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PDF file, 156K, Histopathological characterization of the tumor samples.

PDF file, 923K, Comparison of gene expression between primary tumor sample, early and late xenograft sample.

PDF file, 129K, Table S1: Description of sequenced exons. Table S2: tumor growth observed in the first engraftment. Table S3: Tumor samples for which development of metastasis has been observed after an engraftment into the cecum. Table S4: Ontology analysis of gene differentially expressed between patient tumors and xenografts.

CCR Translation for This Article from 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.

PDF file, 241K, Frequency of abnormalities detected by aCGH among all samples tested across the genome.

PDF file, 67K, Hierarchical clustering of 82 tumors based on Chromosomal abnormalities analyzed by 244k aCGH.

PDF file, 72K.

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. [Cancer Res 2007;67(1):398–407]

Supplementary Material and Methods from Establishment of Human Colon Cancer Cell Lines from Fresh Tumors versus Xenografts: Comparison of Success Rate and Cell Line Features

Genomic alterations in KRAS/BRAF/NRA S wild-type xenografts, according to their response to cetuximab. Tumors were considered to be amplified if the gene copy number was >3. Gene overexpression is defined by an expression superior to average in all PDX panel + 1 SD.

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. Cancer Res; 75(8); 1560–6. ©2015 AACR.

Example of 2 PDXs treated with cetuximab (one red arrow for each injection).

Per literature, patients with epidermal growth factor receptor (EGFR) exon-20 insertions respond poorly to tyrosine kinase inhibitors (TKIs). This study analyzed real-world data to examine the prognostic and predictive value of these mutations.We conducted a retrospective cohort study using Czech TULUNG Registry data, with data on multiple mutation types, collected in 2011-2020.We analyzed 554 (95.85%) patients with EGFR exon-19 deletions or exon-21 L858R substitutions and 24 (4.15%) patients with exon-20 insertions who received first-line high-value therapies. We summarized clinical characteristics and outcomes in all patients and by cohort. The risk of progression was statistically significantly higher (86%) in the exon-20 insertion cohort compared to the cohort with other mutations. Although not statistically significant, the risk of death was 44% higher in patients with exon-20 insertions.Advanced NSCLC patients with rare EGFR exon-20 insertions have a high risk of progression.

Highlights • Immunodeficient mice are susceptible to spontaneous tumors which are rarely reported in patient-derived tumor xenografts (PDX) studies. • Quality control of PDX identity is required at each step on the preclinical assays to avoid erroneous conclusions. • Description of the approaches used for this PDX check should be clearly detailed in material and methods section., Patient-derived tumor xenograft (PDX) is now largely recognized as a key preclinical model for cancer research, mimicking patient tumor phenotype and genotype. Immunodeficient mice, well-known to develop spontaneous lymphoma, are required for PDX growth. As for all animal models used for further clinical translation, a robust experimental design is strongly required to lead to conclusive results. Here we briefly report unintentional co-engraftment of mouse lymphoma during expansion of well-established PDXs to illustrate the importance of systematic check of the PDX identity to avoid misinterpretation. Besides, this quality control based on complementary approaches deserves a more detailed description in materials and methods section to ensure experimental validity and reproducibility., Graphical Abstract Image, graphical abstract

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.

A fully flexible imaging window acting as an implantable transparent “second skin” upgrades in vivo imaging., Intravital microscopy (IVM) is a powerful technique that enables imaging of internal tissues at (sub)cellular resolutions in living animals. Here, we present a silicone-based imaging window consisting of a fully flexible, sutureless design that is ideally suited for long-term, longitudinal IVM of growing tissues and tumors. Crucially, we show that this window, without any customization, is suitable for numerous anatomical locations in mice using a rapid and standardized implantation procedure. This low-cost device represents a substantial technological and performance advance that facilitates intravital imaging in diverse contexts in higher organisms, opening previously unattainable avenues for in vivo imaging of soft and fragile tissues.

Genomic alterations of anal squamous cell carcinoma (ASCC) remain poorly understood due to the rarity of this tumor. Array comparative genomic hybridization and targeted gene sequencing were performed in 49 cases of ASCC. The most frequently altered regions (with a frequency greater than 25%) were 10 deleted regions (2q35, 2q36.3, 3p21.2, 4p16.3, 4p31.21, 7q36.1, 8p23.3, 10q23.2, 11q22.3, and 13q14.11) and 8 gained regions (1p36.33, 1q21.1, 3q26.32, 5p15.33, 8q24.3, 9q34.3, 16p13.3, and 19p13.3). The most frequent minimal regions of deletion (55%) encompassed the 11q22.3 region containing ATM, while the most frequent minimal regions of gain (57%) encompassed the 3q26.32 region containing PIK3CA. Recurrent homozygous deletions were observed for 5 loci (ie, TGFR2 in 4 cases), and recurrent focal amplifications were observed for 8 loci (ie, DDR2 and CCND1 in 3 cases, respectively). Several of the focal amplified genes are targets for specific therapies. Integrated analysis showed that the PI3K/Akt/mTOR signaling pathway was the pathway most extensively affected, particularly in recurrences compared to treatment‐naive tumors (64% vs 30%; P = .017). In patients with ASCC recurrences, poor overall survival (OS) was significantly correlated with a large number of altered regions (P = .024). These findings provide insight into the somatic genomic alterations in ASCC and highlight the key role of the druggable PI3K/Akt/mTOR signaling pathway.

Background: RSPO ligands, activators of the Wnt/β-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). Methods: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT–PCR. The effect of RSPO on the Wnt/β-catenin pathway activity was determined by luciferase assay, western blotting, and qRT–PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/β-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. Results: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10−4). RSPO2 and RSPO4 stimulate Wnt/β-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX. Conclusions: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

International audience; Background: A better understanding of the molecular profile of anal squamous cell carcinomas (ASCCs) is necessary to consider new therapeutic approaches, and the identification of prognostic and predictive factors for response to treatment. Methods: We retrospectively analysed tumours from ASCC patients for mutational analysis of KRAS, NRAS, HRAS, BRAF, PIK3CA, MET, TP53 and FBXW7 genes by HRM and Sanger sequencing analysis. Results: Specimens from 148 patients were analysed: 96 treatment-naive tumours and 52 recurrences after initial radiotherapy (RT) or chemoradiotherapy (CRT). Mutations of KRAS, PIK3CA, FBXW7 and TP53 genes were present in 3 (2.0%), 30 (20.3%), 9 (6.1%) and 7 tumours (4.7%), respectively. The distribution of the mutations was similar between treatment-naive tumours and recurrences, except for TP53 mutations being more frequent in recurrences (P = 0.0005). In patients treated with abdominoperineal resection (APR) after relapse (n = 38, median follow-up of 18.2 years), overall survival (OS) was significantly correlated with HPV16 status (P = 0.048), gender (P = 0.045) and PIK3CA mutation (P = 0.037). The PIK3CA status retained its prognostic significance in Cox multivariate regression analysis (P = 0.025). Conclusions: Our study identified PIK3CA mutation as an independent prognostic factor in patients who underwent APR for ASCC recurrence, suggesting a potential benefit from adjuvant treatment and the evaluation of targeted therapies with PI3K/Akt/mTor inhibitors in PIK3CA-mutated patients.

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

International audience; Anal squamous cell carcinomas (ASCC) are rare tumours in humans. The etiological role of HPV infection is now well established but little is known about the molecular landscape and signalling pathways involved in the pathogenesis of this cancer. Here we report the results from a whole exome sequencing of a homogeneous group of 20 treatment-naive ASCC. A total of 2422 somatic single nucleotide variations (SNV) were found, with an overall moderate rate of somatic mutations per tumour (median 105 relevant SNV per tumour) but a high mutational load in 3 tumours. The mutational signatures associated with age and APOBEC were observed in 100% and 60% of tumours respectively. The most frequently mutated genes were PIK3CA (25%) followed by FBXW7 (15%), FAT1 (15%), and TRIP12 (15%), the two last ones having never been described in ASCC. The main copy number alterations were gains of chromosome 3q (affecting PIK3CA) and losses of chromosome 11q (affecting ATM). The combined analysis of somatic mutations and copy number alterations show that recurrent alterations of the PI3K/AKT/mTOR pathway are frequent (60%) in these tumours, as well as potentially targetable alterations of other signalling pathways that have never been described in ASCC such as chromatin remodelling (45%) and ubiquitin mediated proteolysis (35%). These results highlight the possible implication of these aberrant signalling pathways in anal carcinogenesis and suggest promising new therapeutic approaches in ASCC. The high somatic mutation burden found in some tumours, suggesting an elevated neoantigen load could also predict sensitivity of ASCC to immunotherapy.

International audience; The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis—while surviving hypoxia—, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.

Nature Reviews Cancer 17, 254–268 (2017) In the online html version of this article, Joan Seoane's affiliations were not correct. He is also a member of the EurOPDX Consortium and is at the Vall d'Hebron Institute of Oncology, 08035 Barcelona, the Universitat Autonoma de Barcelona, 08193 Bellaterra,and the Institucio Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain.

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.

Resistance to endocrine therapy is a major complication of luminal breast cancer and studies of the biological features of hormonal resistance are limited by the lack of adequate preclinical models. The aim of this study is to establish and characterize a panel of primary human luminal breast carcinoma xenografts, and to evaluate their response to endocrine therapies. Four hundred and twenty-three tumor fragments obtained directly from patients have been grafted in the interscapular fatpad of Swiss nude mice. After stable engraftment with estradiol supplementation, xenografted tumors have been validated by conventional pathology and immunohistochemistry examination, and additional molecular studies. In vivo tumor growth and response to different endocrine treatments were evaluated. We have engrafted 423 tumors including 314 ER+ tumors, and 8 new luminal breast cancer xenografts have been obtained (2.5%). Tumor take was much lower for luminal tumors than for non-luminal tumors (2.5 vs. 24.7%, P < 0.0001), and was associated with two independent criteria, i.e., ER status (P < 0.0001) and a high grade tumor (P = 0.05). Histological and immunohistochemical analyses performed on patient’s tumors and xenografts showed striking similarities in the tumor morphology as well as in the expression level of ER, PR, and HER2. Response to hormone therapy, evaluated in 6 luminal models, showed different sensitivities, thus exhibiting heterogeneity similar to what is observed in the clinic. We have established a panel of primary human luminal breast cancer xenografts, recapitulating the biological and clinical behaviors of patient tumors, and therefore suitable for further preclinical experiments.

International audience; Background: The sequence of the beta-subunit of human chorionic gonadotropin (hCG beta varies depending on whether hCG beta is encoded by type I or type II genes. Type II genes are upregulated in trophoblast and cancer but hCG beta can be detected in the serum of nonpregnant women and healthy individuals. We aimed to determine whether monoclonal antibody (mAb) FBT11-II specifically detects hCG beta encoded by type II genes (type II hCG beta). Methods: Competitive inhibition assays with synthetic peptides, immunocytochemical and immunohistochemical studies, type II hCG beta dosing immunoassays and sequencing of CGB genes were performed. Results: Competitive inhibition assays determined that mAb FBT11-II recognizes the type II hCG beta derived peptide. CGB mRNA sequencing of JEG-3 (trophoblastic) and 124 (bladder) cell lines confirmed that JEG-3 expresses type II genes while T24 expresses exclusively type I. FBT11-II only recognizes JEG-expressed hCG beta. Placenta immunohistochemical studies confirmed that type II hCG beta expression is restricted to the syncytiotrophoblast. Immunoassays detected type II hCG beta in serum of patients with either nontrophoblastic cancers or fetal Down syndrome. Conclusion: Type II gene expression can be detected using FBT11-II. This specific recognition could improve the clinical usefulness of assays aimed at either managing aggressive tumors or screening for Down syndrome.

Au cours des deux dernieres decennies, le nombre de nouveaux cas de cancers en France a augmente de 63% : en 2000, 278 000 nouveaux cas de cancers ont ete diagnostiques contre 170 000 en 1980 (chiffres de l’Institut national de veille sanitaire, bulletin epidemiologique hebdomadaire du 21 octobre 2003). Si des progres notables ont ete faits dans la prise en charge de la maladie cancereuse tant au niveau du diagnostic que du traitement, le nombre de deces par cancer ne cesse d’augmenter dans notre pays et, en 2000, le cancer a ete responsable de 150 000 deces. Dans ce contexte, il faut rappeler qu’en France comme partout en Europe ou aux Etats-Unis, la survenue de metastases reste la principale cause de deces chez les malades atteints de cancer (1). De ce fait, l’etude de la biologie des metastases devrait etre un des objectifs majeurs de la recherche en cancerologie, et le developpement de nouveaux moyens diagnostiques et therapeutiques des metastases apparait essentiel. Cet article a pour objectif de faire le point sur les avancees recentes de nos connaissances sur la biologie des metastases et de voir comment ces avancees peuvent conduire au developpement de nouveaux examens biologiques pour predire la survenue de metastases.

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. Cancer Res; 75(8); 1560–6. ©2015 AACR.

International audience; BACKGROUND: Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types. METHODS: To characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts. RESULTS: As expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated. CONCLUSIONS: Taken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

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Through 2 examples of digestive tumors, (colorectal cancer, “CRC”, and anal squamous cell carcinoma, “ASCC”), we will present the development and use of patient-derived tumor xenografts. Colorectal cancer remains a major cause of mortality worldwide and CRC patient death is generally attributable to metastasis development. By contrast, ASCC is a rare tumor (< 5% of all lower gastrointestinal tract malignancies in Europe) but with increasing incidence and mutilating surgery for patients with chemoradiotherapy-resistant tumors. Consequently, these both digestive tumors require new therapeutic approaches and relevant preclinical models to identify and test new drugs/combinations. In this context, we develop and characterize patient-derived CRC and ASCC xenografts. We will illustrate the capacity of these PDXs to recapitulate the intratumoral clonal heterogeneity, gene expression profile, key molecular alterations, histology and drug response of the patient tumors through passages in mice. In addition to these now well-known features depicted in a large number of tumor types, we will discuss about practical situations (orthotopic engraftment, impact of mouse host, criteria for measuring drug efficacy). Finally, we will report alternative methods as ex vivo tumor spheres we have developed. Citation Format: Virginie Dangles-Marie. PDX in colorectal cancer and carcinoma of the anal canal. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA05.

Identifying common patterns of regulation in cancer and placental cells might shed new light on cellular programs allowing aggressive tumor development. Indeed, as it has been described for the first time more than a century ago, cancer and placental cells (trophoblasts) share astonishingly similar phenotypes : migration and invasion, immune escape, and angiogenesis induction. These common phenotypes, relying on common genomic sequences and transcriptomic profiles, may result from a shared epigenomic regulation program. This might be especially true for tumor cells leading eventually to metastasis and cytotrophoblasts (a trophoblasts subset) during placental implantation at the beginning of pregnancy. In order to investigate such common epigenomic patterns, we carried out comparisons of DNA methylation in cancer and placental cell genomes. This study involved: cancer samples (primary tumor vs. normal tissue) across various tissues (including breast, colon, liver, lung, prostate, thyroid, uterus), on one hand; and placenta samples (early vs. late term) either heterogeneous (chorionic villi) or homogenous (ex vivo cytotrophoblasts), on the other hand. Cancer data were data downloaded from The Cancer Genome Atlas web portal. Placenta data were downloaded from the Gene Expression Omnibus web portal. In addition, our group generated original data from ex vivo cytotrophoblasts samples. All data were generated on the Illumina Infinium 450K array. Data analysis was carried out thanks to computational methods for epigenomics recently described. This first direct comparison of cancer and placental cells epigenomes, leveraging both published data and original data, led to the identification of large hypomethylated blocks common to cancer and placental cells. Such common patterns have recently been described as a universal defining epigenetic alteration in human solid tumors. These megabase-scale DNA methylation marks differentiate primary tumors from normal tissues. Likewise, they differentiate early term placentas from late term placentas. Moreover, genes belonging to genomic regions displaying common large hypomethylated blocks overlapping in cancers and placentas are enriched for pathways involved in migration and invasion, immune escape, and angiogenesis induction. Common DNA methylation patterns in cancer and placental cells identified in this pilot study might contribute to the epigenomic regulation of cellular programs allowing aggressive tumor development. Further analyses of these common patterns, as well as analyses of differences in cancer and placental cell epigenomes, could eventually lead to the identification of critical epigenomic switches that prevent healthy placentas to degenerate into tumors, while they allow aggressive tumors to develop. Ultimately, such epigenomic switches could also represent innovative targets in oncology. Citation Format: Akpeli V. Nordor, Sophie Richon, Thierry Fournier, Dominique Bellet, Virginie Dangles-Marie, Martin J. Aryee. Common DNA methylation patterns in cancer and placental cells involved in migration and invasion, immune escape, and angiogenesis induction. [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 2762.

e15060Background: A better understanding of the molecular profile of the anal squamous cell carcinomas (ASCCs) is necessary to consider new therapeutic approaches, and the identification of prognos...

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.

Objective— To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. Methods and Results— Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization. Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. Conclusion— DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFC and mesenchymal stem cell angiogenic phenotypes in vivo. DKK1 also increases tumoral angiogenesis. Thus, we demonstrated a major role of DKK1 in angiogenic processes.

Background Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Methods Protein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. Results We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina. Conclusion The present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.

We report here the characterization of an asymmetric ataxia syndrome (head tilt and circling, with death in the most severe cases) demonstrated by profoundly immunodeficient mice housed at the Institut Curie SPF facility. The immune system of the affected mice had been genetically modified so that they were deficient in both B and T cells. Extensive bacteriologic, parasitic, serologic, and histopathologic analysis of the affected animals and their healthy controls led us to identify Ralstonia pickettii as the causative agent of the ataxic syndrome. The outbreak was managed through a test-and-cull process. Even though they also carried Ralstonia pickettii, immunocompetent mice that were kept in the same facility, did not show any of the signs that were expressed by their immunodeficient counterparts. This case highlights the difficulty of maintaining immunocompetent and immunodeficient mice in the same microbiologic unit and the importance of enlarging the spectrum of health monitoring to opportunistic agents when investigating clinical cases in populations of immunocompromised rodents.

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. [Cancer Res 2007;67(1):398–407]

Trophoblast research over the past decades has underlined the striking similarities between the proliferative, migratory and invasive properties of placental cells and those of cancer cells. This review recapitulates the numerous key molecules, proto-oncogenes, growth factors, receptors, enzymes, hormones, peptides and tumour-associated antigens (TAAs) expressed by both trophoblastic and cancer cells in an attempt to evaluate the genes and proteins forming molecular circuits and regulating the similar behaviours of these cells. Among the autocrine and paracrine loops that might be involved in the strong proliferative capacity of trophoblastic and cancer cells, epidermal growth factor (EGF)/EGF receptor (EGFR), hepatocyte growth factor (HGF)/HGF receptor (HGFR) (Met) and vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) loops may play a predominant role. Similar mechanisms of migration and invasion displayed by trophoblastic and malignant cells comprise alterations in the adhesion molecule phenotype, including the increased expression of alpha1beta1 and alphavbeta3 integrin receptors, whereas another critical molecular event is the down-regulation of the cell adhesion molecule E-cadherin. Among proteases that may play an active role in the invasive capacities of these cells, accumulating evidence suggests that matrix metalloproteinase-9 (MMP-9) expression/activation is a prerequisite. Finally, an overview of molecular circuitries shared by trophoblast and cancer cells reveals that the activation of the phosphatidylinositol 3'-kinase (PI3K)/AKT axis has recently emerged as a central feature of signalling pathways used by these cells to achieve their proliferative, migratory and invasive processes.

Since 1997, nine humanized antibodies received the approval of the FDA to be used as drugs for the treatment of various diseases including transplant rejections, metastatic breast and colon cancers, leukaemia, non-Hodgkin lymphomas, allergic conditions or multiple sclerosis. This review describes techniques used to engineer these antibodies and presents the recent evolutions of these techniques : SDRs grafting orabbreviatedCDRs grafting. Based on the illustrative examples of several antibodies, Mylotarg, Herceptin or Xolair, the therapeutic effectiveness of humanized antibodies are underlined and, with the example of Tysabri, the sometimes dramatic adverse effects associated with their clinical use is stressed. In a second part, this review presents some future and realistic avenues to improve the effectiveness of the humanized antibodies, to decrease their immunogenicity and to reduce their cost.

We described previously a CTL clone able to lyse the autologous carcinoma cell line IGR-Heu after specific recognition of an HLA-A2/mutated alpha-actinin-4 peptide complex. Here, we used IGR-Heu, cultured either as standard two-dimensional monolayers or as three-dimensional spheroids, to further analyze the influence of target architecture on CTL reactivity. Interestingly, we found that changes in the tumor structure from two- to three-dimensional induced a dramatic decrease in its capacity to activate autologous CTL, as measured by IFN-gamma and tumor necrosis factor-alpha secretion. These functional alterations were attributable neither to MHC class I expression nor to tumor antigen (Ag) down-regulation, because IGR-Heu, cultured as two- or three-dimensional, expressed similar levels of HLA-A2 and alpha-actinin-4. More importantly, incubation of three-dimensional cells with synthetic epitope completely restored cytokine release by CTL. This defective Ag presentation correlated with a decrease in heat shock protein (hsp)70 expression by three-dimensional tumors compared with two-dimensional cells. Furthermore, transfection of the tumor cells with hsp70 cDNA completely restored the Ag-presenting potential of spheroids and, therefore, cytokine production by T cells. These data strongly suggest that hsp70 down-regulation in three-dimensional cells may result in tumor resistance to the immune response.

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

Objectives. Establishment of pre-clinical models which reflect molecular alterations of primary tumour is a major goal in cancer research. In order to identify novel therapeutic targets and new treatment, xenografts models are powerful tools to analyse pathways and genes involved in carcinogenesis. The aim of this study is to compare gene expression profilings of 4 cancer types (breast, colon, ovary cancer and uveal melanoma) for which primary tumors (PT) and corresponding xenografts (PTDX) are available. We will highlight differentially expressed gene-sets and pathways involved in stromal environment, cancer cells environment, engraftment process and cancer specific development. In addition, this characterisation was associated to survival analysis in breast cancer. Materials and Methods. Gene expression profiling have been determined using Affymetrix technology. Breast cancer dataset was composed of 8 patient/xenograft paired models corresponding to 16 samples, colon, ovary and uveal melanoma datasets was composed of 24, 11 and 12 paired models respectively. Differential analysis between patients and corresponding xenografts was performed on difference matrix (PTDX-PT). Differentially expressed genes were those with a difference unequal to 0. In order to better identified our gene lists, we analysed large public tumors and cell lines datasets. Results. Comparison between PT and corresponding PTDX identified 150 genes always differentially expressed in the different cancer datasets. A large part are down regulated in PTDX. These genes could be divided into two different environments, immunity on one side and angiogenesis, cellular adhesion and development on the other side. Twenty percent are up-regulated and were mainly associated to cell cycle. A large patient and cell lines collections were used for each cancer type in order to differentiate stromal dependant genes and tumor specific genes. A large part of genes were stromal dependant. However some of them presented same expression between patients and cell lines and are lightly deregulated in mice. Survival and response treatment analysis were performed in breast data. In this cancer, genes related to angiogenesis, cellular adhesion and development were associated to survival. Discussion. This descriptive work based on microarray gene-expression profiling allowed to present common genetic background in four different cancer types and increase knowledge in gene micro-environment in carcinogenesis. This study will then focus on breast cancer and we will characterize patient tumors with high probability to grow into mice in order to concentrate on the response to treatment and improve patient management strategies. Citation Format: Cécile Laurent, Elisabetta Marangoni, Philippe Hupé, Fariba Némati, Nathalie Cassoux, David Gentien, Oumou Goundiam, Virginie Dangles-Marie, Sophie Piperno-Neumann, Pascale Mariani, Didier Decaudin, Laurence Desjardins, Xavier Sastre, Sergio Roman-Roman, Fabien Reyal. Identification of gene-expression modifications related to the tumor engraftment in patient-derived xenograft models of solid 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 LB-263. doi:10.1158/1538-7445.AM2013-LB-263