Dalila Labiod

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient‐derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT‐PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple‐negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanibs main targets.

Targeting mTOR pathway inhibits tumor growth in different molecular subtypes of triple-negative breast cancers

Triple-negative breast cancers (TNBC) are characterized by frequent alterations in the PI3K/AKT/mTOR signaling pathway. In this study, we analyzed PI3K pathway activation in 67 patient-derived xenografts (PDX) of breast cancer and investigated the anti-tumor activity of the mTOR inhibitor everolimus in 15 TNBC PDX with different expression and mutational status of PI3K pathway markers. Expression of the tumor suppressors PTEN and INPP4B was lost in 55% and 76% of TNBC PDX, respectively, while mutations in PIK3CA and AKT1 genes were rare. In 7 PDX treatment with everolimus resulted in a tumor growth inhibition higher than 50%, while 8 models were classified as low responder or resistant. Basal-like, LAR (Luminal AR), mesenchymal and HER2-enriched tumors were present in both responder and resistant groups, suggesting that tumor response to everolimus is not restricted to a specific TNBC subtype. Analysis of treated tumors showed a correlation between tumor response and post-treatment phosphorylation of AKT, increased in responder PDX, while PI3K pathway markers at baseline were not sufficient to predict everolimus response. In conclusion, targeting mTOR decreased tumor growth in 7 out of 15 TNBC PDX tested. Response to everolimus occurred in different TNBC subtypes and was associated with post-treatment increase of P-AKT.

SU-G-TeP3-09: Proton Minibeam Radiation Therapy Increases Normal Tissue Resistance

PURPOSE The dose tolerances of normal tissues continue being the main limitation in radiotherapy. To overcome it, we recently proposed a novel concept: proton minibeam radiation therapy (pMBRT) [1]. It allies the physical advantages of protons with the normal tissue preservation observed when irradiated with submillimetric spatially fractionated beams (minibeam radiation therapy) [2]. The dose distributions are such that the tumor receives a homogeneous dose distribution, while normal tissues benefit from the spatial fractionation of the dose. The objective of our work was to implement this promising technique at a clinical center (Proton therapy center in Orsay) in order to evaluate the potential gain in tissue sparing. METHODS Dose distributions were measured by means of gafchromic films and a PTW microdiamond detector (60019). Once the dosimetry was established, the whole brain of 7 weeks old male Fischer 344 rats was irradiated. Half of the animals received conventional seamless proton irradiation (25 Gy in one fraction). The other rats were irradiated with pMBRT (58 Gy peak dose in one fraction). The average dose deposited in the same targeted volume was in both cases 25 Gy. RESULTS The first complete set of dosimetric data in such small proton field sizes was obtained [3]. Rats treated with conventional proton irradiation exhibited severe moist desquamation and permanent epilation afterwards. The minibeam group, on the other hand, exhibited no skin damage and no clinical symptoms. MRI imaging and histological analysis are planned at 6 months after irradiation. CONCLUSION Our preliminary results indicate that pMBRT leads to an increase in tissue resistance. This can open the door to an efficient treatment of very radioresistant tumours. [1] Prezado et al. Med. Phys. 40, 031712, 1-8 (2013).[2] Prezado et al., Rad. RESEARCH 184, 314-21 (2015). [3] Peucelle et al., Med. Phys. 42 7108-13 (2015).

Abstract 1687: Vandetanib as a potential new treatment for ER negative breast cancers

Introduction: Recent studies have shown that the receptor tyrosine kinase RET is involved in the biology of ER positive breast cancers and in the response to endocrine treatment, but its role in ER negative tumors is unknown. Here we investigated the expression of RET in BC patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib in ER negative BC PDX. Methods: RET mRNA expression was analyzed in BC of 446 patients and 57 PDX by RT-PCR analysis. The activity of Vandetanib, a tyrosine kinase inhibitor targeting RET, EGFR and VEGFR2, was tested in three PDX of triple-negative breast cancer (TNBC) and one PDX of HER2+ BC with different levels of RET expression. Protein expression of P-RET, RET, EGFR, P-EGFR and c-KIT were determined by immunohistochemistry (IHC). Analyses of PI3K and MAPK pathways and angiogenesis were performed by IHC and RT-PCR in both untreated and Vandetanib-treated tumors. Results: In both clinical samples and PDX, elevated levels of RET were found in ER+ and HER2+ tumors, and in a subgroup of TNBC tumors. In the HBCx5 (HER2+) and HBCx24 (TNBC) PDX, both with RET over-expression, treatment by Vandetanib resulted in tumor growth inhibition (TGI) of 90% and 98%, respectively. In both models, tumor regressions were observed in 50% of xenografts. The effect of Vandetanib was associated to a marked inhibition of RET phosphorylation. To determine whether the lack of RET over-expression was associate to Vandetanib resistance, we treated two additional TNBC PDX with low and no expression of RET: HBCx4B and HBCx14. In these models, treatment by Vandetanib still inhibited tumor growth with a TGI of 85%. Tumor regressions were registered in 42% of animals in the PDX model with low expression of RET (HBCx4B), while no tumor regression were observed in HBCx14. IHC analyses showed an over-expression of EGFR in the HBCx4B xenograft and inhibition of EGFR phosphorylation in treated tumors, suggesting that tumor response to Vandetanib could depend on EGFR inhibition in this tumor. Further analyses of treated tumors revealed a decreased expression of phospho-ERK in the 4 PDX models, indicating inhibition of MAPK pathway, while the phosphorylation status of the PI3K pathway markers S6 and 4EBP1 was unchanged. Finally, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the 4 PDX tested, indicating angiogenesis inhibition. Conclusions: Treatment by Vandetanib resulted in strong tumor growth inhibition in ER negative PDX with over-expression of RET. This effect was associated to inhibition of RET phosphorylation and MAPK pathway and decreased tumor vascularization. The lack of RET over-expression did not predict Vandetanib resistance, and over-expression of EGFR was also associated to a marked tumor response. These preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers expressing Vandetanib9s targets. Citation Format: Elisabetta Marangoni, Rana Hatem, Dalila Labiod, Sophie Chateau-Joubert, Rania El Botty, Jean-Luc Servely, Ludmilla De Plater, Ivan Bieche. Vandetanib as a potential new treatment for ER negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2015-1687

Abstract 4499: Activation of PI3-kinase pathway and tumor response to everolimus in patient-derived xenografts of triple-negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Patients with triple-negative breast cancer (TNBC) have a poor prognosis and targeted therapies are lacking. Recent studies performed on patients tumors showed and increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway in TNBC. PI3K pathway is critical for cell growth, survival, and angiogenesis. Everolimus is a mTOR inhibitor recently showed to increase survival of patients with metastatic luminal breast cancer. The objectives of this work were to analyze the PI3K activation status in a large cohort of patient-derived xenografts (PDX) of TNBC and to investigate the therapeutic potential of mTOR inhibition. Experimental procedures: this study included a panel of 32 TNBC PDX models previously described (Marangoni et al 2007). Expression of AKT, P-AKT, P-mTOR, S6, P-S6, P-4EBP1, PTEN and INPP4B was analyzed by WB and IHC. Mutations of PIK3CA (exons 9 and 20), PIK3R1 (exons 11-15), and AKT1 (exon 4) were detected by sequencing of cDNA fragments obtained by RT-PCR amplification. The efficacy of the mTOR inhibitor everolimus was investigated in vivo on 10 PDX models with different expressions and mutational status of PI3K markers. Results: INPP4B protein expression was lost in 56% of tumors (n=18) and expressed at low levels in 31% of models, while only 2 models displayed a marked expression. PTEN expression was lost in 78% of tumors. Thirteen PDX models (40%) displayed a concomitant loss of both INPP4B and PTEN proteins. In 67% of tumors, the ratio between phosphorylated and unphosphorylated AKT was greater than 1. S6 was found to be phosphorylated in the great majority of tumors. PI3KCA and AKT1 genes were mutated only in 1 and 2 tumors, respectively. On the 10 PDX models treated with everolimus, 6 models responded to treatment with a tumor growth inhibition (TGI) comprised between 60% and 80%. Four models were classified as resistant or low responder (TGI<50%). Preliminary analysis of treated tumors from 6 models indicates increased level of P-AKT (feedback loop) to occur only in responder models, while inhibition of S6 phosphorylation occurred in treated tumors from both responder and resistant models. Finally, expression of INPP4B or PTEN alone did not predict for tumor response, while a P-AKT/AKT ratio greater than 1 predicted response to everolimus (p<0.05, Fishers exact test). Conclusions: the majority of TNBC PDX models showed loss of PTEN or INPP4B proteins or both, associated with activation of PI3K pathway. Preliminary results obtained from 10 PDX models indicate that mTOR targeting resulted in tumor growth inhibition in several models with AKT phosphorylation. Additional TNBC models will be tested in order to search for robust predictive biomarkers. This large panel of characterized PDX of TNBC models represents a clinical relevant tool to investigate the activity of PI3K-AKT-mTOR inhibitors and identify predictive biomarkers. Citation Format: Elisabetta Marangoni, Rana Hatem, Rania El Botty, Ludmilla De Plater, Dalila Labiod, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bieche. Activation of PI3-kinase pathway and tumor response to everolimus in patient-derived xenografts of triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2014-4499

Abstract A9: Establishment and characterization of residual breast cancer patient-derived xenografts resistant to neo-adjuvant therapy.

Background: In HER2 positive and triple-negative breast cancer subgroups, residual disease after neoadjuvant therapy is associated with higher risk of metastatic recurrence compared to patients achieving a pathological complete response. Residual tumor analysis after neoadjuvant treatment is a major and under-explored field to identify resistance mechanisms. To develop patient-derived xenografts (PDX) of residual breast cancer we started a program of residual tumor engraftment in nude mice, following the same procedures previously published for PDX of human breast cancer (Marangoni et al, 2007 and Reyal et al, 2012). Methods: 26 residual breast tumors and 2 residual metastatic axillary lymph nodes were engrafted in swiss nude mice immediately after surgery. Expression of Ki67, HER2, PTEN, P-AKT, P-S6, MET, RET and KIT were analyzed in xenografts by immunohistochemistry, western blot and RT-PCR analyses. Brain, lungs, liver and bones of xenografts were systematically formalin-fixed to search for human metastasis. The in vivo drug response of established xenografts was determined for the following treatments: adryamicin+cyclophosphamide (AC), docetaxel, capecitabine, cisplatin, irinotecan, everolimus, trastuzumab and lapatinib (for the HER2+ PDX). PDX tumors were additionally mechanically dissociated to establish cell lines. Results: Seven PDX were established (tumor take of 25%), 5 triple-negative and 2 HER2+. Six out of seven PDX were metastatic in the lungs. Two xenografts were established from lymph node metastasis. The in vivo drug responses were concordant with the response to neo-adjuvant treatments in patients. Histological analyses showed that xenografts’ tumors recapitulated the patients’ tumor morphology. Residual tumor xenografts expressed high level of Ki67 protein and tumor latency during the first tumor passages was found to be shorter when compared to tumor latency of non pre-treated breast cancers. In 5/5 triple-negative breast cancer PDX the PTEN protein was lost and the PI3 kinase pathway activated. The mTOR inhibitor Everolimus was tested in 2 triple-negative PDX: one was resistant and one was responding, with a tumor growth inhibition of 80%. Triple-negative PDX show expression of “druggable” tyrosin kinase receptors (MET, RET, KIT) providing relevant models to test new target therapies in these models. One cell line was established from a highly metastatic triple-negative breast cancer xenograft. When re-injected into mice, the cell line was tumorigenic, however the tumor architecture was changed and the xenograft was not metastatic. Conclusions: we have established a panel of metastatic PDX models of breast cancer resistant to neo-adjuvant therapies. These models provide a valuable preclinical tool to investigate mechanisms of resistance to neo-adjuvant treatments and for the preclinical testing of new targeted agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A9. Citation Format: Elisabetta Marangoni, Dalila Labiod, Franck Assayag, Rania El Botty, Rana Hatem, Sophie Richon, Sophie Chateau-Joubert, Marine Carlus, Helene Bonsang-Kitzis, Alice Pinheiro, Cecile Laurent, Ivan Bieche, Fabien Reyal. Establishment and characterization of residual breast cancer patient-derived xenografts resistant to neo-adjuvant therapy. [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 A9.

Abstract LB-111: Mastospheres as a new 3D ex vivo breast cancer microtumors for preclinical drug testing

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL There is increasing evidence that three-dimensional (3D) tissue culture technologies have many uses within the preclinical assays in cancer. The 3D tumor model accurately reproduces the in vivo tumor phenotype and represents an additional tool for studying tumor biology and allowing better preclinical evaluation of anticancer drugs. One of the used models involves small tumor aggregates, termed spheres, that are obtained from mechanical tumor dissociation and that have shown the superiority of 3D culture over standard two-dimensional cell culture for mimicking the tumor biology and drug response observed in vivo. The aim of this study was to generate new ex vivo 3D models from breast (BC) cancer xenografts established from patients’ tumor fragments. These ex vivo mastospheres are easily obtained from mechanically dissociated fresh human BC tissue xenografted in Nude mice, in a similar way as colospheres from human colon cancer (Weiswald et al, Br J Cancer 2009, 101:473). In contrast to mammospheres described in the literature, 3D mastospheres are obtained from tumor fragments, without enzymatic tumor dissociation, without matrix substratum and in SVF supplemented medium. From a large panel of patient-derived BC xenografts already well characterized (Marangoni et al, Clin Cancer Res 2007, 13:3989; Cottu et al, Breast Cancer Res Treat 2011; Reyal et al, Breast Cancer Res 2012, 14:R11), we get mastospheres from 26 out of 36 (72%) xenografts. Mastosphere formation is scored on day 1 after culture according to the number of spheres/mg of dissociated xenograft tissue. Within mastospheres, we clearly distinguish 3 distinct morphologies: round, grape-like and aggregates. Histological analyses show also that mastospheres were formed only with proliferating cancer cells. In a reproducible way, dissociation of a given xenograft leads to a similar score and to a similar morphology. All BC subtypes (luminal, triple negative and HER2+ tumors) give rise to mastospheres. We noted that the 5 out of 5 xenografts (100%) able to metastasise in Nude mice (lung metastasis) form mastospheres, suggesting that the capacity to give mastospheres could be related to tumour aggressiveness, as already reported with colospheres and colon cancer. These different features prompted us to test the potential of these mastospheres in chemosensitivity assays. We first demonstrated that mastospheres can be kept viable a couple of days, consistent with ex vivo assay aim. For this purpose, we tested the sensitivity to cisplatin in two xenograft models with different in vivo response. We found that the paired mastospheres tested in viability assays mimicked these different response profiles (mean of IC50: 1.2 µM versus 15.5 µM). In conclusion, according to these preliminary data, mastospheres deserve additional investigation because of their interest as new ex vivo microtumour model. 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 LB-111. doi:1538-7445.AM2012-LB-111

Abstract A15: Establishment and characterization of a new patient-derived non-small cell lung cancer xenograft panel for pharmacological assessment.

Background: Development of targeted therapies in non-small cell lung carcinoma (NSCLC) is rapidly growing and offers promising treatment perspectives for cancer patients. However, the assessment of new therapeutic compounds requires preclinical models defined by precise tumor molecular features that could impact their antitumor efficacy. The aim of this study was then to develop and characterize a new reliable panel of human NSCLC xenografts. Material and methods: From 28 samples of NSCLC specimens obtained from patient9s surgery and grafted into Swiss nude mice, 18 transplantable xenografts were established (64%). Molecular characterization included histological features, gene mutation status (EGFR, KRAS, and BRAF), and research of ALK translocation in both xenografts and their corresponding patient9s tumors. For determination of the predictive value of the models, a standard pharmacological assessment was then performed in 6 adenocarcinoma (ADK) models using the EGFR inhibitor erlotinib (50 mg/kg/day, 5 days/week, 4 weeks, orally). Tumor Growth Inhibition (TGI) was calculated to measure the efficiency of erlotinib. Finally, to evaluate responses to erlotinib according to individual mouse variability, we decided to consider each mouse as one tumor-bearing entity. In all in vivo experiments, a relative tumor volume variation (RTVV) of each erlotinib-treated mouse was calculated from the following formula: Vt/Vc, where Vt is the volume of the treated mouse and Vc the median volume of the corresponding control group at a time corresponding to the end of treatment. An overall response rate (ORR) was then calculated for both EGFR-mutated and -non mutated tumors. Results: Among the 18 established models, we have diagnosed 9 ADK, 6 epidermoid carcinomas, and 3 undifferientiated large cell cancers, with a high degree of similarity with the originated patient9s tumors in regard to histological characteristics (Morphology and IHC), and a complete concordance for gene mutation status. Among the 18 NSCLC xenografts, 3 were EGFR mutated (exon 18 G719A, exon 19 del18b, and exon 21 L858R) and 3 others were KRAS mutated (G12C). No BRAF mutation nor ALK translocation were found. Erlotinib induced a TGI of 30%, 62%, and 93% for the 3 EGFR-mutated xenografts, and 0% (preliminary result), 37%, and 52% (KRAS-mutated model) for the 3 wild type EGFR tumors. Moreover, the individual mouse ORR ORR ≥ 90% was 26% in the first group and 4% in the second one, respectively (p Conclusions: We have developed and characterized a new panel of primary human NSCLC xenografts that is highly representative of the heterogeneity of the disease and responses to the anti-EGFR targeted therapy, and provides precious tools to assess innovative therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A15.