Gérald Massonnet

Evaluating Patient-Derived Colorectal Cancer Xenografts as Preclinical Models by Comparison with Patient Clinical Data

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2-PI3K and ERBB-RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2-PI3K and ERBB-RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN, and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells’ metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.

Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds

BackgroundThe B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.MethodsUsing a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.ResultsGb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p ≤ 0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p ≤ 0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p ≤ 0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.ConclusionThe enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

Characterization of Breast Cancer Preclinical Models Reveals a Specific Pattern of Macrophage Polarization

Drug discovery efforts have focused on the tumor microenvironment in recent years. However, few studies have characterized the stroma component in patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMs). In this study, we characterized the stroma in various models of breast cancer tumors in mice. We performed transcriptomic and flow cytometry analyses on murine populations for a series of 25 PDXs and the two most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). We sorted macrophages from five models. We then profiled gene expression in these cells, which were also subjected to flow cytometry for phenotypic characterization. Hematopoietic cell composition, mostly macrophages and granulocytes, differed between tumors. Macrophages had a specific polarization phenotype related to their M1/M2 classification and associated with the expression of genes involved in the recruitment, invasion and metastasis processes. The heterogeneity of the stroma component of the models studied suggests that tumor cells modify their microenvironment to satisfy their needs. Our observations suggest that such models are of relevance for preclinical studies.

Abstract B142: Dissociation of preclinical primary human cancer xenografts for cell surface transportome profiling.

Background: Tumor cell metabolism is of growing interest in both basic and clinical cancer research. A better understanding of underlying molecular mechanisms involving metabolite transport in normal and tumor cells should help drug discovery and development. Specific exofacial ligands to metabolite transporters derived from the receptor binding domain (RBD) of retrovirus envelope proteins were developed and used to quantify cell surface metabolite transporters. While cell surface labelling can be readily performed on cultured cell lines, analysis of single cells from solid tumors is more challenging. In this study, we developed a robust method for the disaggregation of tumor cells from human breast cancers grown as xenografts in mice. This procedure was then used to analyse the expression profiles of 4 cell membrane metabolite transporters involved in cell proliferation and tumorigenesis: Glut1, ASCT2, PiT1, and PiT2. Materials and methods: Eight primary human breast cancer xenografts were used for ex vivo experiments (Marangoni et al 2007). We developed an optimized disaggregation protocol to obtain maximum viable cell recovery from the xenografts. The protocol was validated for presence of CD44+ tumor cells and for cell viability using caspase 3 and DAPI exclusion and subsequently, applied to the xenografts for flow cytometry analyses, immunohistochemistry and ex vivo cell culture. Expression profiles of 4 metabolite transporters were assessed in 5 different human breast cancer xenografts. Results: The optimal dissociation protocol developed for these tumors combined mild non-enzymatic (non-enzymatic dissociation buffer) and enzymatic (collagenase III/DNase I) steps, followed by cell purification on a dual density Ficoll gradient. Less than 10% of resulting DAPI negative tumor cells were caspase 3 positive. Dissociated cells showed sustained viability in in vitro cultures for at least 12 days. The numbers of CD44+ cells determined by flow cytometry corresponded to those observed by IHC. The expression profiles of Glut1, ASCT2, PiT1, and PiT2 were distinct for each of the five human breast cancers, and metabolite transporter profiles were highly conserved for xenografts derived from the same tumor. Conclusions: Mouse xenograft implants of human breast cancer tumors were used to optimize and validate a dissociation method for the production of viable single cells. Cell suspensions were then assessed for cell surface metabolite transporters expression by flow cytometry. The expression patterns of four metabolite transporters, Glut1, ASCT2, PiT1, and PiT2 showed distinctive signature profiles for each group of xenografts, indicative of specific metabolic adaptations that can be tracked with our ligands for each tumor. Reference: 1. Marangoni E et al, CCR 2007;13:3989–3998. 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 B142.

Selumetinib-based therapy in uveal melanoma patient-derived xenografts

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.

The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages

Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient’s tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials.

Abstract 2087: 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

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: Bere Diallo, Gerald Massonnet, Rania El-Botty, Chloe Raymondie, Guillaume Carita, Sergio Roman-Roman, Paul Smith, Emma Davies, Didier Decaudin, Fariba Nemati. 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.

Abstract POSTER-THER-1404: in vivo synergism between chemotherapy and the 3C23K monoclonal antibody directed against the anti-müllerian hormone type II receptor in ovarian cancer patient-derived xenografts

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Purpose of the study: Mullerian inhibiting substance, also called anti-Mullerian hormone (AMH), belongs to the TGF-β family and plays a key role during sexual development. It has been shown that AMH inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, and particularly progenitor cancer cells. Moreover, various reports have underlined expression of AMHRII in human gynecologic cancers including ovarian tumors. Based on these considerations, a humanized glyco-engineered monoclonal antibody (3C23K) capable of increasing effector cells engagement has been developed and tested in AMHR2 transfected xenografted cell lines. In order to document its activity in naturalistic models, we have initiated a preclinical study to assess AMHRII expression in a panel of ovarian cancer patient-derived xenografts (PDXs) and evaluate in vivo efficacy of 3C23K. Experimental procedures: AMHRII expression was studied in 14 ovarian cancers PDXs, using both flow cytometry (FC) and immunofluorescence (IF) analyses performed with alexaFluor488-conjugated 3C23K antibody on 300µm slices of xenograft fragments. In vivo efficacy of 3C23K alone (10 or 20 mg/kg twice a week, IP) or in combination with standard chemotherapy (CT) (carboplatin 66 mg/kg IP days 1-22 + paclitaxel 30 mg/kg IP days 1-22) has been planned to be evaluated in 4 AMHRII-positive ovarian cancers PDXs. One undifferentiated highly proliferative serous ovarian adenocarcinoma PDX, the OV54 model, has already been treated. On these 4 selected models, expression and quantification of AMHRII was or will be performed before and after treatments, as well as tumor-associated macrophages (TAMs) infiltrate. Summary of the data: Expression studies showed that over the 14 PDX tested, 6 (43%) were positive with at least 25% of tumor cells being positive, among these 3 expressed more than 50% of positive cells. This expression was confirmed by ex vivo IF with a profile comparable to that observed with fresh primary human ovarian tumors resected from stage IV patients. In vivo experiments (OV54 model) showed an intermediate efficacy of the 3C23K antibody administered alone, as well as a high and significant increase of CT antitumor effect. Synergistic effects were characterized by 50% of complete response (CR) when 3C23K (20mg/kg/inj.) was added to CT vs 10% CR with CT alone. The cumulative number of days under CR was 110 with 3C23K + CT vs 7 on CT alone. Impact on survival was consistent with a 90% survival at D37 with 3C23K + CT vs 60% survival on CT alone. We did not observe modification of AMHRII expression in OV54 tumors after 3C23K administration. Stromal FC analyses showed a high TAMs infiltrate of M2 type compatible with induction of Antibody Dependent Cell Phagocytosis by 3C23K. Conclusions: Using a panel of 14 ovarian cancer PDXs, we confirmed the expression of AMHRII in human ovarian cancers. Targeting this receptor by a specific monoclonal antibody, i.e. 3C23K, dramatically increases the efficacy of a standard chemotherapy. Our results therefore suggest that such combination could be further developed in ovarian cancer patients whose prognosis remains dismal. Citation Format: Fariba Nemati, Jean-Marc Barret, Gerald Massonnet, Houcine Bougherara, Marie-Aude Le Frere-Belda, Jean-Francois Prost, Didier Decaudin. in vivo synergism between chemotherapy and the 3C23K monoclonal antibody directed against the anti-mullerian hormone type II receptor in ovarian cancer patient-derived xenografts [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1404.

Abstract A01: Ex vivo evaluation of an anti-Müllerian hormone type II receptor humanized antibody with optimized Fc effector function in ovarian cancer

Purpose of the study: Epithelial ovarian cancer (OC) is one of the most common gynecologic malignancies and the fifth most frequent cause of cancer death in women. Paclitaxel and Carboplatin chemotherapy (CT) remains the standard treatment, but tumor relapses in approximately 80% of patients, mostly due to drug resistance. Novel therapeutic options are therefore strongly required. Recently, human Anti-Mullerian Hormone type II Receptor (AMHRII) has been identified as a potential target, but up to date the AMHRII expression in ovarian tumors has been poorly documented. A humanized glyco-engineered monoclonal antibody (mAb) called 3C23K with higher Fc effector function has been developed. The aim of this study is to investigate the potential efficacy of this mAb by assessing AMHRII expression and analyzing the localization and amount of mAb responsive immune effectors in OC patient and in a patient-derived xenograft (PDX) model. Experimental procedures: AMHRII expression was prospectively studied in fresh biopsies (primary ovarian tumors, peritoneal carcinomatosis and ascites) from a cohort of 28 patients, by means of both flow cytometry (FC) and immunofluorescence (IF). We have characterized the intratumoral immune infiltrate by FC and determined the localization of immune cells within the tumor by IF. Similar experiments were performed on an undifferentiated highly proliferative serous ovarian adenocarcinoma PDX model, in parallel to in vivo efficacy study of 3C23K alone or in combination with standard CT. The antitumor activity of infiltrating immune cells isolated from OC patients (peritoneal washing) was assessed using red blood cells as target and a glyco-engineered anti-D Rhesus mAb. The antibody-dependent cell cytotoxicity (ADCC) and the antibody-dependent cell phagocytosis (ADCP) abilities of 3C23K against AMHRII+ cells (COV434-RII) were analyzed by IF using monocyte-derived macrophages (MDM) from blood of healthy donors as effector cells. Summary of the data: Here, we show that among the 28 ovarian tumors tested, 19 patients (68%) were positive for AMHRII with at least 15% of tumor cells being positive for AMHRII. OC are largely composed of (CD45+) immune cells mostly composed of tumor associated macrophages (TAMs) and tumor infiltrating lymphocytes (TILs). TAMs highly express M2-like markers, such as CD206, but also strongly express the Fcγ III receptor (CD16). Unlike TILs, which accumulate in the peritumoral stroma, CD16+ TAMs are found in both the stroma and the tumor islets. Moreover, tumors infiltrating immune cells are able to induce a mAb-dependent cytotoxicity ex vivo. In parallel, in vivo experiments have shown an intermediate efficacy of the 3C23K antibody administered alone and a significantly increased antitumor effect in combination with CT including a prolonged survival. IF and FC analyses showed high TAM infiltration compatible with induction of ADCP by 3C23K in this model. Furthermore, 3C23K mAb induces ADCC and/or ADCP of COV434-RII cells by MDM likely through CD16 interaction, either in the absence or presence of saturating concentrations of Ig. Conclusions: We have confirmed the expression of AMHRII in 2/3 of OC patients. MAb responsive immune effectors and particularly macrophages largely infiltrate OC and are found in contact with malignant cells. Targeting AMHRII by the 3C23K mAb dramatically increases the efficacy of a standard CT in vivo. 3C23K appears to act through macrophage-induced ADCC and/or ADCP of tumor cells and current studies are designed to refine its mechanism of action. Our results indicate that most ovarian tumors display optimal conditions for a 3C23K-based immunotherapy. Hence, the combination of 3C23K with CT could bring additional therapeutic benefit in OC patients compared to standard chemotherapy. Citation Format: Houcine Bougherara, Fariba Nemati, Christophe De Romeuf, Jean-Marc Barret, Gerald Massonnet, Marie-Aude Le Frere-Belda, Jean-Francois Prost, Didier Decaudin, Emmanuel Donnadieu. Ex vivo evaluation of an anti-Mullerian hormone type II receptor humanized antibody with optimized Fc effector function in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A01.