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List of genes differentially regulated between resistant and responder PDX.

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

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

Purpose: Uveal melanoma is the most common primary intraocular malignant tumor in adults and is defined by a poor natural outcome, as 50% of patients die from metastases. The aim of this study was to develop and characterize a panel of human uveal melanoma xenografts transplanted into immunodeficient mice.Experimental Design: Ninety tumor specimens were grafted into severe combined immunodeficient mice, and 25 transplantable xenografts were then established (28%). Relationship between tumor graft and clinical, biological, and therapeutic features of the patients included were investigated. Characterization of 16 xenografts included histology, molecular analyses by immunohistochemistry, genetic alteration analysis (single-nucleotide polymorphism), and specific tumor antigen expression by quantitative reverse transcription-PCR. Pharmacologic characterization (chemosensitivity) was also done in four models using two drugs, temozolomide and fotemustine, currently used in the clinical management of uveal melanoma.Results: Take rate of human uveal melanoma was 28% (25 of 90). Tumor take was independent of size, histologic parameters, or chromosome 3 monosomy but was significantly higher in metastatic tumors. Interestingly, in vivo tumor growth was prognostic for a lower metastasis-free survival in patients with primary tumors. A high concordance between the patients' tumors and their corresponding xenografts was found for all parameters tested (histology, genetic profile, and tumor antigen expression). Finally, the four xenografts studied displayed different response profiles to chemotherapeutic agents.Conclusions: Based on these results, this panel of 16 uveal melanoma xenografts represents a useful preclinical tool for both pharmacologic and biological assessments. Clin Cancer Res; 16(8); 2352–62. ©2010 AACR.

Supplementary Data from Establishment and Characterization of a Panel of Human Uveal Melanoma Xenografts Derived from Primary and/or Metastatic Tumors

Supplementary Video 1 from High PTP4A3 Phosphatase Expression Correlates with Metastatic Risk in Uveal Melanoma Patients

Supplementary Table 1 from High PTP4A3 Phosphatase Expression Correlates with Metastatic Risk in Uveal Melanoma Patients

A high percentage of uveal melanoma patients develop metastatic tumors predominantly in the liver. We studied the molecular profiles derived from gene expression microarrays and comparative genomic hybridization microarrays, to identify genes associated with metastasis in this aggressive cancer. We compared 28 uveal melanomas from patients who developed liver metastases within three years of enucleation with 35 tumors from patients without metastases or who developed metastases more than 3 years after enucleation. Protein tyrosine phosphatase type IV A member 3 (PTP4A3/PRL3), was identified as a strong predictor of metastasis occurrence. We demonstrated that the differential expression of this gene, which maps to 8q24.3, was not merely a consequence of 8q chromosome overrepresentation. PTP4A3 overexpression in uveal melanoma cell lines significantly increased cell migration and invasiveness in vivo, suggesting a direct role for this protein in metastasis. Our findings suggest that PTP4A3 or its cellular substrates could constitute attractive therapeutic targets to treat metastatic uveal melanomas. Cancer Res; 71(3); 666–74. ©2010 AACR.

Supplementary Figure 1 from High PTP4A3 Phosphatase Expression Correlates with Metastatic Risk in Uveal Melanoma Patients

Supplementary Video 3 from High PTP4A3 Phosphatase Expression Correlates with Metastatic Risk in Uveal Melanoma Patients

Aim: This short communication aims at providing insights to verify whether common yield sampling protocols (i.e., one round trip within the fields over two representative rows) are optimal whatever the considered fields. In addition, it aims to show how factors like the spatial organisation of the within-field yield variability, the length of the rows, the erratic variance, etc. may affect the optimal sampling route and the error of the yield estimation.Methods and Material: A new algorithm based on constraint programming and stochastic approaches was used to provide optimal sampling routes for vineyards. This algorithm guarantees the representativeness of the measurement sites and a minimization of the walking distance. Practical constraints (trellised structure, starting point, etc.) are considered by the algorithm to optimise the walking distance and the resulting sampling route. The algorithm has been applied to 60 simulated vineyards with known yield variability. Characteristics like yield spatial structure, row length and proportion of erratic variance were controlled during the simulation process and were used to study how they affect the optimal sampling route derived from the algorithm.Results: The row length as well as the spatial organization of the within-field yield variability are the main factors that determine the optimality of a sampling route. Spatial organisation of the yield happens to have a strong incidence; fields with small yield patterns (Range of the semi-variogram = 25 m) showed a yield estimation error of less than 2 % with an optimal sampling route of three minutes with 7 sampling sites, whereas it takes more than 5 minutes (with 9 sampling sites) to achieve the same estimation error for fields with larger spatial patterns (range > 50 m). Results also highlight the relevance of original sampling routes which intend to sample only the beginnings of rows or mixed approaches based on a round trip in two inter-rows and complementary samples on the beginnings of one or more rows.Conclusions: This study shows that an optimal sampling route strongly depends on the field characteristics. The optimal sampling route should therefore be tailored to each field. This approach is a first step which shows how this methodology could be used to identify other factors of influence. It could also apply to real fields to optimise other logistic operations in viticulture.Significance and Impact of the Study: This short communication demonstrates the necessity to tailor sampling strategy to characteristics of each field to provide both an optimised sampling route (minimum walking distance with minimum samples) and the best possible estimate. It also proposes an original approach based on field simulations and an optimal sampling route generation algorithm. This approach makes it possible to produce new insights (and also to validate empirical practices) that can help the wine industry to better manage the logistics at harvest. This paper also gives considerations when it comes to the choice of a sampling route for a given field.

Um die Ernte und das Keltern der Trauben besser vorzubereiten zu konnen, ist eine Abschatzung des Ertrags pro Parzelle sehr wichtig. Dies wird in der Regel durch Probennahmen an einer begrenzten Anzahl von Messpunkten kurz vor der Ernte erreicht. Die Durchfuhrung dieser Stichproben wird durch bestimmte praktische Aspekte stark eingeschrankt. Dies betrifft zum Beispiel die Zeit, die diese Operation in Anspruch nimmt. Sie wird von der Anzahl der Messungen und der jeweiligen Route bestimmt, die gewahlt wurde, um die Messstellen zu erreichen.

International audience; Highlights:• Operational grapevine yield development includes vineyard/winery specific operations.• Operational needs drive the challenges that yield reporting methods have to meet.• Yield reporting methods are related to measurement, sampling and modelling issues.• Yield estimation, prediction and forecast address different levels of uncertainty.• Yield reporting methods should ensure a temporal, local, operational yield monitoring.Abstract: Grapevine yield is defined as the quantity of harvest, expressed as either grape mass or wine volumeunits, which has been collected per surface unit are and per crop cycle. The information about current and future yield, termed a yield assessment in this paper, is an essential decision-making element for the grape and wine industry. Crop management, wine-making, commercial, accounting and strategic operations are all adapted to and all impact on the expected yield of the current vintage. Numerous yield assessment approaches have been proposed in the scientific literature. However, only a few of them have considered their adaptation to the operational context under the constraints, needs and strategies of commercial vineyards and wineries. The few studies that have worked on the operational implementation of yield assessment methods have only partially addressed this issue, concentrating their improvement efforts on a single step in the yield assessment process. This paper first proposes to review the characteristics of yield development in an operational context that must be taken into account by yield assessment methodsThese characteristics are consolidated into three main challenges for yield assessment methods: (i) addressing the complex temporality of yield development, (ii) ensuring a local monitoring of yield development and (iii) fitting to the operational needs and constraints to allow for relevant decision support systems in the field. The approaches of yield estimation, prediction and forecast are discussed in the context of these challenges. In a second step, the paper proposes a generic framework for the yield assessment process, including a review of the variables that are used to explain grapevine yield. Issues and proposals from the literature associated respectively with measurement, sampling and yield modelling are reviewed and the need for improved modelling of relationships between explanatory variables and the desired, reported yield variable is discussed. The yield assessment methods found in the literature are categorised and compared according to measurement, estimation and modelling approaches, and then according to the three challenges identified for yield assessment in operational conditions, such that the yield assessment method is adapted to commercial needs and not to research objectives. In conclusion, concrete proposals for new grape yield assessment methods are discussed in order to investigate the as yet unexplored opportunities for the improvement in yield assessment in operational contexts that have been identified in the paper. These considerations could easily be transposed to other perennial crops.

Background Many countries restrict patient material exchange to central diagnostic laboratories abroad, limiting access to assays like MammaPrint® (MP) and BluePrint® (BP). Both assays are microarray-based, with MP being prognostic for distant recurrence and BP for molecular subtyping of breast cancer (Luminal-, HER2-, and Basal-type). To increase accessibility, decentralization is required with Next Generation Sequencing (NGS) being the preferred testing platform given that most diagnostic laboratories have the technology in place. The aim of this beta testing study is to validate a previously developed and centrally validated MP and BP NGS kit for RNA samples in two large tertiary academic hospitals in Europe. Patients and Methods Patients with early breast cancer diagnosed at the Multidisciplinary Breast Center at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Patients with bilateral breast cancer or presenting with more than 3 positive lymph nodes were excluded. Only patients with invasive ductal and invasive lobular carcinoma were included. Twenty tissue sections were cut from formalin-fixed, paraffin-embedded (FFPE) blocks; 10 tissue sections were analyzed at the local site using the MP and BP NGS kit, and 10 tissue sections were analyzed at Agendia using the same kit and procedure, as well as with the golden standard method (gene expression microarrays). Targeted RNA sequencing of the 70 MP and 80 BP signature genes was performed on Illumina MiSeq instruments. The raw NGS data generated at the local test sites was sent through a secure file transfer protocol server to Agendia for interpretation and comparison with microarray and NGS performed in the Agendia laboratories. We aimed for a minimum concordance rate between MP and BP outcome of 90% between each local site and Agendia's centralized site. Results In this study, 116 early breast cancer patients were included (73 from University Hospitals Leuven and 43 from Curie Institute). Out of these patients, 52% were MP Low Risk and 48% MP High Risk according to microarray. The patients had a BP luminal, HER2 or basal subtype in respectively 83%, 9% and 8%. Concordance between MP microarray obtained from Agendia and MP NGS obtained from the local sites was 91.4%. Concordance between MP High and Low Risk classification between NGS Leuven versus NGS Agendia was 92.1% and between NGS Curie versus NGS Agendia 95.3%. For BP subtype outcomes, the results from microarray versus NGS for all patients combined from both local sites gave a 98.3% concordance and NGS Agendia versus NGS from each local site gave a 100% concordance. Conclusion The MP and BP NGS kit was successfully validated in a decentralized setting, showing high concordance between results obtained at three different sites. There was a clear benefit of having well-trained NGS experienced diagnostic technical teams. The MP and BP NGS kit the first FFPE targeted RNA sequencing based multigene signature for breast cancer care, will provide a high and equal standard of MP and BP gene expression testing for breast cancer in a decentralized setting. Citation Format: Slembrouck L, Laurent C, Delahaye LJ, Mittempergher L, Vanden Bempt I, Vander Borght S, Darrigues L, Vliegen L, Sintubin P, Raynal V, Bohec M, Reyes C, Rapinat A, Helsmoortel C, Jongen L, Hoste G, Neven P, Wildiers H, Smeets A, Nevelsteen I, Punie K, Van Nieuwenhuysen E, Han S, Salomon AV, Faron EL, Cynober T, Witteveen AT, Neijenhuis S, Glas AM, Reyal F, Floris G. Decentralized beta testing of MammaPrint and BluePrint NGS kit at University Hospitals Leuven and Curie Institute Paris [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-25.

International audience; Adult T-cell leukemia/lymphoma (ATL) carries a poor prognosis even in indolent subtypes. We performed targeted deep sequencing combined with mapping of HTLV-1 proviral integration sites of 61 ATL patients of African and Caribbean origin. This revealed mutations mainly affecting TCR/NF-kB (74%), T-cell trafficking (46%), immune escape (29%), and cell cycle (26%) related pathways, consistent with the genomic landscape previously reported in a large Japanese cohort. To examine the evolution of mutational signatures upon disease progression while tracking the viral integration architecture of the malignant clone, we carried out a longitudinal study of patients who either relapsed or progressed from an indolent to an aggressive subtype. Serial analysis of relapsing patients identified several patterns of clonal evolution. In progressing patients, the longitudinal study revealed NF-kB/NFAT mutations at progression that were present at a subclonal level at diagnosis (allelic frequency < 5%). Moreover, the presence in indolent subtypes of mutations affecting the TCR/NF-kB pathway, whether clonal or subclonal, was associated with significantly shorter time to progression and overall survival. Our observations reveal the clonal dynamics of ATL mutational signatures at relapse and during progression. Our study defines a new subgroup of indolent ATLs characterized by a mutational signature at high risk of transformation.

Adult T-cell leukemia/lymphoma (ATL) carries a poor prognosis even in indolent subtypes. We performed targeted deep sequencing combined with mapping of HTLV-1 proviral integration sites of 61 ATL patients of African and Caribbean origin. This revealed mutations mainly affecting TCR/NF-kB (74%), T-cell trafficking (46%), immune escape (29%), and cell cycle (26%) related pathways, consistent with the genomic landscape previously reported in a large Japanese cohort. To examine the evolution of mutational signatures upon disease progression while tracking the viral integration architecture of the malignant clone, we carried out a longitudinal study of patients who either relapsed or progressed from an indolent to an aggressive subtype. Serial analysis of relapsing patients identified several patterns of clonal evolution. In progressing patients, the longitudinal study revealed NF-kB/NFAT mutations at progression that were present at a subclonal level at diagnosis (allelic frequency 5%). Moreover, the presence in indolent subtypes of mutations affecting the TCR/NF-kB pathway, whether clonal or subclonal, was associated with significantly shorter time to progression and overall survival. Our observations reveal the clonal dynamics of ATL mutational signatures at relapse and during progression. Our study defines a new subgroup of indolent ATLs characterized by a mutational signature at high risk of transformation.

Medullary breast carcinoma (MBC) is a rare subtype of triple-negative breast cancer with specific genomic features within the spectrum of basal-like carcinoma (BLC). In this study of 19 MBCs and 36 non-MBC BLCs, we refined the transcriptomic and genomic knowledge about this entity. Unsupervised and supervised analysis of transcriptomic profiles confirmed that MBC clearly differs from non-MBC BLC, with 92 genes overexpressed and 154 genes underexpressed in MBC compared with non-MBC BLC. Immunity-related pathways are the most differentially represented pathways in MBC compared with non-MBC BLC. The proapoptotic gene BCLG (official name BCL2L14) is by far the most intensely overexpressed gene in MBC. A quantitative RT-PCR validation study conducted in 526 breast tumors corresponding to all molecular subtypes documented the specificity of BCLG overexpression in MBC, which was confirmed at the protein level by immunohistochemistry. We also found that most MBCs belong to the immunomodulatory triple-negative breast cancer subtype. Using pan-genomic analysis, it was found that MBC harbors more losses of heterozygosity than non-MBC BLC. These observations corroborate the notion that MBC remains a distinct entity that could benefit from specific treatment strategies (such as deescalation or targeted therapy) adapted to this rare tumor type.

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.

The oncogenic events involved in breast implant-associated anaplastic large cell lymphoma (BI-ALCL) remain elusive. To clarify this point, we have characterized the genomic landscape of 34 BI-ALCLs (15 tumor and 19 in situ subtypes) collected from 54 BI-ALCL patients diagnosed through the French Lymphopath network. Whole-exome sequencing (n = 22, with paired tumor/germline DNA) and/or targeted deep sequencing (n = 24) showed recurrent mutations of epigenetic modifiers in 74% of cases, involving notably KMT2C (26%), KMT2D (9%), CHD2 (15%), and CREBBP (15%). KMT2D and KMT2C mutations correlated with a loss of H3K4 mono- and trimethylation by immunohistochemistry. Twenty cases (59%) showed mutations in ≥1 member of the JAK/STAT pathway, including STAT3 (38%), JAK1 (18%), and STAT5B (3%), and in negative regulators, including SOCS3 (6%), SOCS1 (3%), and PTPN1 (3%). These mutations were more frequent in tumor-type samples than in situ samples (P = .038). All BI-ALCLs expressed pSTAT3, regardless of the mutational status of genes in the JAK/STAT pathway. Mutations in the EOMES gene (12%) involved in lymphocyte development, PI3K-AKT/mTOR (6%), and loss-of-function mutations in TP53 (12%) were also identified. Copy-number aberration (CNA) analysis identified recurrent alterations, including gains on chromosomes 2, 9p, 12p, and 21 and losses on 4q, 8p, 15, 16, and 20. Regions of CNA encompassed genes involved in the JAK/STAT pathway and epigenetic regulators. Our results show that the BI-ALCL genomic landscape is characterized by not only JAK/STAT activating mutations but also loss-of-function alterations of epigenetic modifiers.

International audience; In precision agriculture, most studies focus on spatial crop variability whereas temporal variability and its role in decision-making is equally important. The classical methods for temporal analysis have limitations, potentially resulting in information loss. A novel method based on a Bayesian functional Linear regression with Sparse Steps functions (BLiSS method) is evaluated in this paper to investigate continuous influence analysis when working with time series data. The example of the influence of temperature on the number of clusters per vine during the year before harvest was considered as an example application. The evaluation of the BLiSS results was done by comparing identified critical time periods with traditional viticulture knowledge in the literature. It showed the relevance of the BLiSS method, highlighting already known results and identifying new critical time periods for yield elaboration.

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

Tumors are made of evolving and heterogeneous populations of cells which arise from successive appearance and expansion of subclonal populations, following acquisition of mutations conferring them a selective advantage. Those subclonal populations can be sensitive or resistant to different treatments, and provide information about tumor aetiology and future evolution. Hence, it is important to be able to assess the level of heterogeneity of tumors with high reliability for clinical applications.In the past few years, a large number of methods have been proposed to estimate intra-tumor heterogeneity from whole exome sequencing (WES) data, but the accuracy and robustness of these methods on real data remains elusive. Here we systematically apply and compare 6 computational methods to estimate tumor heterogeneity on 1,697 WES samples from the cancer genome atlas (TCGA) covering 3 cancer types (breast invasive carcinoma, bladder urothelial carcinoma, and head and neck squamous cell carcinoma), and two distinct input mutation sets. We observe significant differences between the estimates produced by different methods, and identify several likely confounding factors in heterogeneity assessment for the different methods. We further show that the prognostic value of tumor heterogeneity for survival prediction is limited in those datasets, and find no evidence that it improves over prognosis based on other clinical variables.In conclusion, heterogeneity inference from WES data on a single sample, and its use in cancer prognosis, should be considered with caution. Other approaches to assess intra-tumoral heterogeneity such as those based on multiple samples may be preferable for clinical applications.

// Guillaume Carita 1,* , Estelle Frisch-Dit-Leitz 2,* , Ahmed Dahmani 1 , Chloe Raymondie 1 , Nathalie Cassoux 3 , Sophie Piperno-Neumann 4 , Fariba Nemati 1 , Cecile Laurent 5 , Leanne De Koning 6 , Ensar Halilovic 7 , Sebastien Jeay 7 , Andrew Wylie 7 , Caroline Emery 7 , Sergio Roman-Roman 2 , Marie Schoumacher 2,* and Didier Decaudin 1,4,* 1 Laboratory of preclinical investigation, Department of Translational Research, PSL University, Institut Curie, Paris, France 2 Department of Translational Research, Institut Curie, PSL University, Paris, France 3 Department of Ophthalmological Oncology, Institut Curie, Paris, France 4 Department of Medical Oncology, Institut Curie, Paris, France 5 Residual Tumor & Response to Treatment Lab, Department of Translational Research, Institut Curie, PSL University, Paris, Paris, France 6 RPPA Platform, Department of Translational Research, Institut Curie, PSL University, Paris, France 7 Novartis Institutes for Biomedical Research, Cambridge, MA USA * These authors have contributed equally to this work Correspondence to: Didier Decaudin, email: // Keywords : xenograft models, cellular response to anticancer drugs, uveal melanoma, AEB071 combinations, synergy Received : April 04, 2016 Accepted : May 10, 2016 Published : May 22, 2016 Abstract Uveal melanoma (UM) is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified. Novel therapeutic approaches are therefore urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071. The most promising approaches were further investigated in vitro using our unique panel of UM cell lines. When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs. Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death. While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease. Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients.

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

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Introduction Uveal melanoma (UM) with an inflammatory phenotype, characterized by infiltrating leukocytes and increased human leukocyte antigen (HLA) expression, carry an increased risk of death due to metastases. These tumors should be ideal for T-cell based therapies, yet it is not clear why prognostically-infaust tumors have a high HLA expression. We set out to determine whether the level of HLA molecules in UM is associated with other genetic factors, HLA transcriptional regulators, or microenvironmental factors. Methods 28 enucleated UM were used to study HLA class I and II expression, and several regulators of HLA by immunohistochemistry, PCR microarray, qPCR and chromosome SNP-array. Fresh tumor samples of eight primary UM and four metastases were compared to their corresponding xenograft in SCID mice, using a PCR microarray and SNP array. Results Increased expression levels of HLA class I and II showed no dosage effect of chromosome 6p, but, as expected, were associated with monosomy of chromosome 3. Increased HLA class I and II protein levels were positively associated with their gene expression and with raised levels of the peptide-loading gene TAP1, and HLA transcriptional regulators IRF1, IRF8, CIITA, and NLRC5, revealing a higher transcriptional activity in prognostically-bad tumors. Implantation of fresh human tumor samples into SCID mice led to a loss of infiltrating leukocytes, and to a decreased expression of HLA class I and II genes, and their regulators. Conclusion Our data provides evidence for a proper functioning HLA regulatory system in UM, offering a target for T-cell based therapies.

PURPOSE To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness. METHODS Cell membrane localization of matrix metalloprotease 14 (MMP14) in uveal melanoma cells expressing protein tyrosine phosphatase A3 (PTP4A3) was assessed by flow cytometry or immunohistochemistry. The vesicular trafficking of MMP14 in the presence of PTP4A3 was evaluated in OCM-1 cells expressing either the wild-type or mutated phosphatase. Finally, MMP14 localization at the cell membrane of OCM-1 cells was impaired using RNA interference, and the PTP4A3-related migration in vitro and invasiveness in vivo of the treated cells were evaluated. RESULTS We found that the membrane-anchored MMP14 is enriched at the cell surface of OCM-1 cells, patient-derived xenograft cells, and human primary uveal melanoma tumors expressing PTP4A3. Moreover, we show that PTP4A3 and MMP14 colocalize and that the vesicular trafficking of MMP14 is faster in the presence of active PTP4A3. Finally, we demonstrate that inhibition of MMP14 expression in uveal melanoma cells expressing PTP4A3 impairs their migration in vitro and invasiveness in vivo. CONCLUSIONS Our observations indicate that PTP4A3 increases cell membrane accumulation of MMP14 as a result of increased cellular trafficking of the metalloprotease. We also show that downregulation of MMP14 expression reduced PTP4A3-induced cell migration and invasiveness. Taken together, our findings suggest that PTP4A3-related subcellular localization of MMP14 is an important event in metastasis induction.

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes., Stem cells in the embryonic mammary gland that express the Notch1 receptor are initially multipotent and highly regenerative, but they progressively restrict their lineage potential to the lumen of the mammary duct, where they may give rise to breast cancer., Author Summary Tissue-specific stem cells are believed to be multipotent, thus able to generate all cell types of their tissue of origin. In the mammary gland epithelium, however, the existence of multipotent versus unipotent adult stem cells is currently under debate. In this study, we have identified and characterized a population of mammary luminal progenitors that express the Notch1 receptor. Using lineage tracing experiments, we found that these cells are self-sustained unipotent adult progenitors with high self-renewal capacity. Although they lack estrogen and progesterone hormone receptors, these cells are highly responsive to hormones. Importantly, Notch1-expressing cells are multipotent during embryonic mammary development, when they can give rise to all mammary cell types, while they become lineage-restricted postnatally. The cells characterized in this study also present extensive plasticity, as they can repopulate the entire mammary gland in transplantation experiments. Our study reveals that the Notch1 receptor is a specific marker for the identification of luminal progenitors that lack expression of hormone receptors and that can be critical for breast cancer initiation.

International audience; Changes in marine environments, induced by the global warming, are likely to influence the prey field distribution and consequently the foraging behaviour and the distribution of top marine predators. Thanks to bio-logging, the simultaneous measurements of fine-scale foraging behaviors and oceanographic parameters by predators allow characterizing their foraging environments and provide insights into their prey distribution. In this context, we propose to delimit and to characterize the foraging environments of a marine predator, the Southern Elephant Seal (SES). To do so, the relationship between oceanographic factors and prey encounter events (PEE) was investigated in 12 females SES from Kerguelen Island simultaneously equipped with accelerometers and with a range of physical sensors (temperature, light and depth). PEEs were assessed from the accelerometer data at high spatio-temporal precision while the physical sensors allowed the continuous monitoring of environmental conditions encountered by the SES when diving. First, visited and foraging environments were distinguished according to the oceanographic conditions encountered in the absence and in presence of PEE. Then, a hierarchical classification of the physical parameters recorded during PEEs led to the distinction of five different foraging environments. These foraging environments were structured according to the main frontal systems of the SO. One was located north to the subantarctic front (SAF) and characterized by high temperature and depth, and low light levels. Another, characterized by intermediate levels of temperature, light and depth, was located between the SAF and the polar front (PF). And finally, the last three environments were all found south to the PF and, characterized by low temperature but highly variable depth and light levels. The large physical and/or spatial differences found between these environments suggest that, depending on the location, different prey communities are targeted by SES over a broad range of water temperature, light level and depth conditions. This result highlights the versatility of this marine predator. In addition, in most cases, PEEs were found deeper during the day than during the night, which is indicative of mesopelagic prey performing nycthemeral migration, a behaviour consistent with myctophids species thought to represent the bulk of Kerguelen SES female diets.

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

Introduction HER2-positive breast cancer (BC) is a heterogeneous group of aggressive breast cancers, the prognosis of which has greatly improved since the introduction of treatments targeting HER2. However, these tumors may display intrinsic or acquired resistance to treatment, and classifiers of HER2-positive tumors are required to improve the prediction of prognosis and to develop novel therapeutic interventions. Methods We analyzed 2893 primary human breast cancer samples from 21 publicly available datasets and developed a six-metagene signature on a training set of 448 HER2-positive BC. We then used external public datasets to assess the ability of these metagenes to predict the response to chemotherapy (Ignatiadis dataset), and prognosis (METABRIC dataset). Results We identified a six-metagene signature (138 genes) containing metagenes enriched in different gene ontologies. The gene clusters were named as follows: Immunity, Tumor suppressors/proliferation, Interferon, Signal transduction, Hormone/survival and Matrix clusters. In all datasets, the Immunity metagene was less strongly expressed in ER-positive than in ER-negative tumors, and was inversely correlated with the Hormonal/survival metagene. Within the signature, multivariate analyses showed that strong expression of the “Immunity” metagene was associated with higher pCR rates after NAC (OR = 3.71[1.28–11.91], p = 0.019) than weak expression, and with a better prognosis in HER2-positive/ER-negative breast cancers (HR = 0.58 [0.36–0.94], p = 0.026). Immunity metagene expression was associated with the presence of tumor-infiltrating lymphocytes (TILs). Conclusion The identification of a predictive and prognostic immune module in HER2-positive BC confirms the need for clinical testing for immune checkpoint modulators and vaccines for this specific subtype. The inverse correlation between Immunity and hormone pathways opens research perspectives and deserves further investigation.

International audience; Triple-negative breast cancer (TNBC) is a heterogeneous group of aggressive breast cancers for which no targeted treatment is available. Robust tools for TNBC classification are required, to improve the prediction of prognosis and to develop novel therapeutic interventions. We analyzed 3,247 primary human breast cancer samples from 21 publicly available datasets, using a five-step method: (1) selection of TNBC samples by bimodal filtering on ER-HER2 and PR, (2) normalization of the selected TNBC samples, (3) selection of the most variant genes, (4) identification of gene clusters and biological gene selection within gene clusters on the basis of String (c) database connections and gene-expression correlations, (5) summarization of each gene cluster in a metagene. We then assessed the ability of these metagenes to predict prognosis, on an external public dataset (METABRIC). Our analysis of gene expression (GE) in 557 TNBCs from 21 public datasets identified a six-metagene signature (167 genes) in which the metagenes were enriched in different gene ontologies. The gene clusters were named as follows: Immunity1, Immunity2, Proliferation/DNA damage, AR-like, Matrix/Invasion1 and Matrix2 clusters respectively. This signature was particularly robust for the identification of TNBC subtypes across many datasets (n = 1,125 samples), despite technology differences (Affymetrix (c) A, Plus2 and Illumina (c)). Weak Immunity two metagene expression was associated with a poor prognosis (disease-specific survival; HR = 2.68 [1.59-4.52], p = 0.0002). The six-metagene signature (167 genes) was validated over 1,125 TNBC samples. The Immunity two metagene had strong prognostic value. These findings open up interesting possibilities for the development of new therapeutic interventions.

Uveal melanoma (UM) is the most frequent eye cancer in the adulthood and it represents 4-5% of all melanomas. Although at diagnosis over 95% of patients have disease limited to the eye, about 50% will develop metastases after a median time of three years. From transcriptomic study, we identified PTP4A3/PRL3 level as an efficient predictor of patient's survival. Overexpression of PTP4A3 in UM OCM-1 increased both cell migration and invasiveness. We then got interested in identifying downstream targets of PTP4A3 and more precisely proteins linked to cytoskeleton. We first highlighted collapsin Response Mediator Protein (CRMP2) by 2-D electrophoresis of phosphorus-enriched UM OCM1 stably-expressing PTP4A3 or an inactive catalytic mutant of PTP4A3 (C104S). CRMP2 was dephosphorylated in presence of PTP4A3. CRMP2 expression in UM is inversely correlated with that of PTP4A3, suggesting an antagonistic role of these proteins in tumor progression. Through the use of shRNA in OCM-1, the downregulation of CRMP2 led to an increase in cell migration of PTP4A3-expressing OCM1. In addition, we focused on ATIP3, a protein encoded by the gene MTUS1, which binds to microtubules. In breast cancer, MTUS1 expression was shown to be downregulated in highly proliferative breast carcinomas of poor clinical outcome. Similarly, our transcriptomic analysis showed that expressions of PTP4A3 and MTUS1 were anti-correlated, suggesting that the reduction of MTUS1 gene and ATIP3 expression in UM would be of bad prognosis. In our OCM1 cells, we demonstrated that ATIP3 overexpression blocks proliferation however effect of ATIP3 overexpression on cell migration still need to be elucidaded.In conclusion, CRMP2 and ATIP3 may play a role in the metastatic process of UM tumors expressing high levels of the phosphatase. Citation Format: Geraldine Liot, Oceane Anezo, Cecile Laurent, Selma Maacha, Nathalie Planque, Simon Saule. Potential targets of PTP4A3 involved in uveal melanoma migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4063. doi:10.1158/1538-7445.AM2014-4063