Late-stage Metastatic Breast Cancer Research Articles

Abstract 3877: Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer

Abstract The estrogen receptor-positive (ER+) subtype accounts for 70% of breast cancer, and endocrine therapies that target ER are effective in the early-stage clinical setting. However, endocrine resistance is ubiquitous in late-stage metastatic breast cancer (MBC). The chief mechanism is the acquisition of ER mutations (ERmuts) in 25% of endocrine-resistant tumors; ERmuts are strongly resistant to aromatase inhibitor (AI) endocrine therapy. We previously identified four novel acquired activating mutations in human epidermal growth factor receptor 2 (HER2) by whole exome sequencing (WES) of paired ER+ MBC in a clinical cohort. These HER2 mutations (HER2muts) were enriched in multiple resistant tumors, and like ERmuts, were resistant to AIs. In contrast to ERmuts, these HER2muts also conferred complete resistance to all other classes of endocrine therapy, as well as cross-resistance to combination therapy with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors; the latter is standard-of-care in the refractory setting in ER+ MBC. At the same time, these HER2muts had variable responses to HER2-targeting therapies used in HER2+ breast cancer. Thus, HER2muts represent only the second validated mechanism of clinical endocrine resistance in ER+ MBC, accounting for ~8% of cases, and that is therapeutically intractable. Systematic phenotypic characterization of HER2muts in ER+ MBC is a prerequisite for effective targeting of this class of resistance. We used cBioPortal to identify >20 novel HER2 variants of unknown significance (VUS) that were frequent pan-cancer, in ER+ breast tumors, or annotated in a domain-restricted HER2 saturation mutagenesis screen, and that had not been previously characterized in ER+ MBC. We also identified a novel HER2 VUS by WES in an ER+/HER2+ tumor with resistance to HER2 therapy, from a clinical cohort of breast cancer patients. We phenotypically characterized all VUS through clinically comprehensive targeted therapy viability assays, and downstream activation assays. In sum, we identified seven novel activating HER2muts, spanning domains, with clinically important phenotypes in ER+ MBC. We found these HER2muts also conferred complete resistance to all classes of endocrine therapies, and cross-resistance to CDK4/6 inhibition. While novel HER2muts had variable responses to HER2-targeting therapies, they could be endocrine re-sensitized by combination with an irreversible tyrosine kinase inhibitor. Lastly, we report that constitutive HER2 homodimerization via reduction-sensitive disulfide bridges that occur as a consequence of mutations in the extracellular and juxtamembrane domains is a more common mechanism of HER2 hyperactivation than previously appreciated. This finding has unique functional consequences, and provides important structure-function insight into HER2 biology, beyond its immediate clinical ramifications in ER+ MBC. Citation Format: Nelson Vicente Guevara, Zirui Zhu, Esha Jain, Jennifer Kavran, Selime Arslan, Nikhil Wagle, Utthara Nayar. Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3877.

Real-time monitoring of circulating stromal cells in the blood to predict responsiveness of new-line therapies in metastatic breast cancer.

e14048 Background: Cancer associated macrophage like cells (CAMLs), a circulating stromal cell subtype, has been shown as an independent prognostic indicator of survival in late stage metastatic breast cancer when cells enlarge to ≥50µm. However, no study has evaluated the clinical relationship between changes in CAML size and their ability to predict treatment response. While CAMLs are prognostic for progression, we hypothesized that monitoring changes in CAMLs after initiation of therapy may be predictive for responsiveness of new treatment regimens. Methods: A prospective 12 months blind multi-institutional pilot study was undertaken to evaluate CAMLs before, and after, induction of a new line of investigational therapy based on CT scans. Patients with progressive metastatic breast cancer (n = 29) who had failed at least 2 prior therapies were recruited. A baseline (BL) blood sample was taken prior to induction of a new therapy and a 2nd sample (T1) taken after initiation (~30 days). Blood was filtered by CellSieve filtration.The quantities and sizes of CAMLs were analyzed based on PFS hazard ratios (HRs) by censored univariate analysis. Results: CAMLs were found in 97% of BL samples and 93% of T1 samples. At first CT scan, after the assigned dose of investigational treatment, 17 of 29 patients had clinical progression with 14/17 (82%) patients having an increased CAML size and 3/17 (18%) having a decreased CAML size. The remaining 12 of 29 patients saw clinically stable, or regression, of disease with 10/12 (83%) having decreased CAML size and 2/12 (17%) having increased CAML size. Overall CAML size change after therapy induction was 83% accurate at predicting response or progression based on CT scans. Further, patients with increasing CAML size at T1 had a 4 month mPFS vs 10 month mPFS for decreasing CAMLs, with a lower 12 month PFS HR = 3.7 (95%CI = 1.5-10.1, p = 0.020). Conclusions: Our data suggests that in metastatic breast cancer, monitoring CAMLs changes over the first 30 days of treatment accurately predicts responsiveness of disease to new treatments. Further, we suggests using blood sampling may increase the clinical value of blood based diagnostics by rapidly predicting the benefit of subsequent therapies.

Reversibly Stabilized Polycation Nanoparticles for Combination Treatment of Early- and Late-Stage Metastatic Breast Cancer.

Metastatic breast cancer is a major cause of cancer-related female mortality worldwide. The signal transducer and activator of transcription 3 (STAT3) and the chemokine receptor CXCR4 are involved in the metastatic spread of breast cancer. The goal of this study was to develop nanomedicine treatment based on combined inhibition of STAT3 and CXCR4. We synthesized a library of CXCR4-inhibiting polymers with a combination of beneficial features that included PEGylation, fluorination, and bioreducibility to achieve systemic delivery of siRNA to silence STAT3 expression in the tumors. An in vivo structure-activity relationship study in an experimental lung metastasis model revealed superior antimetastatic activity of bioreducible fluorinated polyplexes when compared with nonreducible controls despite similar CXCR4 antagonism and the ability to inhibit in vitro cancer cell invasion. When compared with nonreducible and nonfluorinated polyplexes, improved siRNA delivery was observed with the bioreducible fluorinated polyplexes. The improvement was ascribed to a combination of enhanced physical stability, decreased serum destabilization, and improved intracellular trafficking. Pharmacokinetic analysis showed that fluorination decreased the rate of renal clearance of the polyplexes and contributed to enhanced accumulation in the tumors. Therapeutic efficacy of the polyplexes with STAT3 siRNA was assessed in early stage breast cancer and late-stage metastatic breast cancer with primary tumor resection. Strong inhibition of the primary tumor growth and pronounced antimetastatic effects were observed in both models of metastatic breast cancer. Mechanistic studies revealed multifaceted mechanism of action of the combined STAT3 and CXCR4 inhibition by the developed polyplexes relying both on local and systemic effects.

Abstract 3296: SMMART: Serial measurements of molecular and architectural responses to therapy

Abstract SMMART is a precision medicine research program focused on understanding the evolution of actionable biology and mechanisms of resistance in human tumors during therapy. This is accomplished through in depth functional, ‘omic and multiscale image analysis of longitudinal samples acquired during treatment. Here we present a case report detailing the insights that can be gained from the comparative analysis of pre- and post-treatment biopsy specimens in a late-stage metastatic breast cancer patient. To understand the molecular evolution of cancer, we interrogated genomics with targeted and whole exome sequencing, transcriptomics with RNA and gene-fusion sequencing, and proteomics with reverse phase protein arrays. To understand cellular organization and architectural changes, we employed multi-scale imaging tools, including scanning electron microscopy (SEM), cyclic immunofluorescence, immune cell profiling with cyclic immunohistochemistry, and traditional pathological assessment. During the course of treatment, we monitored patient response to therapy with clinical imaging, circulating tumor DNA sequencing and cancer protein assessment. Individual assays revealed key aspects of how this individual's cancer evolved under therapeutic pressure. For example, mutational profiling revealed the patterns of clonal evolution and the acquisition of new genetic driver events. 2D and 3D SEM showed changes in ECM organization, macropinocytosis, mitochondrion size, number and density and number and organization of filopodia-like protrusions. We used a 30-color cyclic immunofluorescence analysis to identify differences in cancer cell proliferation and differentiation state, as well as the composition and organization of infiltrating immune cells. In addition, integrative analyses of multiple data types provided insight into the evolution of actionable biology within this patient's disease. This included changes in the suitability of the patient for immune checkpoint inhibitors as well as specific tyrosine kinase inhibitors. The comprehensive molecular and architectural characterization of an individual patient's cancer at multiple time points provides biologically novel and clinically relevant insight into the ways in which cancers become resistant to treatment. Citation Format: Brett Johnson, Jamie Keck, Max Morris, Kiara Siex, Annette Kolodzie, Swapnil Parmar, Jessica Riesterer, Koei Chin, Summer Gibbs, Laura Heiser, Paul Spellman, Kyle Ellrott, Ozgun Babur, Emek Demir, Adam Margolin, Jeremy Goecks, Lisa Coussens, Raymond Bergan, Joe Gray. SMMART: Serial measurements of molecular and architectural responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3296.

Cofactor of BRCA1: A new genetic marker for common malignant liver cancer

Cofactor of BRCA1: A new genetic marker for common malignant liver cancer

Abstract A182: Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38

Abstract De novo or acquired resistance to anti-cancer drugs represents a major obstacle to successful treatment of cancer patients. In order to improve future cancer drug development we have established the DEN-50R cell line panel which when fully developed will consist of isogenic pairs of drug sensitive and resistant human cancer cell lines representing the 5 most common cancer types. Based on the DEN-50R we have tested a number of novel anti-cancer drugs in this panel of cell lines. One class of drugs that includes the indenoisoquinolines LMP400 and LMP776, which have topoisomerase 1 inhibitory activity and recently passed phase 1 clinical studies, showed very interesting features in the cell line studies. When tested in SN-38 (the active metabolite of irinotecan) -sensitive and -resistant isogenic colorectal cancer cell lines and breast cancer cell lines, LMP400 showed significant dose related cytotoxicity independent of the state of SN-38-resistance (Table). As these resistant cell lines have significant up-regulation of either the BCRP and/or mdr-1 protein, it can be concluded that the indenoisoquinoline LMP400 can target topoisomerase 1 enzyme without being a substrate for these drug efflux pumps. We are now planning to initiate two clinical phase II studies (Simon's two stage design) with LMP400; one in irinotecan-failed metastatic colorectal cancer patients and one in late stage metastatic breast cancer patients. Patients for these studies will be preselected based on an LMP400-responsiveness profile we generated by gene expression data where associations between gene expression profiles and growth inhibition were compared in a panel of cell lines exposed to LMP400. A second step included filtering the identified gene expression profile against mRNA expression from a collection of 3200 human tumors. Only genes being differentially expressed in the clinical tumor material were retained in the model. Sensitivity to LMP776 and LMP400 of human colorectal and breast cancer cell lines with acquired resistance to SN-38 Cell lineCross-resistantSensitiveHCT116-SN-38*LMP400; LMP776HT29-SN-38LMP776LMP400LoVo-SN-38LMP776LMP400MDA-MB-231- SN-38LMP776LMP400MCF-7- SN-38LMP776; LMP400* HCT-116-SN38 resistant cells have gained a mutation in the binding site of SN38 and LMP400 during acquisition of SN-38 resistance. Citation Format: Jan Stenvang, Niels Frank Jensen, Haatisha Jandu, Steen Knudsen, Keli Agama, Thomas Jensen, Anker Hansen, Peter Buhl Jensen, Yves Pommier, Mark Cushman, Nils Brünner. Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A182.

Subsequent endocrine therapy after resistance to ethinylestradiol treatment for the late-stage metastatic breast cancer: A retrospective cohort study.

148 Background: Aromatase inhibitor (AI) is a most commonly used as the endocrine therapy in postmenopausal hormone-dependent breast cancer. Paradoxically, estrogen additive therapy using ethinylestradiol can be useful after long-term estrogen deprivation therapies with AI. Furthermore, there is a possibility of the beneficial effect of AI or fulvestrant as a subsequent endocrine therapy after EE2 failure. Methods: Ethinylestradiol (EE2; 3mg/day, TID) therapy was performed in 20 patients with metastatic breast cancer (median; 62 years-old, the mean observation time: 13.6 months.), who were heavily treated by sequential endocrine therapies (3rd or more line) including cytotoxic chemotherapies. We examined the efficacy of a subsequent endocrine therapy of AI or fulvestrant after becoming resistance to the EE2 therapy in the patients who got the disease control by the prior EE2 therapy. The primary endpoint was clinical benefit rate (CBR) and the secondary endpoint was time to treatment failure (TTF). (Registration number; UMIN 000002831, additional analysis). Results: Median TTF of EE2 treatment was 46 weeks (23-62+, 2 cases were ongoing). The response rate was 41% (9/22), the clinical benefit rate was 50% (11/22). The stable disease (< 6 months) was 18% (4/22) and another 3 cases were judged as progressive disease. Four cases withdrew due to nausea, fatigue and muscle-skeletal pain. In 20 cases progressed after disease control (SD or more) of EE2, a subsequent endocrine therapy, fulvestrant for 10 cases and AI for 10 cases, was performed. Fulvestrant group showed 50% (3 in 6) of CBR and 15 weeks (5-55+) of TTF, and AI-treated group showed 43% (3 in 7) of CBR and 19 weeks (5-33+) of TTF. Two in three cases, who became resistance to anti-estrogen treatment, got long SD by further EE2 re-therapy. Conclusions: Some cases showed clinical benefits of a subsequent therapy by AI or fulvestrant after becoming to EE2 failure. Taken together, sequential use of estrogen and anti-estrogen therapy (vice versa) could be one of the options as a salvage endocrine therapy for the patients with end-stage breast cancer.

Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial–mesenchymal transition (EMT) to mesenchymal–epithelial transition (MET) states

Background:Eribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) in human breast cancer cells were investigated.Methods:Triple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER−)/progesterone receptor (PR−)/human epithelial growth receptor 2 (HER2−) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-β/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting.Results:Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-β induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model.Conclusions:Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.

Abstract P1-14-04: Subsequent endocrine therapy after becoming to resistance of ‘estrogen therapy’ is beneficial as a salvage treatment for the late stage metastatic breast cancer

Abstract BACKGROUND: Aromatase inhibitor (AI) is a most commonly used as the endocrine therapy in postmenopausal hormone-dependent breast cancer. Paradoxically, estrogen additive therapy, high-dose estrogen using ethinylestradiol, can be useful after long-term estrogen deprivation therapies with AI. Furthermore, there is a possibility of the beneficial effect of AI or fulvestrant as a subsequent endocrine therapy after EE2 failure. METHODS: Ethinylestradiol (EE2; 3mg/day, TID) therapy was performed in 22 patients with metastatic breast cancer (median; 62 years-old, the mean observation time: 13.6 months.), who were heavily treated by sequential endocrine therapies (3rd or more line) including cytotoxic chemotherapies. We examined the efficacy of a subsequent endocrine therapy using aromatase inhibitor or fulvestrant after becoming resistance to the EE2 therapy in the patients who got the disease control by the prior EE2 therapy. The primary endpoint was clinical benefit rate (CBR) and the secondary endpoint was time to treatment failure (TTF). (Registration number; UMIN 000002831, additional analysis) RESULTS: Median of TTF of EE2 treatment was 46 weeks (23-62+, 2 cases were ongoing). The response rate was 41% (9/22), the clinical benefit rate was 50% (11/22). The stable disease (< 6 months) was 18% (4/22) and another 3 cases were judged as progressive disease. Four cases withdrew due to nausea, fatigue and muscle-skeletal pain. In 13 cases progressed after disease control (SD or more) of EE2, a subsequent endocrine therapy, fulvestrant for 6 cases and AI for 7 cases, was performed. Fulvestrant group showed 50% (3 in 6) of CBR and 15 weeks (5-55+) of TTF, and AI-treated group showed 43% (3 in 7) of CBR and 19 weeks (5-33+) of TTF. Two in three cases, who became resistance to anti-estrogen treatment, got long SD by further EE2 re-therapy. DISCUSSION: EE2 therapy could be beneficial for heavily treated metastatic breast cancer. Some cases showed clinical benefits of a subsequent therapy by AI or fulvestrant after becoming to EE2 failure. Taken together, sequential use of estrogen and anti-estrogen therapy (vice versa) could be one of the options as a salvage endocrine therapy for the end stage breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-14-04.

Abstract A296: Eribulin mesylate suppresses experimental metastasis in the MX-1 human breast cancer model by reversing phenotypic states between epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET).

Abstract Background: Eribulin mesylate (eribulin), a non-taxane microtubule dynamics inhibitor, has unexpectedly shown greater effects on overall survival compared to progression-free survival in late stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously-unrecognized anti-tumor mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance of EMT/ MET in MX-1 human breast cancer cells were investigated. Methods: MX-1 human breast cancer cells, which are ER-/PR-/HER2- and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes by qPCR and immunoblot, respectively. In addition, the EMT/MET status of MX-1 xenografts in mice treated with eribulin were also examined by qPCR, immunoblot, and immunohistochemical analysis. Proliferation, migration and invasion assays were also conducted. Finally, an experimental lung metastasis model was utilized to gauge the metastatic activity of eribulin treated MX-1 cells. Results: Treatment of MX-1 cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Gene expression analyses showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial marker genes. Furthermore, protein levels of epithelial marker E-cadherin were increased following eribulin treatment, while protein levels of mesenchymal markers N-cadherin and vimentin were decreased. Consistent with these changes, MX-1 cells treated with low levels of eribulin for one week showed decreased capacity for in vitro migration and invasiveness. In the MX-1 xenograft model in vivo, eribulin treatment induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker genes and proteins. Finally, MX-1 cells treated in vitro with low levels of eribulin for one week led to decreased numbers of lung metastases when assessed in an in vivo experimental metastasis model. Conclusions: Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings provide a scientific basis for clinical observations of prolonged overall survival without corresponding effects on progression-free survival in metastatic breast cancer patients treated with eribulin. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A296. Citation Format: Taku Yoshida, Galina Kuznetsov, Takayuki KImura, Naoko H. Sugi, Hajime Shimizu, Yoichi Ozawa, Yoshiaki Sato, Taisuke Uehara, Hideki Watanabe, Kentaro Takahashi, Kentaro Matsuura, Mai Uesugi, Osamu Toyama, Mamoru Yanagimachi, Yasuhiro Funahashi, Junji Matsui. Eribulin mesylate suppresses experimental metastasis in the MX-1 human breast cancer model by reversing phenotypic states between epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A296.

Population pharmacokinetic–pharmacodynamic analysis for eribulin mesilate‐associated neutropenia

Eribulin mesilate is an inhibitor of microtubule dynamics that is approved for the treatment of late-stage metastatic breast cancer. Neutropenia is one of the major dose-limiting adverse effects of eribulin. The objective of this analysis was to develop a population pharmacokinetic-pharmacodynamic model for eribulin-associated neutropenia. A combined data set of 12 phase I, II and III studies for eribulin mesilate was analysed. The population pharmacokinetics of eribulin was described using a previously developed model. The relationship between eribulin pharmacokinetic and neutropenia was described using a semi-physiological lifespan model for haematological toxicity. Patient characteristics predictive of increased sensitivity to develop neutropenia were evaluated using a simulation framework. Absolute neutrophil counts were available from 1579 patients. In the final covariate model, the baseline neutrophil count (ANC0) was estimated to be 4.03 × 10(9) neutrophils l(-1) [relative standard error (RSE) 1.2%], with interindividual variability (IIV, 37.3 coefficient of variation % [CV%]). The mean transition time was estimated to be 109 h (RSE 1.8%, IIV 13.9CV%), the feedback constant (γ) was estimated to be 0.216 (RSE 1.4%, IIV 12.2CV%), and the linear drug effect coefficient (SLOPE) was estimated to be 0.0451 μg l(-1) (RSE 3.2%, IIV 54CV%). Albumin, aspartate transaminase and receival of granulocyte colony-stimulating factor (G-CSF) were identified as significant covariates on SLOPE, and albumin, bilirubin, G-CSF, alkaline phosphatase and lactate dehydrogenase were identified as significant covariates on mean transition time. The developed model can be applied to investigate optimal treatment strategies quantitatively across different patient groups with respect to neutropenia. Albumin was identified as the most clinically important covariate predictive of interindividual variability in the neutropenia time course.

Abstract B71: Syndecan-1 promotes breast cancer metastasis by modulating tissue stiffness

Abstract Tumor-stroma interactions are integral to breast cancer progression and metastasis. It has been well established that women with dense breast tissue have increased risk of developing breast cancer as compared to women with less dense breast tissue; it has also been documented that a stiffer extracellular matrix (ECM) can promote tumor cell invasion both in vitro and in vivo. Our lab has previously shown that Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan, regulates the in vitro organization of a fibroblast-constructed ECM that thus creates an invasion-permissive microenvironment. Therefore, we sought to characterize the role of stromal Sdc1 in cancer progression in vivo. Using the 4T1 model of late stage metastatic breast cancer in both Sdc1 wild-type (WT) and Sdc1 knock-out (KO) mice, we assayed for tumor metastasis from orthotopic tumor cell implants. Sdc1 deficiency in the host did not influence the primary tumor weight or growth rate, but significantly reduced the incidence and the number of metastatic lesions (p<0.001, p<0.0001, respectively). In an effort to dissect out the mechanism by which loss of stromal Sdc1 is inhibiting metastasis, we analyzed the tumor microenvironment for the presence of specific cell types and structural differences. There was no difference in the recruitment of macrophages, the activation of fibroblasts, angiogenesis, or the structure or deposition of collagen between 4T1-induced tumors in the Sdc1 WT and Sdc1 KO mice. Interestingly, preliminary data suggest the tumors from the Sdc1 WT mice are significantly stiffer than the tumors from Sdc1 KO mice, as measured by atomic force microscopy. To determine whether Sdc1 was influencing later stages of metastasis we injected tumor cells via tail vein to specifically assay extravasation and metastatic outgrowth. After 2 weeks, all mice assayed had metastatic lesions but the Sdc1 WT mice had significantly more numerous and larger lesions (p<0.004, p<0.05, respectively). These data indicate that the presence of stromal Sdc1 promotes tumor cell outgrowth in the metastatic target organ, but may not play as large a role in tumor cell extravasation based on the finding that all mice assayed had metastatic lesions. These results further suggest that Sdc1 influences breast cancer metastasis by promoting increased tissue stiffness, which promotes tumor cell invasion and tumor outgrowth at secondary metastatic target organs, and ultimately promotes formation of metastatic lesions. Citation Format: Colleen L. Chute, Patricia Keely, Caroline Alexander, Andreas Friedl. Syndecan-1 promotes breast cancer metastasis by modulating tissue stiffness. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B71.

Abstract 437: Exploring biomarkers of metastatic breast cancer based on nanoporous silica chip for early diagnosis and therapeutic evaluation

Abstract Breast cancer is the most frequently occurring malignancy and the second leading cause of cancer death in the United Stated and other Western countries. In these patients, not the primary tumor, but the metastasis of breast cancer caused to death. There is still a challenging to detect the metastasis in early stage and no effective target therapy at late stage metastatic breast cancer, expect the conventional chemotherapy intervention. Therefore, the identification of biomarkers for the detection of cancer metastasis in the early stage and discovery of new therapy targets may hold the key to the management of metastasis breast cancer. Although high molecular weight proteins in blood have been extensively studied, little is known about the low molecular weight (LMW) proteins and peptides due to difficulties in isolation and purification of this low abundant population. These substances may circulate at very low levels and can be difficult to detect in the presence of large abundance of proteins such as albumin. In our study, we have recently developed nanoporous chip-based technologies to separate small proteins/peptides from the large proteins in serum. We used nanoporous silica chips, with a highly periodic nanostructure and uniform pore size distribution, to isolate LMW proteins and peptides from serum of mouse model with MDA-MB-231 human breast cancer lung metastasis. The matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) were used in profiling the LMW proteins and peptides in three distinct stages (control, early stage and late stage). All data were then carried out by principle component analysis and vertical scatter plot to identify signatures unique to different stages of cancer development. Finally, we found eight peptides significant changes during the tumor development. It is interesting that these eight candidate biomarkers can be divided into three groups. Changes on serum level of group 1 fragments can be detected from both early and late stage mice. These candidates can sever as biomarkers for detection of early stage tumor metastasis. Only one peptide was included in Group 2, which was showed the similar level in control and early stage mice, but significantly elevated in late stage mice. This fragment is indicative of late stage tumors. Level of group 3 fragments increased during tumor development, and reached peak in late stage mice. Group 3 markers could be used to guide physicians to better treat cancer patients based on their tumor progression. More detailed studies in mass sequence are needed to validate the usefulness of these biomarkers. In conclusion, the nanotechnology based silica chips provide a rapid, sensitive, reproducible and inexpensive platform that will lead to significant, personalized diagnosis and new therapies for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 437. doi:1538-7445.AM2012-437

Clinical evidence for the role of eribulin mesylate in the treatment of breast cancer

The development of drugs improving overall survival (OS) in late-stage metastatic breast cancer (MBC) remains a challenge. Eribulin mesylate, a new chemotherapy agent, has shown significant results in this setting. This agent is a synthetic analog of a macrolide isolated from a marine sponge. It inhibits microtubule polymerization, inducing mitotic arrest and apoptosis, and aggregates soluble tubulin in a nonproductive form. In Phase II studies, this drug gave a partial response and stable disease. The EMBRACE study showed that eribulin mesylate improved OS in heavily pretreated (particularly with anthracycline and taxane) MBC with good tolerance. Currently, eribulin mesylate is the first major single-agent that has improved OS in heavily pretreated MBC. These results suggest that this drug could become a new standard in the treatment of advanced breast cancer and should be developed in earlier stages.

Immunoliposomal Delivery of 213Bi for α-Emitter Targeting of Metastatic Breast Cancer

Current treatment for late-stage metastatic breast cancer is largely palliative. alpha-Particles are highly potent, short-range radiation emissions capable of sterilizing individual cells with one to three traversals of the cell nucleus. The alpha-emitter, (213)Bi (T(1/2) = 45.6 min), was conjugated to a 100-nm diameter liposomal-CHX-A''-DTPA construct, upon which the rat HER2/neu reactive antibody, 7.16.4, was grafted. A conjugation time of 10 minutes was achieved giving a specific activity corresponding to 0.1 (213)Bi atom per liposome; stability in vitro and in vivo was confirmed. Efficacy in a rat/neu transgenic mouse model of metastatic mammary carcinoma was investigated. Three days after left cardiac ventricular injection of 10(5) rat HER-2/neu-expressing syngeneic tumor cells, macrophage-depleted Neu-N mice were treated by i.v. injection with (a) 19.2 MBq (520 muCi) of liposome-CHX-A''-DTPA-(213)Bi, (b) 19.2 MBq of liposome-CHX-A''-DTPA-(213)Bi-7.16.4, (c) 4.44 MBq (120 muCi) of (213)Bi-7.16.4, and (d) cold (nonradioactive) liposome-CHX-A''-DTPA-7.16.4 as control. Treatment with (a) increased median survival time to 34 days compared with 29 days for the untreated controls (P = 0.013) and 27 days for treated cold controls. Treatment with the radiolabeled antibody-conjugated liposome (b) increased median survival time to 38 days (P = 0.0002 relative to untreated controls). The radiolabeled antibody-treated group (c) gave a median survival of 39 days, which was similar to that for the radiolabeled antibody-conjugated liposome-treated group (P = 0.5). We have shown that the (213)Bi radiolabeled immunoliposomes are effective in treating early-stage micrometastases, giving median survival times similar to those obtained with antibody-mediated delivery of (213)Bi in this animal model.

Abstract 1558: STX140 but not paclitaxel inhibits mammary tumor initiation, progression and metastasis in C3(1)/SV40 T/t-antigen transgenic mice

Abstract New chemotherapeutic options for late-stage metastatic breast cancer are urgently required. Currently, the taxanes are considered one of the most effective treatments for late-stage cancer treatment. Paclitaxel and the novel cytotoxic agent STX140, compounds that have shown significant activity against numerous xenograft mouse models of cancers, are here investigated in the C3(1)/SV40 T/t-antigen mouse model of spontaneous breast cancer. This mouse model recapitulates important histopathological and molecular alterations in mammary cancer development over a highly predictable time-course. At 8 weeks of age, the female animals develop low-grade mammary intra-epithelial neoplasia (MIN) lesions. By 12 to 14 weeks of age this progresses to high-grade MIN, which is similar to human ductal carcinoma in situ. From 15 weeks of age onwards invasive, metastatic carcinomas are observed. STX140 and paclitaxel were administered to these mice at 12 weeks of age (early intervention) and when tumors had reached 0.5 cm (late intervention). Results demonstrated that all mice in the control and paclitaxel treated groups at early intervention developed palpable mammary cancer which grew to 3112 mm3 by 25 weeks of age and led to the termination of these animals at week at this time point. However, STX140, when administered at week 12 onwards, blocked disease progression and no palpable tumor develop in 47% of tumors and significantly inhibited the growth of tumors that did develop. This resulted in a significant (P < 0.01) survival advantage for animals treated with STX140. Conversely, in C3(1)/SV40 mice, paclitaxel failed to inhibit tumor growth and therefore did not confer a survival benefit. In the late treatment groups, STX140 significantly (P < 0.05) inhibited primary mammary cancer growth and also resulted in a significant (P < 0.001) survival advantage compared to control animals. Furthermore, it was observed that paclitaxel treatment increased liver metastases in these mice. To conclude, STX140 has a greater anti-cancer efficacy and conferred an improved survival outcome compared to paclitaxel in a clinically relevant transgenic mouse model of metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1558.

TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer

The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%-80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-beta1. Whether TGF-beta1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-beta1-activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-beta1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-beta1-mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-beta1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.

Alterations in p53-specific T cells and other lymphocyte subsets in breast cancer patients during vaccination with p53-peptide loaded dendritic cells and low-dose interleukin-2

We have previously established a cancer vaccine using autologous DCs, generated by in vitro stimulation with IL-4 and GM-CSF, and pulsed with six HLA-A*0201 binding wild-type p53 derived peptides. This vaccine was used in combination with low-dose interleukin-2 in a recently published clinical Phase II trial where 26 HLA-A2+ patients with progressive late-stage metastatic breast cancer (BC) were included. Almost 1/3rd of the patients obtained stable disease or minor regression during treatment with a positive correlation to tumour over-expression of p53. In the present study, we performed a comprehensive analysis of the effector stage of the p53-specific CD8+ T cells by the use of Dextramer Technology and multicolour FACS. Pre- and post-treatment blood samples from eight BC patients were analysed. Independent of clinical outcome p53-specific T cells were phenotypic distinctly antigen experienced (CD44 high, CCR-7 low and CD62L low). Furthermore, fresh blood from 18 cancer patients included in the vaccination trial were prospectively examined for more general treatment associated quantitative and qualitative changes in T cell subpopulations. We found that the frequency of CD4+ CD25 high regulatory T cells was almost doubled after only 4 weeks of weekly vaccination and low-dose IL-2. In addition, a decrease in the percentage of CD27 highCCR-7 high CD4/CD8 naïve T cells was measured particularly in patients with progressive disease during vaccination. Finally, prior to immunotherapy a higher percentage of both CD28 and CD27 positive CD8 naïve/early effector memory T cells were present in chemotherapy-treated patients.