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The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

List of genes differentially regulated between resistant and responder PDX.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX.Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605–15. ©2018 AACR.

Boxplot of the expression of genes encoding proteins involved in capecitabine metabolism (TYMP, TYMS, TK1, DPYD) and cell-cycle control (RB1, CDKN2A, CCND1).

Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC.Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays.Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival.Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Supplementary Table S7. Upstream regulators identified by the Upstream Regulator Analysis in HBCx22 TamR and HBCx34 TamR xenografts.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient's outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

Chordomas are rare neoplasms characterized by a high recurrence rate and a poor long-term prognosis. Considering their chemo-/radio-resistance, alternative treatment strategies are strongly required, but their development is limited by the paucity of relevant preclinical models. Mutations affecting genes of the SWI/SNF complexes are frequently found in chordomas, suggesting a potential therapeutic effect of epigenetic regulators in this pathology. Twelve PDX models were established and characterized on histological and biomolecular features. Patients whose tumors were able to grow into mice had a statistically significant lower progression-free survival than those whose tumors did not grow after in vivo transplantation (p = 0.007). All PDXs maintained the same histopathological features as patients’ tumors. Homozygous deletions of CDKN2A/2B (58.3%) and PBRM1 (25%) variants were the most common genomic alterations found. In the tazemetostat treated PDX model harboring a PBRM1 variant, an overall survival of 100% was observed. Our panel of chordoma PDXs represents a useful preclinical tool for both pharmacologic and biological assessments. The first demonstration of a high antitumor activity of tazemetostat in a PDX model harboring a PBRM1 variant supports further evaluation for EZH2-inhibitors in this subgroup of chordomas.

Malignant adenomyoepithelioma (AME) of the breast is an exceptionally rare form of breast cancer, with a significant metastatic potential. Chemotherapy has been used in the management of advanced AME patients, however the majority of treatments are not effective. Recent studies report recurrent mutations in the HRAS Q61 hotspot in small series of AMEs, but there are no preclinical or clinical data showing H-Ras protein as a potential therapeutic target in malignant AMEs. We performed targeted sequencing of tumours’ samples from new series of 13 AMEs, including 9 benign and 4 malignant forms. Samples from the breast tumour and the matched axillary metastasis of one malignant HRAS mutated AME were engrafted and two patient-derived xenografts (PDX) were established that reproduced the typical AME morphology. The metastasis-derived PDX was treated in vivo by different chemotherapies and a combination of MEK and BRAF inhibitors (trametinib and dabrafenib). All malignant AMEs presented a recurrent mutation in the HRAS G13R or G12S hotspot. Mutation of PIK3CA were found in both benign and malignant AMEs, while AKT1 mutations were restricted to benign AMEs. Treatment of the PDX by the MEK inhibitor trametinib, resulted in a marked anti-tumor activity, in contrast to the BRAF inhibitor and the different chemotherapies that were ineffective. Overall, these findings further expand on the genetic features of AMEs and suggest that patients carrying advanced HRAS-mutated AMEs could potentially be treated with MEK inhibitors.

Background Endocrine therapies targeting estrogen signaling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signaling, estrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumor response in vivo. Methods The interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analyzed their anti-proliferative effects on 6 ERα+ and 3 ERα− PDX models. Genomic and non-genomic estrogen signaling were assessed by measuring ERα/PI3K interaction by PLA and the expression of estrogen target genes by RT-QPCR, respectively. Results We confirmed that ERα/Src and ERα/PI3K interactions were associated with a trend to poorer survival, the latter displaying the most significant effects. In ERα+ tumors, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status, or ERα/PI3K targeting. Remarkably, resistance to fulvestrant was associated with non-genomic ERα signaling, since genomic degradation of ERα was unaltered in these tumors, whereas the treatment did not diminish the level of ERα/PI3K interaction. Interestingly, in 2 ERα− models, fulvestrant alone impacted tumor growth, and this was associated with a decrease in ERα/PI3K interaction. Conclusions Our results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant in ERα+ tumors was associated with a lack of impairment of ERα/PI3K interaction in the cytoplasm. In addition, an efficient targeting of ERα/PI3K in ERα− tumors could constitute a promising therapeutic option.

Background: Chordomas are considered as rare bone neoplasms characterized by a high level of recurrence and a poor long-term prognosis. Considering their chemo-radio-resistance, alternative treatment strategies are therefore strongly requested, but their development is limited by the paucity of relevant preclinical models. Mutations affecting genes of the SWI/SNF complex including PBMR1 are frequently found in chordomas, suggesting a potential therapeutic effect of epigenetic regulators in this indication. Methods: Thirty-eight chordoma specimens derived from primary patients’ tumors were grafted into nude mice. Relationship between grafted tumors and clinical, biological and therapeutic features of corresponding patients were investigated. We subjected the established PDX models to histopathological and genetic examination; finally, a therapeutic program focused on the EZH2-inhibition was performed in one PDX model. Results: Twelve PDX models were established and characterized on histological and biomolecular features. Patients whose tumors were able to grow into mice had a statistically significant lower progression-free survival than those whose tumors did not grow after in vivo transplantation (p=0.007). All PDXs maintained the same histopathological features as patients’ tumors. Homozygous deletions of CDKN2A/2B (58.3%) and PBRM1 (25%) variants were the most common genomic alterations found. In the tazemetostat treated PDX model harboring a PBRM1 variant, an overall survival of 100% was observed. Conclusion: Our panel of chordoma PDXs represents a useful preclinical tool for both pharmacologic and biological assessments. The first demonstration of a high antitumor activity of tazemetostat in a PDX model harboring a PBRM1variant supports further evaluation of EZH2-inhibitors in this subgroup of chordomas. Citation Format: Thibault Passeri, Ahmed Dahmani, Julien Masliah-Planchon, Adnan Naguez, Marine Michou, Rania El-Botty, Sophie Vacher, Rachida Bouarich, André Nicolas, Marc Polivka, Coralie Franck, Anne Schnitzler, Fariba Némati, Sergio Roman-Roman, Franck Bourdeaut, Homa Adle-Biassette, Hamid Mammar, Sébastien Froelich, Ivan Bièche, Didier Decaudin. Dramatic in vivo efficacy of the EZH2-inhibitor tazemetostat in PBRM1-mutated human chordoma xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB567.

Background: Onapristone (ONA) is a progesterone receptor (PR) antagonist that prevents PR-mediated DNA transcription. ONA is currently being evaluated in metastatic breast cancers (MBC), as well as other hormone-dependent cancers. Recently, therapeutic options for metastatic estrogen receptor (ER) positive breast cancers have been expanded by the introduction of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), administered in combination with endocrine treatments, as first line therapy. Moreover, the selective PI3K inhibitor alpelisib (ALP) has been approved for the treatment of PIK3CA-mutated endocrine resistant MBC. The objective of this study was to evaluate the efficacy of ONA in combination with fulvestrant (FUL) and palbociclib (PAL) or ALP in different PDX models established from ER and PR positive breast cancers. Methods: PDX models were established from primary tumors or biopsies from bone metastases from endocrine therapy patients with progressing tumors. ER and PR expression were analyzed by immunohistochemistry (IHC) and western blot (WB) analysis in 17 PDX models. The anti-tumor activity of onapristone alone or combined with FUL and PAL was tested in 2 ER+/PR+ PDX studies, while the combination with ALP+FUL was tested in one PIK3CA-mutated PDX study. Phosphorylation of both PR and ER-PR interactions were analyzed in treated tumors by WB and by the in situ Proximity Ligation Assay (PLA), respectively. Results: PR expression was found in 1/9 PDX established from primary breast tumors and 4/8 PDX established from bone metastases. ONA in vivo activity was tested in 2 PDX of bone metastases: PDX BC1101 (PR low) and BC1117 (PR high). BC1101 showed amplification of FGFR1 and CCND1 genes, while BC1117 has an activating mutation of PIK3CA gene. In the low PR PDX BC1101, treatment with ONA was ineffective in both the monotherapy and combination setting. Conversely, in the PDX with high expression of PR, BC1117, ONA treatment, given as monotherapy, decreased tumor growth. The anti-tumor activity of ONA+FUL+PAL combination was significantly increased as compared to FUL+PAL, with the majority of xenografts showing tumor regression. Expression of PR and phospho-PR were inhibited in the tumors treated by FUL, ONA+FUL, and ONA+FUL+PAL. Interaction between ER and PR, analyzed by the in situ PLA assay, was inhibited in the ONA+FUL treated xenografts ±PAL. Finally, treatment with the triple combination of ONA+FUL+ALP was also highly effective and significantly greater than the combination of FUL+ALP. RNAseq analysis of treated xenografts is ongoing to identify transcriptomic changes in the different treatment arms. In conclusion, our study demonstrates that ONA improved the response to endocrine treatment using CDK4/6 or PI3K inhibitors in a MBC-derived PDX model with high PR expression. Citation Format: Martin Lehr, Ahmed Dahmani, Léa Huguet, Rania El-Botty, Charlène Thiebaut, Muriel Romancer, Paul Cottu, Elisabetta Marangoni. Targeting progesterone receptor (PR) with the antiprogestin onapristone in patient-derived xenograft (PDX) models of estrogen receptor positive (ER+), PR positive (PR+) bone metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 649.

International audience; Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient’s outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment., Identifying novel therapies for the treatment of CDK4/6 inhibitor-resistant patients is of great importance. Here, the authors demonstrate that PLK1 inhibition is a potential therapeutic target in CCND1-driven and in RB-positive Palbociclib-resistant breast cancers.

Background Endocrine therapies targeting oestrogen signalling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signalling, oestrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumour response in vivo.Methods The interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analysed their anti-proliferative effects on 6 ERα + and 3 ERα- PDX models. Genomic and non-genomic oestrogen signalling were assessed by measuring ERα/PI3K interaction by PLA or the expression of key oestrogen target genes by RT-QPCR, respectively.Results We confirmed that ERα/Src and especially ERα/PI3K interaction were associated with a trend to poorer survival. In ERα + tumours, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status or ERα/PI3K targeting. Interestingly, in some ERα- models, fulvestrant alone impacted tumour growth and this was associated with a decrease in ERα/PI3K interaction.Conclusions Our results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant was clearly associated with a lack of ERα/PI3K targeting in the cytoplasm.

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma. IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.

Breast cancer is a complex disease in which each patient could present several genetic alterations that are therapeutically relevant in cancers. Here we explored the therapeutic benefit of combining PARP and mTOR inhibitors in a context of DNA repair deficiency and PI3K pathway activation. The combination of everolimus and olaparib was tested in BRCA2-mutated patient-derived xenografts (PDX) carrying alterations in the PI3K/AKT/mTOR pathway. An RPPA analysis of different signalling pathways was performed in untreated and treated xenografts. Everolimus and olaparib showed marked anti-tumor activities in the monotherapy setting and high efficacy when given in combination with 100% of mice showing tumor regressions. The fraction of P-H2AX positive cells was increased in both monotherapy arms and strongly increased in the combination setting. Everolimus given as monotherapy resulted in downregulation of different proteins involved in DNA damage repair, including FANCD2, RAD50 and SUV39H1. In the combination setting, expression of these proteins was almost completely abolished, suggesting convergence of PARP and mTOR in downregulation of DNA damage repair components. In conclusion, our results suggest that combining mTOR and DNA repair inhibition could be a successful strategy to treat a subset of breast cancer with BRCA2 mutation and alterations in the PI3K/AKT/mTOR pathway.

The prognosis of metastatic uveal melanoma (UM) is among the worst of all human cancers. The identification of near-ubiquitous GNAQ/GNA11 mutations and the activation of MAPK signaling in UM have raised hopes of more effective, targeted therapies, based on MEK inhibition, for example. We evaluated the potential of drug combinations to increase the efficacy of the MEK inhibitor selumetinib (AZD6244, ARRY-142886), in UM cell lines and Patient-Derived Xenografts. We first evaluated the combination of selumetinib and DTIC. We found that DTIC did not improve the in vitro or in vivo antitumor efficacy of selumetinib, consistent with the outcome of the SUMIT clinical trial assessing the efficacy of this combination in UM. We then tested additional selumetinib combinations with the chemotherapy agent docetaxel, the ERK inhibitor AZ6197, and the mTORC1/2 inhibitor, vistusertib (AZD2014). Combinations of selumetinib with ERK and mTORC1/2 inhibitors appeared to be the most effective in UM PDX models.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with BAP-1 (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient&rsquo, s tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM.

Summary Nanodiamonds (NDs) are emerging delivery systems with biomedical applications and interesting perspectives in oncology. Their use has been proposed to assist the internalization of anticancer drugs and to decrease administered drug doses. The pro-apoptotic peptide ERα17p, which is issued from the hinge/N-terminus parts of the AF2 region of the human estrogen receptor α (ERα), is active at a concentration of 10 μM on breast cancer cells and particularly on those cancer cells that are ERα-positive. We have synthesized ND@ERα17p conjugates by physisorption of the cationic peptide ERα17p on the surface of anionic NDs. Resulting ND@ERα17p suspensions were characterized by far-UV electronic circular dichroism (ECD), dynamic light scattering (DLS) and zetametry. We then tested the anti-proliferative action of ND@ERα17p on ERα-positive MCF-7 breast carcinoma cells. ND@ERα17p allowed a decrease of the active concentration to 0.1 nM (ND@ERα17p), revealing unambiguously that NDs could be used to improve the anti-proliferative action of this peptide. This preliminary study proposes a novel approach for enhancing the apoptotic action displayed by ERα17p, in the context of breast cancer.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.

Patient-Derived Xenografts (PDXs) are preclinical models largely used to study tumor biology and drug response. Recent literature highlighted the possibility that growth of human tumors in a mouse microenvironment imposes a selection driving mouse-specific genetic evolution of PDXs, which may compromise their reliability as human cancer models. Conversely, independent studies observed a conservation of the genomic landscape during PDX engraftment and passaging. We noticed that PDX genetic evolution was particularly evident in studies based on copy number aberration (CNA) inferred from gene expression data, while it was negligible when DNA-based CNA profiles were employed. Therefore, in a joint international effort of the EurOPDX and PDXNet consortia, we assembled a dataset of 37 hepatocellular and 54 gastric carcinoma tumor or PDX samples with matched RNA-based and DNA-based CNA profiles. We found that DNA-based CNA profiles invariably yield higher concordance between patient's tumor and derived PDXs than those inferred from RNA. RNA-based profiles displayed poor concordance with matched DNA-based profiles, and much lower resolution, so that they missed many focal copy number events detected by DNA-based methods. These results revealed that CNA measurements cannot be accurately estimated by expression data and that a systematic reassessment of CNA dynamics in PDXs based on DNA data is required. To this aim, we generated CNA profiles by low-pass whole genome sequencing (WGS) of 87 colorectal and 43 breast cancer triplets, each composed of matched patient's tumor (PT) and PDX at early (PDX-early) and later (PDX-late) passage. In this way, for each tumor type, we generated three perfectly matched PT, PDX-early and PDX-late cohorts and performed CNA recurrence analysis by GISTIC in each cohort. The hypothesis was that if the mouse host induces a selective pressure capable of shaping the CNA landscape during PDX engraftment and propagation, GISTIC analysis would highlight systematic and progressive changes, from the PT to the PDX-early cohort, and then to the PDX-late cohort. Notably instead, the CNA profiles of the PT and PDX-early/late cohorts were virtually indistinguishable, with no progressive accumulation or loss of CNA during PDX passage and only minor changes not functionally related or associated to cancer-driver or actionable genes. These results were not consequence of insufficient capture of the CNA repertoire, since the GISTIC profiles recapitulated those generated by TCGA for colorectal and breast cancer. In summary, our analyses highlighted that while RNA-based CNA inferences have inadequate resolution and accuracy to study genomic evolution in PDXs, DNA-based CNA profiles confirm retention of CNAs in PTs and PDXs, excluding a systematic mouse driven selection via copy number changes. Ultimately, these results support the robustness of PDXs as preclinical models for predicting drug response. Citation Format: Jessica Giordano, Xing Yi Woo, Anuj Srivastava, Zi-Ming Zhao, Michael W. Lloyd, Roebi de Bruijn, Yun-Suhk Suh, Francesco Galimi, Andrea Bertotti, Adam Lafferty, Alice C. O'Farrell, Elodie Modave, Diether Lambrechts, Petra ter Brugge, Violeta Serra, Elisabetta Marangoni, Rania El Botty, Jong-Il Kim, Han-Kwang Yang, Charles Lee, Dennis A. Dean, Brandi Davis-Dusenbery, Yvonne A. Evrard, James H. Doroshow, Alana L. Welm, Bryan E. Welm, Michael T. Lewis, Bingliang Fang, Jack Roth, Funda Meric-Bernstam, Meenhard Herlyn, Michael Davies, Li Ding, Shunqiang Li, Ramaswamy Govindan, Jeffrey A. Moscow, Carol J. Bult, Claudio Isella, Livio Trusolino, Annette T. Byrne, Jos Jonkers, Jeffrey H. Chuang, Enzo Medico, EurOPDX consortium & PDXNET consortium. Absence of mouse-specific tumor evolution in patient-derived cancer xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1118.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma

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 Vandetanib's main targets.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments. Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX. Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment. Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605–15. ©2018 AACR.

Background: Metastatic estrogen receptor positive (ER+) breast cancers (BC) are genetically heterogeneous and a significant proportion of patients display intrinsic or acquired resistance to endocrine treatments, mTOR and CDK4/6 inhibitors. The objective of this study was to identify new therapies in clinically relevant PDX models of metastatic ER+ BC progressing on endocrine treatment and/or palbociclib. Methods: PDX models were generated by engraftment of spinal bone metastases from 120 BC patients. PDXs were molecularly characterized using SNP-arrays, targeted exome sequencing and global gene expression (GEX) analysis. The anti-tumor efficacy of the PLK1 inhibitor volasertib was evaluated in vivo compared to fulvestrant and palbociclib in two PDX models. PLK1 abundance was analyzed by immunohistochemistry in PDXs and compared with the original primary tumor and bone metastasis from each patient. PLK1 expression measured by RT-PCR was assessed in a cohort of 441 BC patients with 15-year follow-up. GEX analysis of PLK1was assessed in patients treated with neoadjuvant anastrazole. Mechanistic studies were performed in a panel of endocrine resistant BC cell lines. Results: Ten PDX have been established, 8 were ER+ and 2 triple-negative (TN). Genomic alterations included mutations in AKT1, PI3KCA, BRCA2, GATA3, NF1, and amplifications of FGFR1, CCND1 and CCNE2. Comparative pathway analysis of bone metastases derived PDX and patients' primary tumors showed enrichment for pathways associated with mitotic nuclear division, chromatid segregation and G2/M transition. PLK1 was the top commonly up-regulated gene within these pathways. Treatment of an endocrine-resistant ER+ PDX, harboring amplification of CCND1, CCNE2, FGFR1 and high expression of CDK1, showed only partial response to palbociclib and resistance to the FGFR inhibitor, AZD4547. However, PLK1 inhibitor volasertib induced rapid tumor shrinkage and complete response within 5 weeks. Importantly, volasertib was also highly effective when tested as second line therapy in palbociclib pre-treated xenografts. In-vitro inhibition of PLK1 by siRNA or volasertib inhibited tumor proliferation without affecting the expression of ER-regulated genes, suggesting an ER-independent function of PLK1 in regulating cell proliferation. Assessment of the clinical validity of PLK1 expression, revealed a strong association with poor metastases free survival (p Conclusion: We show that inhibition of PLK1 is a new potential treatment strategy for metastatic ER+ BC. Additional experiments are ongoing in PDX and cell lines to investigate G2/M phase dependence of ER+ BC and to identify predictive biomarkers. Citation Format: Elodie Montaudon, Joanna Nikitorowicz-Buniak, Laura Sourd, Rania El Botty, Ahmed Dahmani, Léa Huguet, Ludivine Morriset, Pierre Painsec, Fariba Nemati, Sophie Vacher, Sophie Chateau-Joubert, Sunil Pancholi, Camilla Rega, Ricardo Ribas, André Nicolas, Didier Meseure, Anne Salomon, Zakia Tariq, Keltouma Driouch, Florence Coussy, Guillaume Dutertre, Paul Cottu, Ivan Bièche, Lesley-Ann Martin, Elisabetta Marangoni. PDX models of ER+ endocrine-resistant metastatic breast cancer identify Polo-like kinase 1 (PLK1) as a therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 925.

Background: Acquired resistance to endocrine treatments (ET) occurs in more than 70% of cases of luminal breast cancer (LBC). We used patient derived xenografts (PDX) models of LBC to study molecular changes associated with acquired resistance to different ET modalities. Methods: A PDX model of LBC, established from an early stage BRCA2-mutated breast cancer, was treated with different ET (tamoxifen, fulvestrant, oophorectomy and letrozole) during several months. Tumors escaping to therapies were re-engrafted and maintained under therapy. ET-resistant and parental hormono-responders tumors were analyzed with immunohistochemistry (IHC), RT-PCR and Affymetrix Gene Expression Arrays. Hormono-resistant tumors were additionally studied for their in vivo response to ET, mTOR and PARP inhibitors. Results: From the initially ET sensitive HBCx22 xenograft model (Cottu, BCRT 2012) two resistant models were obtained respectively to tamoxifen (HBCx22-TamR) and to estrogen deprivation (HBCx22-OvaR). Unsupervised clustering of gene expression showed a clear cut separation between parental, TamR and OvaR tumors. Genes differentially expressed in TamR and OvaR tumors compared to parental HBCx22 were only partially overlapping. Up-Regulated genes in both TamR and OvaR tumors (n = 302) were involved in response to wounding, nucleotide metabolism, immune system, adhesion and cell growth. Biological Processes (BP) specifically deregulated in OvaR tumors (n = 380) included embryonic development, antigen presentation, amino acid and lipid metabolism. The top BP specifically regulated in TamR tumors (n = 1059) were response to estrogen and steroid hormones, TGF-b signaling, hypoxia, regulation of cell proliferation, with several strongly up-regulated genes of the histone clusters 1 and 3. Ingenuity Transcription Factor Analysis predicted activation of NFKB, SP1, AP-1 and JUN, and inhibition of ESR1. RT-PCR and IHC analyses confirmed the down regulation of ER controlled genes in the TamR tumors. Expression of ER co-regulators determined by RT-PCR showed that GREB1 was strongly reduced in TamR, while PBX1, GATA3 and FOXA1 were inhibited in OvaR. IHC analysis showed a loss of PTEN expression in HBCx22, with high levels of p-AKT and p-RPS6 in both parental and TamR and OvaR tumors. In vivo ET showed that the TamR xenograft was resistant to all modalities of ET, while OvaR was resistant to estrogen deprivation while retaining some sensitivity to tamoxifen and fulvestrant. Treatment with the mTOR inhibitor RAD001 arrested tumor growth but did not show any additive effect when combined to ET in TamR or OvaR tumors. Conversely, the combination of RAD001 with Olaparib was highly synergistic and induced complete tumor response in 70% of mice. Conclusions: According to the therapeutic selection, tumors derived from a PDX model of ER+ breast cancer show specific resistance patterns and gene expression profiles including disruption in the ER transcriptional program. The analysis of additional resistant tumors established from a second ER+ PDX will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-07.

Patients with triple-negative breast cancer (TNBC) are unresponsive to endocrine and anti-HER2 pharmacotherapy, limiting their therapeutic options to chemotherapy. TNBC is frequently associated with abnormalities in the PI3K/AKT/mTOR signaling pathway; drugs targeting this pathway are currently being evaluated in these patients. However, the response is variable, partly due to heterogeneity within TNBC, conferring a need to identify biomarkers predicting response and resistance to targeted therapy. In this study, we used a metabolomics approach to assess response to the mTOR inhibitor everolimus in a panel of TNBC patient-derived xenografts (PDX) (n = 103 animals). Tumor metabolic profiles were acquired using high-resolution magic angle spinning magnetic resonance spectroscopy. Partial least-squares-discriminant analysis on relative metabolite concentrations discriminated treated xenografts from untreated controls with an accuracy of 67% (p = 0.003). Multilevel linear mixed-effects models (LMM) indicated reduced glycolytic lactate production and glutaminolysis after treatment, consistent with PI3K/AKT/mTOR pathway inhibition. Although inherent metabolic heterogeneity between different PDX models seemed to hinder prediction of treatment response, the metabolic effects following treatment were more pronounced in responding xenografts compared to nonresponders. Additionally, the metabolic information predicted p53 mutation status, which may provide complementary insight into the interplay between PI3K signaling and other drivers of disease progression. Copyright © 2017 American Chemical Society. This is the authors' accepted and refereed manuscript to the article. Locked until 14 March 2018 due to copyright restrictions

Purpose: The prognosis of metastatic uveal melanoma (UM) patients remains one of the worst observed in human cancers. The identification of frequently mutated GNAQ/GNA11 and the activation of MAPK signaling in UM raise the possibility of using efficient targeted therapies such as MEK inhibition. The aim of our study was to evaluate drug combinations that might enhance the efficacy of the MEK inhibitor, selumetinib (AZD6244, ARRY-142886), using patient-derived xenografts. Materials: Five UM PDXs were used in this study, i.e., MP34, MP46, MP55, MP77, and MM26, all mutated for GNAQ or GNA11. Selumetinib was administered alone or combined with chemotherapies (dacarbazine and docetaxel) or targeted therapies: the ERK inhibitor, AZ6197 and the mTORC1/2 inhibitor, vistusertib (AZD2014). All targeted therapies were administered orally five days/week at the following doses: selumetinib at 25 mg/kg/day BID, AZ6197 at 50 mg/kg QD, AZD2014 at 15mg/kg QD. Dacarbazine was administered at 40 mg/kg for 5 consecutive days every 28 days and docetaxel at 15 mg/kg weekly. Both were administered intraperitoneally. Results: The combination of selumetinib and dacarbazine did not show improvement of antitumor efficacy in MP34, MM26 and MP55.The results showed a slight increase of overall response rate when selumetinib was combined with docetaxel, in comparison to the treatment with selumetinib or docetaxel alone. Importantly, we observed a significant antitumor effect when selumetinib was used in combination with AZ6197 or AZD2014 with respect to the monotherapies. Preliminary pharmacodynamics studies showed variation of the expression of p-ERK and also p-AKT and/or p-S6 when UM PDX were treated with AZ6197 or AZD2014 alone or in combination with selumetinib. Conclusion: Our data suggest that ERK and mTORC1/2 targeting could represent promising therapeutic approaches for the treatment of metastatic UM patients. Citation Format: Didier Decaudin, Rania EL Botty, Béré Diallo, Gerald Massonnet, Justine Fleury, Adnan Naguez, Marine Dubois, Emma J. Davies, Aaron Smith, Joanne Wilson, Colin Howes, Paul Smith, Nathalie Cassoux, Pascale Mariani, Sophie Piperno-Neumann, Sergio Roman Roman, Fariba Nemati. Selumetinib-based therapy in uveal melanoma patient-derived xenografts [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 5782.

Background: triple-negative breast cancer (TNBC) patients with residual disease following neoadjuvant chemotherapy have a high risk of relapse and poor survival. Conventional treatments for relapsed patients are limited, particularly, because standard chemotherapeutic regimens containing anthracyclines and taxanes have usually already been given in the neoadjuvant settings. Our objectives were 1) to identify efficient chemotherapies in patient-derived xenografts (PDX) established from residual TNBC and 2) to determine feasibility of PDX establishment and drug testing before tumor recurrence in patients. Methods: tumors from 51 patients with residual disease at surgery were transplanted in nude mice. Established TNBC PDX were treated with different chemotherapies used in early stages and metastatic settings: anthracyclines combined to cyclophosphamide (AC), taxanes, platins, capecitabine and gemcitabine. Drug responses in PDX were compared to responses in patients who recurred after surgery and were treated in the metastatic setting. Results: overall tumor take of residual tumors was 40% and 75% for TNBC, with 15/20 PDX established, more than twice the tumor take of treatment naïve TNBC (34%). Median latency time, defined as time from implantation till first tumour growth, was only 60 days and was further reduced to 3-4 weeks during successive tumor passages. On the 8 TNBC PDX evaluated for chemosensitivity, 7 exhibited a multidrug-resistance phenotype with resistance or limited response to AC, taxanes and platins. Capecitabine, a chemotherapy given in the advanced stage as second or third line, was efficient in 5 out of 8 PDX tested with 3 models showing stable disease and 2 models durable tumor regressions. Interestingly, capecitabine efficacy was decreased when xenografts were pre-treated with a first line containing platins, suggesting that in some tumors capecitabine might have superior activity when given in the adjuvant setting or as a first line. In one third of TNBC patients, time to recurrence, comprised between 7 and 12 months after surgery, was compatible with xenograft establishment and drug testing. Conclusions and perspectives: we established a unique panel of PDX models from patients with residual disease after neoadjuvant chemotherapy. These aggressive PDX recapitulate the resistance phenotype of patients’ tumors to treatments given in neo-adjuvant and metastatic settings. We identified capecitabine as efficient first line chemotherapy for residual chemoresistant PDX. In 30% of cases, PDX models could have been used to evaluate chemotherapy responses before tumor recurrence occur in patients. In order to identify predictive biomarkers of capecitabine response, additional experiments are ongoing in 25 supplementary TNBC PDX, established from treatment-naïve patients. Citation Format: Elisabetta Marangoni, Cécile Laurent, Florence Coussy, Rania El Botty, Ludmilla de Plater, Franck Assayag, Sophie Vacher, Ivan Bièche, Fabien Reyal. Efficacy of capecitabine in chemoresistant PDX established from triple-negative breast cancers with residual disease after neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2017-3859

Purpose: DNA repair deficiencies and activation of PI3K/AKT/mTOR pathway are common events in breast cancer. BRCA1/2 mutations have been associated with sensitivity to PARP1 inhibitors (synthetic lethality), while alterations in component of the PI3K/AKT/mTOR pathway might confer sensitivity to PI3KCA and mTOR inhibitors. Here we explored the therapeutic benefit of combining a mTOR and a PARP inhibitor in BRCA2-mutated patient-derived xenografts (PDX) with alterations in the PI3K/AKT/mTOR pathway. Experimental design: the combination of the mTOR inhibitor everolimus and the PARP inhibitor olaparib was tested in two BRCA2-mutated PDX established from a luminal B tamoxifen-resistant and a basal-like breast cancers. Both carried alterations in the PI3K/AKT/mTOR pathway: PIK3R1 mutation and PTEN loss for the luminal B PDX and PTEN loss for the basal-like PDX. To identify putative crosstalk events between mTOR and DNA repair, a Reverse Phase Protein Array (RPPA) analysis of multiple signaling pathways and DNA repair processes was performed on untreated and treated xenografts. Gene and protein expression changes were confirmed by RT-PCR and Western Blot analyses. The capacity to repair DNA damage was measured by P-H2AX immunostaining. Results: in both PDX, everolimus and olaparib showed marked anti-tumor activity with a growth inhibition comprised between 78% and 86% in the monotherapy setting and 96% in the combination arm, where 100% of mice showed tumor regressions. In the luminal B tumor this combination was more efficient than the combination of everolimus and endocrine therapies (fulvestrant or tamoxifen). In both PDX, the fraction of P-H2AX (marker of unrepaired DNA double-strand breaks) positive cells was increased after everolimus treatment, suggesting a link between mTOR and DNA damage, and strongly increased in the combination setting. RPPA analysis of tumors treated by everolimus alone revealed a marked downregulation of different proteins involved in DNA repair, including FANCD2, RAD50 and SUV39H1, a chromatin compactor factor essential in homologous recombination. In the combination setting, expression of these proteins was almost completely abolished, suggesting convergence of PARP and mTOR in downregulation of DNA repair components. Conclusions: our results suggest that combining mTOR and DNA repair inhibitors could be a successful strategy to treat a subset of breast cancer with BRCA2 mutation and alterations in the PI3K/AKT/mTOR pathway. Further experiments with mTOR and PARP inhibitors combinations are ongoing in sporadic breast cancer PDX showing a BRCAness phenotype. Citation Format: Florence Coussy, Rania El Botty, Rana Hatem, Franck Assayag, Ahmed Dahmani, Marine Huppé, Sophie Chateau Joubert, Jean -Luc Servely, Virginie Bernard, Sophie Vacher, Berangere Ouine, Aurelie Cartier, Leanne De Koning, Paul Cottu, Ivan Bieche, Elisabetta Marangoni. Inhibition of mTOR downregulates expression of DNA repair proteins and is highly efficient against BRCA2-mutated breast cancer when combined to PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1853. doi:10.1158/1538-7445.AM2017-1853

Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Uveal melanomas (UM) constitute the most common primary intraocular tumors in adults and are characterized by a constitutive activation of the MAPK pathway due to mutations of the GTPase genes GNAQ or GNA11 in almost 80% of cases. The most commonly used treatments for UM are alkylating agents such as dacarbazine (DTIC) and temozolomide (TMZ). The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) has shown clinical activity compared to DTIC/TMZ in a recent Phase II clinical trial and has recently completed a Phase III clinical trial in combination with DTIC (NCT01974752). In parallel with this trial we sought to evaluate the efficacy of DTIC + selumetinib in UM patient-derived xenografts (PDXs). Three models were included in the study (MP34, MP55, and MM26), all bearing a GNAQ or GNA11 mutation. Selumetinib was administered orally at 25 mg/kg/day, 5 days a week, and DTIC at a dose of 40 mg/kg/day on Days 1 to 5 every 4 weeks. A significant tumor growth inhibition (TGI) of 54% was observed in the MP34 model but not in the two remaining PDXs. In one model, MM26, DTIC induced a strong TGI of about 99% with 6/9 complete remissions (CRs). The combination of selumetinib + DTIC did not significantly increase efficacy compared to monotherapy in any of the models; in the MM26 PDX, the combination induced a similar TGI (99%) and CR rate (5/9) as DTIC alone. In this experiment, after two courses of DTIC + selumetinib, selumetinib was continued alone, showing a significant increased growth delay (p In conclusion, we have observed that response of UM PDX models to DTIC was not increased when combined with selumetinib; these results are similar to those seen in the Phase III study of this drug combination. The observation that MEK inhibition was effective in delaying progression in the DTIC-sensitive PDX and published clinical studies demonstrating MEK inhibitor monotherapy activity indicate that MEK inhibition may have value as a treatment for UM; perhaps in the adjuvant setting in two specific clinical situations, i.e. patients with irradiated or enucleated high-risk primary intraocular or surgically resected metastatic UM. Citation Format: Béré Diallo, Gerald Massonnet, Rania El-Botty, Chloé Raymondie, Guillaume Carita, Sergio Roman-Roman, Paul Smith, Emma Davies, Didier Decaudin, Fariba Némati. The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) appears as an efficient targeted therapy when used in an adjuvant setting in patient-derived xenografts of uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2087.

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 Vandetanib's targets. Citation Format: Elisabetta Marangoni, Rana Hatem, Dalila Labiod, Sophie Chateau-Joubert, Rania El Botty, Jean-Luc Servely, Ludmilla De Plater, Ivan Bièche. 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

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 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 Bièche. 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

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, Hélène Bonsang-Kitzis, Alice Pinheiro, Cécile Laurent, Ivan Bièche, 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.

The addition of everolimus (Rad) to endocrine therapies (ET) in luminal advanced breast cancers has shown to increase overall survival in randomized studies. However drug resistance is an emerging problem and most patients eventually experience progression of disease. We aimed to establish and characterize patient-derived xenografts (PDX) of luminal breast cancers with acquired resistance in vivo to both ET and Rad. Two PDX xenografts (HBCx22 and HBCx34), established from early stage ER+ breast cancers, were treated in vivo with different ET (tamoxifen, fulvestrant, ovariectomy and letrozole) during 12 months and tumors escaping to therapies were re-engrafted and maintained under treatment. Four hormono-resistant (HR) xenografts were established, 2 resistant to Tamoxifen (HBCx22TamR and HBCx34TamR) and 2 resistant to ovariectomy (HBCx22OvaR and HBCx34OvaR). Expression of estrogen receptor (ER) was maintained, while expression of several ER-controlled genes (MYB, PS2 and PR) was decreased in HR tumors indicating modifications of ER transcriptional activity. The expression of ER co-regulators was additionally studied: GREB1 was reduced specifically in TamR, while FOXA1 were increased specifically in TamR. Expression analysis of P-AKT, P-mTOR and P-S6 showed similar levels of PI3 kinase pathway activation between parental and HR xenografts. Gene expression profiling, performed with Affymetrix Gene Expression Arrays, showed that genes differentially expressed in TamR and OvaR tumors were only partially overlapping, suggesting treatment-specific mechanisms of hormono-resistance. In addition, TamR and OvaR signatures were tumor-specific. Gene ontology enrichment and pathway analyses of gene expression datasets will be presented at the meeting. HR xenografts were then treated with Rad alone and combined to ET. In HBCx22OvaR and TamR, resistant to all ET, treatment with Rad arrested tumor growth in vivo but did not show any additive effect when combined to ET. Conversely, the HBCx34TamR model was still responding to fulvestrant, and the combination Rad+Fulvestrant resulted in tumor regressions. In the HBCx34TamR xenograft, tumor escape to Rad treatment occurred in one mouse after 8 months of continuous treatment. The tumor was re-engrafted and maintained as tumorgraft resistant to both tamoxifen and Rad (HBCx34Tam-RadR). Western blot and IHC studies showed that mTOR signaling was still inhibited in this tumor, but it was associated to MAP kinase activation and increased IGF-1R expression. In conclusion, we have developed PDX models of luminal breast cancer with cross-resistance to ET and everolimus in vivo. These models provide a valuable preclinical tool to investigate molecular mechanisms of acquired resistance and to test new targeted therapies and new combinations in patients’ tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C174. Citation Format: Rania El Botty, Paul Cottu, Franck Assayag, Pierre de la Grange, David Gentien, Aurélie Thuleau, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bièche, Elisabetta Marangoni. Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus. [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 C174.