bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of switching aromatase inhibitors on arthralgia: the ATOLL study.
Authors: Briot K , Bastit L , Rotarsky M , Spaeth D , Tubiana M , Mauresan Kloos I , Bourdeix I , Roux C
Published: December 2010

Abstract #1142

Background: Aromatase inhibitors (AIs) are standard adjuvant therapy in postmenopausal women (PMW) with hormone receptor-positive breast cancer (HR+BC). The estrogen-deficient environment induced by AIs may predispose women to increased bone loss and arthralgia. Switching from one AI to another has been shown to improve arthralgia. However, clinical trial data documenting the course of joint pain symptoms in switching studies has been sparse. The Articular Tolerance of Letrozole (ATOLL) study evaluates the tolerability of letrozole (L) in patients who were switched from anastrozole (A) due to severe joint pain.  Objectives: This study included PMW with HR+BC who experienced severe joint pain with A requiring discontinuation and then switched to L. The aim of the study was to (i) determine the incidence of L discontinuation due to joint pain (ii) identify predictive factors for L tolerance.  Methods: Only PMW with HR+ BCwho experienced severe joint pain on A requiring discontinuation were eligible for this 6-month prospective open-label, multi-centre trial. Treatment with L was initiated following a 1 month washout period. Pain assessment was done by a oncologist just prior to L initiation and after six months of follow up. Outcomes were based on patient reports of joint pain after initiating L. A central review was done by a rheumatologist. Statistical evaluations were determined on a 95% CI, and correlations were performed using a Cox model.  Results: 179 PMW with HR+ breast cancer (mean age 61.3 ± 8.4) were eligible. The mean duration of A treatment prior to discontinuation was 14.6 ± 10.59 months with 126 (73.7%) on A for more than 6 months. At the end of the 6 month follow-up, 128 patients (71.5%) remained on L and continued by choice while 51(28.5%) patients discontinued L due to worsening of joint pain;73.5% patients discontinued due to arthralgia and 21% due to muscle pain . Hands, knees and spine were the most commonly affected joints. There was no association between L discontinuation with age, duration of menopause, socio-demographic status or previous joint disorders including osteoarthritis. The predictor of L discontinuation was significant only for the duration of the previous A treatment (p=0.04).  Conclusion: These data suggest switching from anastrozole to letrozole may help to maintain the benefit of AI therapy in patients who complain with severe joint pain and support the reported findings of Renshaw et al that more than 50% of women obtained relief of joint pain by switching from one AI to another. Symptomatic arthralgia due to prolonged use of A reduced the usefulness of switching to L in this patient cohort, therefore, switching from one AI to the other at the onset of symptoms may help to maintain the benefit of AI therapy in patients who complain of severe joint pain.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1142.
A randomised study of the effects of anastrozole (A), letrozole (L) and exemestane (E) on bone turnover.
Authors: McCaig FM , Renshaw L , Williams L , Young O , Murray J , Macaskill EJ , McHugh M , Hannon R , Dixon J
Published: December 2010

Abstract #1145

Background:  Aromatase inhibitors (AIs) reduce circulating oestrogen which increases bone turnover. L decreases circulating oestrogen levels to a greater degree than A. This study compares the effects of the non-steroidal aromatase inhibitors, A and L and the steroidal inactivator, E on bone turnover.  Patients and Methods:  This was an open, randomized pharmacodynamic study. Bone turnover markers were measured in 162 postmenopausal women with invasive ER +ve breast cancer. As part of their adjuvant hormone therapy, patients were randomized to receive either:  16 weeks of A, 16 weeks of L or 16 weeks of E  Fasting blood and urine samples were collected at entry and after 12 and 16 weeks of each drug. Bone remodelling releases breakdown products of type 1 collagen including the terminal peptide fragments N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx). An increase in these indicates bone resorption. Increases in type 1 pro-collagen peptides (N-terminal – PINP) and bone specific alkaline phosphatase (ALP) indicate bone formation. These were measured as was parathyroid hormone (PTH) which is an indirect measure of overall bone turnover.  Results:  A vs E vs L  A, E and L significantly increased the bone turnover markers - PINP, CTX and bone ALP.  E showed a greater change in bone turnover but there were no significant differences between the drugs.        E resulted in a greater increase in PINP and uNTX compared to the non-steroidal AIs however these were of borderline significance.  PINP, CTX and bone ALP all have significantly higher than zero percentage changes, regardless of drug or drug type.  Conclusions:  AIs cause a significant increase in both bone breakdown and bone formation.  There is no evidence to suggest E has less effects on bone turnover than A or L.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1145.
Endocrine effects of adjuvant letrozole versus tamoxifen in hormone responsive postmenopausal early breast cancer patients: results from the HOBOE randomized trial.
Authors: Morabito A , Rossi E , Di Rella F , Esposito G , Gravina A , Labonia V , Landi G , Nuzzo F , Pacilio C , De Maio E , Piccirillo M , De Feo G , D'Aiuto G , Botti G , Gallo C , Perrone F , de Matteis A
Published: December 2010

Abstract #1150

Purpose. We compared the endocrine effects of 6 and 12 months of adjuvant letrozole versus tamoxifen in postmenopausal patients with hormone responsive early breast cancer, within an ongoing phase 3 trial (HOrmonal adjuvant treatment BOne Effects – HOBOE, ClinicalTrial.gov id: NCT00412022).  Patients and Methods. Patients were randomised to receive tamoxifen or letrozole ± zoledronate. Serum values of 17-b-estradiol, FSH, LH, testosterone, dehydroepiandrosterone-solphate, progesterone, and cortisol were measured at baseline, after 6 and 12 months of treatment. For each hormone, baseline, 6 and 12-month values were compared between treatment groups, by the exact Wilcoxon-Mann-Whitney test.  Results. At December 31, 2006, 157 postmenopausal patients had been enrolled into the study; baseline data were available for 139 patients (88.5%), 43 assigned to tamoxifen and 96 assigned to letrozole. Median age was 61 and 62 years in the two groups, respectively. Baseline values were similar between the two groups for all hormones. At 6 and 12 months, levels of 17-b-estradiol were significantly lower with letrozole as compared with tamoxifen (p=0.0003 and p<0.0001, respectively). Patients treated with letrozole also showed higher levels of progesterone and testosterone at 6 (p=0.001 and p=0.01, respectively) and 12 months (p=0.004 and p=0.02, respectively) than those treated with tamoxifen. FSH and LH were lower (all p<0.0001 for both hormones), while cortisol was higher (p=0.003 at 6 and 0.001 at 12 months) with tamoxifen than with letrozole.  Conclusion. To our knowledge, this is the first study reporting on endocrine effects of letrozole as adjuvant treatment of postmenopausal early breast cancer and allowing a prospective comparison with tamoxifen. Adjuvant letrozole and tamoxifen result in significantly distinct endocrine effects. Such differences can explain the higher efficacy of letrozole as compared to tamoxifen. Long-term impact needs to be studied.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1150.
BIG 1-98: A randomized double-blind phase III study evaluating letrozole and tamoxifen given in sequence as adjuvant endocrine therapy for postmenopausal women with receptor-positive breast cancer.
Authors: Mouridsen HT , Giobbie-Hurder A , Mauriac L , Paridaens R , Colleoni M , Thuerlimann B , Forbes JF , Gelber RD , Wardley A , Smith I , Price KN , Coates A , Goldhirsch A ,   BIG I-98 Collaborative and the International Breast Cancer Study Group
Published: December 2010

Abstract #13

Background: BIG 1-98 compares letrozole (Let), tamoxifen (Tam) and sequences of Let and Tam as adjuvant endocrine therapy for postmenopausal women with receptor-positive breast cancer. Initial results (published in 2005) demonstrated the superiority of Let over Tam in significantly prolonging disease-free survival (DFS) and reducing the risk of relapse in distant sites. Following these results, investigators were informed of the treatment allocation for patients randomized to Tam alone, and about one third of these patients opted to receive Let. The three Let-containing treatments remained blinded. We now evaluate Let and Tam given in sequence compared with Let alone and update the comparison of Let alone and Tam alone.  Methods: 6182 patients were randomized to the 4-arm option (entry Sep99-May03) [Tam x 5 years (Tam), Let x 5 (Let), Tam x 2→Let x 3 (Tam→Let), or Let x 2→Tam x 3 (Let→Tam)]. Another 1828 patients were randomized to the 2-arm option (Tam vs. Let; entry Mar98-Mar00). The primary endpoint was DFS (time from randomization to first occurrence of invasive breast cancer recurrence, invasive contralateral breast cancer, second non-breast malignancy, or death from any cause). The presentation will focus on the clinically-relevant comparisons evaluating Tam→Let vs. Let and Let→Tam vs. Let, assessed from the time of randomization. Acknowledging multiple comparisons, the hazard ratios are presented with 99% confidence intervals (CIs).  Results: The median follow-up time is 71 months. Hazard ratios comparing treatments from time of initial randomization in terms of DFS, overall survival, and time to distant recurrence are displayed below.    With this long term follow-up, adverse events for Let and Tam are consistent with the known safety profile of both agents.  A protocol-specified update of the previously-reported comparison of Let x 5 vs. Tam x 5 (i.e., the monotherapy arms, including patients in both the 2-arm and 4-arm options; N=4922) suggests improved survival for patients treated with Let (HR=0.87, 95% CI=0.75-1.02, p=0.08, 76 months median follow-up). In this comparison, about a quarter of the patients in the Tam arm crossed over to Let after 3-5 years of Tam.  Conclusion: The sequential treatments did not improve DFS compared with Let alone. Trends support initial use of Let in patients at higher risk of relapse. Patients commenced on Let can be switched to Tam if required. Updated results of the monotherapy comparison suggest superior overall survival with Let compared with Tam.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 13.
Tamoxifen and anastrozole as a sequencing strategy in postmenopausal women with hormone-responsive early breast cancer: updated data from the Austrian breast and colorectal cancer study group trial 8.
Authors: Jakesz R , Gnant M , Griel R , Tausch C , Samonigg H , Kwasny W , Kubista E , Stierer M , Luschin G , Rüecklinger E , Mittlböck M
Published: December 2010

Abstract #14

Background: Historically, standard endocrine treatment for postmenopausal women with hormone-sensitive early breast cancer (EBC) was 5 years' tamoxifen. However, the inclusion of an aromatase inhibitor (AI) is now considered standard practice. There is increasing evidence that whether as initial adjuvant treatment or as a switch treatment following 2-3 years of tamoxifen therapy, Ais improve patients' outcomes compared with 5 years' tamoxifen monotherapy.  To date, there is limited evidence comparing a prospective sequencing strategy of 2 years of tamoxifen followed by 3 years of an AI compared with 5 years' tamoxifen. Previously presented data from ABCSG Trial 8 (SABCS 2005), at a median follow-up of 55 months, revealed a 24% reduction in the risk of recurrence in favor of a sequencing strategy compared with 5 years' tamoxifen (hazard ratio [HR] 0.76; 95% confidence interval [CI] 0.56-1.02), although the difference was not statistically significant (p=0.07). Here we present updated data from ABCSG Trial 8.  Methods: Postmenopausal women with histologically verified G1 or G2, locally radically treated invasive or minimally invasive hormone receptor-positive breast cancer were eligible for inclusion. Newly diagnosed patients were randomized to receive 5 years' tamoxifen therapy or 2 years' tamoxifen followed by anastrozole for 3 years. The primary endpoint was event-free survival, including locoregional and distant metastatic recurrences and new contralateral breast cancers.  Results: This final analysis will report the data cut-off point, the number of eligible patients, and the median follow-up period of the trial, which will exceed 5 years. The incidence of first events, local recurrences, distant metastases, and contralateral breast cancers for the two treatment arms of the study will be presented. The effect of treatment on the trial endpoint will be analyzed using a Cox proportional hazards model and data will be presented as estimated HRs with two-sided 95% CIs and p-values.  Conclusions: The updated analyses of ABCSG Trial 8 will provide evidence regarding 2 years' treatment with tamoxifen followed by 3 years' anastrozole in comparison to 5 years' tamoxifen for postmenopausal women with newly diagnosed hormone-sensitive EBC.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 14.
Scoring system to predict under-evaluation in patients diagnosed with benign papilloma at ultrasound-guided core needle biopsy (CNB).
Authors: Ko E , Han W , Lee J , Kim E , Moon H , Yu J , Noh D
Published: December 2010

Abstract #2001

Background: The aim of this study was to determine factors that predict under-evaluation of atypical lesion or malignancy in patients diagnosed with benign papilloma at ultrasound-guided core needle biopsy (CNB), and to develop a prediction algorithm for scoring the possibility of a diagnosis upgrade to atypical lesion or malignancy based on clinical, radiological and pathological factors. Methods: The study enrolled patients diagnosed with benign papilloma at ultrasound-guided CNB who subsequently underwent surgical excision of the lesion. Multivariate analysis was used to identify relevant clinical, radiological and pathological factors that may predict malignancy. Results: A total of 262 patients with benign papilloma at CNB were identified. Of the 110 patients who underwent subsequent surgical excision, 22 (20%) were diagnosed with atypical papilloma or malignancy. Multivariate analysis revealed that mass or calcification on mammography, size on imaging >15 mm, peripheral location, and a palpable lesion were independent predictors of malignancy. A scoring system was developed based on logistic regression models and beta coefficients for each variable. The area under the ROC curve was 0.868 (95% CI: 0.794-0.941), and the negative predictive value was 100% for a score of 0 or 1 (Of the 110 patients, 58 patients had the score of 0 or 1). Conclusions: A scoring system to predict malignancy in patients diagnosed with benign papilloma at CNB was developed based on four factors: peripheral location, palpable lesion, mass or calcification on mammography, size on imaging. This system was able to identify a subset of patients with lesions likely to be benign, indicating that imaging follow-up rather than surgical excision may be appropriate.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2001.
Using custom protein microarrays to identify autoantibody biomarkers for the early detection of breast cancer.
Authors: Anderson KS , Sibani S , Wong J , Hainsworth E , Mendoza EA , Eugene R , Raphael J , Logvinenko T , Ramachandran N , Godwin A , Marks J , Engstrom P , LaBaer J
Published: December 2010

Abstract #2003

Background: Cancer patients make antibodies to tumor-derived proteins that are potential biomarkers for early detection. To detect autoantibodies to tumor antigens in patient sera, we have adapted novel high-density custom protein microarrays (NAPPA) expressing 6,500 candidate tumor antigens for biomarker detection. These arrays are probed with sera from patients with early stage breast cancer and healthy women. Using this approach, we identified antibodies in the sera of breast cancer patients.  Methods: 6,500 full-length human antigens were expressed using mammalian reticulocyte lysate and captured onto NAPPA protein microarrays. Protein expression (>90%) was confirmed with anti-GST antibodies. Patient sera were added, and bound IgG detected with secondary antibodies. Serum samples were obtained from 103 patients with stages I-III breast cancer, and 103 age-matched control women, all undergoing routine mammography.  Results: Using high-density protein microarrays, sera from breast cancer patients (n=53) and healthy donor sera (n=53) were screened for autoantibodies to 6,500 protein antigens. Antigens were selected for further analysis if the 95th percentile of signal of cases and controls were significantly different (p<0.05) and if the number of cases with signals above the 95th percentile of controls was larger than the number expected due to random chance (p<0.05). Known tumor antigens, such as p53, were detected, as well as novel antigens such as DCC1, Rab7L and USP44. In total, 768 unique antigens were selected for further analysis with an independent set of breast cancer patient (n=50) and healthy donor sera (n=50).  Conclusions: Custom in-situ protein microarrays can be used to detect serum tumor antigen-specific antibodies and enables the rapid, simultaneous detection of immunogenic tumor antigens from patient sera. These autoantibodies are being evaluated as potential biomarkers for the early diagnosis of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2003.
Evaluation of human secretoglobins and MIC-1 as serum diagnostic biomarkers for breast cancer.
Authors: Bistrup A , Cabaltica C , Jalilie M , Wolfert RL , Kim NW
Published: December 2010

Abstract #2005

Using microarray and quantitative RT-PCR analysis, we found mRNA corresponding to several members of the human secretoglobin family and MIC-1 to be highly expressed in various cancer tissues, as compared to the corresponding normal tissues. Our target ECM2, known in the literature as Lipophilin B (LipB), was found to be upregulated in endometrial, uterine, breast and prostate cancer tissues. Our target ECM3, also known as Mammaglobin B (MamB) or Lipophilin C, was found to be upregulated in endometrial and uterine cancer tissues. DD-X065, also known in the literature as macrophage inhibitory cytokine-1 (MIC-1), a member of the transforming growth factor-beta (TGF-b) family, was found to be upregulated in breast, colon, lung, ovarian, and prostate cancer tissues.  There is considerable evidence suggesting that members of the secretoglobin family exist predominantly as heterodimers and higher order complexes. Mammaglobin A (MamA) protein has been found to be present in breast tissue in a complex with LipB protein. We generated a series of mouse monoclonal antibodies directed against different complexes composed of members of the family, including MamA, LipB, and MamB. Employing these specific antibodies, we have developed sensitive immunoassays to measure serum levels of several of the complexes.  In the current study, we evaluated serum from breast cancer patients for levels of two secretoglobin family complexes: MamA/LipB and MamB/LipB, as well as for MIC-1, utilizing our previously established ELISA assay2.  The study comprised serum samples from 150 normal healthy individuals and 250 breast cancer patients. The sensitivity and specificity of the assays for detection of breast cancer were quantified by receiver operating characteristic (ROC) analysis. Compared to normal healthy individuals, the median serum concentrations of MamA/LipB was increased by approximately 2.8 fold in breast cancer, irrespective of stage. ROC analysis of breast cancer patients versus normal healthy individuals resulted in an AUC of 0.67 in all stages. For MamB/LipB, the median serum concentration in cancer serum was increased over normals by approximately 1.8 fold and the corresponding AUC was 0.62. The median serum concentration for MIC-1 in cancer serum was increased over normals by approximately 1.1 fold and the corresponding AUC was 0.61. The sensitivity of the MIC-1 assay was greater than that of any of the secretoglobin family complex assays at 90%, 95% and 98%.  Our study suggests that MIC-1 and members of the secretoglobin family may be promising serum markers for breast cancer. Their utility may be improved when used in combination with other novel diagnostic biomarkers. Further studies are underway to validate the clinical potential of this family of markers.  2Bistrup, A, et al., (2007) Proceedings of 98th AACR Annual Meeting, Los Angeles, CA.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2005.
CDK2 is required for LMW-cyclin E mediated murine mammary tumorigenesis.
Authors: Akli S , Van Pelt CS , Bui TN , Keyomarsi K
Published: December 2010

Abstract #2007

Cyclin E is a regulatory protein with multiple functions that activates CDK2, controls centrosome duplication, and regulates histone gene transcription. Our previous studies have shown that the presence of Low-molecular-weight (LMW) forms of cyclin E correlates strongly with decreased survival in patients with breast cancer. Transgenic mice overexpressing LMW cyclin E had increased incidence of mammary tumors and distant metastasis when compared to full length cyclin E. The critical role of LMW cyclin E in mammary tumorigenesis may be the result of the ability of LMW cyclin E to bind and activate the cyclin-dependent kinase 2 (CDK2). To specifically test the importance of LMW cyclin E-associated CDK2 kinase activity in mammary tumorigenesis, we generated double and triple transgenic mice LMW cyclin E-T1;CDK2-/-, and LMW cyclin E-T1;p53+/-;CDK2-/-. We found that: (i) The development of the mammary glands proceeds relatively normally in these animals demonstrating that CDK2 kinase activity is largely dispensable for mammary gland development; (ii) These mice were resistant to breast cancers initiated by LMW cyclin E overexpression. Whereas LMW-cyclin E-T1 mice in CDK2+/- or CDK2+/+ background succumb to mammary tumors with mean latencies of 16 and 19.5 months respectively, their LMW cyclin E-T1; CDK2-/- littermates do not form mammary tumors up to 24 months (P=0.05 CDK2+/+ versus CDK2-/-; P=0.0252 CDK2+/- versus CDK2-/-; P=0.1457 CDK2+/+ versus CDK2+/-, Log rank test). Similarly, while MMTV-LMW cyclin E-T1/p53+/- transgenic mice in CDK2+/- or CDK2+/+ background succumb to mammary gland tumors between 7 and 13 months with 100% penetrance, their MMTV-LMW cyclin E-T1;p53+/-; CDK2-/- littermates are resistant to tumor formation (P<0.01 CDK2+/+ versus CDK2-/-; P=0.011 CDK2+/- versus CDK2-/-, P=0.5072 CDK2+/+ versus CDK2+/-, Log rank Test). In fact none of the CDK2-/- mice developed mammary tumors when crossed with cyclin E LMW transgenic mice. However, 2 out of the 12 MMTV-LMW cyclin E; p53+/-; CDK2-/- mice generated salivary gland tumors. Hence, these LMW cyclin E; p53+/-; CDK2-/- are susceptible to LMW cyclin E induced salivary tumors in the absence of CDK2. We concluded that CDK2 is critically required for LMW cyclin E induced mammary tumorigenesis and consequently, the loss of CDK2 renders CDK2-/- mice resistant to breast cancer induced by LMW cyclin E expression. In clear contrast to mammary epithelial cells, we found that in salivary gland cells LMW cyclin E continued to elicit malignant transformation even in the absence of CDK2. Our work suggests that due to the requirement of CDK2 for LMW-cyclin E mediated mammary tumorigeneis, these tumors are prime candidates for anti-CDK2 therapy.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2007.
Preservation of lymphatic drainage from arm in breast cancer surgery: is it safe?.
Authors: Kang S , Choi J , Jeon Y , Lee S , Bae Y
Published: December 2010

Abstract #201

Purpose: Sentinel lymph node biopsy (SLNB) for breast cancer was introduced to prevent the high morbidity seen with Axillary lymph node dissection (ALND). Although multiple comparison studies have confirmed that SLNB consistently has lower morbidity and lymphedema rates than ALND; it is still clinically significant ranging from 0∼13%. The aim of this study was to confirm the feasibility of the technique, so called Axillary reverse mapping (ARM), and to test the hypothesis that arm lymphatics are never involved by the metastatic process of breast cancer.  Methods: We reviewed prospectively maintained database of 129 patients who operated for primary breast cancer since June 2007. Sentinel lymph nodes (SLNs) were identified by using a radiolabeled isotope (TC99m-phytate or human serum albumin). Under general anesthesia, 2.5∼3 mL of blue dye was injected in the upper inner arm along the medial intramuscular groove of ipsilateral arm in order to locate the draining lymphatics or lymph node from the arm. We then proceeded as usual with the SLNB or ALND. During axillary procedure, we take the blue node (ARM node) and SLN (hot node), and both were sent to pathology department for frozen section analysis. If SLNs were positive, ALND was performed and axillary contents were sent to pathology. Histological results of ARM node were compared with that of the other nodes of the SLNB or ALND.  Results: The mean age of patients was 48.3 years old (rang 27∼73). Surgeries included 41 mastectomies, 80 breast conserving surgeries and eight skin sparing mastectomies with immediate reconstruction. Of the 129 procedures, 81 were in SLNB (62.8%), 43 SLNB+ALND (33.3%), and five ALND. One hundred one of 129 (78.3%) blue ARM nodes were identified in the axilla. The average number of removed ARM node was 1.5 (range 1∼5). In 19 of 96 SLNB cases, the ARM nodes were hot, yielding 18.9% of concordance rate between ARM node and SLN. Among these 19 cases, seven ARM nodes contained metastasis. In one SLNB case, the metastatic ARM node was not hot. In all five ALND cases, ARM nodes were identified and one of five ARM nodes was positive for metastasis. Thus, ARM node metastasis rate of identified ARM node during ARM procedure was 8.9% (9/101).  Conclusion: According to our study, it is thought that lymphatic drainage from arm share common lymphatic channel in axilla with lymphatic flow from breast. Therefore, intentional preservation of lymphatics or lymph node drained from arm during performing axillary surgery in breast cancer could be a dangerous one.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 201.
Development of a breast cancer brain metastases model to study 131I radioablative therapy.
Authors: Renier C , De A , Hou L , Dunkel J , Sun A , Prugpichailers T , Gambhir SS , Tse V , Wapnir IL
Published: December 2010

Abstract #2011

Background: An increasing number of women develop brain metastases (BM) after breast cancer (BC) treatment. A large proportion of these are estrogen/progesterone receptor-negative (ER-/PR-) and/or Her-2/neu overexpressing tumors. 131I radioablative therapy may provide a therapeutic alternative to treat metastases at this anatomic sanctuary since over 70% of invasive breast cancers, including a majority of ER- tumors and some brain metastases (unpublished data) express the sodium-iodide symporter (NIS). This approach relies on the success of radioiodide as a targeted treatment for thyroid cancers. To test this concept, we developed a BC BM model using tumor cells engineered to express NIS.  Methods: MDAMB231 and SKBr3 cell lines were transduced with a lentiviral vector carrying a bicistronic cassette with NIS and the firefly luciferase (Fluc) genes separated by an internal ribosomal entry site. Single cell clones were selected and characterized for iodide uptake and bioluminescence. NIS-Fluc-MDAMB231 or NIS-Fluc SKBr3 cells (2.5 x 106 cells) were implanted subcutaneously (sc) in the mammary fat pad (mfp) of nude mice (NCr nude; 5-6 weeks old; n=5). NIS-Fluc mfp tumor xenografts were then explanted, 1x1 mm pieces excised and inserted stereotactically into the basal ganglia of the animal. All tumor development was monitored by serial in vivo bioluminescent imaging. Once established, brain tumors were excised, dissociated, established in tissue culture and re-implanted sc in the mfp of a new set of mice. Successive passages in the mfp then in the brain were performed in an attempt to increase tumor take. A second strategy tested with MDAMB231 cells consisted of direct implantation of cells into the basal ganglia. NIS expression was evaluated on tissue sections with a polyclonal antibody raised against the C-terminus of the human NIS.  Results: All mice survived and were healthy in appearance. Intracranial implantation of mfp xenografts was highly successful with 66% take in both MDAMB231 (after two passages) and SKBr3 (after first passage). Bioluminescent imaging revealed sustained growth of tumors for more than 4 weeks. Microscopically, the explanted brain tumors had a cellular appearance without stromal cell or lymphocytic infiltration and were congruent with the histology of mfp xenografts. However, the tumor cell population was heterogeneous as NIS expression was present with plasma membrane staining in about 50% of SKBr3 and 15% of MDAMB231 cells. Direct cell implantations failed as no discernible bioluminescence was noted over a period of 3 weeks and no visible tumor at necropsy.  Conclusions: A BCBM model has been developed by implanting intracranially mfp xenografts obtained with ER-/PR- +/- Her-2/neu overexpressing cells. Using this model, it will be possible to evaluate the effects of 131I on NIS-expressing BCBM.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2011.
Antitumor activity of chemoendocrine therapy in premenopausal (capecitabine [CAP] + tamoxifen [TAM]) and postmenopausal (CAP + letrozole [LET]) human breast cancer [BC] xenograft models.
Authors: Kataoka M , Yamaguchi Y , Sawada N , Moriya Y , Evans DB , Yasuno H , Mori K , Hayashi S
Published: December 2010

Abstract #2012

Background: The preferential treatment for estrogen receptor (ER)-positive metastatic or recurrent breast cancer is endocrine therapy unless it is life-threatening. CAP, an oral fluoropyrimidine, is activated to 5-FU by thymidine phosphorylase (TP) in tumors. We have evaluated the efficacy of endocrine therapy in combination with CAP in premenopausal and postmenopausal ER-positive human BC xenograft models.  Methods: In a premenopausal BC model, ER-positive MCF7 cells were subcutaneously (sc) inoculated into nude mice which had been implanted sc with 0.25 mg 17β-estradiol pellets. The mice received 6 weeks oral therapy with vehicle, CAP (days 1-14, every 3 weeks) at the maximal tolerated dose (MTD) of 539 mg/kg or 2/3 MTD +/- TAM at 100 or 30 mg/kg/day. In a postmenopausal BC model, nude mice were ovariectomized and implanted sc with 1.5 mg androstenedione pellets the day before tumor cell inoculation. MCF7A25F3 cells, an aromatase gene transfectant of MCF7, were inoculated sc into the mice. The mice received 6 weeks' oral administration of vehicle, CAP (days 1-14, every 3 weeks) at 2/3 MTD +/- LET at 0.1 mg/kg/day. TP level in tumors was measured on the final day of the first treatment cycle by ELISA. In vitro ER reporter gene assays were performed by co-culturing MCF7 cells transfected with estrogen response element-GFP gene with stromal cells isolated from human BC tissues. Testosterone was added to the culture medium as a substrate for aromatase and the expression of GFP was measured.  Results: In the premenopausal model, the antitumor activity of the combination therapy with CAP and TAM was significantly superior to that of each monotherapy (p<0.001). In the postmenopausal model, the antitumor activity of the combination therapy with CAP and LET was also significantly superior to that of each monotherapy (p<0.05). No augmentation of toxicity in terms of body weight loss was observed with the combination therapy in each xenograft model. In the premenopausal model, the tumor levels of TP were up-regulated (p<0.05) by TAM treatment indicating that TAM might potentiate the antitumor activity of CAP. On the other hand, the tumor levels of TP remained unchanged by LET treatment in the postmenopausal model. In ER reporter gene assays, GFP expression was decreased by the addition of either 5'-deoxy-5-fluorouridine (5'-DFUR, an intermediate of CAP), 4-hydroxytamoxifen (4-OHT, active form of TAM) or LET in a concentration-dependent manner. 5'-DFUR showed at least additive inhibitory effect in combination with 4-OHT or LET.  Conclusion: Chemoendocrine therapy using CAP might be a useful treatment modality for BC patients regardless of the menopausal status.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2012.
The role of receptor tyrosine kinase and activated PI3K signaling in response/resistance to HER-2 targeted therapy.
Authors: O'Brien NA , Browne BC , Chow L , Ginther C , Bensadigh B , Arboleda J , Duffy MJ , Crown J , O'Donovan N , Slamon DJ
Published: December 2010

Abstract #2013

Background: Trastuzumab and lapatinib therapy provide proven clinical benefits to women with HER-2 positive breast tumors. However, not all patients that have high HER-2 levels respond. Here we describe the development and characterization of models of both clinical forms of trastuzumab resistance, i.e., de novo and acquired resistance.  Materials and Methods: Models of acquired resistance were established by culturing BT-474 and SKBR3 cells in high dose trastuzumab (105 μg/ml) for nine months. De novo resistance was examined in a panel of 17 HER-2 amplified cell lines. Response to the dual EGFR-HER2 inhibitor, lapatinib, was also measured. For each agent, response was measured by two independent assays; cell count and colony formation assay. Levels of HER-2, p-HER-2, EGFR, p-EGFR, IGF-1R, p-IGF-1R, p95, PTEN, and p27, and the presence of PIK3CA activating mutations were assessed in each cell line.  Results: Both BT-474 and SKBR3 conditioned cell lines have a reduced response to trastuzumab but remain sensitive to lapatinib. Analysis of the panel of HER-2 amplified cell lines revealed that SUM-225, HCC-1419, HCC-1954, HCC-1569 and JIMT-1 are models of de novo resistance to trastuzumab. The SUM-225 and HCC-1419 cell lines, despite being trastuzumab resistant, were exquisitely sensitive to lapatinib. The UACC-893, UACC-732, HCC-1569, MDA-MB-361 and JIMT-1 cell lines were identified as being resistant to lapatinib (IC50 > 1 μM), whereas the HCC-1569 and JIMT-1 were resistant to both trastuzumab and lapatinib. Resistance to trastuzumab correlated significantly with active PI3K signaling (p = 0.003), as assessed by combined analysis of PTEN status and PIK3CA mutation status. Response to lapatinib correlated significantly with levels of HER-2 (p = 0.013), pHER-2 (p = 0.086), p95 (p = 0.033), p-EGFR (p = 0.019) and p-IGFR (p = 0.046).  Conclusion: We have identified reliable, HER-2 overexpressing, cell line models of response and resistance to trastuzumab and lapatinib. Our analysis of these models indicates that activated PI3K signaling plays a crucial role in de novo resistance to trastuzumab while high levels of RTK signaling appears to sensitize HER-2 positive cells to lapatinib treatment.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2013.
Ixabepilone plus capecitabine improves progression free survival in patients with metastatic breast cancer resistant to taxanes: a pooled analysis from two phase III trials.
Authors: Roche H , Li R , Ro J , Vrdoljak E , Rahman ZU , Medina C , Conte P , Volles LA , Poulart V , Sparano JA
Published: December 2010

Abstract #2015

Background:  Patients (pts) with MBC whose tumors are resistant to taxanes (T) have limited therapeutic options. Ixabepilone (ixa), the first in a new class of antineoplastic agents, showed clinical benefit in combination with capecitabine (C) in 2 large clinical trials in metastatic breast cancer (MBC) pts resistant to (study 046, JCO, 2007), or pretreated with anthracycline (A) and T (study 048). Pts whose tumors were resistant to A/T were also allowed to enter the 048 trial. In 048, ixa + C, compared to C alone, also demonstrated significant increases in progression-free survival (PFS, HR 0.79 [0.69-0.90)]) and ORR (43% vs 29%). A trend toward increased OS was seen both in 048 (HR 0.90 [0.78-1.03]) and in 046 (HR 0.90 [0.77-1.05]), which did not reach statistical significance. Here we present a pooled analysis from the 2 studies of clinical outcomes (ORR, PFS, and OS) in patients with a strict definition of resistance to T.  Methods:  1973 pts with MBC previously treated with an A and T, were randomized in phase III trials to receive either ixa (40 mg/m2 IV over 3h Q3w) + C (1000 mg/m2 PO BID x14d Q3w) or C alone (1250 mg/m2 PO BID x14d Q3w). For both studies, resistance was defined as disease progression up to 4 months following T in the metastatic setting and up to 12 months following adjuvant T therapy. Due to the similarity of the study populations, individual pt data from both studies was pooled to better evaluate treatment effect within pre-planned patient subgroups.  Results:  1337 pts from the 2 studies were T resistant and randomized to ixa + C or C alone. ORR and PFS favored ixa + C in this pt population, and there was a trend towards increased OS, which did not reach statistical significance (see table).    Conclusion:  Ixa + C is the first combination to show a clinical benefit of PFS in pts with MBC resistant to T (following a strict definition of resistance). The benefit seen in this pooled analysis was consistent with that observed in the resistant pt population from the individual studies as well.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2015.
Transcriptional profiling of patient-matched ER-positive breast cancer tissue from post-menopausal women treated with neoadjuvant RAD001.
Authors: Sabine VS , Sims AH , Macaskill EJ , Renshaw L , Thomas JS , Dixon JM , Bartlett JM
Published: December 2010

Abstract #2035

Background: RAD001 (everolimus) is a rapamycin derivative that inhibits mTOR and its downstream substrates and has shown promising results in Phase I clinical trials. This is the first clinical study of RAD001 administered alone in breast cancer. The aim of this study was to characterise the effects of RAD001 treatment on tumour gene expression and to assess whether we can predict which patients will respond to RAD001.  Materials & Methods: 32 post-menopausal women with operable estrogen receptor positive early breast cancer received 5mg RAD001 pre-operative treatment daily for 14 days prior to primary surgery. 25 patients completed treatment, 6 withdrew and 1 did not start medication. Paired biopsies were taken at diagnosis and at surgery from each of the 31 patients who received RAD001. Half of the biopsy was immediately flash frozen in liquid nitrogen and RNA extracted, amplified in duplicate and analysed using Illumina Human Ref.8 chip v2, the other half of the biopsy was embedded in paraffin for the analysis of biological response markers, including changes in proliferation (Ki67) by immunohistochemistry (Macaskill et al., 2006: Breast Cancer Research & Treatment: 100: S1: S286). 15 patients were classified as responders to RAD001 and 10 patients non-responders (responders were defined by a fall in % Ki-67 positive cells). Gene expression changes were compared before and after treatment with RAD001 and between the responders and non-responders using Bioconductor programs implemented in the R statistical programming language.  Results & Discussion: All pre- and post-treatment biopsies were available for microarray processing and assessment of biological response markers for patients completing RAD001 treatment. Analysing data from duplicate samples enabled us to vigorously evaluate the reproducibility of the Illumina beadarray platform and revealed some slight batch-to-batch variation which can be accounted for in the analysis. This dataset has enabled us to begin to identify genes and pathways associated with RAD001 treatment and to generate a gene expression classifier for predicting which patients will benefit from RAD001 treatment. Genes of interest will be validated by quantitative real time PCR and key pathways reported.  Acknowledgements: This work was supported by a grant from Breast Cancer Research & Treatment, UK.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2035.
Role of KCNMA1 gene in breast cancer invasion and metastasis to brain.
Authors: Khaitan D , Sankpal UT , Ningaraj NS
Published: December 2010

Abstract #2043

Background: The prognosis for patients with brain metastasis from breast cancer is extremely poor. A significant number of breast tumor patients despite aggressive therapy develop metastatic disease in brain. The mechanism or the cause of breast cancer metastasis to brain is unknown. Therefore, it is critical to identify prognostic molecular markers of the metastatic process in order to design therapeutic modalities for reducing the occurrence of metastasis. In this study we investigated KCNMA1 gene, which encodes alpha subunit of calcium activated potassium (KCa) channels. Altered KCa channels, are described in a wide variety of tumor cell types and up-regulation of KCa channels was shown to be a novel mechanism for the malignant phenotype of human tumor cells.  Material and Methods: We performed Global GeneChip exon arrays to study the expression of KCNMA1 gene in metastatic breast cancer in brain and compared it with primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immuno-histochemistry was performed to study the expression and localization of alpha subunit of KCa channel protein in primary and metastatic breast tumor tissues and breast cancer cell lines. We performed Matrigel invasion, transendothelial migration and membrane potential assays in normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether KCa channel inhibition attenuates breast tumor invasion and metastasis to brain.  Results: The Global GeneChip exon array and RT-PCR showed higher KCNMA1 gene expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased KCa channel activity, leading to greater invasion and trans-endothelial migration, which could be attenuated by blocking the KCNMA1 gene with siRNA and biochemical inhibition with IBTX.  Discussion: Recent investigations in patients with metastatic lung and breast cancers reported alarming rates of brain metastases. Unraveling the genetic aberrations that drive the metastatic events in these cancer cells offer insights into how to limit or prevent this lethal process. For instance, determining the relative abundance of KCNMA1 gene in breast cancer metastasis to brain and understanding the mechanism of brain metastasis provides a unique opportunity to identify specific genetic markers that could differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This would in turn allow for the appropriate and efficient application of treatments to obtain the greatest benefit while sparing low risk patients for metastasis from the toxic side effects of chemotherapy.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2043.
CXCR4 alters ID family and ITF2B/E2-2/TCF-4 dynamics to confer invasive phenotype to breast cancer cells.
Authors: Appaiah HN , Bhat-Nakshatri P , Nakshatri H , Badve S
Published: December 2010

Abstract #2045

Introduction: Breast cancer is characterized by a distinct metastatic pattern involving lymph nodes, bone marrow, lung, liver and brain. The chemokine CXCL12 and its receptor CXCR4 are suggested to play a role in invasion and organ-specific metastasis. Signaling pathways downstream of CXCL12:CXCR4 involved in invasion and/or metastasis are not known.  Methods: MDA-MB-231 breast cancer cells were sorted for CXCR4-positive and CXCR4-negative subpopulation by FACS and subjected to microarray gene expression analysis. Differentially expressed genes were further verified by RT-PCR or qRT-PCR. siRNA or shRNA expression studies were used to evaluate the role of genes overexpressed in CXCR4-positive cells in invasion through Matrigel. Orthotopic xenograft model is being used to evaluate primary tumor growth and metastasis pattern of CXCR4-positive and CXCR4-negative subpopulation with various manipulations.  Results: ITF2B, a class I bHLH transcription factor, is expressed at ∼4-fold higher level in CXCR4-positive cells. ITF2B mRNA level was lower in CXCR4 siRNA treated or CXCR4shRNA expressing CXCR4-positive cells suggesting that ITF2B is the direct target of CXCL12:CXCR4. ITF2B siRNA reduced invasion of CXCR4-positive cells whereas ITF2B overexpression increased invasive capacity of CXCR4-negative cells. ITF2B is a Wnt β-catenin regulated gene with transforming ability and controls the function of Inhibitor of DNA binding family of transcription factors (ID1 to ID4). ID1 is essential for lung metastasis whereas ID2 induces differentiation of breast cancer cells and higher levels of ID2 correlates with favorable prognosis in breast cancer. ID2 expression was higher in CXCR4-negative cells and its overexpression in CXCR4-positive cells reduced invasion. Both CXCR4 and ITF2B are involved in reducing ID2 expression as siRNA against both of these genes increased ID2 mRNA in CXCR4-positive cells. Interestingly, CXCR4-negative cells appear to have adapted to alternative mode of invasion/metastasis as these cells express higher levels of metastasis-associated genes such as TWIST and MT1-MMP. Xenograft studies with cells overexpressing ITF2B and ID2 are currently underway.  Conclusions: CXCL12:CXCR4 activated signaling pathways target ID family/bHLH transcription factor network to promote invasion of breast cancer cells. Inducers of ID2 may help to overcome CXCR4- mediated invasion and/or metastasis of cancer cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2045.