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Abstract Background Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood. Methods The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses. The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer. Results In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer. Conclusions The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer.

Background: Resistance to chemotherapy, combined with heterogeneity among resistant tumors, represents a significant challenge in the clinical management of triple negative breast cancer (TNBC). By dissecting molecular pathways associated with treatment resistance, we sought to define patient sub-groups and actionable targets for next-line treatment., Methods: Bulk RNA sequencing and reverse phase protein array profiling were performed on isogenic patient-derived xenografts (PDX) representing paclitaxel-sensitive and -resistant tumors. Pathways identified as upregulated in the resistant model were further explored as targets in PDX explants. Their clinical relevance was assessed in two distinct patient cohorts (NeoAva and MET500)., Results: Increased activity in signaling pathways involving SRC-family kinases (SFKs)- and MAPK/ERK was found in treatment resistant PDX, with targeted inhibitors being significantly more potent in resistant tumors. Up-regulation of SFKs- and MAPK/ERK-pathways was also detected in a sub-group of chemoresistant patients after neoadjuvant treatment. Furthermore, High SFK expression (of either SRC, FYN and/or YES1) was detected in metastatic lesions of TNBC patients with fast progressing disease (median disease-free interval 27 vs 105 months)., Conclusions: Upregulation of SFK-signaling is found in a subset of chemoresistant tumors and is persistent in metastatic lesions. Based on pre-clinical results, these patients may respond favorably to treatment targeting SFKs., (© 2024. The Author(s).)

Treatment with the anti-angiogenic drug bevacizumab in addition to chemotherapy has shown efficacy for breast cancer in some clinical trials, but better biomarkers are needed to optimally select patients for treatment. Here, we present an omics approach where DNA methylation profiles are integrated with gene expression and results from proteomic data in breast cancer patients to predict response to therapy and pinpoint response-related epigenetic events. Fresh-frozen tumor biopsies taken before, during, and after treatment from human epidermal growth factor receptor 2 negative non-metastatic patients receiving neoadjuvant chemotherapy with or without bevacizumab were subjected to molecular profiling. Here, we report that DNA methylation at enhancer CpGs related to cell cycle regulation can predict response to chemotherapy and bevacizumab for the estrogen receptor positive subset of patients (AUC = 0.874), and we validated this observation in an independent patient cohort with a similar treatment regimen (AUC = 0.762). Combining the DNA methylation scores with the scores from a previously published protein signature resulted in a slight increase in the prediction performance (AUC = 0.784). We also show that tumors receiving the combination treatment underwent more extensive epigenetic alterations. Finally, we performed an integrative expression-methylation quantitative trait loci analysis on alterations in DNA methylation and gene expression levels, showing that the epigenetic alterations that occur during treatment are different between responders and non-responders and that these differences may be explained by the proliferation-epithelial-to-mesenchymal transition axis through the activity of grainyhead like transcription factor 2. Using tumor purity computed from copy number data, we developed a method for estimating cancer cell-specific methylation to confirm that the association to response reflects DNA methylation in cancer cells. Taken together, these results support the potential for clinical benefit of the addition of bevacizumab to chemotherapy when administered to the correct patients., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is an aggressive subtype of this disease. Targeted treatment has improved outcome, but there is still a need for new therapeutic strategies as some patients respond poorly to treatment. Our aim was to identify compounds that substantially affect viability in HER2+ breast cancer cells in response to combinatorial treatment. We performed a high-throughput drug screen of 278 compounds in combination with trastuzumab and lapatinib using two HER2+ breast cancer cell lines (KPL4 and SUM190PT). The most promising drugs were validated in vitro and in vivo, and downstream molecular changes of the treatments were analyzed. The screen revealed multiple drugs that could be used in combination with lapatinib and/or trastuzumab. The Src-inhibitor dasatinib showed the largest combinatorial effect together with lapatinib in the KPL4 cell line compared to treatment with dasatinib alone (p < 0.01). In vivo, only lapatinib significantly reduced tumor growth (p < 0.05), whereas dasatinib alone, or in combination with lapatinib, did not show significant effects. Protein analyses of the treated xenografts showed significant alterations in protein levels compared to untreated controls, suggesting that all drugs reached the tumor and exerted a measurable effect. In silico analyses suggested activation of apoptosis and reduced activity of survival pathways by all treatments, but the opposite pattern was observed for the combinatorial treatment compared to lapatinib alone., Competing Interests: Dr Mads Haugland Haugen, Dr Gunhild Mari Mælandsmo and Dr Olav Engebraaten report an international patent application PCT/EP2021/052016 published at WO 2021/156137 A1. The authors report no other conflicts of interest in this work., (Copyright: © 2023 Normann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The purpose of the present study is to investigate if consumption and supply hypoxia (CSH) MR-imaging can depict breast cancer hypoxia, using the CSH-method initially developed for prostate cancer. Furthermore, to develop a generalized pan-cancer application of the CSH-method that doesn't require a hypoxia reference standard for training the CSH-parameters. In a cohort of 69 breast cancer patients, we generated, based on the principles of intravoxel incoherent motion modelling, images reflecting cellular density (apparent diffusion coefficient; ADC) and vascular density (perfusion fraction; fp). Combinations of the information in these images were compared to a molecular hypoxia score made from gene expression data, aiming to identify a way to apply the CSH-methodology in breast cancer. Attempts to adapt previously proposed models for prostate cancer included direct transfers and model parameter rescaling. A novel approach, based on rescaling ADC and fp data to give more nuanced response in the relevant physiologic range, was also introduced. The new CSH-method was validated in a prostate cancer cohort with known hypoxia status. The proposed CSH-method gave estimates of hypoxia that was strongly correlated to the molecular hypoxia score in breast cancer, and hypoxia as measured in pathology slices stained with pimonidazole in prostate cancer. The generalized approach to CSH-imaging depicted hypoxia in both breast and prostate cancers and requires no model training. It is easy to implement using readily available technology and encourages further investigation of CSH-imaging in other cancer entities and in other settings, with the goal being to overcome hypoxia-induced resistance to treatment.

HER2 is a predictive biomarker for HER2-targeted therapeutics. For antibody-drug conjugates (ADCs; e.g., trastuzumab emtansine (T-DM1)), HER2 is utilized as a transport gate for cytotoxic agents into the cell. ADC biomarkers may therefore be more complex, also reflecting the intracellular drug transport. Here we report on a positive correlation between the early endosome marker RAB5A and T-DM1 sensitivity in five HER2-positive cell lines. Correlation between RAB5A expression and T-DM1 sensitivity is confirmed in breast cancer patients treated with trastuzumab emtansine/pertuzumab in the I-SPY2 trial (NCT01042379), but not in the trastuzumab/paclitaxel control arm. The clinical correlation is further verified in patients from the KAMILLA trial (NCT01702571). In conclusion, our results suggest RAB5A as a predictive biomarker for T-DM1 response and outline proteins involved in endocytic trafficking as predictive biomarkers for ADCs., (© 2021. The Author(s).)

Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple-negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR-Cas9 loss-of-function screen targeting a 2240-gene 'druggable genome' to identify phenotype-specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR-RAS-MAPK signaling, while the mesenchymal-like cells had increased sensitivity to knockout of G 2 -M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype-specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype-associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G 2 -M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR-Cas9 loss-of-function screen enables the identification of phenotype-specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A subset of breast cancer patients benefits from preoperative bevacizumab and chemotherapy, but validated predictive biomarkers are lacking. Here, we aimed to evaluate tissue-based angiogenesis markers for potential predictive value regarding response to neoadjuvant bevacizumab treatment in breast cancer. In this randomized 1:1 phase II clinical trial, 132 patients with large or locally advanced HER2-negative tumors received chemotherapy ± bevacizumab. Dual Factor VIII/Ki-67 immunohistochemical staining was performed on core needle biopsies at baseline and week 12. Microvessel density (MVD), proliferative microvessel density (pMVD; Factor VIII/Ki-67 co-expression), glomeruloid microvascular proliferation (GMP), and a gene expression angiogenesis signature score, were studied in relation to pathologic complete response (pCR), clinico-pathologic features and intrinsic molecular subtype. We found that high baseline MVD (by median) significantly predicted pCR in the bevacizumab-arm (odds ratio 4.9, P = 0.012). High pMVD, presence of GMP, and the angiogenesis signature score did not predict pCR, but were associated with basal-like (P ≤ 0.009) and triple negative phenotypes (P ≤ 0.041). pMVD and GMP did also associate with high-grade tumors (P ≤ 0.048). To conclude, high baseline MVD significantly predicted response to bevacizumab treatment. In contrast, pMVD, GMP, and the angiogenesis signature score, did not predict response, but associated with aggressive tumor features, including basal-like and triple-negative phenotypes.

Purpose: Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer (BC), there is an unmet need to identify patients who benefit from such treatment., Patients and Methods: In the NeoAva phase II clinical trial, patients (N = 132) with large (≥ 25 mm) human epidermal growth factor receptor 2 (HER2)-negative primary tumors were randomly assigned 1:1 to treatment with neoadjuvant chemotherapy (CTx) alone or in combination with bevacizumab (Bev plus CTx). The ratio of the tumor size after relative to before treatment was calculated into a continuous response scale. Tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse-phase protein arrays (RPPA) for expression levels of 210 BC-relevant (phospho-) proteins. Lasso regression was used to derive a predictor of tumor shrinkage from the expression of selected proteins prior to treatment., Results: We identified a nine-protein signature score named vascular endothelial growth factor inhibition response predictor (ViRP) for use in the Bev plus CTx treatment arm able to predict with accuracy pathologic complete response (pCR) (area under the curve [AUC] = 0.85; 95% CI, 0.74 to 0.97) and low residual cancer burden (RCB 0/I) (AUC = 0.80; 95% CI, 0.68 to 0.93). The ViRP score was significantly lower in patients with pCR ( P < .001) and in patients with low RCB ( P < .001). The ViRP score was internally validated on mRNA data and the resultant surrogate mRNA ViRP score significantly separated the pCR patients ( P = .016). Similarly, the mRNA ViRP score was validated ( P < .001) in an independent phase II clinical trial (PROMIX)., Conclusion: Our ViRP score, integrating the expression of nine proteins and validated on mRNA data both internally and in an independent clinical trial, may be used to increase the likelihood of benefit from treatment with bevacizumab combined with chemotherapy in patients with HER2-negative BC., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/cci/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Mads H. HaugenPatents, Royalties, Other Intellectual Property: Patent application 62/969770 to USPTOOle Christian LingjærdeConsulting or Advisory Role: NovartisThomas HatschekConsulting or Advisory Role: Roche, Pfizer, Pierre Fabre Research Funding: Roche, Pfizer Travel, Accommodations, Expenses: RocheAnne-Lise Børresen-DaleEmployment: Arctic Pharma AS, PubGene Stock and Other Ownership Interests: Arctic Pharma ASGordon B. MillsStock and Other Ownership Interests: Catena, SignalChem, Tarveda Therapeutics, ImmunoMET Honoraria: Nuevolution: AstraZeneca, Tarveda Therapeutics, Tesaro, Symphogen, PDX Pharmacy, ImmunoMET, Lilly Consulting or Advisory Role: AstraZeneca, SignalChem, Tarveda Therapeutics, Symphogen, Takeda/Millennium, PDX Pharmacy, ImmunoMET, Lilly, Turbine, ION Pharma, Zentalis Research Funding: Adelson Medical Research Foundation, AstraZeneca, NanoString Technologies, Breast Cancer Research Foundation, Karus Therapeutics, Pfizer, Prospect Creek Foundation, Tarveda Therapeutics, Ions Pharmaceuticals, ImmunoMET Patents, Royalties, Other Intellectual Property: HRD assay to Myriad Genetics, DSP technology patent with Nanostring Travel, Accommodations, Expenses: AstraZeneca, Pfizer, Symphogen, Chrysalis Biomedical Advisors, ImmunoMET, Michigan Primary Care ConsortiumGunhild M. MælandsmoPatents, Royalties, Other Intellectual Property: Patent application submitted for a nine-protein/gene panel predicting response to anti VEGF therapies in combination with chemotherapyOlav EngebraatenPatents, Royalties, Other Intellectual Property: Patent application pending for a biomarker for antibody drug conjugates, Patent application submitted for a nine-protein/gene panel predicting response to anti VEGF therapies in combination with chemotherapy No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Antiangiogenic drugs are potentially a useful supplement to neoadjuvant chemotherapy for a subgroup of patients with human epidermal growth factor receptor 2 (HER2) negative breast cancer, but reliable biomarkers for improved response are lacking. Here, we report on a randomized phase II clinical trial to study the added effect of bevacizumab in neoadjuvant chemotherapy with FEC100 (5-fluorouracil, epirubicin and cyclophosphamide) and taxanes (n = 132 patients). Gene expression from the tumors was obtained before neoadjuvant treatment, and treatment response was evaluated by residual cancer burden (RCB) at time of surgery. Bevacizumab increased the proportion of complete responders (RCB class 0) from 5% to 20% among patients with estrogen receptor (ER) positive tumors (P = .02). Treatment with bevacizumab was associated with improved 8-year disease-free survival (P = .03) among the good responders (RCB class 0 or I). Patients treated with paclitaxel (n = 45) responded better than those treated with docetaxel (n = 21; P = .03). Improved treatment response was associated with higher proliferation rate and an immune phenotype characterized by high presence of classically activated M1 macrophages, activated NK cells and memory activated CD4 T cells. Treatment with bevacizumab increased the number of adverse events, including hemorrhage, hypertension, infection and febrile neutropenia, but despite this, the ECOG status was not affected., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Introduction: In a recent phase I trial in a heterogeneous group of carcinoma patients with advanced disease, we did not observe objective responses by CT at 8 weeks in patients treated with either the anti-EpCAM immunotoxin MOC31PE alone or administered in combination with the immunosuppressor cyclosporin (CsA). We have now assessed overall survival (OS) data for the two groups to reveal potential differences, and to elucidate putative underlying mechanisms. Material and methods: The OS time of MOC31PE monotherapy (34 patients) and MOC31PE in combination with CsA (23 patients), was assessed. Pre- and post-treatment patient sera were analyzed in a multiplex immunoassay, and the immunogenic effects of MOC31PE were studied in vitro and in a dendritic cell maturation assay. Results: When the data were analyzed for all treated patients regardless of cancer type, the MOC31PE alone group had a median OS of 12.7 months (95% CI = 5.6-19.8 months) compared to 6.2 months (95% CI = 5.6-6.8 months) ( p =.066) for the patients treated with MOC31PE + CsA group. For the subgroup of patients with colorectal cancer, the median OS survival was 16.3 months (95% CI = 5.6-27.0) for the MOC31PE only cohort ( n  = 15), compared to 6.0 months (CI = 5.8-6.2) ( p  < .001) for the combination group. The cytokine profile in patient sera and the in vitro immunological studies indicate that MOC31PE induced an immunogenic response leading to T-cell activation; a response that was suppressed in patients treated with MOC31PE + CsA. Conclusions: The results reveal a promising clinical benefit of anti-EpCAM immunotoxin treatment in patients with advanced disease, an effect apparently explained by a previously unknown immunogenic effect of MOC31PE.

Angiogenesis is necessary for tumor growth and has been targeted in breast cancer; however, it is unclear which patients will respond and benefit from antiangiogenic therapy. We report noninvasive monitoring of patient response to neoadjuvant chemotherapy given alone or in combination with anti-vascular endothelial growth factor (bevacizumab) in a randomized clinical trial. At four time points during neoadjuvant chemotherapy ± bevacizumab of receptor tyrosine-protein kinase erbB-2-negative breast cancers, we measured metabolites and inflammation-related markers in patient's serum. We report significant changes in the levels of several molecules induced by bevacizumab, the most prominent being an increase in pentraxin 3 (PTX3) and von Willebrand factor (VWF). Serum levels of AXL, VWF and pulmonary and activation-regulated cytokine (PARC/CCL18) reflected response to chemotherapy alone or in combination with bevacizumab. We further analyzed serum cytokines in relation to tumor characteristics such as gene expression, tumor metabolites and tumor infiltrating leukocytes. We found that VWF and growth-differentiation factor 15 tumor mRNA levels correlated with their respective serum protein levels suggesting that these cytokines may be produced by tumors and outflow to the bloodstream while influencing the tumor microenvironment locally. Finally, we used binomial logistic regression which allowed to predict patient's response using only 10 noninvasive biomarkers. Our study highlights the potential of monitoring circulating levels of cytokines and metabolites during breast cancer therapy., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Patients with locally advanced breast cancer have a worse prognosis compared to patients with localized tumors and require neoadjuvant treatment before surgery. The aim of this study was to characterize the systemic metabolic effect of neoadjuvant chemotherapy in patients with large primary breast cancers and to relate these changes to treatment response and long-term survival. This study included 132 patients with large primary breast tumors randomized to receive neoadjuvant chemotherapy with or without the addition of the antiangiogenic drug Bevacizumab. Tumor biopsies and serum were collected before and during treatment and, serum additionally 6 weeks after surgery. Samples were analyzed by nuclear magnetic resonance spectroscopy (NMR). Correlation analysis showed low correlations between metabolites measured in cancer tissue and serum. Multilevel partial least squares discriminant analysis (PLS-DA) showed clear changes in serum metabolite levels during treatment ( p -values ≤ 0.001), including unfavorable changes in lipid levels. PLS-DA revealed metabolic differences between tissue samples from survivors and nonsurvivors collected 12 weeks into treatment with an accuracy of 72% ( p -value = 0.005); however, this was not evident in serum samples. Our results demonstrate a potential clinical application for serum-metabolomics for patient monitoring during and after treatment, and indicate potential for tissue NMR spectroscopy for predicting patient survival.

One of the hallmarks of cancer is sustained angiogenesis. Favorable results have been reported in some breast cancer (BC) patients receiving antiangiogenic therapy with bevacizumab (Bev) in combination with chemotherapy, and further knowledge on how Bev can be optimally combined with conventional treatment to increase efficacy is strongly needed. In this randomized, neoadjuvant phase II clinical trial, 132 patients with HER2-negative, nonmetastatic BC were treated with Bev in combination with sequential chemotherapy. Biopsies were sampled before treatment, after 12 weeks with anthracycline and after taxane therapy at week 25. MicroRNA (miRNA) expression profiling was performed on biopsies from each time point. Altogether, 241 biopsies were analyzed with the aim of identifying miRNA-based biomarkers of response to therapy. Results from the miRNA analyses were reported for the ER-positive cohort, which were previously demonstrated to benefit from antiangiogenic therapy in this study. For both treatment arms of this cohort, significantly different expression was observed for 217 miRNAs between objective responding and nonresponding patients before treatment initiation. These miRNAs have been linked to regulation of epithelial-mesenchymal transition, metastasis, and tumor growth, among other processes. Bev in combination with chemotherapy resulted in similar miRNA changes to chemotherapy alone. However, the deregulation of miRNA expression occurred earlier in the Bev arm. In both arms, tumor suppressor miRNAs were found upregulated after treatment, while oncogenic miRNAs were downregulated in the Bev arm. Patients responding to Bev showed a strong correlation between deregulated miRNAs and decreased proliferation score during the course of treatment, with downregulation of miR-4465 as the strongest indicator of reduced proliferation. Integrative analyses at miRNA-, gene-, and protein expression further indicated a longitudinal decrease in proliferation. Altogether, the results indicate that proliferation might represent a predictive factor for increased Bev sensitivity, which may aid in the identification of patients who could potentially benefit from Bev., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The usefulness of mechanistic models to disentangle complex multiscale cancer processes, such as treatment response, has been widely acknowledged. However, a major barrier for multiscale models to predict treatment outcomes in individual patients lies in their initialization and parametrization, which needs to reflect individual cancer characteristics accurately. In this study, we use multitype measurements acquired routinely on a single breast tumor, including histopathology, MRI, and molecular profiling, to personalize parts of a complex multiscale model of breast cancer treated with chemotherapeutic and antiangiogenic agents. The model accounts for drug pharmacokinetics and pharmacodynamics. We developed an open-source computer program that simulates cross-sections of tumors under 12-week therapy regimens and used it to individually reproduce and elucidate treatment outcomes of 4 patients. Two of the tumors did not respond to therapy, and model simulations were used to suggest alternative regimens with improved outcomes dependent on the tumor's individual characteristics. It was determined that more frequent and lower doses of chemotherapy reduce tumor burden in a low proliferative tumor while lower doses of antiangiogenic agents improve drug penetration in a poorly perfused tumor. Furthermore, using this model, we were able to correctly predict the outcome in another patient after 12 weeks of treatment. In summary, our model bridges multitype clinical data to shed light on individual treatment outcomes. SIGNIFICANCE: Mathematical modeling is used to validate possible mechanisms of tumor growth, resistance, and treatment outcome., (©2019 American Association for Cancer Research.)

The effect of poly(2-ethyl-butyl cyanoacrylate) nanoparticles containing the cytotoxic drug cabazitaxel was studied in three breast cancer cell lines and one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice. Nanoparticle-encapsulated cabazitaxel had a much better efficacy than similar concentrations of free drug in the basal-like patient-derived xenograft and resulted in complete remission of 6 out of 8 tumors, whereas free drug gave complete remission only with 2 out of 9 tumors. To investigate the different efficacies obtained with nanoparticle-encapsulated versus free cabazitaxel, mass spectrometry quantification of cabazitaxel was performed in mice plasma and selected tissue samples. Nanoparticle-encapsulated drug had a longer circulation time in blood. There was approximately a three times higher drug concentration in tumor tissue 24 h after injection, and two times higher 96 h after injection of nanoparticles with drug compared to the free drug. The tissue biodistribution obtained after 24 h using mass spectrometry analyses correlates well with biodistribution data obtained using IVIS® Spectrum in vivo imaging of nanoparticles labeled with the fluorescent substance NR668, indicating that these data also are representative for the nanoparticle distribution. Furthermore, immunohistochemistry was used to estimate infiltration of macrophages into the tumor tissue following injection of nanoparticle-encapsulated and free cabazitaxel. The higher infiltration of anti-tumorigenic versus pro-tumorigenic macrophages in tumors treated with the nanoparticles might also contribute to the improved effect obtained with the nanoparticle-encapsulated drug. Tumor infiltration of pro-tumorigenic macrophages was four times lower when using nanoparticles containing cabazitaxel than when using particles without drug, and we speculate that the very good therapeutic efficacy obtained with our cabazitaxel-containing particles may be due to their ability to reduce the level of pro-tumorigenic macrophages in the tumor. In summary, encapsulation of cabazitaxel in poly(2-ethyl-butyl cyanoacrylate) nanoparticles seems promising for treatment of breast cancer., (Copyright © 2018 Oslo University Hospital. Published by Elsevier B.V. All rights reserved.)

Cancer cells' phenotypic plasticity, promoted by stromal cells, contributes to intra-tumoral heterogeneity and affects response to therapy. We have disclosed an association between fibroblast-stimulated phenotype switching and resistance to the clinically used BRAF inhibitor (BRAFi) vemurafenib in malignant melanoma, revealing a challenge in targeting the fibroblast-induced phenotype. Here we compared molecular features and drug sensitivity in melanoma cells grown as co-cultures with fibroblasts versus mono-cultures. In the presence of fibroblasts, melanoma cells switched to the dedifferentiated, mesenchymal-like, inflammatory phenotype that showed reduced sensitivity to the most of 275 tested cancer drugs. Fibroblasts, however, sensitized melanoma cells to PI3K inhibitors (PI3Ki) and particularly the inhibitor of GSK3, AR-A014418 (GSK3i), that showed superior efficacy in co-cultures. The proteome changes induced by the BRAFi + GSK3i combination mimicked changes induced by BRAFi in mono-cultures, and GSK3i in co-cultures. This suggests that the single drug drives the response to the combination treatment, depending on fibroblast presence or absence, consequently, phenotype. We propose that the BRAFi and GSK3i (or PI3Ki) combination exemplifies phenotype-specific combinatorial treatment that should be beneficial in phenotypically heterogeneous tumors rich in stromal interactions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Chemotherapeutic agents such as anthracyclines and taxanes are commonly used in the neoadjuvant setting. Bevacizumab is an antibody which binds to vascular endothelial growth factor A (VEGFA) and inhibits its receptor interaction, thus obstructing the formation of new blood vessels., Methods: A phase II randomized clinical trial of 123 patients with Her2-negative breast cancer was conducted, with patients treated with neoadjuvant chemotherapy (fluorouracil (5FU)/epirubicin/cyclophosphamide (FEC) and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, after 12 weeks of treatment with FEC ± bevacizumab, and after 25 weeks of treatment with taxane ± bevacizumab. A time course study was designed to investigate the genomic landscape at the three time points when tumor DNA alterations, tumor percentage, genomic instability, and tumor clonality were assessed. Substantial differences were observed with some tumors changing mainly between diagnosis and at 12 weeks, others between 12 and 25 weeks, and still others changing in both time periods., Results: In both treatment arms, good responders (GR) and non-responders (NR) displayed significant difference in genomic instability index (GII) at time of diagnosis. In the combination arm, copy number alterations at 25 loci at the time of diagnosis were significantly different between the GR and NR. An inverse aberration pattern was also observed between the two extreme response groups at 6p22-p12 for patients in the combination arm. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. Increase in subclonal amplification was observed at 6p21.1, a locus which contains the VEGFA gene for the protein which are targeted by the study drug bevacizumab. Of the 13 pre-treatment samples that had a gain at VEGFA, 12 were responders. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 weeks, with the peak occurring at TMEM100, an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of VEGFA and TMEM100 are particularly sensitive to this treatment regime., Conclusions: Taken together, these results suggest that heterogeneity and subclonal architecture influence the response to targeted treatment in combination with chemotherapy, with possible implications for clinical decision-making and monitoring of treatment efficacy., Trial Registration: NCT00773695 . Registered 15 October 2008.

Cancers elicit an immune response by modifying the microenvironment. The immune system plays a pivotal role in cancer recognition and eradication. While the potential clinical value of infiltrating lymphocytes at the tumor site has been assessed in breast cancer, circulating cytokines - the molecules coordinating and fine-tuning immune response - are still poorly characterized. Using two breast cancer cohorts (MicMa, n = 131, DCTB, n = 28) and the multiplex Luminex platform, we measured the levels of 27 cytokines in the serum of breast cancer patients prior to treatment. We investigated the cytokine levels in relation to clinicopathological characteristics and in perspective of the tumor infiltrating immune cells predicted from the bulk mRNA expression data. Unsupervised clustering analysis of the serum cytokine levels in the MicMa cohort identified a cluster of pro-inflammatory, pro-angiogenic, and Th2-related cytokines which was associated with poor prognosis. Notably high levels of platelet derived growth factor BB (PDGF) reflected a more aggressive tumor phenotype and larger tumor size. A significant positive correlation between serum levels of interferon gamma-induced protein 10 (IP10) and its mRNA expression at the tumor site suggested that tumor-IP10-production may outflow to the bloodstream. High IP10 serum levels were associated with a worse prognosis. Finally, we found serum levels of both PDGF and IP10 associated with enrichment scores of specific tumor infiltrating immune cells. Our study suggests that monitoring cytokine circulating levels in breast cancer could be used to characterize breast cancers and the immune composition of their microenvironment through readily available biological material.

A high concentration of circulating vascular endothelial growth factor (VEGF) in cancer patients is associated with an aggressive tumor phenotype. Here, serum levels of 27 cytokines and blood cell counts were assessed in breast cancer patients receiving neoadjuvant chemotherapy with or without bevacizumab (Bev) in a randomized cohort of 132 patients with non-metastatic HER2-negative tumors. Cytokine levels were determined prior to treatment and at various time-points. The cytotoxic chemotherapy regimen of fluorouracil, epirubicin, and cyclophosphamide (FEC) had a profound impact on both circulating white blood cells and circulating cytokine levels. At the end of FEC treatment, the global decrease in cytokine levels correlated with the drop in white blood cell counts and was significantly greater in the patients of the Bev arm for cytokines, such as VEGF-A, IL-12, IP-10 and IL-10. Among patients who received Bev, those with pathological complete response (pCR) exhibited significantly lower levels of VEGF-A, IFN-γ, TNF-α and IL-4 than patients without pCR. This effect was not observed in the chemotherapy-only arm. Certain circulating cytokine profiles were found to correlate with different immune cell types at the tumor site. For the Bev arm patients, the serum cytokine levels correlated with higher levels of cytotoxic T cells at the end of the therapy regimen, which was indicative of treatment response. The higher response rate for Bev-treated patients and stronger correlations between serum cytokine levels and infiltrating CD8T cells merits further investigation.

Background: We conducted a meta-analysis in nonmetastatic breast cancer patients treated by neoadjuvant chemotherapy (NCT) to assess the clinical validity of circulating tumor cell (CTC) detection as a prognostic marker., Methods: We collected individual patient data from 21 studies in which CTC detection by CellSearch was performed in early breast cancer patients treated with NCT. The primary end point was overall survival, analyzed according to CTC detection, using Cox regression models stratified by study. Secondary end points included distant disease-free survival, locoregional relapse-free interval, and pathological complete response. All statistical tests were two-sided., Results: Data from patients were collected before NCT (n = 1574) and before surgery (n = 1200). CTC detection revealed one or more CTCs in 25.2% of patients before NCT; this was associated with tumor size (P < .001). The number of CTCs detected had a detrimental and decremental impact on overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P < .001), but not on pathological complete response. Patients with one, two, three to four, and five or more CTCs before NCT displayed hazard ratios of death of 1.09 (95% confidence interval [CI] = 0.65 to 1.69), 2.63 (95% CI = 1.42 to 4.54), 3.83 (95% CI = 2.08 to 6.66), and 6.25 (95% CI = 4.34 to 9.09), respectively. In 861 patients with full data available, adding CTC detection before NCT increased the prognostic ability of multivariable prognostic models for overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P = .008)., Conclusions: CTC count is an independent and quantitative prognostic factor in early breast cancer patients treated by NCT. It complements current prognostic models based on tumor characteristics and response to therapy.

Using our recently developed high-throughput automated platform, N-glycans from all serum glycoproteins from patients with breast cancer were analysed at diagnosis, after neoadjuvant chemotherapy, surgery, radiotherapy and up to 3 years after surgery. Surprisingly, alterations in the serum N-glycome after chemotherapy were pro-inflammatory with an increase in glycan structures associated with cancer. Surgery, on the other hand, induced anti-inflammatory changes in the serum N-glycome, towards a noncancerous phenotype. At the time of first follow-up, glycosylation in patients with affected lymph nodes changed towards a malignant phenotype. C-reactive protein showed a different pattern, increasing after first line of neoadjuvant chemotherapy, then decreasing throughout treatment until 1 year after surgery. This may reflect a switch from acute to chronic inflammation, where chronic inflammation is reflected in the serum after the acute phase response subsides. In conclusion, we here present the first time-course serum N-glycome profiling of patients with breast cancer during and after treatment. We identify significant glycosylation changes with chemotherapy, surgery and follow-up, reflecting the host response to therapy and tumour removal., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Purpose: Chemotherapy-induced alterations to gene expression are due to transcriptional reprogramming of tumor cells or subclonal adaptations to treatment. The effect on whole-transcriptome mRNA expression was investigated in a randomized phase II clinical trial to assess the effect of neoadjuvant chemotherapy with the addition of bevacizumab. Experimental Design: Tumor biopsies and whole-transcriptome mRNA profiles were obtained at three fixed time points with 66 patients in each arm. Altogether, 358 specimens from 132 patients were available, representing the transcriptional state before treatment start, at 12 weeks and after treatment (25 weeks). Pathologic complete response (pCR) in breast and axillary nodes was the primary endpoint. Results: pCR was observed in 15 patients (23%) receiving bevacizumab and chemotherapy and 8 patients (12%) receiving only chemotherapy. In the estrogen receptor-positive patients, 11 of 54 (20%) treated with bevacizumab and chemotherapy achieved pCR, while only 3 of 57 (5%) treated with chemotherapy reached pCR. In patients with estrogen receptor-positive tumors treated with combination therapy, an elevated immune activity was associated with good response. Proliferation was reduced after treatment in both treatment arms and most pronounced in the combination therapy arm, where the reduction in proliferation accelerated during treatment. Transcriptional alterations during therapy were subtype specific, and the effect of adding bevacizumab was most evident for luminal-B tumors. Conclusions: Clinical response and gene expression response differed between patients receiving combination therapy and chemotherapy alone. The results may guide identification of patients likely to benefit from antiangiogenic therapy. Clin Cancer Res; 23(16); 4662-70. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Doxorubicin, a widely used chemotherapeutic drug, has several potential high-risk side effects including cardiomyopathy. Furthermore, cellular resistance to this drug develops with time. By using liposomes as carrier vesicles both the side effects and drug resistance might be avoided. In this study we have investigated the cytotoxic effect of doxorubicin encapsulated in liposomes with and without ceramides containing 6 or 12 carbon atoms in the N-amidated fatty acyl chains. The short-chain ceramide species were included in the liposomal compositions due to their pro-apoptotic properties, which might cause a synergistic anticancer effect. We demonstrate that the ceramide species enhance the liposomal doxorubicin toxicity in a cell-specific manner. The C6-ceramide effect is most pronounced in cervical cancer cells (HeLa) and colon cancer cells (HCT116), whereas the C12-ceramide effect is strongest in breast cancer cells (MDA-MB-231). Moreover, the study reveals the importance of investigating cell toxicity at several time points and in different cell-lines, to assess drug-and formulation-induced cytotoxic effects in vitro . Furthermore, our data show that the cytotoxicity obtained with the nanocarriers in vitro , does not necessarily reflect their ability to inhibit tumor growth in vivo . We speculate that the larger effect of Caelyx® than our liposomes in vivo is due to a greater in vivo stability of Caelyx®., Competing Interests: CONFLICTS OF INTEREST None.

Purpose: Non-invasive response monitoring can potentially be used to guide therapy selection for breast cancer patients. We employed dynamic 2-deoxy-2-[ 18 F]fluoro-D-glucose positron emission tomography ([ 18 F]FDG PET) to evaluate changes in three breast cancer xenograft lines in mice following three chemotherapy regimens., Procedures: Sixty-six athymic nude mice bearing bilateral breast cancer xenografts (two basal-like and one luminal-like subtype) underwent three 60 min [ 18 F]FDG PET scans. Scans were performed prior to and 3 and 10 days after treatment with doxorubicin, paclitaxel, or carboplatin. Tumor growth was monitored in parallel. A pharmacokinetic compartmental model was fitted to the tumor uptake curves, providing estimates of transfer rates between the vascular, non-metabolized, and metabolized compartments. Early and late standardized uptake values (SUV E and SUV L , respectively); the rate constants k 1 , k 2 , and k 3 , and the intravascular fraction v B were estimated. Changes in tumor volume were used as a response measure. Multivariate partial least-squares regression (PLSR) was used to assess if PET parameters could model tumor response and to identify PET parameters with the largest impact on response., Results: Treatment responders had significantly larger perfusion-related parameters (k 1 and k 2 ) and lower metabolism-related parameter (k 3 ) than non-responders 10 days after the start of treatment. These findings were further supported by the PLSR analysis, which showed that k 1 and k 2 at day 10 and changes in k 3 explained most of the variability in response to therapy, whereas SUV L and particularly SUV E were of lesser importance., Conclusions: Overall, rate parameters related to both tumor perfusion and metabolism were associated with tumor response. Conventional metrics of [ 18 F]FDG uptake such as SUV E and SUV L apparently had little relation to tumor response, thus necessitating full dynamic scanning and pharmacokinetic analysis for optimal evaluation of chemotherapy-induced changes in breast cancers.

Context: Chronic fatigue (CF) in breast cancer (BC) survivors is multifactorial and may be caused by immune activation triggered by BC or its treatment. In the Neoadjuvant Avastin in Breast Cancer study, BC patients received neoadjuvant chemotherapy (FEC100→taxane) ± bevacizumab, a monoclonal antibody with fatigue as a potential side effect., Objectives: To examine fatigue levels and prevalence of CF before and during chemotherapy and at follow-up, and their associations with C-reactive protein (CRP) and clinical variables., Methods: Eighty-four HER2-negative patients with cT2-4N0-3M0 BC responded to questionnaires and had CRP measured before treatment (T0), after FEC100 (T1), after taxanes before surgery (T2), and at two-year follow-up (T3)., Results: The prevalence of CF increased from 8% at T0 to 36% at T3, P < 0.0001. Fatigue levels peaked during chemotherapy from 12.0 at T0 to 20.0 at T2, and declined to 16.7 at T3, P < 0.001. Women with CF at T3 had higher fatigue levels at T0, T2, and T3 than those without CF (P ≤ 0.01). Psychological distress (P = 0.03) and pain (P = 0.04) at T3 were associated with CF at T3. Only psychological distress remained a significant predictor in multivariate analysis. CRP increased from T0 to T1 (P < 0.01) and declined to baseline values at T3, but changes were not associated with bevacizumab treatment. No association was found between bevacizumab or CRP, and fatigue levels or CF., Conclusion: Neither bevacizumab treatment nor low-grade systemic inflammation as measured by CRP was associated with the increased fatigue levels and raised prevalence of CF, observed during and after BC therapy. Increased fatigue levels at baseline and psychological distress at T3 were associated with CF at T3., (Copyright © 2017 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)