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Characterization of a novel dormant, drug resistant, stem cell subpopulation in acute lymphoblastic leukemia
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Conclusions: MSOT is a direct method to image blood vasculature. It is able to distinguish histological and functional differences in xenograft mouse models. Xenografts from the breast cancer cell line MCF-7 showed a functional vasculature. In the later tumour stages of MDA-MB-231 xenograft model an increase in vascular density was detected independently from the cytokine VEGF and the endothelial regulator NO. This vascular network is inefficient in oxygenating the tumour. In the next steps, we will investigate the utility of MSOT biomarkers to monitor response to anti-angiogenic therapies, hence establishing the potential of the technique as a companion diagnostic. No conflict of interest. 890 Characterization of a novel dormant, drug resistant, stem cell subpopulation in acute lymphoblastic leukemia S. Ebinger1 , E. Ozdemir1 , S. Tiedt1 , C. Ziegenhain2 , C. Castro-Alves1 , W. Enard2 , I. Jeremias1 . 1 Helmholtz Zentrum Munchen, ¨ AGV, Munich, ¨ Munchen, Germany, 2 Faculty of Biology- Ludwig-Maximilians-Universitat ¨ Anthropology and Human Genetics, Munich, Germany Introduction: Treatment-resistant cells determine the prognosis of cancer patients as they might induce disease relapse associated with poor outcome. Novel therapeutic options are urgently needed to eradicate the chemo-resistant subpopulation. Acute lymphoblastic leukemia (ALL) is a hematologic neoplasia with dismal prognosis upon disease relapse. The aim was here to identify and characterize the drug resistant subpopulation in patients’ ALL. Material and Method: We engrafted primary patients’ ALL cells in NSG mice and used genetic engineering to express the transgenes NGFR, a red fluorochrome and a luciferase. A combined MACS/FACS procedure based on the expression of the transgenes enabled the ALL enrichment out of the mouse bone marrow with a factor above 10,000. CFSE staining of ALL cells was used to characterize the proliferation in vivo. Results and Discussion: All 9 tested ALL samples showed a heterogeneous growth behavior in mice with a small subpopulation of long-term dormant PDX cells, called “label retaining cell” (LRC). Re-transplantation into secondary recipient mice converted LRC into fast proliferative ALL cells (non-LRC) and vice versa, suggesting a high plasticity of ALL cells, which are able to convert from dormant into proliferating cells and vice versa. We next studied the drug sensitivity of patient-derived ALL cells and performed preclinical in vivo treatment trials with cytotoxic drugs on patients ALL cells growing in mice. While highly proliferative, non-LRC were decreased by the in vivo treatment by more than 1 order of magnitude, the amount of dormant LRC remained completely unchanged, suggesting increased in vivo treatmentresistance of LRC. Treatment resistant LRC revealed stem cell potential upon re-transplantation into next recipient mice. We next aimed at characterizing the expression profile of LRC and non-LRC by single cell RNA sequencing. Principle component analysis showed two distinct populations with an overlap revealing the plasticity between dormant and proliferating cells. Gene set enrichment analysis of published gene signatures demonstrated the clinical significance of LRC as the LRC expression profile resembles high risk ALL cells, the subpopulation of CD34 positive CML cells, leukemic stem cells and hematopoietic stem cells. Conclusion: Taken together and using the cutting edge technology of genetic engineering in individual patient-derived xenograft cells, we showed that ALL contains a rare subpopulation of dormant and therapy resistant cells. These cells show the challenging characteristics of relapse inducing cells which determine patients’ prognosis. Our model might be used to develop novel therapies which eliminate treatment resistant cells in ALL patients, prevent relapse and increase patients’ outcome. No conflict of interest. 892 Clinicopathologic significance of androgen receptor expression in invasive breast cancer: A comprehensive analysis in large cohorts M.A. Aleskandarany1 , R. Abduljabbar2 , S. Sonbul1 , C.F. Lai3 , S. Ali3 , M. Diez-Rodriguez1 , C. Nolan1 , A.R. Green1 , I.O. Ellis1 , E.A. Rakha1 . 1 University of Nottingham, Pathology, Nottingham, United Kingdom, 2 Azadi Teaching Hospital- Duhok- Iraq, Department of Oncology-, Duhok, Iraq, 3 Imperial College London, Department of Surgery and Cancer, London, United Kingdom Introduction: Differential prognostic roles of Androgen Receptor (AR) have been proposed in breast cancer (BC) depending on tumour oestrogen receptor (ER) status. This study aimed to evaluate the prognostic and/or predictive significance of androgen receptor (AR) expression in invasive BC. Material and Methods: In this study AR expression was studied on a large (n = 1141) consecutive series of early-stage (I-III) BC using tissue microarray and immunohistochemistry (IHC). AR mRNA expression was assessed in a matched subset of cases included into the multicentre METABRIC study (n = 285) with relevance to AR protein expression, clinicopathological parameters and patients’ outcome. The prognostic impact of AR mRNA expression was externally validated using the online BC gene expression data sets (n = 25 datasets, 4,078 patients).
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Results: Nuclear AR IHC expression was significantly associated with features of good prognosis including older age, smaller tumour size, lower grade and lobular histology particularly in the ER-positive tumours. AR was associated with ER-related markers GATA3, FOXa1, RERG and BEX1. Negative association was observed with HER2, p53, Ki67, TK1, CD71 and AGTR1. AR Overexpression was associated with longer survival (p < 0.001), independent of tumour size, grade, and axillary nodal stage (p = 0.033, hazard ratio (HR) = 0.80 95% CI 0.64–0.98). Similar associations were maintained in ER+ tumours in univariate and multivariate analysis (p < 0.01) both in patients with and without adjuvant endocrine or chemotherapy. AR mRNA expression showed significant association with tumour grade, molecular subtypes, and longer 10 and 15 years survival in luminal BC. In the external validation cohorts, AR gene expression data was associated with improved patients’ outcome (p < 0.001, HR = 0.84, 95% CI 0.79−0.90). Conclusion: AR is abundantly expressed in invasive BC with significant association with favourable prognostic parameters both at protein and transcriptomic levels. It is an independent prognostic factor and can further stratify the patient into distinct prognostic subgroups significantly different in outcome. There is interaction between AR and ER and proliferation, which may explain the differential prognostic effect of AR in BC. Understanding these biological interactions may help in utilising AR as a molecular therapeutic target in invasive BC. No conflict of interest. 893 Photo-acoustic molecular imaging advancement by unmixing and optical flow method with unfocused ultrasound sonoporation method as improvement in tumor treatment P. Giustetto1 , D. Fuchs2 , P. Trochet2 . 1 Freedom Waves S.R.L., Research, Turin, Italy, 2 FUJIFILM Visualsonics, Pre Clinical Applications Manager, Amsterdam, Netherlands Background: Photoacoustic imaging (PAI) demonstrated a wide potential to obtain simultaneously structural, functional and molecular information. Liposomes are clinically used as nanocarriers for many drugs, including chemotherapeutics as theranostic agents. The aim of this work was to assess changes in tumor vascular dynamics with the use of PAI, followed by the systemic injection liposomes, and to show the differences on nanosystem’s flows with and without sonoporation treatments applied on tumoral masses In the image analysis, an important role is made by spectral Unmixing Software Tools (UTS) and optical flow (OF). OF can estimate the entropy behavior, optic flow vectors, and apparent velocities of the molecules in a time dependent manner. Material and Method: Ten Balb/C bearing a subcutaneous syngeneic breast cancer (TS/A) were enrolled in the study: five of them have been exposed to sonoporation treatment immediately after liposome injection (t = 0). Sonoporation was carried out by using a system with a non-focused 1 MHz transducer and low acoustic intensity (Freedom Waves S.R.L.) with an insonation time of 40 sec. The mice were injected i.v. with 150 ml of PEGylated liposomes (120 nm diameter). The liposomes were labeled with near-infrared Cy5.5 dye in order to pick up the PAI signal. The image acquisition with VEVO LAZR system (VisualSonics Inc.), equipped with a highly sensitive photo-acoustic transducer at a frequency of 21 MHz, consisted in: (1) application of sequential laser pulses at 750 and 850 nm to obtain PAI of the tissue vasculature: this method allowed for quantification of Oxygen saturation (sO2) and total hemoglobin (HbT); (2) scanning tumor in the range 680–970 nm to get a whole spectra profile. The maximum liposomes flow value, measured between 1 and 90 minutes after injection and it was quantified using an OF Matlab code. Results: An increase in the values for OF with sonoporation apply can be observed. An obvious increment of the flow can be directly observed from the images obtained post Matlab code processing, applied in order to calculate the OF. Moreover, thanks to the analysis of sO2 and HbT, we have been able to observe changes of these two parameters in the total tumor mass and in particular, in the hypoxic areas. Significant variation showed borne by microenvironment behavior, in particular, for divergence and vorticity of tumor blood flow. Conclusion: The results show a great improvement in oxygenation and flow only when sonoporation treatment was applied. Liposome flow has increased by 51.2% in the tumoral mass and increasing values sO2, HbT in hypoxic portion are observed. Non-invasive ultrasound sonoporation as tumor treatment method causing transient cell membranes poration and PAI, represents a powerful combination with great potential as clinical applications to improve effectiveness of cancer treatment. No conflict of interest. 894 Mathematical biology framework for exploring heterogeneous drug responses within a patient H. Mistry1 . 1 University of Manchester, Pharmacometrics, Manchester, United Kingdom Background: In the metastatic disease setting it is well known that there is heterogeneity within each tumour within each patient. This level of heterogeneity makes developing preclinical animal models of disease
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Results: Nuclear AR IHC expression was significantly associated with features of good prognosis including older age, smaller tumour size, lower grade and lobular histology particularly in the ER-positive tumours. AR was associated with ER-related markers GATA3, FOXa1, RERG and BEX1. Negative association was observed with HER2, p53, Ki67, TK1, CD71 and AGTR1. AR Overexpression was associated with longer survival (p < 0.001), independent of tumour size, grade, and axillary nodal stage (p = 0.033, hazard ratio (HR) = 0.80 95% CI 0.64–0.98). Similar associations were maintained in ER+ tumours in univariate and multivariate analysis (p < 0.01) both in patients with and without adjuvant endocrine or chemotherapy. AR mRNA expression showed significant association with tumour grade, molecular subtypes, and longer 10 and 15 years survival in luminal BC. In the external validation cohorts, AR gene expression data was associated with improved patients’ outcome (p < 0.001, HR = 0.84, 95% CI 0.79−0.90). Conclusion: AR is abundantly expressed in invasive BC with significant association with favourable prognostic parameters both at protein and transcriptomic levels. It is an independent prognostic factor and can further stratify the patient into distinct prognostic subgroups significantly different in outcome. There is interaction between AR and ER and proliferation, which may explain the differential prognostic effect of AR in BC. Understanding these biological interactions may help in utilising AR as a molecular therapeutic target in invasive BC. No conflict of interest. 893 Photo-acoustic molecular imaging advancement by unmixing and optical flow method with unfocused ultrasound sonoporation method as improvement in tumor treatment P. Giustetto1 , D. Fuchs2 , P. Trochet2 . 1 Freedom Waves S.R.L., Research, Turin, Italy, 2 FUJIFILM Visualsonics, Pre Clinical Applications Manager, Amsterdam, Netherlands Background: Photoacoustic imaging (PAI) demonstrated a wide potential to obtain simultaneously structural, functional and molecular information. Liposomes are clinically used as nanocarriers for many drugs, including chemotherapeutics as theranostic agents. The aim of this work was to assess changes in tumor vascular dynamics with the use of PAI, followed by the systemic injection liposomes, and to show the differences on nanosystem’s flows with and without sonoporation treatments applied on tumoral masses In the image analysis, an important role is made by spectral Unmixing Software Tools (UTS) and optical flow (OF). OF can estimate the entropy behavior, optic flow vectors, and apparent velocities of the molecules in a time dependent manner. Material and Method: Ten Balb/C bearing a subcutaneous syngeneic breast cancer (TS/A) were enrolled in the study: five of them have been exposed to sonoporation treatment immediately after liposome injection (t = 0). Sonoporation was carried out by using a system with a non-focused 1 MHz transducer and low acoustic intensity (Freedom Waves S.R.L.) with an insonation time of 40 sec. The mice were injected i.v. with 150 ml of PEGylated liposomes (120 nm diameter). The liposomes were labeled with near-infrared Cy5.5 dye in order to pick up the PAI signal. The image acquisition with VEVO LAZR system (VisualSonics Inc.), equipped with a highly sensitive photo-acoustic transducer at a frequency of 21 MHz, consisted in: (1) application of sequential laser pulses at 750 and 850 nm to obtain PAI of the tissue vasculature: this method allowed for quantification of Oxygen saturation (sO2) and total hemoglobin (HbT); (2) scanning tumor in the range 680–970 nm to get a whole spectra profile. The maximum liposomes flow value, measured between 1 and 90 minutes after injection and it was quantified using an OF Matlab code. Results: An increase in the values for OF with sonoporation apply can be observed. An obvious increment of the flow can be directly observed from the images obtained post Matlab code processing, applied in order to calculate the OF. Moreover, thanks to the analysis of sO2 and HbT, we have been able to observe changes of these two parameters in the total tumor mass and in particular, in the hypoxic areas. Significant variation showed borne by microenvironment behavior, in particular, for divergence and vorticity of tumor blood flow. Conclusion: The results show a great improvement in oxygenation and flow only when sonoporation treatment was applied. Liposome flow has increased by 51.2% in the tumoral mass and increasing values sO2, HbT in hypoxic portion are observed. Non-invasive ultrasound sonoporation as tumor treatment method causing transient cell membranes poration and PAI, represents a powerful combination with great potential as clinical applications to improve effectiveness of cancer treatment. No conflict of interest. 894 Mathematical biology framework for exploring heterogeneous drug responses within a patient H. Mistry1 . 1 University of Manchester, Pharmacometrics, Manchester, United Kingdom Background: In the metastatic disease setting it is well known that there is heterogeneity within each tumour within each patient. This level of heterogeneity makes developing preclinical animal models of disease