Patricia Vrignaud

Characterization of a Large Panel of Patient-Derived Tumor Xenografts Representing the Clinical Heterogeneity of Human Colorectal Cancer

Purpose: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. Experimental Design: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. Results: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. Conclusions: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients. Clin Cancer Res; 18(19); 5314–28. ©2012 AACR.

Preclinical Antitumor Activity of Cabazitaxel, a Semisynthetic Taxane Active in Taxane-Resistant Tumors

Purpose: Taxanes are important chemotherapeutic agents with proven efficacy in human cancers, but their use is limited by resistance development. We report here the preclinical characteristics of cabazitaxel (XRP6258), a semisynthetic taxane developed to overcome taxane resistance. Experimental Design: Cabazitaxel effects on purified tubulin and on taxane-sensitive or chemotherapy-resistant tumor cells were evaluated in vitro. Antitumor activity and pharmacokinetics of intravenously administered cabazitaxel were assessed in tumor-bearing mice. Results: In vitro, cabazitaxel stabilized microtubules as effectively as docetaxel but was 10-fold more potent than docetaxel in chemotherapy-resistant tumor cells (IC50 ranges: cabazitaxel, 0.013–0.414 μmol/L; docetaxel, 0.17–4.01 μmol/L). The active concentrations of cabazitaxel in these cell lines were achieved easily and maintained for up to 96 hours in the tumors of mice bearing MA16/C tumors treated with cabazitaxel at 40 mg/kg. Cabazitaxel exhibited antitumor efficacy in a broad spectrum of murine and human tumors (melanoma B16, colon C51, C38, HCT 116, and HT-29, mammary MA17/A and MA16/C, pancreas P03 and MIA PaCa-2, prostate DU 145, lung A549 and NCI-H460, gastric N87, head and neck SR475, and kidney Caki-1). Of particular note, cabazitaxel was active in tumors poorly sensitive or innately resistant to docetaxel (Lewis lung, pancreas P02, colon HCT-8, gastric GXF-209, mammary UISO BCA-1) or with acquired docetaxel resistance (melanoma B16/TXT). Conclusions: Cabazitaxel is as active as docetaxel in docetaxel-sensitive tumor models but is more potent than docetaxel in tumor models with innate or acquired resistance to taxanes and other chemotherapies. These studies were the basis for subsequent clinical evaluation. Clin Cancer Res; 19(11); 2973–83. ©2013 AACR.

Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography.

Objectives:To evaluate the early tumor vasculature disrupting effects of the AVE8062 molecule and the feasibility of dynamic contrast-enhanced ultrasonography (DCE-US) in the quantitative assessment of these effects. Material and Methods:AVE8062 was administered at a single dose (41 mg/kg) to 40 melanoma-bearing nude mice, which were all imaged before and after drug administration (5 + 15 minutes, 1, 6, and 24 hours). Using an ultrasound scanner (Aplio, Toshiba), intratumor vessels were counted in power Doppler mode and tumor microvasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software for contrast-uptake quantification (Sonovue, Bracco). The peak intensity (PI), time-to-PI (TPI), and full-width at half maximum (FWHM) were extracted from the time-intensity curves expressed as linear raw data. Histologic analysis evaluated microvessel density (MVD) and necrosis at each time point studied. Statistical significance was estimated (paired sum rank and Mann–Whitney tests) to evaluate drug activity and to compare its efficacy at the different time points. Results:In power Doppler mode, intratumoral vessels depletion started 15 minutes postinjection (32%, P = 0.004) and the decrease was maximal at 6 hours (51%, P = 0.002). PI decreased by 3.5- and 45.7-fold at 1 and 6 hours, respectively, compared with preinjection values (P = 0.016 and P = 0.008). The decrease at 6 hours was significantly different from the variation at 1 hour (P = 0.0012) and at 24 hours (P = 0.0008). TPI and FWHM showed a significant increase exclusively at 6 hours (P = 0.0034, P = 0.0039). Histology revealed significantly decreased MVD and increased necrosis at 24 hours (P < 0.01). Conclusion:DCE-US allowed quantitative in vivo evaluation of the functional effects of AVE8062, which was found most effective on tumoral microvasculature 6 hours after its administration. A clinical phase-1 study of AVE8062 is ongoing using the same ultrasonography methodology before and 6 and 24 hours postadministration.

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.

Antitumor activity of intoplicine (RP 60475, NSC 645008), a new benzo-pyrido-indole: evaluation against solid tumors and leukemias in mice.

SummaryIntoplicine (RP 60475, NSC 645008) is a new 7H-benzo[e]pyrido[4,3-b] indole derivative which interacts with DNA and inhibits both topoisomerases I and II.In vitro it was found cytotoxic against various cell types with greater cytotoxicity towards solid tumor cells. We report here the anticancer activity of RP 60475 against a variety of transplantable tumors of mice, and also its cross-resistance profile in leukemias. The end points used were % T/C (median tumor weight of the Treated over the Control × 100) and logCK (log10 cell kill total). RP 60475 administered i.v. was found schedule-independent with a peak plasma level problem. It had a good therapeutic index and host recovery usually occurred 7.5 days post last treatment. RP 60475 was found to be highly active against early stage colon 38 (T/C=0%, 2.9 logCK) and could induce 5/5 complete regressions of advanced stage tumor. It was found active against colon adenocarcinoma 51 (T/C=3.6%, 1.9 logCK) and colon carcinoma 26 (T/C=11.7%, 1.2 logCK). Most of the mammary adenocarcinomas were found very responsive, MA16/C (T/C=0%, 2.8 logCK), MA14/A (T/C=0%, 1.4 logCK), MA13/C (T/C=0%, 3.1 log CK) and MA44 (T/C=34%). Excellent activity was also observed against early stage pancreatic ductal adenocarcinoma 03 (T/C=0%) and RP 60475 could achieve 5/5 complete regressions of upstaged tumor. Activity was also obtained on Glasgow osteogenic sarcoma (T/C=0%, 3.3 logCK), on B16 melanoma (T/C=14%, 1.3 logCK) and to a lesser extent on Lewis lung carcinoma (T/C=33.2%). Evaluation of RP 60475 against leukemia sublines with acquired resistance, revealed that L1210/cisplatin and L1210/BCNU were not cross-resistant to RP 60475 whereas P388/vincristine was partially cross-resistant to RP 60475 and P388/doxorubicin was cross-resistant to RP 60475. Based on RP 60475 broad activity against transplantable tumors of mice, its effectiveness against some resistant sublines, its original mechanism of action and its acceptable toxicological profile, this compound was selected for clinical trials.

RPR 130401, a Nonpeptidomimetic Farnesyltransferase Inhibitor with in Vivo Activity

A nonpeptidomimetic farnesyltransferase inhibitor (FTI) was identified through an enzymatic Ras screening, leading to the synthesis of benzo[f]perhydroisoindole derivatives, a new class of competitive inhibitors with the farnesyl pyrophosphate substrate.1 One compound of this family, exhibiting enzymatic and cellular activities at a micromolar level, was evaluated in vivo, but was found inactive.2 Thereafter, new chemical modifications were performed. RPR 130401 was selected for its cytostatic in vitro activity in a colony formation assay against human Ki-Ras carcinoma cells, inhibiting concentration 50% (IC50) ranging from 0.045 to 0.48 μM. The goal of the work presented herein was to evaluate its in vivo activity. According to the chemotherapy methods previously described,3 the in vivo antitumor efficacy of RPR 130401 was evaluated against human Ki-Ras carcinomas obtained from the American Type Culture Collection and xenografted in Swiss nude mice: two colons (HCT 116 and SW620), one pancreas (MIA PaCa-2), and one lung (H460). Swiss nude mice were grafted sc with tumor implants on day 0. Tumors were measured with a caliper 2–3 times a week, and tumor weight was evaluated using the following formula: tumor weight (mg) = length (mm) × width2 (mm2)/2. RPR 130401 was suspended in water containing 0.5–1% polysorbate 80 and 0.5% methyl cellulose. It was administered orally twice a day for 19 consecutive days to mice bearing palpable sc tumors at dosages of 248, 400, and 645 mg/kg/administration. A dosage producing 20% body weight loss or drug-related death was declared toxic. The end points were tumor growth delay (T-C difference in median times required for treatment and control groups to reach a predetermined size) and log10 cell kill net (lck net = tumor growth delay − treatment duration/3.32 × tumor doubling time). Cytostatic activity was obtained for a tumor growth delay corresponding at least to the treatment duration. A negative lck net indicated that tumor grew under treatment. Pharmacokinetics of RPR 130401 was determined following a single administration of 250 mg/kg in nontumor-bearing B6D2F1 mice (FIG. 1). The area under the curve (AUC0–24h) was 820.3 h.μg/ml, with a 2.8-hour half-life of elimination. Peak plasma concentration was 142.1 μg/ml 2 hours postdose, a concentration 100 times greater than the in vitro IC50 (∼0.091 μg/ml in HCT 116 colony formation assay), and 19.9 μg/ml 8 hours postdose. Therefore, for efficacy studies, this compound was administered orally twice a day, 7 hours apart, for 3 consecutive weeks.

Abstract 2522: Therapeutic synergism of cabazitaxel in combination with cisplatin in tumor-bearing mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cabazitaxel (Jevtana), a semi-synthetic taxane derived from 10-deacetyl baccatin III, is as efficient as docetaxel in promoting tubulin assembly in vitro and stabilizing microtubules against cold-induced depolymerization. Cabazitaxel was selected for clinical development based on better antiproliferative activity than docetaxel against chemotherapy resistant tumor-cell lines (Bissery M.C. et al, AACR 2000) and a broad spectrum of in vivo antitumor activity (Vrignaud P. et al, AACR 2000), not only in docetaxel-sensitive tumor models, such as the human gastric N87 xenograft, but also in tumors models in which docetaxel was either poorly active or inactive. As an example, cabazitaxel was highly active in mice bearing UISO-BCA1 tumors, a human mammary xenograft expressing Her-2, which is poorly sensitive to ixabepilone and not sensitive to docetaxel. In addition, cabazitaxel penetrates the blood brain barrier (Cisternino S. et al, British J. Pharm. 2003) and has marked antitumor activity in nude mice bearing intracranial glioblastomas (Dykes D.J. et al, AACR 2000). Cabazitaxel was recently approved by the FDA for patients with metastatic hormone-refractory prostate cancer previously treated with docetaxel-based therapy. In order to explore other therapeutic indications, the cabazitaxel-cisplatin combination was evaluated in C51 tumor-bearing mice. A 5-arm dose-response study was performed with cabazitaxel and cisplatin administered IV on days 5 and 12 after tumor implantation as single agents and in combination using 3 different sequences of administration: simultaneous in group A and sequentially separated by a 24h interval in groups B (cabazitaxel first) and C (cisplatin first). At their highest nontoxic doses (HNTD), both single agents were found active with tumor growth delays (T-C) of 15.2 days for cabazitaxel at 20 mg/kg/inj and 22.1 days for cisplatin at 4.3 mg/kg/inj. The 3 combination arms showed greater antitumor activity at their respective HNTD with T-C of 38.4 days in group A, 37.8 days in B, and 36.2 days in C. Of interest, 2 (A) and 3 (B & C) dose levels were found more active than the single agents in the combination arms. In conclusion, a therapeutic synergism was achieved when cabazitaxel was combined with cisplatin in C51 tumor-bearing mice. These data are supporting the ongoing Phase I clinical study of cabazitaxel-cisplatin combination in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2522. doi:10.1158/1538-7445.AM2011-2522

Abstract LB-34: Similar PI3K and RTK-Ras status in patient derived colorectal cancer-xenografts and patients.

There is a recent increase in the use of patient-derived tumor xenografts (PDX) engrafted into immunodeficient mice as improved preclinical models of patient tumors. An important component in the validation of disease-specific PDXs for clinical relevance is comparing the genomic alterations and the response to standard agents. The CReMEC collection of 54 colorectal PDX has been recently reported to mimic the clinical situation for histopathological and molecular diversity of colorectal cancer. We further analyze here this colorectal PDX collection in regard to robust human patient molecular features: 1) Alterations in both PI3K and RTK-Ras pathways; 2) Role of oncogenic activation of EGFR-Ras downstream signaling on response to cetuximab. The Cancer Genome Atlas Network (TCGA) newly reported a large genome-scale analysis of 276 colorectal cancer tissue samples, showing common genetic alterations in the PI3K and RTK-RAS pathways, with mutual exclusions in the PI3K pathway. The analysis of the PDX panel by CGH array, RNA expression (microarray, real-time qRT-PCR) and exome sequencing confirmed activation with mutual exclusion in PI3K pathway (IGF2 focal amplification/overexpression; IRS2 overexpression, mutation of PIK3CA, PIK3R1 and PTEN homozygous deletion). In RTK-Ras pathway, frequencies of genomic abnormalities (ERBB2 mutation/amplification; mutation of ERBB3, NRAS, KRAS and BRAF) in PDXs are fully in line with the TCGA patient collection. The response to cetuximab of 52 subcutaneous engrafted PDX (including 24 KRAS mutated PDX) has been analyzed according to translated clinical criteria: xenograft regression as been defined as a partial response (decrease of at least 70% of the tumor volume measured at the beginning of the treatment) or as a complete response. In this unselected population, tumor regression occurred in 8 out of 52 cases (15%); all were KRAS wild type tumors. The percentage of responders was enriched up to 30% (7/23) when PTEN deletion and mutations of KRAS, BRAF, and PIK3CA are concomitantly excluded. These results completely fit with recent publication of data in patients treated with anti-EGFR antibodies. Taken together, these results demonstrate that colorectal PDXs are representative clinical colorectal tumor models. They also underline their interest as appropriate tools to identify and test new targeted therapeutics. Citation Format: Manoel Nunes, Louis-Bastien Weiswald, Patricia Vrignaud, Sophie Vacher, Edouard Turlotte, Sophie Richon, Dominique Bellet, Sergio Roman-Roman, Ivan Bieche, Virginie Dangles-Marie. Similar PI3K and RTK-Ras status in patient derived colorectal cancer-xenografts and patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-34. doi:10.1158/1538-7445.AM2013-LB-34

Abstract 5055: Efficacy of cabazitaxel in mouse models of pediatric brain tumors.

Successful treatment of pediatric brain tumors is limited by significant long-term toxicities associated with surgery and radiation therapy on the developing central nervous system There is an unmet need for chemotherapy with improved efficacy in difficult to treat tumors, such as supratentorial primitive neuroectodermal tumors (sPNET) and atypical teratoid/rhabdoid tumors (ATRT), as well as diagnoses that currently have good long-term survival, such as medulloblastoma. Overcoming the blood brain barrier is a large hurdle in effectively treating brain tumors, particularly when there is a need, as with very young patients, to avoid radiation therapy. Cabazitaxel is a promising taxane derivative shown to effectively kill tumor cells in pre-clinical in vitro and in vivo experiments. Similar to docetaxel, cabazitaxel has favorable drug properties and a manageable safety profile in patients with advanced solid tumors, with the advantage of being able to cross the blood brain barrier. Additionally, cabazitaxel has shown efficacy in docetaxel resistant tumors. The experiments presented here, tested the relative efficacy of cabazitaxel and docetaxel in mouse models of pediatric brain tumors. A spontaneous mouse model of medulloblastoma driven by Smo activation was evaluated by magnetic resonance imaging for tumor growth and blood brain barrier integrity before and after treatment with cabazitaxel or docetaxel. Efficacy of cabazitaxel and docetaxel against patient-derived pediatric brain tumors was tested in flank and orthotopic xenograft models of medulloblastoma, sPNET, ATRT, and ependymoma. We observed regression in some flank xenograft tumors after treatment with cabazitaxel or docetaxel, with evidence of superior action of cabazitaxel. These data support further testing of cabazitaxel as a therapy to treat human pediatric brain tumors. This work has been funded by Sanofi Inc. Citation Format: James M. Olson, Emily Girard, Sally Ditzler, Andrew Richards, Doonhoon Lee, Patricia Vrignaud. Efficacy of cabazitaxel in mouse models of pediatric brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5055. doi:10.1158/1538-7445.AM2013-5055

Abstract 2785: Development program of patient tumor tissue bank to support the drug and target discovery.

Background: The humanization of mice with various tissues named Chi-mice® aimed to reproduce the human situation to be more predictive than conventional models. Despite significant progress in identifying malignancy of cancer cells, a more detailed understanding of tumor generation is needed. Xenograft of tumor cells into immunodeficient rodents has constituted the major preclinical screen for the development of new drugs. These models have identified efficacious agents, but their chemosensitivity, genetic drift and clonal selection induced by cell culture have been part of the high attrition rate observed in the clinical development. Patient-derived tumor xenograft (PDX) obtained in xenografting fresh patient tumor samples in mice are reported as being more predictive to the clinical situation in maintaining the histopathology and molecular diversity of the patient tumors. Methods: The PDXs collection has been set up under ethical agreement with informed consent of patients. The patients have been screened for absence of HIV, HBV and HCV. The anonymized patient9s clinical history and tissue banking (including normal tissue when available) are centralized in our internal biological resource center. Tumor samples were freshly implanted in nude or SCID mice. Results: Cryopreservation of the PDX is performed at early passages allowing using these PDX only at low passage. The histopathology, EGFR, PSA, AR, HER, ER and PR statute for lung, prostate and breast carcinoma and tumor growth characteristics of these PDX are being performed. Lymphoma characterization was performed using immunohistochemistry (hCD20, m/hKi67). Lymphoma detection, probably related to EBV infection, leads to switch to nude mice for xenografting.Conclusion: To create a highly diversified panel of PDX, we organized a global process from multiple centers. These PDX are currently being used in preclinical development of new therapies and clinical positioning including biomarkers identification. Citation Format: Marc Hillairet de Boisferon, Francis Bichat, Caroline Mignard, Loic Morgand, Lydia Blot, Corinne Venot, Ana Merino-Trigo, Patricia Vrignaud. Development program of patient tumor tissue bank to support the drug and target discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2785. doi:10.1158/1538-7445.AM2013-2785