed every cycle after the start of treatment. Primary endpoint is comparison the rates of nail and skin toxicity between protected and control hand. Secondary endpoints are feasibility of adjuvant TC regimen and adverse events by frozen glove in Japanese women.Results: Fifty two patients were enrolled from September 2007 to June 2008. The nail changes (grade 1 and 2) was occurred in 10 events (19.2%) and 19 events (36.5%) at protected and control hand, respectively. The skin toxicity was occurred in 14 events (26.9%, G1:8, G2:6) and 21 events (40.4%, G1:14, G2:7) at protected and control hand, respectively. The appearance of nail and skin toxicity was significantly decreased at protected hand than control hand (p=0.01). Overall, 41 patients (78%) completed the planned four cycles of TC without dose reductions or study discontinuation. One patient discontinued because of hematological toxicity defined as discontinuation by protocol. The dose reductions were necessary in 10(19%) patients because of hematological (febrile neutropenia) and non hematological toxicity by TC. Grade 3 and 4 adverse events (AEs) were neutropenia (G3:3.9%, G4:21.6%), febrile neutropenia (21.2%), fatigue (1.9%) and dermatitis (3.8%). Grade 1 or 2 AEs frequently observed (more than 20%) were dermatitis such as rash (45%, G1:29.4%, G2:15.7%), pigmentation, nausea, anorexia, disturbance of taste, tear dropping, fatigue, edema and peripheral sensory disturbance. However, there are no adverse events by frozen glove.Conclusion: TC is feasible regimen for early Japanese breast cancer patients after surgery. Frozen glove may be useful procedure which protected the nail and skin toxicity. Furthermore, it is necessary to protect the severe dermatitis due to completely performed planned TC treatment after surgery in Japan.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 808.
Extra-Nuclear Estrogen Receptors: Functions in Breast Cancer.
Authors: Levin E. , Levin E.
Published: December 2010

Pools of classical estrogen receptors alpha and beta exist in various cellular locations including the nucleus, plasma membrane, and in cytoplasmic organelles (e.g.-mitochondria). The roles and importance of the extra-nuclear receptors are under investigation but in some women with breast cancer, membrane ERalpha greatly exceeds the nuclear ER pool. To understand how these receptors traffic to the plasma membrane and function, we took a proteomic approach that identified Heat Shock Protein 27 (Hsp27) as an important promoter of membrane ER localization. Hsp27 physically interacts with the ERalpha monomer, promoting palmitoylation of the sex steroid receptor, leading to enhanced association with caveolin-1 in cytoplasm and subsequent trafficking to the membrane. Using RNAi for Hsp27, this protein was shown to be necessary for ERalpha to localize to the membrane and signal through ERK and PI3 kinase, accounting for about 50% of total cell proliferation induced by estradiol. Membrane ERalpha and not nuclear ERalpha also blocks DNA repair signaling through ATR and ATM kinases in both normal breast epithelial cells and breast cancer cells (Mol Biol Cell 20:3374,2009).This occurs in the setting of radiation or chemotherapy-induced cell damage, in-vitro. Inhibiting kinase signaling leads to loss of cell cycle checkpoints, delay of DNA repair, inhibition of p53 function, and enhanced chromosomal damage upon exposing cells to gamma radiation. As for ERbeta, we find that this receptor exists mainly in extra-nuclear, mitochondrial locations in breast cancer cell lines. There is evidence that the cytoplasmic ERbeta 2 isoform correlates to a poor prognosis in women with breast cancer (Clin Canc Res, 2008). We found that mitochondrial ERbeta regulates reactive oxygen species formation. This function importantly participates in the response to tamoxifen, sensitivity (apoptosis) versus resistance (survival and growth), in several breast cancer cell lines. Mechanisms involved will be explored at the meeting. In summary, new and potentially important functions of extra-nuclear ERalpha and ERbeta could contribute to breast pathobiology.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr BS2-1.
Value of axillary ultrasound and cytology in assessing nodal status preoperatively.
Authors: Absar MS , Barr N , Wilson M , Beetles U , Knox F , Morris J , Bundred NJ
Published: December 2010

Abstract #1007

Preoperative identification of axillary node involvement in early breast cancer allows correct surgical procedures to be performed and avoids second axillary operations. We compared preoperative axillary assessment using ultrasound ± cytology with the eventual histopathological results to identify the accuracy of preoperative axillary node assessment.  Methods: Preoperative axillary ultrasound was used to identify the presence of suspicious or malignant nodes which were then confirmed with cytological assessment to allow management decisions regarding axillary clearance (or sentinel node biopsy (SNB) if nodes appeared negative).  Results: Overall 79/365 (21%) early breast cancer patients had suspicious or malignant nodes on ultrasound, of which 78 were confirmed on cytology, thus avoiding an unnecessary SNB operation in these patients. Ultrasound and cytology accurately identified patients with ER negative (P=≤0.001), high grade (P=≤0.001) and large size tumours ≤20mm (P=≤0.001), with involved nodes. Only 11% ER negative compared to 46% ER positive tumours were incorrectly classified (P=0.002). In contrast ER positive, low grade, small tumours were most likely to have a false negative axillary assessment. Specificity of cytological assessment of nodes was 99% and sensitivity 54%.  Conclusions: Axillary ultrasound combined with cytological assessment of suspicious nodes accurately identifies majority of women who require axillary clearance.   

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1007.
The value of preoperative axillary ultrasound staging in the management of early breast cancer.
Authors: O'Mahony DM , Quinn LM , Hafeez A , Dunne BM , Griffin M , Wilson GF , McDermott RA , Boyle TJ , Connolly EM
Published: December 2010

Abstract #1023

Background: The prognosis of breast cancer is strongly associated with the presence of metastatic disease in the axillary lymph nodes at the time of diagnosis. Sentinel lymph node biopsy (SLNB) is the current standard of care for staging a clinically negative axilla in early breast cancer. Approximately 25% of women with early stage breast cancer undergoing SLNB are node positive and require completion axillary lymph node dissection (ALND). Ultrasound guided fine needle aspiration cytology (FNAC) of axillary lymph nodes is a minimally invasive technique that can be used preoperatively to stage the axilla in breast cancer patients, reducing the number of operative procedures required for staging and local control of disease. Aim: To determine the sensitivity and specificity of ultrasound guided axillary FNAC to detect positive axillary lymph nodes in breast cancer patients and to determine the number of patients who avoided unnecessary SLNB. Methods: Newly diagnosed invasive breast cancer patients were prospectively followed from January 2007 to June 2008. Patients with stage IV breast cancer, clinically node positive disease and patients who did not proceed to surgery were excluded. Criteria for a suspicious sonographic axillary node included diffuse cortical thickening >3mm, focal nodular cortical thickening and a mass-like appearance with loss of the fatty hilum. Results: 133 patients who met the inclusion criteria were diagnosed with invasive breast cancer between January 2007 and June 2008. Median age was 60 [21-87]. 49 patients had T1 tumours, 77 patients had T2, and 7 patients had T3 tumours. 126 (95%) patients had axillary ultrasounds, 44 ( 35%) of which showed abnormal axillary lymph nodes. 38 ( 86%) of these 44 patients proceeded to ultrasound guided axillary FNAC and 27 (71%) of these were positive for malignant cells. All patients with positive axillary nodal FNAC underwent axillary clearance and all had positive nodes on final histopathology. The median number of positive nodes was 2, range was 1 to 21. 26 (20%) patients avoided unnecessary SLNB. 82 patients had a negative axillary ultrasound and 27 of these had axillary node positive disease. The median number of positive nodes was 1, the range was 1 to 8.    Conclusion: Ultrasound guided axillary FNAC has a high specificity and positive predictive value in radiologically suspicious nodes. A positive FNAC allows definitive treatment of the axilla in radiological node positive patients and avoids the unnecessary step of SLNB.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1023.
Anti-angiogenic effects of tamoxifen in breast cancer by decreased secretion and reduced nuclear accumulation of angiogenin.
Authors: Nilsson UW , Dabrosin C
Published: December 2010

Abstract #1033

Angiogenin, is a 14.2 kD polypeptide member of the RNase A superfamily of enzymes with potent angiogenic activity. Angiogenin binds to endothelial cells and induces proliferation, cell migration, and invasion. Nuclear accumulation of angiogenin is essential for its angiogenic activity. In breast cancer, increased angiogenin expression has been associated with the transition from normal to invasive breast carcinoma and to ER expression. We have previously reported a pro-angiogenic action of estradiol and anti-angiogenic activity of tamoxifen in breast cancer by regulation of VEGF secretion and generation of endostatin. In this paper we investigated if estradiol and/or tamoxifen affected angiogenin. We show that estradiol increased and tamoxifen decreased angiogenin secretion of human breast cancer cells in culture. In vivo, in solid human breast cancers in nude mice, tamoxifen therapy decreased angiogenin extracellularly, sampled using microdialysis. Moreover, estradiol-induced angiogenin derived from cancer cells significantly increased human endothelial cell (HUVEC) proliferation. Adding tamoxifen to the estradiol treatment as well as an antibody against angiogenin, reversed this increase. Additionally, exposure of tamoxifen to HUVEC in culture significantly decreased the endogenous secretion of angiogenin as well as the translocation of angiogenin to the nucleus. We conclude that angiogenin expression is estrogen-regulated in human breast cancer cells. In addition, decreased secretion of angiogenin by cancer and endothelial cells, and a restrained nuclear accumulation of angiogenin, may suggest novel anti-angiogenic mechanisms of tamoxifen. This may be a target for anti-estrogen therapeutic options in the future.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1033.
Angiogenesis in breast cancer: comparison between three vascular endothelial markers.
Authors: da Silva BB , Lopes-Costa PV , dos Santos AR , Sousa-Júnior EC , Facina G , Nazário AC
Published: December 2010

Abstract #1037

Background: Controversial results have been reported on the three vascular endothelial markers most commonly evaluated in breast carcinomas. The objective of this study was to evaluate the quantification of angiogenesis by comparing the performance of anti-Factor VIII related antigen, anti-CD31 and anti-CD34 monoclonal antibodies to assess microvessel density in breast cancer. Methods: Twenty-three postmenopausal women diagnosed with stage II breast cancer and submitted to mastectomy or segmentectomy were evaluated. The monoclonal antibodies used were anti-Factor VIII, anti-CD31 and anti-CD34. Microvessels were counted in the areas of highest microvessel density using a magnification of 200x in 10 random fields. Microvessel density was defined as the mean number of microvessels counted in 10 fields. The data were analyzed using the Kruskal-Wallis nonparametric test (p<0.05). Results: Mean microvessel densities with anti-Factor VIII, anti-CD31 and anti-CD34 were 4.16, 4.09 and 6.59, respectively. Microvessel density as detected by anti-CD34 was significantly greater than that detected by anti-CD31 or anti-factor VIII (p<0.0001). There was no statistically significant difference between microvessel density detected by anti-CD31 and anti-Factor VIII (p=0.4889) (see Table 1 and Figure 1).    [figure1]Discussion: In the present study, despite the current controversies, a higher density of microvessels and a greater intensity of staining were found when anti-CD34 was used in comparison with anti-CD31 and anti-Factor VIII related antigen in paraffin-fixed samples of infiltrating ductal breast carcinoma from postmenopausal women. These findings justify the increasing use of this monoclonal antibody in the study of angiogenesis, not only in breast cancer but also in other neoplasias.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1037.
Differences in outcome of young breast cancer patients according to BRCA1 mutation status.
Authors: Gabrys D , Trela K , Boratyn-Nowicka A , Suwinski R , Grzybowska E , Behrendt K , Galwas K , Wojcieszek P , Idasiak A , Thames H
Published: December 2010

Abstract #1044

Introduction  5 to 10 % of all breast cancer and 25 % of early onset breast cancer are inheritable, which may be caused by mutations of tumour suppressor genes such as BRCA1. Moreover, the issue of a familial breast cancer has raised much attention in recent years due to numerous medical and social implications. In the present paper we investigate the clinical characteristic and outcomes of younger (<50 years old) breast cancer patients with BRCA1 mutation in comparison to patients without this germline mutation.  Methods and Materials  This is an ongoing study and patients will be enrolled till end of 2008. Till now we followed 495 breast cancer patients who were diagnosed before age 50 and were asked to provide a blood sample for BRCA1 mutation screening. We compared contralateral breast cancer and ovarian cancer incidence, disease free, metastases free, and overall survival, between BRCA1 mutation carriers and non-carriers.  Results  BRCA1 mutations were detected in 90 breast cancer patients (5382insC in 60 patients, 300T/G in 18 pts, 185delAG in 10 pts, and 4153delAin 2 pts); the remaining 405 women did not carry the mutation. BRCA1 related tumours showed higher grade, higher Ki67 protein expression, more frequent negative oestrogen, progesterone, HER2-neu receptor status. Patients with BRCA1 mutation had a higher incidence of bilateral breast (40% compared to 10% in non mutated patients) and ovarian cancer (16% compared to less than 1% in non mutated patients. 10-years OS (overall survial) was 81% in no BRCA1 mutation patients compare to 88% in mutated BRCA1 patients (p=0.7), 10-years DSF (local-regional and distant failure) was 81% vs. 67% (p=0.8), 10-years MFS (metastases free survival) was 85% vs 71% (p=0.1), and 10-years RFS (recurrence free survival) was 90% vs. 80% (p=0.1) respectively.  Multivariate Cox analysis for DFS showed that node ratio > 12% (HR=2.12), increasing tumour diameter (HR=1.23), experienced significantly decreased DFS. Furthermore, overall survival analysis also showed that node ratio > 12% (HR=2.65), increasing tumour diameter (HR=1.23), experienced significantly decreased OS. There is an insignificant trend for patients without BRCA1 mutation and younger to have worse DFS (HR=1.28) and worse OS (HR=2.23).  Conclusions  Patients with BRCA1 mutations have higher incidence of bilateral breast and ovarian cancer which imposes the need for frequent and careful follow-up after therapy. Node ratio and tumour diameter are the strongest prognostic factors. A final conclusion will require more patients and longer follow up.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1044.
Lobular breast carcinoma as a possible candidate phenotype for recessively inherited breast cancer.
Authors: Olsson HL
Published: December 2010

Abstract #1045

Background:There are reasons to believe that beside dominant inheritance of breast cancer other modes of inheritance as recessive inheritance ought to exist. Studies have however failed to find recessive inheritance.Patients developing lobular carcinoma who often show multifocal and bilateral presentation could be a candidate phenotype for such a recessive disorder. Further a previous hypothesis suggests that lobular carcinomas develop from a more mature breast tissue later in reproductive life than f.i. tumours of ductal origin. Therefore lobular carcinomas ought to present at a later age and breast cancer without other cancer sites would be the predominant tumour occurring in such families.  Objective: To study breast cancer patients and relate their type of family history (vertical, horizontal or combined) to tumour histology and then especially the number of lobular carcinomas in relation to age at presentation in order to possibly identify a recessive phenotype for breast cancer.  Material and methods: Two different sets of patients have been used. First from a consecutive series of breast cancer patients(n=1682) seen at the Department of Oncology, University Hospital, Lund between 1980-2002, randomnly seen by one physician, a comprehensive standardized family history was taken. Family history was classified into 4 groups; vertical (sister-sister), horizontal (mother-daughter), combined or none. Family history was related to whether the patient had developed lobular carcinoma and the age at tumour presentation (≤50 years or above 65 years). Secondly in patients with ≥4 malignant tumours of which at least one is a breast cancer, n=67, (identified through the population based cancer registry) extended family trees were used and related to the tumour histology of the proband (lobular cancer or not).  Results: Patients with a horizontal family history (sister-sister) had more lobular carcinomas (18.8%) compared with the other groups (11.3%), p=0.05. The relationship was strongest for patients with disease above 65 years of age (31% lobular carcinoma for horizontal vs 10.9% for the others), p=0.0003, but not significant for disease presentation below age 50.  Conclusion: A horizontal family history (sister-sister) was significantly associated with lobular carcinoma developing at higher ages. These type of families may be the phenotype for studies of recessive genes in breast cancer, while in patients with multiple tumours of various types lobular carcinoma is not associated with a horisontal family history. This indicates that the possible phenotype for a recessive disorder encompassing lobular carcinoma should exhibit breast cancer as the only tumor type in the family.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1045.
TP53 mutation is an early event in breast cancer progression.
Authors: Zhou W , Muggerud AA , Vu P , Due EU , Sørlie T , Børresen-Dale A , Wärnberg F , Langerød A
Published: December 2010

Abstract #1047

Background: In breast cancer, previous studies have shown that somatic TP53 mutations cause a more aggressive disease with poor clinical outcome and may impact treatment response. Although TP53 mutation is considered to be an early event in breast cancer, the timing of TP53 mutations is not known, and there are controversies regarding the cellular origin and linear model of breast cancer. The purpose of this study was to investigate whether TP53 mutations are early events in breast cancer progression.  Methods: From a population-based cohort of women diagnosed between 1986 and 2004 either with a pure ductal carcinoma in situ (DCIS), a pure invasive cancer (<15mm) or a mixed lesion (i.e. invasive cancer with a DCIS component), we included 118 women with stored frozen tissue. Mixed lesions were microdissected using LCM (laser capture microdissection) on a PALM slide to separate in situ and invasive tumor cells. DNA was isolated using phenol-chloroform extraction. The entire coding sequence of TP53 was analyzed for mutations by direct sequencing on a 3730 DNA analyzer.  Results: Of 118 tumor samples, 19 were detected with a TP53 mutation; five 5 of 32 (15.6%) pure DCIS, 4 of 38 (10.5%) pure invasive cancers and 10 of 48 (20.8%) mixed lesions. In the mixed lesions, both the invasive and the DCIS component showed the same mutation in all 5 cases where we successfully could microdissect the two components separately. Pure DCIS demonstrated missense mutations (4/5, 80%) more frequently than pure invasive cancers (2/4, 50%) and mixed lesions (4/10, 40%), although this difference was not statistically significant (p=0.3). Also, the frequency of missense mutations in the DNA binding domain was not statistically different between the three groups.  Conclusion: TP53 mutation is likely an early event in breast cancer, occurring previous to or in the in situ stage. Presence of the same mutation in both DCIS and invasive component from the same tumor indicates same cellular origin. The role of mutant TP53 in the progression into invasive cancer is less clear and may vary between subtypes of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1047.
Novel inflammatory breast cancer cell line, MDA-IBC-1 expresses and secretes WISP3, a putative tumor suppressor in inflammatory breast cancer.
Authors: Hall CS , Debeb BG , Xu W , Ueno NT , Reuben JM , Robertson F , Lucci A , Buchholz TA , Hortobagyi GN , Cristofanilli M , Woodward WA
Published: December 2010

Abstract #1051

Wnt-1-induced secreted protein 3 (WISP3) is a member of the CCN family of proteins, which also includes connective tissue growth factor (CTGF), Cy61, Nov, WISP1, and WISP2. WISP3, as well as the other CCN family members, plays important roles in cellular processes such as proliferation, migration, and survival. WISP3 was originally isolated as Lost in Inflammatory Breast Cancer (LIBC) based on its expression in only 20% of human IBC tumors. Lack of WISP3 expression has also been reported in the IBC-derived cell line SUM-149. In SUM149 cells WISP3 has been reported to function as a tumor suppressor capable of decreasing proliferation and maintaining an epithelial phenotype. Here we examine the expression of WISP3 in a novel aggressive IBC cell line with epithelial-mesenchymal transition (EMT) morphology and abundant WISP3 expression. MDA-IBC-1 cells were isolated from the pleural fluid of a patient with ER+, PR-, HER2/neu- IBC. In 2-dimensional (2D) adherent culture, MDA-IBC-1 cells exhibit fibroblast-like EMT morphology and express EMT-associated proteins such as N-cadherin, vimentin, and fibronectin. Correspondingly, E-cadherin protein expression is absent in 2D cultures. MDA-IBC-1 cells derived from 2D cultures form mammospheres when cultured in 3-dimensional (3D) progenitor promoting conditions. Interestingly, the expression of EMT molecular markers is absent in 3D progenitor promoting mammosphere culture. MDA-IBC-1 cells exhibit robust WISP3 protein expression in both 2D and 3D culture (1.8-fold higher in 3D). In addition, MDA-IBC-1 cells secrete WISP3 as indicated by the presence of WISP3 protein in MDA-IBC-1 derived conditioned media. WISP3 binding is reported to decrease insulin-like growth factor-1 (IGF-1) activation of the IGF1R signaling cascade in SUM-149 cells. IGFR1 protein is present in 2D and 3D cultured MDA-IBC-1 cells. IGF-1 protein overlay assays using conditioned media demonstrate a WISP3:IGF-1 interaction, suggesting functional folding. Serum-free MDA-IBC-1 conditioned media did not increase basal SUM149 proliferation and it inhibited IGF-1 stimulated proliferation to serum-free (basal) levels (p≤0.05). In addition, culturing SUM-149 cells in conditioned media obtained from IBC-1 cells significantly alters SUM-149 to a completely fibroblastic morphology. These data demonstrate that MDA-IBC-1 cells represent an IBC cell line with novel expression and regulation of WISP3. MDA-IBC-1 cells may better represent the cohort of IBC patients whose tumors express WISP3. This data suggest WISP3 may not function as a tumor suppressor in all IBC patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1051.
Immunohistochemistry-based marker profile accurately predicts risk in operable, hormone receptor-positive patients.
Authors: Linke SP , Bremer TM , Jacquemier J , Charafe-Jauffret E , Viens P , Birnbaum D
Published: December 2010

Abstract #1062

Background: The performance of current prognostic breast cancer tests is limited, because they only address simple additive molecular risks, and the results are often difficult to interpret in the context of overlapping clinicopathologic risks. Gene expression-based tests can be further confounded by contamination with non-tumor cells. Our goal was to develop a protein-based profile that addresses these limitations. Methods: ER, PR, Her2, EGFR, BCL2, p27/Kip1, and p53 (IHC) and MYC (FISH) were scored in tumor cells of FFPE tissue from patients with operable, pN0-2, hormone receptor-positive breast cancer at two clinical sites. A consensus prognostic model was trained using robust cross-validation in 290 hormone therapy (HT)-treated patients using statistical pattern recognition methods to account for complex marker interactions. Tumor grade, pT, and pN were directly incorporated into the model to the extent they were not replaced by the molecular markers. Continuous risk scores ranging from 0 to 10+ were generated for the 290 HT-treated patients, 90 untreated patients, and 119 patients treated with chemotherapy and HT. Results: A predetermined threshold of 3.8 separated HT-treated patients into high and low risk groups with hazard ratios of 8.8 (p<1E-4) for metastasis and 13.0 (p<1E-4) for disease-specific survival (DSS) without the need for an equivocal intermediate risk group. NPV/PPV were 96%/35% for metastasis at 10 yr and 97%/27% for DSS at 8 yr. In multivariate analyses, the profile was independent and fully replaced the significance of all individual prognostic factors and clinical treatment guideline combinations. Within treatment guideline elevated risk groups (Adjuvant! relapse risk >15%, NPI >3.4, and St. Gallen intermediate/high), the profile reclassified as low risk 84%, 81%, and 85% of pN0 patients, and 43%, 43%, and 32% of pN1 patients, respectively. The reclassified patients had <10% 10-yr metastasis and <5% 8-yr DSS event rates. In HT-treated patients with profile risk scores >7.0, addition of chemotherapy produced a >20% DSS benefit at 8 years (p=0.04). Conclusion: This 8-marker profile with super-additive algorithm achieved significantly higher classification accuracy than treatment guidelines and could aid selection of the most appropriate level of adjuvant therapy in patients with operable breast cancer.   

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1062.
Comparison of risk stratification by OncotypeDX versus HOXB13/IL17BR index and molecular grade index in 166 estrogen receptor positive patients from multiple institutions.
Authors: Sgroi D , Goss P , Steffel L , Lester S , Brock J , Hameed O , Hattab E , Ma X , Erlander M
Published: December 2010

Abstract #1072

Background  We have previously reported two complementary prognostic markers in breast cancer for predicting risk of recurrence (Ma et al, Clin Cancer Res, 2008): a molecular grade index (MGI) that recapitulates grade 1 and 3 tumors and stratifies grade 2 tumors, and a gene expression index (HOXB13/IL17BR or H/I) that predicts clinical outcome in estrogen receptor positive (ER+) patients and endocrine benefit (Jerevell et al, Breast Cancer Res Treat, 2007). In combination, MGI and H/I define three risk groups: Low risk (low MGI), intermediate risk (low H/I, high MGI) and high risk (high H/I, high MGI). We conducted a study to compare risk stratification by MGI and H/I within samples for which a previous recurrence score (RS) by Oncotype Dx was reported.  Materials and Methods  RNA was extracted from formalin-fixed paraffin-embedded sections from all obtainable samples of patients (pts) from five institutions in which a RS was reported MGH, 77pts; Brigham, 48pts; IU, 19pts; UAB, 13pts; Scripps, 9pts. Real-time RT-PCR assays for MGI and H/I were completed and binary results of low and high for both were determined using pre-defined cutpoints. Risk stratification via MGI + H/I for each sample was compared to previously reported RS's (low, intermediate or high).  Results  For the 166 pts, 48% had a low, 45% intermediate and 7% high RS's. Overall, risk stratifications by RS versus MGI + H/I were significantly correlated (p < 0.001) with 81% of patients with low RS also having low risk (low MGI). Risk stratification by H/I + MGI reduced intermediate risk via RS by 2.5-fold (45% versus 18%).    Discussion  In ER+ pts, MGI + H/I produces a significantly smaller group of patients with intermediate risk by re-stratifying a large number of patients with intermediate RS into low and high risk groups. Risk stratification by H/I + MGI in ER+ pts is by measurement of two discrete parameters: endocrine responsiveness (H/I) and proliferative status (MGI). This may allow for more informed treatment decisions by the treating oncologist. Further studies with clinical outcome are required to establish the clinical significance of these different risk stratification methods.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1072.
Is the prognostic value of Ki-67 dependent on ER status and histological grade?.
Authors: Klintman M , Bendahl P , Grabau D , Lövgren K , Malmström P , Fernö M
Published: December 2010

Abstract #1076

Purpose: Histological grade, tumor size, age, hormone receptor, and HER2 are established prognostic indicators for distant recurrences in node negative breast cancer. However, the role of the proliferation marker Ki-67 as a prognostic factor is still under debate. The aim of this study is to evaluate the additional prognostic value of Ki-67 in lymph node negative breast cancer.  Patients and methods: In 200 consecutive premenopausal women with lymph node negative breast cancer, Ki-67 was determined using the monoclonal MIB-1 antibody on tissue microarray. High Ki-67 proliferation index was defined as >20% positive tumor cells. Cox proportional hazards regression was used to model the impact of the prognostic factors on distant disease-free survival (DDFS). Due to non-proportional hazards, the analysis was restricted to the first 5 years after diagnosis, a time period during which 38 patients developed distant recurrences.  Results: Strong positive correlations were observed between Ki-67, histological grade, S-phase, and HER-2. In univariate analysis, Ki-67 was a prognostic factor for DDFS (HR 2.9 95%CI: 1.5-5.7, p=0.002), as well as HER2, histological grade, age and ER. Tumor size was not a significant factor. In multivariate analysis, Ki-67 was an independent prognostic factor for DDFS (HR 2.3 95% CI: 1.0-5.0, p=0.044) together with HER2 and age, whereas tumor size and ER were not significant factors. Models including both Ki-67 and histological grade were not fitted due to colinearity. When stratifying for ER status Ki-67 was a significant prognostic factor in the ER positive group only (HR 5.1 95% CI: 2.0-13, p=0.001, univariate analysis) and significant interaction between the two markers was found (p=0.02). When stratifying for histological grade, Ki-67 could divide the patients with histological grade 2 (n=69; 10 distant recurrences) into two separate groups with significant difference in DDFS (HR 15 95% CI: 3.7-57, p=0.0001). This remained significant after adjustment for age and HER2 status. In histological grade 1 and 3, Ki-67 provided no additional prognostic information.  Conclusion: This study demonstrates that Ki-67 is an independent prognostic factor for premenopausal patients with lymph node negative breast cancer. It is noteworthy that Ki-67 adds prognostic information in ER positive patients, but not in ER negative, and in patients with histological grade 2, but not in patients with histological grade 1 and 3. Taken together, Ki-67 may be clinically helpful for prognostic considerations and for selection of adjuvant treatment.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1076.
Expression of fatty acid synthase as a prognostic marker in triple negative breast cancer.
Authors: Kang S , Jeon Y , Lee S , Bae Y
Published: December 2010

Abstract #1082

Background: Fatty acid synthase (FAS), the key enzyme required for fatty acid biosynthesis, is minimally expressed in most normal human tissue. Recent studies have demonstrated that hyperactivity or overexpression of FAS is a common feature in subset of sex steroid-related tumors, including breast carcinoma. Moreover, up-regulation of FAS gene expression is more pronounced in more advanced tumors, and strong expression of FAS seems to be associated with a poor prognosis of breast cancer. We examined the expression of FAS in invasive breast carcinomas to investigate the prognostic value of FAS expression in a large cohort of breast carcinomas with long term follow up data.  Methods: Five hundred and ninety six cases of invasive breast carcinoma, retrospectively selected from 1987-2004, were incorporated into tissue microarrays (TMAs). We performed immunohistochemical stains for FAS, ER, progesterone receptor (PR) and Her-2/neu. Triple-negative breast cancers (TNBCs) were defined as those with ER-, PR- and Her-2/neu-negative results. The results of immunohistochemical stains were correlated with other known clinicopathologic parameters and evaluated for an effect on estimating prognosis.  Results: The mean age at diagnosis was 47.2 (ranges 20∼85 years) with a mean tumor size of 2.5cm (ranges 0.4∼11cm). The expression of FAS was positive in 437 (73.3%) cases. There was no significant correlation between the expression of FAS and clinicopathologic characteristics of the tumor (age, tumor size, lymph node metastasis and lymphovascular invasion) (p>0.05). FAS expression was inversely correlated with histologic grade (p=0.009) and positively correlated with the expression of ER (p<0.001), PR (p<0.001) and Her-2/neu (p<0.001). One hundred thirty eight of 596 cases (23.1%) were TNBCs. FAS-expressing breast invasive carcinomas had a higher tendency to have triple-negative phenotype when compared to FAS-negative cancers (p<0.001) with a mean follow up of 72.3 months, there was no significant survival difference according to the FAS expression (p>0.05). However, FAS-positive group showed shorter disease free survival (DFS) (p=0.014) and overall survival (OS) (p=0.021) than FAS-negative group in patients with TNBC.  Conclusion: This study showed that FAS expression is strongly associated with the expression of hormonal receptors and Her-2/neu in invasive breast carcinomas. Especially FAS could be a potential prognostic marker in TNBCs.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1082.
The importance of updating the family history of breast cancer survivors.
Authors: Madlensky L , Wasserman L , Parker BA , Pierce JP
Published: December 2010

Abstract #1097

BACKGROUND: The cancer family history is the most important tool in identifying women who may be at high risk of having the hereditary breast-ovarian cancer syndrome (HBOCS) caused by mutations in the BRCA1 and BRCA2 genes. While many breast cancer patients may be asked about their family history at the time of their initial diagnosis, it is not known how many women initially classified as low risk of HBOCS become high risk of HBOCS over time, as new cancers are diagnosed in their relatives.  PURPOSE: 1)To determine how the prevalence of "high risk" cancer family histories changes over time in cancer patients, and; 2) To determine how many breast cancer survivors at high risk for HBOCS self-report having had genetic testing for BRCA1/2.  METHODS: Breast cancer survivors in the Women's Healthy Eating and Living (WHEL) study provided detailed family history data at baseline and again at study closure. The Myriad prevalence tables were used to categorize survivors as "high risk" of HBOCS if their risk of having a mutation in BRCA1/2 was 10% or greater.  RESULTS: At baseline, 8.2% of 2508 survivors were classified as high risk. At follow-up (avg. 7.5 years later), an additional 5.8% of women became high risk due to new cancers in their families. Among high-risk survivors, 15% reported that they had never heard of BRCA testing. Of the 85% who had heard of testing, 23% reported that they had been tested.  CONCLUSIONS: The prevalence of "high risk" cancer family histories increased by 70% in a cohort of breast cancer survivors over 7.5 years. It is important for clinicians to identify high-risk families not only at initial diagnosis, but to update the family history periodically. A substantial proportion of high-risk women had never heard of BRCA1/2 testing, and could benefit from genetic counseling to discuss the cancer risk in their families.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1097.
BRCA1 c.5181_5183delGTT (p.Val1688del) is a true pathogenic mutation that affects both protein stability and function: implications for genetic counseling.
Authors: De Nicolo A , Pathania S , Parisini E , Branda KJ , Stoeckert K , Garber JE
Published: December 2010

Abstract #1106

Germ line mutations in the BRCA1 tumor suppressor gene are associated with a significantly increased lifetime risk of breast and/or ovarian cancers. Since genetic testing has become a routinely step in the clinical practice, the discrimination between pathogenic mutations and benign variants appears a mandatory task as it can facilitate risk estimation and surveillance, as well as implementation of cancer prevention and treatment strategies. Most of the deleterious BRCA1 mutations identified so far are frameshift or nonsense ones that result in a premature translational truncation of the protein; only a small percentage is represented by missense mutations residing in the N-terminal RING finger or in the C-terminal BRCT domains. Interestingly, a growing number of variants of unknown significance (VUSs) are being described, which lack ad hoc functional assays thus remaining unclassified with respect to their pathogenic or neutral effect.  Recently, disease-causality was predicted, based upon a multifactorial likelihood approach, for a BRCA1 VUS, c.5181_5183delGTT (p.Val1688del), recurrent in Italian breast/ovarian cancer families (Malacrida et al., 2008). The same VUS was also identified in one of the families recruited at the Dana-Farber Cancer Institute: pedigree analysis revealed early onset breast cancer in both the proband and her mother; family history confirmed Italian descent.  To gain insights into the specific risk associated with the BRCA1 c.5181_5183delGTT (p.Val1688del) VUS, we investigated its impact on BRCA1 function. The sequence variant causes a single amino acid in frame deletion that falls within the first of the two tandem BRCA1 BRCTs. A series of approaches, including protein stability assays and co-immunoprecipitation experiments, as well as structural modeling based on sequence alignment, were employed to characterize both the full-length mutant protein (ΔValBRCA1) and its C-terminal fragment.  We observed that ΔValBRCA1 displayed decreased stability when compared to the wild type counterpart. Moreover, when tested, interactions with known BRCA1 partners, BRIP1 and CtIP, were completely abrogated. In addition, ΔValBRCA1 failed to interact with Rap80 and was deficient in DNA damage response and repair by homologous recombination. These results demonstrate that BRCA1 c.5181_5183delGTT (p.Val1688del) is a true pathogenic sequence alteration and provide the first evidence of its effect on the stability and function of the encoded protein. We believe that our findings will impact the genetic counseling of those patients who harbor this newly defined mutation and their family members thus allowing them to benefit of appropriate risk assessment, prevention and therapeutic strategies.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1106.
Decreased TGFβ signaling and increased COX2 expression in high risk women with increased mammographic breast density.
Authors: Yang WT , Lewis MT , Wong H , Hess K , Tsimelzon A , Karadag N , Cairo M , Meric-Bernstam F , Sahin A , Chang JC
Published: December 2010

Abstract #1107

Background  High mammographic density is associated with up to a 6-fold increased risk of breast cancer. Pathways responsible for this increased density are unknown. We hypothesize that specific molecular pathways exist that are associated with increased mammographic density and breast cancer risk, and may therefore be used to identify potential targets for chemoprevention.  Methods  Histologically confirmed normal breast tissue was collected from women undergoing breast surgery who had available demographic data and mammograms for review. Breast parenchymal density was classified according to the American College of Radiology's Breast Imaging – Reporting and Data System reporting system. Quantitative classification of mammographic parenchyma was performed using thresholding method and percent density. Women with low (less than 50%) versus high (greater than 50%) breast density were compared. Double-stranded cDNA was synthesized from the normal breast tissue using an oligo-dT primer containing a T7 RNA polymerase promoter, followed by in vitro transcription with biotinylated ribonucleotides. The labeled cRNA was hybridized to Affymetrix HG U133Plus2 chips which comprise ∼28,600 genes to determine gene expression patterns. Immunohistochemical analysis (IHC) of estrogen receptor, progesterone receptor, proliferation (Ki67) and COX2 expression was performed.  Results  Sixty-two women were identified, 26 (42%) had high, and 36 (58%) had low mammographic density. Neither age, menopausal nor hormone receptor status influenced the gene expression pattern. Seventy-three genes had differential expression between normal breast tissue with high and low mammographic density (p<0.001, fold change>1.5) and had a low false discovery rate (<10%). Of these 73 differentially expressed genes, network and canonical pathway analysis demonstrate decreased TGFβ signaling (TGFBR2, SOS, SMAD3 and CD44 expression) in dense breast relative to non-dense breast. By IHC, Ki67 (stroma) and COX2 expression were significantly higher in dense breasts (p<0.05) on univariate analysis, and only COX2 expression in the stroma was statistically significant at (p<0.01) on multivariate analysis.  Conclusion  TGFβ ligands are currently the only growth factors known to prevent mammary epithelial cell proliferation. TGFβ has been reported to influence COX-2 expression, and these molecules are highly differentially expressed in individuals at high risk of developing breast cancer. These results suggest that COX2 inhibition should be further investigated for breast cancer prevention despite possible increase in cardiovascular risk.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1107.