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Background Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity but remains hindered by technical challenges associated with CTC rarity and heterogeneity. Objective To evaluate CTCs as surrogate samples for genomic analyses in metastatic castration-resistant prostate cancer (mCRPC). Design, setting, and participants Three isolation strategies (filter laser-capture microdissection, self-seeding microwell chips, and fluorescence-activated cell sorting) were developed to capture CTCs with various epithelial and mesenchymal phenotypes and isolate them at the single-cell level. Whole-genome amplification (WGA) and WGA quality control were performed on 179 CTC samples, matched metastasis biopsies, and negative controls from 11 patients. All patients but one were pretreated with enzalutamide or abiraterone. Whole-exome sequencing (WES) of 34 CTC samples, metastasis biopsies, and negative controls were performed for seven patients. Outcome measurements and statistical analysis WES of CTCs was rigorously qualified in terms of percentage coverage at 10× depth, allelic dropout, and uncovered regions. Shared somatic mutations between CTCs and matched metastasis biopsies were identified. A customized approach based on determination of mutation rates for CTC samples was developed for identification of CTC-exclusive mutations. Results and limitations Shared mutations were mostly detected in epithelial CTCs and were recurrent. For two patients for whom a deeper analysis was performed, a few CTCs were sufficient to represent half to one-third of the mutations in the matched metastasis biopsy. CTC-exclusive mutations were identified in both epithelial and nonepithelial CTCs and affected cytoskeleton, invasion, DNA repair, and cancer-driver genes. Some 41% of CTC-exclusive mutations had a predicted deleterious impact on protein function. Phylogenic relationships between CTCs with distinct phenotypes were evidenced. Conclusions CTCs can provide unique insight into metastasis mutational diversity and reveal undiagnosed genomic aberrations in matched metastasis biopsies. Patient summary Our results demonstrate the clinical potential of circulating tumor cells to provide insight into metastatic events that could be critical to target using precision medicine.

Background The role of chemotherapy in early luminal breast cancer remains challenged. The NEOPAL trial (NCT 02400567; Cottu et al, ESMO 2017 LBA09) compared sequential chemotherapy (CT) and letrozole-palbociclib (LP) as neoadjuvant treatment in PAM50 defined high-risk luminal breast cancer patients, showing that LP might be as efficient as CT with regard to breast conserving surgery and pathological response. We report here extended exploratory pathological results, focusing on tumor infiltrating lymphocytes (TILs), proliferative response and preoperative endocrine prognostic index (PEPI) scores. Material and Methods Tumor blocks from baseline biopsy and surgical specimens were available for centralized review from the 106 randomized patients (53 in each arm). TILs quantification, KI67 staining and counting, and ER quantification were performed according to standard methods. Residual proliferative cancer burden (RPCB) and PEPI scores were computed according to published algorithms. Wilcoxon rank sum test and Mann Whitney test were used to compare paired and unpaired data. The chi-square and Fisher exact tests were used for categorical variables. Results Overall, median TILs count did not differ between LP and CT patients, both at baseline (p=0.37) and at the end of treatment (p=0.42). Median TILs count climbed from 5% (0-60) to 10% (1-60) in the LP arm (p=0.0026) and from 2% (0-30) to 10% (0-60) in the CT arm (p=0.0023). Median Ki67 dropped sharply in both arms, from 30% (1-80) to 1% (0-30) in the LP arm (p=1.10e-8) and from 30% (2-80) to 5% (0-30) in the CT arm (p=3.10e-9). Decrease in the Ki67 geometric mean was as sharp. Of note, while baseline Ki67 was similar in both arms (p=0.315), decrease in the LP arm was significantly more profound than in the CT arm (p=0.00075). Pathological response according to RPCB were as follows, in the LP and CT arm, respectively: class 0: 9.6%/10.2%; class I: 84.6%/73.5%; class II: 5.8%/16.3%. The relapse free survival PEPI scores were as follow in the LP and CT arm, respectively: class I: 13.5%/16.3%; class II: 59.6%/46.9%; class III: 28.9%/36.8% (p=0.504). Breast cancer specific survival PEPI scores were as follow in the LP and CT arm, respectively: class I: 18.9%/8.2%; class II: 54.7%/40.8%; class III: 26.4%/51%. These results were significantly better in the LP arm (p=0.027). There was no correlation between final TILs quantification and the RPCB or PEPI scores. Conclusions In this prospective multicenter study with centralized pathological review, neoadjuvant letrozole-palbociclib combination generates impressive proliferative and endocrine specific response features. It compared well with chemotherapy. The LP combination also significantly increased lymphocytic infiltration. Its clinical significance and utility remain to be elucidated, but it potentially adds new prognostic and theranostic information. Citation Format: Vincent-Salomon A, Mathieu M-C, Bataillon G, Arnould L, Verrièle V, Ghnassia J-P, Haudebourg J, Penault-Llorca F, Lefebvre C, Maran-Gonzalez A, Guinebretière J-M, Duprez R, Berghian A, Blanc-Fournier C, Calès V, Galant C, Delrée P, Lemonnier J, Delaloge S, Cottu PH. TILs variations, proliferative response and PEPI scores in patients with luminal breast cancer receiving neoadjuvant letrozole-palbociclib or chemotherapy: An extended analysis of the NEOPAL trial [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 P4-15-02.

International audience; But: Les inhibiteurs du point de contrôle immunitaire (ICI) dirigés par le ligand antiprogrammé de la mort (PD1/PD-L1) sont largement utilisés pour traiter les patients atteints d’un cancer du poumon non à petites cellules (CPNPC) avancé qui progressent après une chimiothérapie de première intention. La meilleure stratégie après une progression précoce en première ligne n’a pas été spécifiquement étudiée.Patients et méthodes: Nous avons mené une étude rétrospective multicentrique incluant tous les patients consécutifs atteints de CPNPC progressant dans les 3 premiers mois suivant l’introduction de la chimiothérapie de première intention et traités par ici de deuxième ligne en monothérapie ou chimiothérapie entre mars 2010 et novembre 2017. Nous avons analysé les données clinicopathologiques et les résultats de la chimiothérapie de deuxième intention par rapport à l’ICI de deuxième ligne : taux de réponse objective (ORR), survie sans progression (SSP), survie globale (SG).Résultats: Nous avons identifié 176 patients atteints d’une maladie réfractaire, 99 qui ont reçu une immunothérapie ultérieure et 77 ont subi une chimiothérapie. Les 2 populations étaient comparables en ce qui concerne les principaux critères pronostiques, l’âge médian était de 60 ans, l’histologie principale était l’adénocararcimome (68,2%). La SSP n’était pas significativement différente entre les deux traitements 1,9 [1,8-2,1] contre 1,6 mois [1,4-2,0] (P = 0,125). Par rapport à la chimiothérapie, les patients traités par ICI avaient une SG supérieure (P = 0,03) (IC médiane [IC à 95 %)] OS 4,6 [2,8-6,7] contre 4,2 mois [3,4-5,9] et une amélioration non significative de la RRO (17,2 % contre 7,9 %, respectivement, P = 0,072). Un mauvais état de performance (ECOG PS≥2) et un nombre plus élevé de sites métastatiques (≥3) ont été associés à un pronostic plus sombre. Les patients mutés par KRAS ne semblaient pas bénéficier davantage de l’ICI que de la chimiothérapie.Conclusions: L’ICI semble être le traitement de deuxième intention préféré des patients réfractaires à la chimiothérapie de première intention.

GANEA2 study was designed to assess accuracy and safety of sentinel lymph node (SLN) after neo-adjuvant chemotherapy (NAC) in breast cancer patients. Early breast cancer patients treated with NAC were included. Before NAC, patients with cytologically proven node involvement were allocated into the pN1 group, other patient were allocated into the cN0 group. After NAC, pN1 group patients underwent SLN and axillary lymph node dissection (ALND); cN0 group patients underwent SLN and ALND only in case of mapping failure or SLN involvement. The main endpoint was SLN false negative rate (FNR). Secondary endpoints were predictive factors for remaining positive ALND and survival of patients treated with SLN alone. From 2010 to 2014, 957 patients were included. Among the 419 patients from the cN0 group treated with SLN alone, one axillary relapse occurred during the follow-up. Among pN1 group patients, with successful mapping, 103 had a negative SLN. The FNR was 11.9% (95% CI 7.3–17.9%). Multivariate analysis showed that residual breast tumor size after NAC ≥ 5 mm and lympho-vascular invasion remained independent predictors for involved ALND. For patients with initially involved node, with negative SLN after NAC, no lympho-vascular invasion and a remaining breast tumor size 5 mm, the risk of a positive ALND is 3.7% regardless the number of SLN removed. In patients with no initial node involvement, negative SLN after NAC allows to safely avoid an ALND. Residual breast tumor and lympho-vascular invasion after NAC allow identifying patients with initially involved node with a low risk of ALND involvement.

The development of precision medicine for the management of metastatic breast cancer is an appealing concept; however, major scientific and logistical challenges hinder its implementation in the clinic. The identification of driver mutational events remains the biggest challenge, because, with the few exceptions of ER, HER2, PIK3CA and AKT1, no validated oncogenic drivers of breast cancer exist. The development of bioinformatic tools to help identify driver mutations, together with assessment of pathway activation and dependency should help resolve this issue in the future. The occurrence of secondary resistance, such as ESR1 mutations, following endocrine therapy poses a further challenge. Ultra-deep sequencing and monitoring of circulating tumour DNA (ctDNA) could permit early detection of the genetic events underlying resistance and inform on combination therapy approaches. Beside these scientific challenges, logistical and operational issues are a major limitation to the development of precision medicine. For example, the low incidence of most candidate genomic alterations hinders randomized trials, as the number of patients to be screened would be too high. We discuss these limitations and the solutions, which include scaling-up the number of patients screened for identifying a genomic alteration, the clustering of genomic alterations into pathways, and the development of personalized medicine trials.

The pathogenesis and nosology of HIV-associated neurological disease (HAND) remain incompletely understood. Here, to provide new insight into the molecular events leading to neurocognitive impairments (NCI) in HIV infection, we analyzed pathway dysregulations in gene expression profiles of HIV-infected patients with or without NCI and HIV encephalitis (HIVE) and control subjects. The Gene Set Enrichment Analysis (GSEA) algorithm was used for pathway analyses in conjunction with the Molecular Signatures Database collection of canonical pathways (MSigDb). We analyzed pathway dysregulations in gene expression profiles of patients from the National NeuroAIDS Tissue Consortium (NNTC), which consists of samples from 3 different brain regions, including white matter, basal ganglia and frontal cortex of HIV-infected and control patients. While HIVE is characterized by widespread, uncontrolled inflammation and tissue damage, substantial gene expression evidence of induction of interferon (IFN), cytokines and tissue injury is apparent in all brain regions studied, even in the absence of NCI. Various degrees of white matter changes were present in all HIV-infected subjects and were the primary manifestation in patients with NCI in the absence of HIVE. In particular, NCI in patients without HIVE in the NNTC sample is associated with white matter expression of chemokines, cytokines and β-defensins, without significant activation of IFN. Altogether, the results identified distinct pathways differentially regulated over the course of neurological disease in HIV infection and provide a new perspective on the dynamics of pathogenic processes in the course of HIV neurological disease in humans. These results also demonstrate the power of the systems biology analyses and indicate that the establishment of larger human gene expression profile datasets will have the potential to provide novel mechanistic insight into the pathogenesis of neurological disease in HIV infection and identify better therapeutic targets for NCI.

Objectives Bioinformatics analyses of pathways and genes differentially expressed between malignant and benign lesions could allow discovering new therapeutic targets. Here, we identified Checkpoint kinase 1 (Chk1) as a potent therapeutic target in triple-negative breast cancer (TNBC). Materials and methods Differential gene expression between TNBC, other malignant and benign lesions was performed on two breast cancer datasets. Chk1 was targeted using RNA interference or chemical inhibitor in several TNBC cell lines. Results DNA repair pathway was identified as one mostly deregulated pathway in TNBC as compared to benign lesions. Chk1 was identified as candidate target among the 35 genes included in this pathway. Gene expression analysis revealed that Chk1 gene was significantly overexpressed in TNBC as compared to non-TNBC and benign lesions. Depletion of Chk1 protein expression induced a marked reduction of cell viability and led to mitotic catastrophe in TNBC cells. Chemical Chk1 inhibitor decreased survival in TNBC cells, and transcriptome analyze revealed a modulation of gene expression profile in response to Chk1 treatment. Conclusion These findings suggest that Chk1 may represent a therapeutic target in TNBC, and provide a rationale to evaluate Chk1 inhibitors in breast cancer patients.

The relationship between ovarian cysts and infertility is a subject of debate, mainly because it is difficult to determine the real impact of the cyst and its treatment on later fertility. For a long time it was hoped that surgical treatment could prevent potential complications (such as rupture or malignancy). For presumed benign ovarian tumors, fertility sparing should be the main concern. The goal of this survey of current knowledge on the subject is to thoroughly explore the potential relationship between cysts, their treatment, and infertility. Our study is based on a review of the literature dealing with the epidemiology of ovarian cysts and the effects of their surgical management in relation to infertility. Analysis of the epidemiologic data, drawn mainly from comparative studies and cohorts, shows that the role of cysts in infertility is controversial and that the effects of surgical treatment are often more harmful than the cyst itself to the ovarian reserve. Surgery does not seem to improve pregnancy rates. When a surgical option is nonetheless chosen, a conservative laparoscopic approach is more suitable. Besides excision, sclerotherapy and plasma vaporization are promising, offering a greater preservation of the ovarian parenchyma, especially in endometriomas. These techniques must be better defined. The context of the infertility is essential, and surgeons and specialists in reproductive medicine should decide management jointly.

Purpose: Liquid biopsies based on circulating cell-free DNA (cfDNA) analysis are described as surrogate samples for molecular analysis. We evaluated the concordance between tumor DNA (tDNA) and cfDNA analysis on a large cohort of patients with advanced or metastatic solid tumor, eligible for phase I trial and with good performance status, enrolled in MOSCATO 01 trial (clinical trial NCT01566019). Experimental Design: Blood samples were collected at inclusion and cfDNA was extracted from plasma for 334 patients. Hotspot mutations were screened using next-generation sequencing for 50 cancer genes. Results: Among the 283 patients with tDNA–cfDNA pairs, 121 had mutation in both, 99 in tumor only, 5 in cfDNA only, and for 58 patients no mutation was detected, leading to a 55.0% estimated sensitivity [95% confidence interval (CI), 48.4%–61.6%] at the patient level. Among the 220 patients with mutations in tDNA, the sensitivity of cfDNA analysis was significantly linked to the number of metastatic sites, albumin level, tumor type, and number of lines of treatment. A sensitivity prediction score could be derived from clinical parameters. Sensitivity is 83% in patients with a high score (≥8). In addition, we analyzed cfDNA for 51 patients without available tissue sample. Mutations were detected for 22 patients, including 19 oncogenic variants and 8 actionable mutations. Conclusions: Detection of somatic mutations in cfDNA is feasible for prescreening phase I candidates with a satisfactory specificity; overall sensitivity can be improved by a sensitivity score allowing to select patients for whom cfDNA constitutes a reliable noninvasive surrogate to screen mutations. Clin Cancer Res; 22(12); 2960–8. ©2016 AACR.

How dying tumor cells get noticed Besides killing tumor cells directly, some chemotherapies, such as anthracyclines, also activate the immune system to kill tumors. Vacchelli et al. discovered that in mice, anthracycline-induced antitumor immunity requires immune cells to express the protein formyl peptide receptor 1 (FPR1). Dendritic cells (DCs) near tumors expressed especially high amounts of FPR1. DCs normally capture fragments of dying tumor cells and use them to activate nearby T cells to kill tumors, but DCs lacking FPR1 failed to do this effectively. Individuals with breast or colon cancer expressing a variant of FPR1 and treated with anthracyclines showed poor metastasis-free and overall survival. Thus, FPR1 may affect anti-tumor immunity in people, too. Science , this issue p. 972

Background A substantial proportion of cancer cases present with a metastatic tumor and require further testing to determine the primary site; many of these are never fully diagnosed and remain cancer of unknown primary origin (CUP). It has been previously demonstrated that the somatic point mutations detected in a tumor can be used to identify its site of origin with limited accuracy. We hypothesized that higher accuracy could be achieved by a classification algorithm based on the following feature sets: 1) the number of nonsynonymous point mutations in a set of 232 specific cancer-associated genes, 2) frequencies of the 96 classes of single-nucleotide substitution determined by the flanking bases, and 3) copy number profiles, if available. Methods We used publicly available somatic mutation data from the COSMIC database to train random forest classifiers to distinguish among those tissues of origin for which sufficient data was available. We selected feature sets using cross-validation and then derived two final classifiers (with or without copy number profiles) using 80 % of the available tumors. We evaluated the accuracy using the remaining 20 %. For further validation, we assessed accuracy of the without-copy-number classifier on three independent data sets: 1669 newly available public tumors of various types, a cohort of 91 breast metastases, and a set of 24 specimens from 9 lung cancer patients subjected to multiregion sequencing. Results The cross-validation accuracy was highest when all three types of information were used. On the left-out COSMIC data not used for training, we achieved a classification accuracy of 85 % across 6 primary sites (with copy numbers), and 69 % across 10 primary sites (without copy numbers). Importantly, a derived confidence score could distinguish tumors that could be identified with 95 % accuracy (32 %/75 % of tumors with/without copy numbers) from those that were less certain. Accuracy in the independent data sets was 46 %, 53 % and 89 % respectively, similar to the accuracy expected from the training data. Conclusions Identification of primary site from point mutation and/or copy number data may be accurate enough to aid clinical diagnosis of cancers of unknown primary origin. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0130-0) contains supplementary material, which is available to authorized users.

Background: MATCH-R is a prospective molecular characterization trial (NCT02517892) aiming at defining the molecular basis of acquired resistance to targeted agents and immune checkpoint blockers. RNA sequencing (RNAseq) has been used to identify mechanisms of secondary resistance to immunotherapy. Patients and methods: Patients’ metastatic tumors were multi-site biopsied at relapse under immunotherapies after a period of clinical benefit, defined by a partial response or a stable disease of more than 6 months. Genome-wide RNAseq counts were intra-patient normalized and a score of each gene’s expression was computed in comparison to a cohort of 450 metastatic cancer patients with RNAseq available at the time of analysis. Results: To date, 10 patients treated by immunotherapies have had a successful RNAseq in the MATCH-R trial. Five patients were treated with PD-1 inhibitors and 5 with PD-L1 inhibitors. Three patients had NSCLC, 2 MSI high endometrial carcinoma, 2 anal carcinoma, 2 urothelial carcinoma and 1 TNBC. Eight out off ten patients had an expression of IDO1 higher than the median expression of IDO1 in our 450 controls (p value = 0.005). A patient with endometrial carcinoma had one of the highest expressions of IDO1 in the cohort. Consistently, IDO1 activation has previously been reported as a mechanism of secondary resistance to immunotherapies. A 40 year old smoker NSCLC patient with a TP53 mutation has been treated during 11 months with anti-PD1. RNAseq analysis on the biopsy of a progressive lesion showed decreased expression of different actors of the JAK-STAT pathway (biopsy composed of 40% tumor cells and 60% microenvironment). Of the 78 genes signatures used (including 52 immunogenes signatures), the interferon gamma signature had the lowest expression (p value = 0.004), consistent with a previous report of JAK-STAT-induced resistance to immunotherapy. Two more patients had an altered immune profile that could be involved in resistance to immunotherapies, but was not yet reported in the litterature. Confirmation of the RNAseq analysis with immunohistochemistry is currently ongoing. The gene signatures of the 10 patients, composed of immunogenes, DNA repair genes and epigenes, were compared to the whole cohort in order to deduce corresponding false discovery rates. As such we could identify 2 gene clusters, one enriched in T cells, dendritic cells and macrophages, and the other enriched in epigenes and DNA repair genes. Analysis of more patients is currently ongoing in order to cluster the results with clinical characteristics. Conclusion: Gene expression in the biopsy of patients that relapsed after initial benefit to immunotherapy is informative and helps to identify the mechanism of acquired resistance. Citation Format: Loic Verlingue, Linda Mahjoubi, Sandrine Aspeslagh, Marion Pedrero, Giulia Buzzatti, David Brandao, Zsofia Balogh, Etienne Rouleau, Ludovic Lacroix, Rastislav Bahleda, Christophe Massard, Antoine Hollebecque, Anas Gazzah, Céline Lefebvre, Serge Koscielny, Jean Yves Scoazec, Eric Angevin, Fabrice André, Aurélien Marabelle, Jean Charles Soria. RNAseq analysis obtained from on-purpose tumor biopsies of patients in the MATCH-R trial allows the identification of potential mechanisms of acquired resistance to PD(L)1 therapies [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 1011. doi:10.1158/1538-7445.AM2017-1011

148 Background: Molecular characterization of metastatic castration resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTC) offers an attractive noninvasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in mCRPC patients. Methods: Blood samples were drawn from 11 enzalutamide or abiraterone pre-treated mCRPC patients enrolled in the clinical program MOSCATO (NCT02613962). CTC enrichment, immunofluorescent detection and single cell isolation were performed using three methods (ISET filtration, CellSearch and the VyCap puncher system and RosetteSep enrichment) to obtain pools of 1-10 CTCs with distinct epithelial or mesenchymal phenotypes. After Whole Genome Amplification (WGA), WES was performed on the Illumina HiSeq 2000 platform. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. Results: 189 WGA of CTC pools were performed. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5-10 cells. 17/34 (50%) CTC samples had shared sSNV with the paired tumor sample (range 0.35%-68%) Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large non epithelial CTC pointing out a high level of genetic heterogeneity between CTC. Overall, 89 deleterious protein-coding mutations were found only in pools of CTC, including mutations affecting oncogenic drivers such as MAPK1, HSP90AB1 or KDM5B. Conclusions: We present single cell WES of CTCs harboring distinct phenotypes. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity.

Background: Major advances have been achieved in the understanding of breast cancer biology using high throughput technologies at the DNA and RNA levels. Most of these advances were made in early breast cancers (eBC) which are cured in about 80% of the cases. Nevertheless, little is known about metastatic breast cancers (mBC) which remain lethal in most of the cases. Methods: Whole-exome sequencing was performed on 216 tumour-normal pairs from mBC patients who underwent a biopsy in the context of the SAFIR01/SAFIR02/SHIVA or MOSCATO prospective trials. TCGA dataset (n = 772) was used to assess frequency of mutations in eBC. Findings: Twelve genes (TP53, PIK3CA, GATA3, ESR1, MAP3K1, CDH1, AKT1, MAP2K4, RB1, PTEN, CBFB, CDKN2A) were identified as drivers using MutSig algorithm (FDR150 non-synonymous mutations). This subset was observed in 2% of early breast cancers (TCGA, p = 7.1e?06). The prevalence of this subset increased with the time from diagnosis of metastasis to biopsy. Highly mutated HR+/Her2- mBC (n = 17) presented higher rate of PIK3CA mutations (n = 12, 70%), high number of neoantigens, an APOBEC mutational signature and a poor outcome (multivariate analysis, HR = 4.68, 95%CI: 1.8-12.1, p = 0.001). Interpretation: Whole exome sequencing of metastatic breast cancers identifies a subset of HR+/Her2- mBC who present a high mutational load. RB1, PALB2 and TSC1/2 mutations were found enriched in either mBC or HR+ mBC. Fundings: Breast Cancer Research Foundation, Fondation ARC, Fondation Lombard-Odier “Philanthropia”, Odyssea, Operation Parrains Chercheurs, Dassault Foundation, French NCI: INCa-DGOS-INSERM 6043 Citation Format: Maud Kamal, Celine Lefebvre, Thomas Bachelot, Thomas Filleron, Marion Pedrero, Mario Campone, Jean-Charles Soria, Christophe Massard, Christelle Levy, Monica Arnedos, Julie Garrabey, Yannick Boursin, Marc Deloger, Yu Fu, Frederic Commo, Veronique Scott, Ludovic Lacroix, Emmanuel Martin, Veronique Dieras, Anthony Goncalves, Jean-Marc Ferrero, Gilles Romieu, Laurence Vanlemmens, Marie-Ange Mouret-Reynier, Jean-christophe Thery, Pierre Kerbrat, Severine Guiu, Florence Dalenc, Gilles Clapisson, Hervé Bonnefoi, Martha Jimenez, Christophe Le Tourneau, Fabrice Andre. Mutational profile of metastatic breast cancers using whole-exome sequencing: a retrospective analysis of 216 samples from SAFIR01 / 02 / SHIVA / MOSCATO trials. [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 LB-353.

Background: Although several studies have reported genomic characterisation of primary breast tumors, little is known about the genomic alterations of the metastatic tissues. Breast cancer patients who were prospectively enrolled in a trial (SAFIR01) underwent a biopsy of metastasis. The primary aim of this biopsy was to drive the treatment according to the results of whole genome CGH arrays and sanger sequencing on two genes (PIK3CA and AKT1). The results of the clinical trial were previously reported (André et al ASCO 2013). The secondary goal of the biopsies was to perform genomic characterisation of metastases in breast cancer patients. Here we report the analyses of such metastatic samples using next generation sequencing (NGS) approaches. Patients and methods: Two approaches were applied. In order to describe the incidence of targetable genomic alterations, we performed in depth targeted sequencing on 100 genes (200x coverage) using Illumina HiSeq 2000 (DNA vision). This analysis was performed on 240 metastatic samples. The second approach was more exploratory and aimed at discovering new genes involved in the metastatic process and/or resistance to therapies. In order to achieve this goal, we performed whole exome sequencing in 100 pairs of metastatic tissue (100x) and normal DNA (Integragen Inc, Hiseq platform). Finally, phosphor-S6K staining was performed in 300 samples in order to explore the activation status of mTOR pathway in metastatic disease, and to correlate with genomic data. Results: Targeted sequencing was performed on 240 metastatic samples in order to report the prevalence of targetable genomic alterations in metastatic breast cancer. Results are available for the first 159 samples. In addition to the already reported PIK3CA (26%) and AKT1 mutations (4%), NGS identified mutations of PTEN (4%), ERBB2 (2%), K-Ras (1%), ATM (2%), CDH1 (2%), GATA3 (2%), PTPN11 (1%), PTPRD (1%), ROS1 (1%). Results on the 81 samples, together with whole exome sequencing (n = 100) and phosphoproteins are being analysed and results will be available mid-november 2013. Conclusion: This is the first large study that aims at defining the genomic landscape of metastatic samples. Results will provide insights into the prevalence of targetable genomic alterations in metastatic tissue, together with candidate genes involved in the metastatic process and resistance to therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-07.

Protein disulfide isomerase (PDI) is a chaperone protein that regulates oxidative protein folding as well as cell viability. Increased PDI levels have been documented in a variety of human cancers, including ovarian, prostate, and lung cancers. Inhibition of PDI activity leads to apoptosis in cancer, suggesting that PDI is a promising druggable target. XCE853 was originally identified within a family of synthetic compounds displaying a preferential antiproliferative activity on drug-resistant human cancer cells (Gutmann et al, AACR annual meeting, 2013). XCE853 inhibits in vitro recombinant PDI activity and blocks in vitro proliferation of human tumor cells with IC50s in the nanomolar range through an irreversible cytolysis (Prevost et al, AACR annual meeting, 2014). By contrast, XCE853 has no or limited antiproliferative activity on several human non-tumoral immortalized cells (MCF10, RPE1 or RWPE1) even at high concentrations. The ex-vivo approach using fresh human hepatocellular carcinoma explants incubated 48h with XCE853 (100nM) has shown the decrease of the proliferation (KI-67 labeling) with an apoptosis induction (Caspase-3 labeling). In addition, XCE853 which displayed an excellent oral bioavailability in mouse blocks growth of human ovarian cancer using the in vivo OVCAR-3 xenograft model leading to complete tumor growth arrest even after the cessation of treatment. To optimize the preclinical and clinical development, we are now characterizing new biomarkers to 1) stratify the tumors which are sensitive and non-sensitive to XCE853 and to 2) identify the optimal dose regimen. Altogether, these data support further efforts on XCE853 preclinical studies to prepare the Phase-I clinical study entry. Citation Format: Gregoire Pierre Prevost, Marine Garrido, Shili Xu, Celine Lefebvre, Anne Chauchereau, Denis Carniato, Jean François Briand, Mathieu Gutmann, Maria Serova, Annemilai Tijeras-Raballand, Armand De Grammont, Eric Raymond, Christian Gespach, Michèle Sabbah, Nouri Neamati, Marc-Henry Pitty, Paul Foster. New biomarkers to optimize preclinical development of the PDI inhibitor XCE853. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2015-4466

Background Half of the patient treated with neoadjuvant chemotherapy (NAC) for a large operable breast cancer has no axillary lymph node involvement at the time of surgery. Sentinel lymph node detection (SLND), performed after NAC, must select patient who should be spared of an axillary lymph node dissection (ALND). The application of SLND for staging the axilla after NAC for patient who initially had a proven axillary lymph node involvement remains controversial because of a low detection rate (DR) and a high false negative rate (FNR). Objective The aim of GANEA 2 trial was to assess the DR and the FNR of SLND after NAC in the particular case of patients with a proven axillary lymph node involvement. Patients and Method GANEA 2 was validated by scientific and ethical national boards. Inclusion criteria: FIGO stage T2-T3 infiltrating breast carcinoma, indication of NAC, surgery (radical or conservative) after NAC and signed consent form, Exclusion criteria: inflammatory cancer, local relapse, previous surgical removal of the tumour, mental disorder, pregnancy or no contraceptive method, contra-indication to NAC, NAC interrupted due to progressive disease. Design: Diagnosis and indication to plan a NAC, control of inclusion and exclusion criteria, consent form signature, axillary sonography with fine needle cytology before NAC to select patients with a proven lymph node involvement. After NAC patients underwent both SLND, with the combined technique Blue dye and radiolabeled colloid, and complementary ALND. Pathological procedure: Pathological analysis, of sentinel and non sentinel nodes, carried out according to standard methods and classified according the last American Joint Committee staging system and Sataloff classification. Studied parameters were detection rate, false negative rate and Sataloff grading on tumor and lymph nodes. We evaluated particularly the likelihood that the FNR in patients with one or more SLN examined was greater than 10%. Patients with no lymph node involvement before NAC underwent only a SLND with an ALND only in the case of SLN macro-metastasis with a rigorous follow up. They are not part of this abstract. Results From July 2010, to February 2014, 242 patients from 19 institutions were enrolled, with a proven axillary lymph node involvement before NAC. After NAC, 1/3 had metastasis free axillary lymph node (80/142). Detection rate was 83.1% (201/242). Half of the patients with a detection failure had an involved ALND. The false negative rate was 14.2% in the whole series but 24.5% in the case of only 1 SLN resected, and 8% in case of more than 1 SLN resected. In case of involved SLN, half of the patients had involved ALND. Considering the node Sataloff scoring, 18 of the 20 false negative cases were grade C or D (n=15 grade C, metastatic disease and therapeutic effect; n = 3 grade D, metastasis and no therapeutic effect). Conclusion Among patients treated by NAC for a large operable breast cancer with proven involved lymph node before NAC, who had only 1 SLN examined, the false negative rate was 24.5%. SLND with the combined technique, provides a FNR of less than 10% only in the case of 2 or more SLN resected. Citation Format: Jean-Marc Classe, Loic Campion, Severine Alran, Christine Tunon de Lara, Pierre Francois Dupre, Christelle Faure, Nicolas Paillocher, Serge Lasry, Marie Pierre Chauvet, Gilles Houvenaeghel, Marian Gutowski, Pascaline De Blaye, Charlotte Ngo, Emmanuel Barranger, Jean Luc Veraeghe, Celine Lefebvre, Jean Francois Rodier, Virginie Bordes, Helene Charitansky, Gwenael Ferron, Pierre Gimbergues. Sentinel node surgery after neoadjuvant chemotherapy in patient with axillary node involvement: The French GANEA 2 prospective multi-institutional trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-01.

Celine Lefebvre is a computational biologist with research goals directed towards the identification of biomarkers and therapeutic targets for cancer by developing innovative statistical and computational methods. Specialist of the inference and interrogation of gene regulatory networks, she has developed a method for the identification of master regulators of physiological and pathological cellular phenotypes [1, 2]. The application of the method successfully retrieved key regulators of proliferation of germinal center B cells [2] and synergistic regulators of aggressive prostate cancers [3]. At Gustave Roussy Cancer Institute, she is leading the bioinformatics team of the translational research unit where she is focusing her research on deciphering the relations between tumor molecular alterations and associated response to treatments. To this aim, the research group is integrating different data types including molecular and pharmacological profiles for cell lines and molecular profiles and clinical data for patients from public databases and from Gustave Roussy personalized medicine clinical trials.

316 Background: Although several studies have reported genomic characterisation of primary urothelial carcinoma (UC), little is known about the genomic alterations of the metastatic cisplatin-resistant UC. We sought to define the prevalence and co-occurrence of actionable genomic alterations in patients with metastatic cisplatin-resistant lethal UC. Methods: Patients with metastatic UC, who progressed after at least one line of standard therapy, were enrolled in a prospective molecular screening trial (MOSCATO 01) at Gustave Roussy. CT-Scan or ultrasound-guided biopsies were performed in metastases to carry out a comprehensive molecular characterization. DNA was extracted from fresh tumor samples and analyzed by comparative genomic hybridization (CGH) (≥ 30% tumor cells) and by Next Generation Sequencing (NGS) for up to 74 target genes (≥ 10% tumor cells). Whole-exome (WES) and RNA seq sequencing were performed retrospectively in selected cases. Results: From 12/2011 to 12/2013, 30 heavily pretreated patients with metastatic UC were included. Median age was 61 (37-73); all patients had been treated with platin-based chemotherapy with a median number of lines of 2.5 (1-5). A tumor biopsy could be performed in 26 patients (87%). CGH and targeted exome sequencing profiles were assessed in 19 (73%) and 23 (88%) of them, respectively. A total of 19 patients (73%) were profiled for both sequencing and CGH. WES and RNA seq was performed in 16 (62%) pairs of metastatic tissue and normal DNA (Integragen Inc, Hiseq platform). Our analyses identified potential therapeutic targets in 61 % of the tumours, including 31% with targets in the PI3K/AKT/mTOR pathway, 35% with targets in the FGF/FGFR pathway and 15% with targets in the RTK/MAPK pathway (including ERBB2). Others frequent aberrations were found in the chromatin regulatory genes (MLL gene family) and cell-cycle regulatory genes (E2F3, CDKN2A/B genes). FGFR gene fusions were found in 1 out of 16 screened patients (6%) Conclusions: Lethal platin-resistant UC exhibit high-level of genomic heterogeneity. The majority of these tumors harbour actionable genomic alterations that can be targeted with selective agents currently in clinical development (phase I trials) or registered.

Aim: So far, little is known about immune and genomic landscape in metastatic breast cancer (mBC). Methods: Patients were retrospectively identified from SAFIR01 and MOSCATO studies that performed molecular screening from a biopsy of a metastatic lesion. Whole exome sequencing (WES) was performed using Hi-Seq technology. Coverage was 50x and 100x for normal and tumoral tissue respectively. Bioinformatic analyses reported mutations and copy number alterations. Immune characterisation was determined by the presence of intratumoral and stromal TILs with PD1 and PDL1 expression assessed by IHC (internal protocol, Medimmune). We correlated tumor characteristics and immune and genomics data. Results: 280 samples were stained for immune analyses. Using a 50% cut-off, few tumors presented intratumoral (n = 3/244, 1%) and stromal (n = 11/244, 5%) TILs. This rate was significantly higher in HER2+ tumors (stromal TIL, 16%; p = 0.0002). Positivity for PD1 (n = 14/252, 5%) and PDL1 (n = 7/255, 3%) were rare, compared to reported in other tumor types. A trend towards higher PDL1 was observed in HER2+ mBC (8.3%; p = 0.0653). Ninety-three samples were analysed for WES with 17 genes found mutated in >3 samples (p Conclusions: This is the first study that assesses both immune and genomic landscapes in mBC. We observed that mBC dramatically differs from primary tumors, and is enriched in genes potentially involved in resistance mechanisms (ESR1, TSC1) or migration process (FRAS1, SCAPER). TILs, PD1, PDL1 are at very low frequency in metastatic lesions, except for Her2 + ++ mBC. Our results suggest that other immune suppressor networks are involved in mBC. Therefore, CD73, CD39 and FoxP3 are being analysed and will be presented. Disclosure: P.B. Robbins: Salary from Medimmune, Inc. All other authors have declared no conflicts of interest.

11057 Background: Although several studies have reported genomic characterisation of primary urothelial carcinoma (UC), little is known about the genomic alterations of the metastatic cisplatin-res...

Background: Half of the patient treated with neoadjuvant chemotherapy (NAC) for a large operable breast cancer have no axillary lymph node involvement at the time of surgery. Sentinel lymph node detection (SLND), performed after NAC, would select patient who might be spared of an axillary lymphadenectomy (AL). In a previous study, we assessed the feasibility of SLND after NAC in the case of patients without axillary involvement1. Previous published series have shown that, for patients with an axillary lymph node involvement before treatment, SLND after NAC bring a low detection rate and a high false negative rate (FNR), making this technique contra indicated in this situation. The aim of GAEA 2 study is to assess the FNR of SLND after NAC in the particular case of patients with a proven axillary lymph node involvement before NAC. Patients and Method: Prospective study validated by scientific and ethical National boards. Inclusion criteria: FIGO stage T2-T3 infiltrating breast carcinoma, indication of NAC, surgery (radical or conservative) after NAC and signature of the consent form, Exclusion criteria: locally advanced, inflammatory breast cancer, local relapse, previous surgical removal of the tumour, mental disorder, pregnancy or no contraceptive method, contra-indication to NAC, NAC interrupted due to progressive disease. Design of the study: Indication to plan a NAC, control of inclusion and exclusion criteria, consent form signature, axillary sonography before NAC to select the patient in group 1 (patient with a proven lymph node involvement treated with SLND and complementary AL) or 2 (no involvement proven treated with SLND + AL only if detection failure or involvement). Surgery, breast and axilla, performed 4 to 6 weeks after NAC. Pathological procedure: No intraoperative histopathological examination. Pathological analysis, of sentinel and non sentinel nodes, carried out according to standard methods and classified according the last American Joint Committee staging system and Sataloff classification. FNR is defined as the ratio of patients with a false negative case of SLNB to the patients with at least one involved node, SLN or not, among patients with SLN detected. The hypothesis: Taking into account results of lymph node involvement rate found in GANEA 1, to estimate our hypothesis of a FNR between 10 and 15% with a 95% confidence interval will require to include 858 patients in order to obtain 260 patient with a proven axillary lymph node involvement (group 1). A standard follow up is planned for each patient, with a clinical breast and axillary examination two times/ year and an annual mammography, for five years. In case of clinical axillary relapse a fine needle aspiration must be performed guided with sonography. Results: On May 31, 2012, 341 patients were included from 16 French institutions; 130 patients with a proven SLN involvement before NAC and 211 with SLN free of metastasis. 1Classe JM, Bordes V, et al. Sentinel lymph node biopsy after neoadjuvant chemotherapy for advanced breast cancer: results of Ganglion Sentinelle et Chimiotherapie Neoadjuvante, a French prospective multicentric study. J Clin Oncol. 2009 Feb. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT2-1-01.

Background: Large multi institutional studies have pointed that previous surgical resection of breast tumours before axillary sentinel node detection (ASLND) was the main criteria of failure of this technique. Screening campaigns provide small tumours and despite efforts to obtain a diagnosis of early breast cancer, this is not always obtained, due to small tumours or false negative results of micro biopsies. The aim of our series was to assess identification rates and false negative rates of ASLND after previous surgical resection of breast tumours. Material and Methods: In a prospective multi institutional setting (14 multidisciplinary teams), we have included patients with a previous breast tumour surgical resection for the diagnosis of infiltrative breast adenocarcinoma. Patients with only a core biopsy and no surgical removal of the tumor before axillary surgery were not included. Each patient underwent a secondary surgical procedure for ASLND and axillary lymphadenectomy, and sometimes a breast secondary surgical procedure for margins. ASLND was performed with the combined method, with blue dye and technetium. Pathology was performed with serial sectioning, eosin safron and immune histo chemistry (IHC). Results: From July 2006 to November 2011, 138 patients where included. The median tumor size was 9mm. Identification rate was 86% (118/138). A macrometastasis was found in 11 cases, in a sentinel node (9), or in a non sentinel node(2). False negative rate was 9% (1 false negative sentinel node with macrometastasis in non sentinel node from lymphadenectomy/11 cases with a macrometastasis in either a sentinel node or a non sentinel node). In 1 case a micrometastasis was found in a sentinel node through IHC, with a macrometastasis in a non sentinel node from lymphadenectomy. Without IHC or without the decision of performing a complementary lymphadenectomy in the case of micrometastasis, the false negative rate would have been 18%. Conclusions: After previous surgical resection of early breast cancer, ASLND remains feasible with a low identification rate of 86%, despite the use of the combined method. The False negative rate is acceptable with the use of IHC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-21.

Our laboratories have been investigating novel mechanisms of cancer progression, as well as new therapeutic approaches for early intervention and treatment of advanced disease by integrating analyses of human clinical data with molecular and functional studies of genetically engineered mutant (GEM) mice. In our analyses of prostate cancer, we have generated GEM mice that recapitulate the entire spectrum of disease progression from preinvasive lesions, termed prostate intraepithelial neoplasia (PIN), to castration-resistant and metastatic disease, which are the lethal forms of the disease. These GEM models are based on perturbation of the Akt/mTOR and MAP kinase signaling pathways, which are often co-activated in human prostate cancer and which function synergistically to promote castration-resistant metastatic prostate cancer. We have found that combinatorial inhibition of these signaling pathways in GEM mice that display castration-resistant metastatic prostate cancer results in abrogation of primary tumors, improved survival and reduced metastasis. Using computational approaches for comparative analyses of large-scale gene expression profiling data for mouse and human prostate cancer, we have been assembling molecular networks, called interactomes, to elucidate conserved cancer pathways. We have been investigating the consequences of drugs perturbations on the transcriptional network with the ultimate goal of identifying new druggable targets. In summary, our comparative investigations of prostate cancer in humans and GEM mice have revealed promising new molecular targets and new therapuetic approaches for the treatment of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY22-01. doi:10.1158/1538-7445.AM2011-SY22-01