bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3-14-23: Affitoxin — A Potent Therapeutic Agent for Treatment of HER2- Overexpressing Tumors
Authors: Zielinski R , Lyakhov I , Hassan M , Kuban M , Shafer-Weaver K , Capala J.
Published: April 2011

Introduction: Cancers overexpressing HER2 gene are usually described as more aggressive and are associated with poor prognosis. Although introduction of trastuzumab, a HER2-specific humanized antibody, significantly improved the outcome, there are still number of patients who do not respond or acquire resistance to HER2-targeted therapy. We have previously described the construction, expression, and in vitro characterization of Affitoxin, a recombinant protein combining HER2- specific Affibody and modified Pseudomonas exotoxin A (PE 38) that blocks protein synthesis by ADP ribosylation of eEF-2 (Zielinski et al. 2009). In this report we present in vivo data.

Materials and Methods. Recombinant Affitoxins were expressed and purified from the soluble fraction of E.coli. HER2 specificity was confirmed in vitro by measurement of the residual ATP level in N87 cells treated with Affitoxin and “Non-specific” toxin. containing “off-target” instead of HER2 - Affibodies. Acute toxicity and immunogenicity studies were performed in female Balb/c mice. Affitoxin clearance rate was determined by ELISA and residual serum toxicity. Biodistribution in subcutaneous BT474 tumors model was determined by NIR optical imaging. In vivo efficacy of the drug was verified in subcutaneous (BT474, SKOV3) and intraperitoneal (SKOV3-luc-D3) tumor models.

Results. In this work we have confirmed that HER2-specific Affitoxin is a potent agent eliminating HER2-overexpressing cells in vitro at low picomolar concentration range, while non-specific toxin-containing offtarget Affitoxin is four orders of magnitude less efficient. Acute toxicity studies showed that Affitoxin is well tolerated by mice with LD50 = 0.572±0.051 mg/kg. Injection of six doses every third day neither lead to significant weigh loss of the animal nor induced severe liver problems. Pharmacokinetic data indicated fast clearance of Affitoxin from the circulation with half-life shorter than 10 min. Using human tumor xenograft models, we have shown that Affitoxin accumulates in HER2- overexpressing BT474 tumors, leading not only to reduction of growth rate but also permanent shrinkage of relatively large (<1000mm3) xenografts. Treatment with non-specific toxin did not affect the BT474 tumors. Affitoxin was also shown to delay the growth of both SKOV3 subcutaneous and intraperitoneally spread tumors.

Immunogenicity studies indicated that Affitoxin is less immunogenic than toxin containing antibody fragments fused to PE38 (HA22).

Conclusions. Affitoxin is a novel recombinant protein, using high affinity Affibody molecules to target HER2 receptors. Our in vitro and in vivo data strongly suggest that Affitoxin might be an attractive alternative or complement for existing HER2 targeted therapies

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-23.
Abstract P3-14-24: Overall Survival Benefit Observed with Lapatinib (L) Plus Paclitaxel (P) as First-Line Therapy in Patients with HER2-Overexpressing Metastatic Breast Cancer (MBC)
Authors: Guan Z-z , Xu B-h , Arpornwirat W , Tong Z-s , Lorvidhaya V , Wang L , Newstat B , DeSilvio M , Moore Y , Shen Z-Z.
Published: April 2011

Background: Lapatinib (L) is a potent, oral selective, reversible, inhibitor of EGFR (ErbB1) and HER2 (ErbB2) receptor tyrosine kinases. EGF104535 is a phase III, international, randomized, double-blind study comparing lapatinib plus paclitaxel (L+P) versus P alone as 1st linetreatment for pts with HER2-positive+ (MBC). The study was focused in the Asia Pacific region.

Methods: Pts with centrally confirmed HER2-positive (by FISH) MBC and measurable disease were randomized to L 1500 mg daily + P 80mg/m2/wk 3 out of 4 wk or to P 80mg/m2/wk 3 out of 4 wk alone. Pts were administered P for 6 cycles with additional cycles at investigator discretion, and continued on blinded randomized therapy until disease progression (PD). At time of PD, there was an optional monotherapy extension to allow pts on P alone arm to receive L monotherapy. The primary endpoint of the study was overall survival (OS); secondary endpoints included progressionfree survival (PFS), overall response rate (ORR), clinical benefit rate (CBR) and safety. Analyses were conducted on the intent-to-treat population.

Results: 444 pts (N=222/arm) across 43 centers were randomized: 67% of P pts received L monotherapy. Baseline demographics were well-balanced between treatment groups. OS was significantly longer in the combination therapy (L+P) as compared with P alone. [Cox Regression Analysis: Treatment Hazard Ratio (HR) (95% CI)=0.64(0.49,0.82), p=0.0005, Kaplan-Meier estimates: median OS 27.8 mos (95% CI=23.2,32.2) vs 20.5 mos (95% CI=17.9,24.3), respectively]. Median PFS increased to 9.7 mos (95% CI=9.2, 11.1) compared with 6.5 mos (95% CI=5.5, 7.3), in the L+P vs P, respectively [HR (95%CI)=0.52(0.42,0.64), stratified log rank P<0.0001]. ORR for L+P was 69% (95% CI=63%,75%) compared with 50% (95% CI=43%,56%) for P alone [Odds Ratio=2.3 (1.54, 3.47), P<0.0001]. CBR improved with L+P (75%, 95% CI=69%,80%) compared with P alone (56%, 95% CI=49%,63%) [Odds Ratio=2.34 (1.54, 3.58), P<0.0001]. The most frequent adverse events (AEs) were diarrhea (L+P, 77%, Grade 3/4 20/0; P, 29% Grade 3/4, <1/<1) and neutropenia (L+P, 77% Grade 3/4, 35/16; P, 47%, Grade 3/4, 15/5). The incidence of withdrawal from treatment due to AEs (13% vs 10%) was a similar. Conclusion: The combination of L+P showed a statistically and clinically meaningful survival advantage. Secondary endpoints support the observed clinical benefit. The combination of L+P was well tolerated.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-24.
Abstract P3-15-02: Underreporting of Myelotoxicity with Emerging Breast Cancer Regimens
Authors: Vandenberg T , Verma S , Loibl S , Crawford J , Choi MR , Dreiling L , Chan A.
Published: April 2011

Background: Although many emerging regimens that incorporate targeted agents may produce increased myelosuppressive side-effects, clinical data guiding colony-stimulating factor (CSF) use with these regimens are not readily available. This review assessed the reporting diligence around febrile neutropenia and the related use of CSF and antibiotics in published clinical trials evaluating emerging regimens for breast cancer treatment.

Methods: We searched Medline, EMBASE, and Cochrane databases to identify randomized, controlled phase 3 breast cancer studies published between Jan 2005 and June 2009. After excluding trials of vaccines, hormonal therapy alone, and stem cell transplantation/mobilization, corresponding publications were retrieved and data extracted on the incidence of neutropenia and its complications and CSF/antibiotic use. We then calculated the percentage of these publications that reported each outcome.

Results: Of 463 trials identified from the search, only 73 met the inclusion criteria. Overall, 70% and 55% of trials reported the incidence of grade 3/4 neutropenia and febrile neutropenia, respectively (Table). Neutropenia-related hospitalizations were reported in 3% of trials. Prophylactic use of CSF and antibiotics was defined in the methods section of 59% and 23% of trials, respectively; and only reported in the results section of 22% and 5% of trials, respectively.

Conclusion: Clinically significant neutropenia and neutropenia-related events (including febrile neutropenia) were generally described in the studies evaluated; however, the reported use of CSF and/or antibiotics was infrequent and inconsistent in the published literature of emerging regimens. A standardized approach to reporting neutropenic outcomes as well as the use of supportive care measures can assist cliniciansto prospectively manage the relevant toxicities associated with these emerging regimens and thereby facilitate their safe and effective use in clinical practice.

Table: Reporting of myelotoxicity data in published phase 3 trials

CSF = colony-stimulating factor

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-15-02.
Abstract P3-15-09: Symptomatic Congestive Heart Failure after Neo/Adjuvant Dose-Dense Epirubicin: A Retrospective Analysis of Two Trials with Long-Term Follow-Up
Authors: Gajria D , Pokuri V , Steingart R , Hudis C , Dang C.
Published: April 2011

Background:

Dose-dense (dd) anthracycline-taxane (A/T) based regimens (Rxs) have led to incremental benefits in breast cancer outcomes. Long-term incidence of symptomatic cardiac events for A/T Rxs (conventional and dd) has been ≥2.5%. The risk of long-term cardiac dysfunction related to total anthracycline dose and schedule remains a clinical concern. Additionally, the significance of asymptomatic short-term changes in left ventricular ejection fraction (LVEF) on the development of clinically significant congestive heart failure (CHF) is uncertain. Here we examine the incidence of symptomatic CHF, as per the New York Heart Association (NYHA) functional classification, for 82 patients (pts) exposed to neo/adjuvant dd epirubicin.

Methods:

We conducted a retrospective review of pts treated on two clinical trials: trial A-pts received fluorouracil, epirubicin, and cyclophosphamide (FEC) (500/100/500 mg/m2 every 2 weeks x 6) followed by weekly paclitaxel (80 mg/m2) alternating with docetaxel (35 mg/m2) x18; trial B-pts received EC (100/600 mg/m2 every 2 weeks x 6) followed by paclitaxel (175 mg/m2 every 2 weeks x 6). Total E dose was 600 mg/m2 for each trial. Pts with a known history of unstable angina, myocardial infarction, and/or CHF were excluded. All pts received a baseline LVEF evaluation, and in trial B pts received a repeat evaluation within 3 months after Rx completion.

Results:

Trial A enrolled 44 pts with a median age of 46 years (range, 26-63), 9 pts with pre-existing diagnosis of hypertension (HTN), and a median baseline LVEF of 66% (55-80%); 16/44 (36%) pts received left-sided radiation. At a median follow-up of 83 months, one patient (2.2%) developed NYHA class III CHF; 12 months after Rx completion her repeat LVEF was stable and 75 months after completion, an echocardiogram showed an LVEF of 21%.

Trial B enrolled 38 pts with a median age of 47 years (30-72), 5 pts with pre-existing HTN, and a median baseline LVEF of 69% (57-85%); 18/38 (47%) pts received left-sided radiation. Within 3 months of Rx completion, 28/38 (74%) pts had a repeat LVEF assessment with a median LVEF of 65% (40-76%). At a median follow-up of 56 months, one patient (2.6%) developed anthracycline-induced symptomatic class II CHF three months after Rx completion (baseline LVEF 61%, 3 months LVEF 42%). One patient (2.6%) suffered class III CHF one week after completing Rx with LVEF of 40%; this was in the setting of cocaine use and a repeat LVEF evaluation one month after the event showed a return to baseline LVEF. Overall, 2/82 (2.4%) pts had NYHA class III CHF.

Conclusion:

With long term follow-up our two studies demonstrate an acceptable 2.4% incidence of NYHA class III CHF after dose-dense epirubicin therapy. These findings are consistent with those of larger randomized trials evaluating the anthracyclines in breast cancer treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-15-09.
Abstract P4-01-03: The Homeobox Protein H0XC10 Is Overexpressed in Breast Cancer and Confers Resistance to Chemotherapy
Authors: Sadik H , Shah N , Gupta RA , Chang HY , Sukumar S.
Published: April 2011

Background: Breast cancer is the second leading cause of cancer deaths in women worldwide. Although chemotherapy is effective, resistance to drugs develops over time and can account for treatment failure in over 90% of metastatic breast cancer patients. HOX genes are homeobox-containing transcription factors well-known for their role in morphogenesis. However, accumulating evidence has emphasized their importance during carcinogenesis and metastasis. The goal of this study is to understand the role of HOXC10 in breast cancer and the consequence of its overexpression in conferring chemotherapy resistance.

Methods: We conducted a tiling array of all four HOX clusters to identify dysregulated HOX genes in a panel of primary and metastatic breast cancer tissues, and validated the overexpression of HOXC10 in metastatic breast cancer. Next we established cell lines that stably overexpress HOXC10 and others where endogenously overexpressed HOXC10 was silenced by shRNAs, and determined phenotypic and biochemical changes resulting from these manipulations.

Results: HOXC10 is overexpressed in 67% of primary breast tumors (n=31), in 82% of the metastatic tissues (n=49) and in most breast cancer cell lines. When compared to cancers at other sites, overexpression tends to be significantly higher in breast cancer, and correlates with increasing grade and tumor size. Cell survival assays (MTT and colony formation) after drug treatment show that overexpression of exogenous HOXC10 in MCF10A conferred drug resistance. Further molecular examination revealed that overexpression of HOXC10 led to a dysregulation in the Rb-E2F pathway, and therefore the G1/S checkpoint was affected. In addition, the tendency for the formation of 8N cells and the concomitant protection from apoptosis suggested that HOXC10 overexpression might lead to genomic endoreduplication and instability. This fact along with the strikingenhanced recovery of the cells from pactitaxel treatment (as compared to the recovery from any other chemotherapeutic drug) and the protection from mitotic catastrophe implied a role of HOXC10 in the mitotic checkpoint. This was confirmed by a dysregulation of the expression of the genes involved in this checkpoint. All these experiments were further validated by stably expressing a shRNA to HOXC10 in SUM159 breast cancer cells with high endogenous HOXC10 levels. Conclusion: Our studies show for the first time that HOXC10, a homeobox protein previously shown to be regulated during the cell cycle and to have a positive effect on proliferation, is overexpressed in the majority of breast cancers. This upregulation may have some clinical implications since cells with higher expression of HOXC10 tend to have more invasive properties, more genomic instability, and are more resistant to some chemotherapy drugs.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-03.
Abstract P4-01-04: The Impact of the Retinoblastoma Tumor Suppressor Pathway in Her2 Positive Breast Cancer Pathogenesis and Therapeutic Response
Authors: Rivadeneira DB , Ertel A , Witkiewicz AK , Mercier I , Lisanti MP , Knudsen E.
Published: April 2011

Extensive studies have shown that breast cancer is associated with molecular aberrations that underlie the distinct subtypes of disease that exhibit differential prognosis and susceptibility to targeted therapeutic. A critical oncogenic event used to classify breast cancers is the amplification or overexpression of the human epidermal growth factor receptor 2 (Her2). Tumors harboring Her2 overexpression present aggressive characteristics related to metastatic spread and poor overall disease outcome. However, such tumors are also susceptible to targeted therapeutic agents which effectively impinge on this oncogene. Strikingly, those events that cooperate with Her2 and modulate susceptibility to targeted therapeutics remain poorly understood. Here the significance of the retinoblastoma tumor suppressor (RB) pathway in the context of Her2 positive breast cancer pathogenesis and therapeutic response was evaluated. In silico and histological analyses suggested that a subset of Her2 positive tumors exhibit compromised RB function. To model interactions between Her2 and the RB-pathway genetic deletion and knockdown strategies were employed in the context of Her2 driven tumors. These studies revealed that RB loss accelerated disease progression of such models and further enhanced the aggressive tumor phenotype.

To delineate the functionality of the RB-pathway downstream from therapeutic interventions targeting Her2, two complimentary approaches were used. First, the ability to effectively intercede in Her2-positive tumors using drugs that specifically activate the RB-pathway was interrogated. The data revealed that specific CDK4/6 targeted therapies were highly effective at limiting the growth of Her2-positive tumor cells. Second, the impact of RB status on the response the Her2/EGFR antagonist lapatinib was evaluated. These studies demonstrated that loss of RB compromises the effectiveness of lapatinib in the treatment of Her2 positive breast cancer cell lines. Ongoing studies are evaluating the spontaneous mechanisms of resistance to Her2 targeted therapeutics and the significance of reestablishing RB function as a therapeutic alternative in such cancers. Together, these studies underscore the importance of the interplay between Her2 signaling and cell cycle control in breast cancer, and how this crosstalk influences the response to targeted therapeutics in the treatment of breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-04.
Abstract P4-01-06: The Role of the PI3K/AKT, RAS/MAPK and Novel Signaling Pathways in Resistance to HER2-Targeted Therapy
Authors: O'Brien NA , Wilcox K , McDermott M , Crown J , O'Donovan N , Slamon DJ.
Published: April 2011

Despite the proven clinical benefits of HER2-targeted therapy, resistance to trastuzumab and lapatinib exists. Understanding the molecular mechanisms responsible for resistance is critical to improving the survival rates of patients with this aggressive subtype of breast cancer. Currently, there are no clinically validated biomarkers of resistance to therapy. To address this issue, we measured the responses of a panel of 18 HER2-amplified breast cancer cell lines to both trastuzumab and lapatinib and evaluated the activity of PI3K/AKT/mTOR and RAS/MAPK/ERK signaling in resistance. Eight of the cell lines were identified as resistant to trastuzumab and seven as resistant to lapatinib by both two and three dimensional response assays. Five of the trastuzumab resistant cell lines remained sensitive to lapatinib (IC50 < 1 μM), indicating that these two agents have at least partially non-overlapping mechanisms of resistance. The combination of low PTEN or the presence of activating mutations of PI3K, and thus increased activity of PI3K/AKT signaling, is a strong predictor of resistance to trastuzumab. However, this is not the case with lapatinib, two of the cell lines with activating mutations of PI3K (MDA-361 and HCC-202) were resistant to lapatinib whereas two other PI3K mutant cells lines (SUM-190 and HCC-1954) were lapatinib sensitive, indicating that lapatinib resistance can occurindependently of increased PI3K/AKT signaling. To further investigate these mechanisms, we measured the effects of trastuzumab and lapatinib treatment on AKT/mTOR and ERK activation. Cell lines sensitive to trastuzumab or lapatinib showed a marked reduction in pAKT, pS6K and pERK in response to either agent, whereas the resistant cells maintained high pAKT, pS6K and pERK levels in the presence of trastuzumab and lapatinib. In cell lines resistant to trastuzumab yet sensitive to lapatinib, treatment with lapatinib significantly reduced the phosphorylation of AKT, S6K and ERK. Thus in a subpopulation of cell lines lapatinib can overcome trastuzumab resistance via continued deactivation of PI3K/AKT/mTOR and MAPK/ERK signaling. However, there still remains a subgroup of HER2-positive cells that are resistant to both trastuzumab and lapatinib. We used global expression analysis by both mRNA microarray and proteomic screening to identify a set of genes and proteins that are consistently altered in these innately resistant cells relative to the sensitive cell lines. In vitro validation of these targets will reveal which are playing a functional role in resistance and are thus potentially druggable targets to be used in improving the survival of patients with HER2-positive disease.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-06.
Abstract P4-01-07: Endoplasmic Reticulum-Associated Calcium Regulates Resistance of the Anti-Apoptotic Protein Bcl-2 to Taxol-Induced Apoptosis in Breast Cancer Cells
Authors: Pan Z , Gollahon L
Published: April 2011

Background: The unique anti-apoptotic function of Bcl-2 results in cellular resistance to many chemotherapeutic agents used in solid tumor treatments. However, there is a controversy regarding the relationship between Bcl-2 resistance and the mechanism of action for Taxol, an anticancer agent commonly used in breast cancer treatment. Although previous studies showed both Bcl-2 and Taxol may have effects on calcium homeostasis independently, no studies have addressed whether calcium is the keystone in the relationship between Bcl-2 resistance and Taxol action. Objective: Since most internal calcium is stored in the endoplasmic reticulum (ER), this study focused on the role of ER calcium changes in Bcl-2-activated resistance to Taxol-induced apoptosis. The ER calcium channel IP3R and its relationship with Bcl-2 and Taxol were investigated in order to clarify the mechanism underlying calcium regulation.

Methods and Materials: For this study, MDA-MB-468, a Bcl-2 negative breast carcinoma cell line, its Bcl-2 stable transfectant and MCF-7, a Bcl-2 positive breast carcinoma cell line were used. The dynamic calcium changes induced by Taxol in these cells were determined by measuring free cytosolic calcium fluctuations using Fluo4-AM and ER calcium changes by the calcium cameleon, D1ER. Taxol-induced apoptosis was measured using the Annexin V-FITC assay. The calcium interfering agents BAPTA-AM and 2-APB were applied to evaluate whether Taxol-induced calcium changes were related to this apoptotic event. Western blot was used to detect changes induced by Taxol treatment in Bcl-2 and IP3R protein levels.

Results: Taxol induced a rapid ER calcium release followed by a gradual ER calcium depletion in both Bcl-2 positive and negative cell lines, indicating that ER calcium store provides a direct target for Taxol, independently of Bcl-2 expression. Interfering with these calcium changes inhibited the Taxol-induced apoptosis, suggesting ER calcium release promotes Taxol-induced apoptosis. Our results also showed that Bcl-2 inhibited the basal ER calcium release. Taxol stimulated ER calcium release and thus attenuated Bcl-2-initiated resistance to apoptosis. Relevance: This research addressed the question of whether ER calcium changes play a critical or marginal role in mediating resistance of Bcl-2 to Taxol-induced apoptosis. Elucidating the role of calcium in this process will not only help to clarify the mechanism of drug resistance associated with Bcl-2, but also aid in more effective application of Taxol in breast cancer treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-07.
Abstract P4-02-03: Gene Copy Number Alterations Related to mRNA and miRNA Expression in Endocrine Resistant Breast Cancers
Authors: Larionov A , Reis-Filho JS , Lambros MBK , Sproul D , Dixon JM.
Published: April 2011

Background

Endocrine resistance is a major clinical issue in breast cancer. Multiple studies provide data relating molecular features of primary tumours on their response to endocrine treatment. However, there is a paucity of data on molecular profiles of breast tumours at time when endocrine resistance has already manifested.

Aim

Provide multilayer molecular profiling of a series of breast tumours at time of relapse or progression on endocrine treatment.

Results

High density microarray-comparative genomic hybridisation (aCGH) analysis was performed in 39 endocrine treated breast tumours at time of progression or relapse. 25 of these tumours were also profiled using whole genome mRNA and miRNA arrays (HT-12 mRNA array and DASL Sentrix Array Matrix micro-RNA array, Illumina).

Overall the aCGH profiles of resistant tumours were similar to aCGH profiles of un-selected luminal breast tumours and cell lines published earlier. The most common gene copy gain areas (>10M of length) were located in 1q23.1-44, 5p13.1-33, 8p11.23-q24.3, 16p12.1-13.3, 17q21.33-24.1 and 20q13.2-13.33, while the most common losses were in 1p33-36.32, 8p12-23.3, 11q22.3-24.1, 16q12.2-24.3 and 17p11.2-13.3. Unsupervised hierarchical clustering by gene copy number profiles split of the resistant tumours into 3 groups. As expected, on average mRNAs and microRNAs were higher expressed in tumours carrying gains of their genes and were decreased in tumours carrying the losses. Copy number gains in this series were associated with the upregulation of multiple RNAs, including C8orf76, TATDN1, CYC1, ZBTB10, C16orf63, THUMPD1, TFB2M, miR-454, miR-301a and miR-296-3p/5p. Losses were associated with the downregulation of RNAs including ACADVL, BNIP3L, UBE4A, REXO2, ATP5L, PPP2CB, NBL1 and PCM1. Of the above genes UBE4A and ATP5L are closely co-localised in 11q23.3; miR454-miR301a are located closely in 17q22. Such associations suggest that changes in expression of these genes in breast cancers may be driven by alterations in the gene copy number. Analysis of the recurrent amplification regions on this dataset is underway.

Conclusions

aCGH profiles of endocrine resistant tumours at time of relapse or progression are similar to those found in un-selected and non-treated luminal tumours. The study provides the first dataset integrating gene copy number with mRNA and microRNA gene expressions in clinical specimens of endocrine resistant breast cancers.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-03.
Abstract P4-02-06: Beyond CYP2D6 Genotyping: Personalized Dosing of Tamoxifen (Tam) Based on Low Serum Endoxifen Levels
Authors: Barginear MF , Jaremko M , Yu C , Peter I , Kasai Y , Kemeny M , Adelson K , Desnick R , Raptis G.
Published: April 2011

Background: The efficacy of Tam varies widely. Tam is biotransformed to endoxifen and 4-OH-Tam by the cytochrome P450 2D6 enzymes. Studies on 2D6 variants as a predictor of Tam's efficacy show conflicting results. Compared to Tam, endoxifen and 4-OH-Tam have a 100-fold greater affinity for the estrogen receptor (ER) and in suppressing estrogen-dependent cell proliferation. However, endoxifen is present at concentrations up to 20-fold higher and is pharmacologically distinct from 4-OH-Tam. Endoxifen's effect on ERα-degredation and transcription is concentration dependent; serum endoxifen levels ≥40nM block ERα-mediated transcriptional activation and inhibit estrogen-induced breast cancer cell proliferation.

Methods: Pts taking Tam 20 mg/day, for any reason, were potentially eligible. Serum Tam, Tam metabolites and nineteen 2D6 alleles were analyzed. 2D6 genotyping was performed by PCR-based procedures and metabolites were assessed on Liquid Chromatography/Tandem Mass Spectrometry. Pts were stratified by 2D6 function into 3 groups: extensive metabolizers (EM) who had normal enzyme activity; intermediate metabolizers (IM) who had reduced enzyme activity; and poor metabolizers (PM) who had no enzyme activity. Tam was increased to 30 mg/day if serum endoxifen < 40nMol. Serum Tam and Tam metabolites were measured every 2 weeks for 90 days.

Results: 121 patients have enrolled to date. Age, ethnicity, BMI, concomitant medications, and adherence were recorded. 25/117 (21%) pts had low serum endoxifen; all of these pts were increased to Tam 30 mg/day. Of this cohort, 36% were PMs (PM/PM and PM/IM), 32% were IMs (EM/PM and IM/IM) and 32% were EMs (EM/EM and EM/IM). All patients’ serum endoxifen increased to >40nMol by day 30 (Diagram below).

Conclusions: 1) Sub-therapeutic serum endoxifen can be found in any 2D6 phenotype. 2) It is possible to achieve therapeutic serum endoxifen/Tam metabolite levels by optimizing the dose of Tam. 3) Subsequently, it is likely that other gene variants are involved in Tam metabolism hence why 2D6 testing may lead to conflicting results. 4). Further investigation is warranted as personalizing the dose of Tam is feasible and may potentially improve the outcome of pts taking Tam regardless of 2D6 genotype. Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-06.
Abstract P4-04-01: Proliferation, Migration, Stem Cell Activity and the Role of Cyclin D1 in Breast Cancer
Authors: Lamb RM , Lehn S , Tobin N , Berglund P , Landberg G.
Published: April 2011

Background: Proliferation, migration, and stem cell (SC) activity have all been implicated in the development and progression of breast cancer. Growing evidence suggests a relationship between these cellular characteristics, which are tightly regulated by complex signalling pathways. The oncogene cyclin D1 is an essential cell cycle protein that regulates G1 to S phase transition and is overexpressed in many breast cancers. Despite the proliferation activating properties of cyclin D1 it has been linked to a less malignant phenotype.

Aim: The aim is to investigate the relationship between migration, proliferation and SC activity in breast cancer and to determine the molecular mechanisms that control these processes focusing on cyclin D1. Methods and Results: To establish a link between proliferation and migration two breast cancer cell lines (MDAMB231 and MDAMB468) were quiesced and proliferation assessed by Ki67 and migration by transwell migration assays. G0/G1 cells displayed an increased migratory capacity (2.9 ±0.15 fold) compared to cycling cells. Next we downregulated cyclin D1 in actively cycling cells using siRNA which resulted in significantly decreased proliferation and the novel observation of increased migration (1.7±0. 19 fold). Having established a link between proliferation and migration the next aim was to investigate relationships with SC activity. To achieve this, cells were sorted following Hoechst 33342 and 7AAD labelling into three populations G0/G1, S, and G2/M. SC-like activity was assessed using a non-adherent mammosphere culture system where single cells are seeded at a density of 500 cells per cm2 for cultured for 7 days. The data clearly demonstrated that G0/G1 cells had increased migratory capacity and also SC-like activity (1.5±0.2 fold) compared to actively cycling cells. Interestingly, down-regulation of cyclin D1 increased both migratory and SC-like activity with most significant effects observed in G0/G1 and S phase of the cell cycle indicating that cyclin D1 is a key protein in the regulation of these cellular processes. When downregulating the cyclin D1 associated kinases CDK4/6 no effect on migration or SC activity was observed suggesting that cyclin D1 affects these cellular processes independent of CDK activation. Finally, when analysing a large set of premenopausal breast cancers we observed an inverse, proliferation-independent link between cyclin D1 and cancer recurrence as well as tumour size (p=0.03), validating that the protein might abrogate infiltrative and malignant behaviour in vivo.

Conclusion: These novel functions of cyclin D1 illustrates the interplay between tumor proliferation and migration and SC activity and may explain the lessened malignant behaviour in cyclin D1 high breast cancers.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-01.
Abstract P4-04-03: Transcriptome Analyses of Mouse and Human Mammary Cell Subpopulations Reveals Multiple Conserved Genes and Pathways
Authors: Lim E , Wu D , Pal B , Bouras T , Asselin-Labat M-L , Vaillant F , Yagita H , Lindeman GJ , Smyth GK , Visvader JE.
Published: April 2011

INTRODUCTION: Molecular characterization of the normal epithelial cell types that reside in the mammary gland is an important step toward understanding pathways that regulate self-renewal, lineage commitment, and differentiation along the hierarchy. We previously reported distinct stages in human mammary epithelial cell development and linked them to breast tumor subtypes previously defined by gene expression profiling. Here we determined the gene expression signatures of four distinct subpopulations isolated from the mouse mammary gland. The epithelial cell signatures were used to interrogate mouse models of mammary tumorigenesis and to compare with their normal human counterpart subsets to identify conserved genes and networks.

METHODS: RNA was prepared from freshly sorted mouse mammary cell subpopulations (mammary stem cell (MaSC)-enriched, committed luminal progenitor, mature luminal and stromal cell) and used for gene expression profiling analysis on the Illumina platform. Gene signatures were derived and compared with those previously reported for the analogous normal human mammary cell subpopulations. The mouse and human epithelial subset signatures were then subjected to Ingenuity Pathway Analysis (IPA) to identify conserved pathways.

RESULTS: The four mouse mammary cell subpopulations exhibited distinct gene signatures. Comparison of these signatures with the molecular profiles of different mouse models of mammary tumorigenesis revealed that tumors arising in MMTV-Wnt-1 and p53-/- mice were enriched for MaSC-subset genes, whereas the gene profiles of MMTV-Neu and MMTV-PyMT tumors were most concordant with the luminal progenitor cell signature. Comparison of the mouse mammary epithelial cell signatures with their human counterparts revealed substantial conservation of genes, whereas IPA highlighted a number of conserved pathways in the three epithelial subsets.

CONCLUSIONS: The conservation of genes and pathways across species further validates the use of the mouse as a model to study mammary gland development and highlights pathways that are likely to govern cell-fate decisions and differentiation. It is noteworthy that many of the conserved genes in the MaSC population have been considered as epithelialmesenchymal transition (EMT) signature genes. Therefore, the expression of these genes in tumor cells may reflect basal epithelial cell characteristics and not necessarily cells that have undergone an EMT. Comparative analyses of normal mouse epithelial subsets with murine tumor models have implicated distinct cell types in contributing to tumorigenesis in the different models.

Figures available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-03.
Abstract P4-04-05: Histone Deacetylase Inhibitors Increase Markers of the Dedifferentiated Cancer Stem Cell Phenotype in Human Breast Cancer Cells
Authors: Debeb BG , Xu W , Lacerda L , Hortobagyi GN , Robertson F , Ueno NN , Lucci A , Reuben JM , Buchholz TA , Woodward WA.
Published: April 2011

It has been suggested that differentiated cancer cells can de-differentiate into the cancer stem cell phenotype (Meyers et al., 2009; Gupta et al., 2009) and that Histone Deacetylase (HDAC) inhibitors enable efficient induction of pluripotent stem cells from adult fibroblasts (Huangfu et al., 2009). Moreover, we have recently demonstrated that the HDAC inhibitor valproic acid radiosensitized breast cancer cells grown on plastic with serum while it radioprotected breast cancer cells grown in stem cell promoting culture conditions (Debeb et al., 2010). Based on these data, we hypothesized that HDAC inhibitors increase the cancer stem cell population via dedifferentiation of differentiated cancer cells. To examine our hypothesis, Sum159 breast cancer cells were FACS-sorted based on ALDH activity and subsequently treated with one of two HDAC inhibitors, valproic acid or SAHA (suberoylanilide hydroxamic acid). After sorting, ALDH-negative cells were treated either with valproic acid, SAHA, or vehicle. After a week, the percentage of ALDH-positive cells (passage 0, P0) was examined with flow cytometry while the remaining cells were passaged and incubated with and without valproic acid or SAHA for a week and the percentage of ALDH-positive cells again evaluated (P1). This was repeated for the third time (P2). Samples from each generation were also collected to examine the protein expression. On average, a 3-fold increase in ALDH positive cells was seen in valproic acid-treated cells (35.6% vs.12.6%) and a 1.5-fold increase in SAHA-treated cells (41% vs. 28%) compared to vehicle-treated controls. This effect was maintained through multiple passages. Moreover, the expression of Beta-catenin and EMT associated genes like vimentin, fibronectin, n-cadherin, which have been implicated in generating cancer stem cells was significantly increased withtreatment in initial and passaged cells. Further functional endpoint studies are needed to validate these in vitro marker-based findings.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-05.
Abstract P4-04-06: Histone H2A Inhibits Breast Cancer Stem Cell Growth
Authors: Tourville Poirier AM , Balicki D. CRCHUM
Published: April 2011

Introduction: Recent studies have identified epigenetic abnormalities as the underlying cause of many breast cancers. In particular, the aberrant acetylation of core histones may be a pivotal source of dysregulated expression of both oncogenes and tumour suppressor genes. Histone deacetylase inhibitors are known to restore histone acetylation and to induce cellular differentiation and growth arrest in breast cancer cells and are the object of extensive research for novel anticancer treatments. We hypothesize that acetylated proteins, in particular histone H2A, could also serve as an alternative substrate for histone deacetylase, thereby promoting cellular histone acetylation and arrest of cancer cell growth. Indeed, our previous results indicate that exogenous calf thymus histone H2A penetrates cells and their nuclei, and induces differentiation and senescence in human breast cancer cells. In contrast, human recombinant histone H2A and H2AX, which lack post-translational modifications, fail to yield these results in spite of the fact that human and bovine histone H2A are virtually identical in their amino acid sequences. However, these recombinant molecules were obtained from bacteria, which lack the intracellular machinery that is necessary to generate post-translational modifications. Thus, post-translational histone modifications are pivotal to the inhibition of breast cancer cell proliferation. Objective: We have extended our study to establish if exogenous calf thymus histone H2A could also induce growth arrest of breast cancer stem cells, and subsequently to determine which portion (s) of the histone H2A molecule is responsible for this action, using overlapping synthetic peptides.

Materials and Methods: Breast cancer stem cells were incubated with exogenous calf thymus histone H2A and seven synthetic peptides corresponding to overlapping portions of the entire protein and include all of its post-translational modifications. Cellular morphology was observed and viable cells were counted using trypan blue exclusion. Control experiments were performed with normal breast cells, cells from fibrocystic breast tissues, and breast cancer cells that were not significantly enriched in cancer stem cells.

Results and Discussion: Histone H2A inhibits breast cancer stem cell growth and increases their adhesion. Stem cells have recently been identified in many malignant tumours, including breast cancer. Since cancer stem cells are thought to be an important source of therapeutic resistance, they provide a novel research focus for alternative treatment strategies. Our results are very encouraging and may lead to the development of new pharmacological molecules targeting epigenetic abnormalities in breast cancer stem cells.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-06.
Abstract P4-05-01: Molecular Interactions between Human Adipocytes and Breast Cancer Cells May Underline Increased Breast Cancer Incidence in Obese Postmenopausal Women
Authors: Siriwardhana N , Moustaid-Mossa N , Wimalasena J.
Published: April 2011

There is an increasing body of evidence that obesity, metabolic syndrome, insulin resistance, and diabetes may substantially increase the risk and modify the outcome of a variety of cancers including postmenopausal breast cancer (BC). In has been proposed that stromal adipose tissue is activated in these metabolic diseases and that the activated stroma regulates function of cancer cells. Much of the stroma in the breast is made of adipocytes (AD) and AD and resident immune cells, such as macrophages, secrete cytokines/chemokines which have direct regulatory actions on BC cells. The latter may, in turn, regulate the behavior of AD. We have initiated studies where cultured AD from obese individuals is co-cultured with BC cells (such as MCF-7 and MDA MB 231 cells) in a 3 dimensional (3D) Matrigel co-culture system or non tumorigenic MCF10A cells.We quantitatively measured the changes in 26pro-inflammatory/anti-inflammatory cytokines(using Millipore multiplex ELISA) secreted into culture media. Of the 12 differentially regulated cytokines the expression levels of 6 cytokines (Eotaxin, G-CSF, and IL1α. IL-6, IL-8 and MCP-1) were significantly different between treatments. Secretion of Eotaxin, G-CSF, IL-6 and MCP-1) cytokines from obese adipocytes were significantly higher than the lean adipocyte and the opposite was recorded for IL-8. Further, the secretion of G-CSF, IL-6 and MCP-1 was significantly different in the order of MCF10A <MCF7 < MDA MB 231. In the co-cultures with adipocytes IL-6, IL-8 and MCP-1 showed different secretion levels when lean or obese adipocytes were cocultured with BC cells(e.g. the IL-6 secretion was increased in the order of MCF10A <MCF7 < MDA MB 231 in lean cells but in obese cells the order was changed to MDA MB <MCF10A < MCF7). Further, we measured the differences in 3D acini sizes in the 3D co-cultures, MCF7 cell formed irregular and growth unrestricted structures and when compared to the lean adipocytes diameter measured was 17% higher in the presence of obese adipocytes. The same pattern was observed when monolayer culture media from obese adipocytes were transferred into growing MCF7 cells and growth measured by MTT assay. These data suggest that adipocytes from lean and obese women regulate BC functions differently and such differences may underlie the promotional effect of obesity in BC. Further studies are underway to dissect the molecular mechanisms by which differentially regulated cytokines influence the behavior of BC cells.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-01.
Abstract P4-05-03: Quantification of an Individual's Response to Drug Therapy Utilising a Novel Three-Dimensional Assay with Primary Breast Cancer Explants
Authors: Leeper AD , Thomas JSJ , Dixon JM , Harrison DJ , Katz E.
Published: April 2011

Background: Breast cancer is not a single disease but one that displays both inter-and intra-tumoral heterogeneity. The principal models for breast cancer research are currently immortalised breast cancer cell lines as they are easily propagated, tractable to genetic modification and yield reproducible results However, it is not possible to use a cell line to predict the disease course of an individual patient. Moreover, epithelial cells grown on plastic in two dimensional cultures fail to recapitulate their three dimensional interaction with the tumour microenvironment, which is believed to be integral to tumour progression.

Purpose: Using a collagen based three dimensional assay system and fresh breast cancer biopsies obtained at surgical resection, we have developed a novel assay which demonstrates ongoing proliferation and invasion of an individual's breast cancer. The proliferation and invasion of the breast cancer can be modified using currently available anti-cancer drugs. The effect of these drugs on an individual patient's cancer may then be quantified.

Results: The model is amenable to investigation both in real-time and following formalin fixation. Using live microscopy and time delay image capture techniques we are able to study proliferation and invasion over a timecourse. Subsequent formalin fixation and sectioning enables two dimensional immunofluorescence quantification of drug effect at a cellular level. Three dimensional scanning and quantification, utilising Optical Projected Tomography and Velocity software enables one to demonstrate the effect of a drug on invasion at a macroscopic level. Conclusions: This assay has provided a method of sustaining an individual's primary breast tissue in long-term culture. Moreover, it provides a method of observing different patterns of invasion. Through the addition of currently available and novel therapeutics we are able to quantify how residual disease may respond to a battery of different treatments. In turn this may improve the ability of the clinician to decide on optimal adjuvant therapy, resulting in individually tailored therapy.

Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-03.
Abstract P4-06-04: Rho-Family GTPase Activity in Invading Mammary Tumor Cells Visualized by Two-Photon Excitation Laser Scanning Microscopy
Authors: Kamioka Y , Matsuda M
Published: April 2011

Background: Rho-family GTPases play critical roles in the regulation of tumor cell motility and invasion. Despite the vast accumulated knowledge of Rho-family GTPase regulation in two-dimensional culture models, their three-dimensional spatio-temporal regulation in vivo remains poorly understood because of technical difficulties. The ability of tumor cells to migrate in vivo depends on both physical and biochemical characteristics of the tumor microenvironment, i.e., extracellular matrix, stromal cells and myeloid cells as well as tumor cell properties. Therefore, development of methods for directly measuring the activity of Rho-family GTPases in vivo is required for further understanding the mechanism of tumor invasion. Material and Methods: To visualize the activity of Rho-family GTPases in vivo, we developed fluorescence resonance energy transfer (FRET)- based probes comprising of fluorescent proteins. The probes were stably expressed in 4T1 mouse mammary tumor cells by means of retrovirus-mediated gene transfer. 4T1 mouse mammary tumor cells highly expressing FRET probes were selected by fluorescence activated cell sorting (FACS) and implanted subcutaneously in nude mice. The 4T1 cells formed tumors within 5 to 10 days. The activity of Rho-family GTPases was examined by two-photon excitation laser scanning microscopy (TPLSM). The collagen matrix surrounding tumors was visualized by second harmonic signal. TexasRed-dextran was injected into nude mice intravenously before observation for the visualization of blood vessels. Results: We found that 4T1 mouse mammary tumor cells assumed an elongated, mesenchymal phenotype in vivo and in three-dimensional culture models. Cell motility in vivo was significantly slower than in 2D cell culture models. In the in vivo models, the elongated tumor cells demonstrated cell movement along aligned collagen fibers away from the primary tumor mass. Furthermore, we observed that Rac1 activity was higher at the tumor-stroma borders than at the tumor cores, using FRET imaging.

Discussion: In this study, we successfully monitored the activity of Rho-family GTPases in vivo by FRET imaging. We also confirmed that 4T1 mouse mammary tumor cells mainly exhibited a mesenchymal phenotype when these tumor cells invaded into the surrounding stromal tissue. Recent studies have shown that some tumor cells exhibit two different modes of individual cell movement in 3D cell culture models: mesenchymal movement is characterized by an elongated cell shape and a dependence upon ECM proteolysis, whereas in amoeboid movement, cells have a rounded morphology, and are less dependent on proteases. We hereby demonstrate the utility of a three-dimensional, in vivo imaging technique toward understanding the mechanisms underlying tumor invasion.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-04.