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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.

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.

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.

Comprehensive genomic profiling using high-throughput sequencing brings a wealth of information, and its place in the clinical setting has been increasingly prominent. This review emphasizes the utility of whole-exome sequencing (WES) and transcriptome sequencing (RNAseq) in patient care and clinical research, based on published reports as well as our experience with the MOSCATO-01 (MOlecular Screening for CAncer Treatment Optimization) molecular triage trial at Gustave Roussy Cancer Center. In this trial, all contributive samples of patients with advanced solid tumors were analyzed prospectively with targeted gene sequencing (TGS) and comparative genomic hybridization. In addition, 92 consecutive metastatic patients with contributive biopsies were sequenced for WES and RNAseq and compared with TGS and comparative genomic hybridization. Whole-exome sequencing allowed the reporting of additional variants in relevant genes in 38% of patients. Mutation detection sensitivity of WES was 95% compared with TGS. Additional information derived from WES and RNAseq could influence clinical decision, including fusion transcripts, expression levels, allele-specific expression, alternate transcripts, RNA-based pathogen diagnostic, tumor mutation load, mutational signatures, expression signatures, HLA genotyping, and neoepitope prediction. The current challenge is to be able to process the large-scale data from these comprehensive genome-wide technologies in an efficient way.

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.

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.

Background Major advances have been achieved in the characterization of early breast cancer (eBC) genomic profiles. Metastatic breast cancer (mBC) is associated with poor outcomes, yet limited information is available on the genomic profile of this disease. This study aims to decipher mutational profiles of mBC using next-generation sequencing. Methods and Findings Whole-exome sequencing was performed on 216 tumor–blood pairs from mBC patients who underwent a biopsy in the context of the SAFIR01, SAFIR02, SHIVA, or Molecular Screening for Cancer Treatment Optimization (MOSCATO) prospective trials. Mutational profiles from 772 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as a reference for comparing primary and mBC mutational profiles. Twelve genes (TP53, PIK3CA, GATA3, ESR1, MAP3K1, CDH1, AKT1, MAP2K4, RB1, PTEN, CBFB, and CDKN2A) were identified as significantly mutated in mBC (false discovery rate [FDR] < 0.1). Eight genes (ESR1, FSIP2, FRAS1, OSBPL3, EDC4, PALB2, IGFN1, and AGRN) were more frequently mutated in mBC as compared to eBC (FDR < 0.01). ESR1 was identified both as a driver and as a metastatic gene (n = 22, odds ratio = 29, 95% CI [9–155], p = 1.2e-12) and also presented with focal amplification (n = 9) for a total of 31 mBCs with either ESR1 mutation or amplification, including 27 hormone receptor positive (HR+) and HER2 negative (HER2−) mBCs (19%). HR+/HER2− mBC presented a high prevalence of mutations on genes located on the mechanistic target of rapamycin (mTOR) pathway (TSC1 and TSC2) as compared to HR+/HER2− eBC (respectively 6% and 0.7%, p = 0.0004). Other actionable genes were more frequently mutated in HR+ mBC, including ERBB4 (n = 8), NOTCH3 (n = 7), and ALK (n = 7). Analysis of mutational signatures revealed a significant increase in APOBEC-mediated mutagenesis in HR+/HER2− metastatic tumors as compared to primary TCGA samples (p < 2e-16). The main limitations of this study include the absence of bone metastases and the size of the cohort, which might not have allowed the identification of rare mutations and their effect on survival. Conclusions This work reports the results of the analysis of the first large-scale study on mutation profiles of mBC. This study revealed genomic alterations and mutational signatures involved in the resistance to therapies, including actionable mutations., Fabrice Andre and colleagues describe the mutations occurring in a large group of patients with metastatic breast cancer., Author Summary Why Was This Study Done? Breast cancer often results in poor outcomes after it has metastasized to distant organs, but, while primary breast tumors have been extensively characterized at the molecular level, metastatic lesions are poorly understood. This study aims to characterize the mutational landscape of metastatic breast cancer by performing and analyzing whole-exome sequencing of a large collection of metastatic breast tumors and corresponding blood samples. Understanding of the mutational landscape of metastatic tumors should open new avenues for assessing resistance to therapy and developing better treatments. What Did the Researchers Do and Find? The authors generated a large collection of whole-exome sequencing data from the DNA of breast cancer metastases and from each patient’s corresponding unmutated DNA in order to identify mutations and gene copy number alterations specific to the tumors. The bioinformatics analyses identified recurrently mutated genes in metastatic tumors and revealed the genes specifically involved in metastatic disease by comparing their mutational frequency to those of primary breast tumors. The study allowed identification of the affected genes and of mutational signatures that were more prevalent in metastatic as compared with primary tumors and that may be involved in the resistance to therapies. What Do These Findings Mean? The identification of mutational and copy number alterations specifically involved in breast cancer metastasis demonstrated that tumors evolve under the pressure of therapy. Characterization of mutations and copy number alterations in metastatic lesions in addition to primary tumors should help to tailor treatment for patients, with the potential for improved clinical outcomes.

DNA extracted from cancer patients' whole blood may contain somatic mutations from circulating tumor DNA (ctDNA) fragments. In this study, we introduce cmDetect, a computational method for the systematic identification of ctDNA mutations using whole-exome sequencing of a cohort of tumor and corresponding peripheral whole-blood samples. Through the analysis of simulated data, we demonstrated an increase in sensitivity in calling somatic mutations by combining cmDetect to two widely used mutation callers. In a cohort of 93 breast cancer metastatic patients, cmDetect identified ctDNA mutations in 54% of the patients and recovered somatic mutations in cancer genes EGFR, PIK3CA, and TP53. We further showed that cmDetect detected ctDNA in 89% of patients with confirmed mutated cell–free tumor DNA by plasma analyses (n = 9) within 46 pan-cancer patients. Our results prompt immediate consideration of the use of this method as an additional step in somatic mutation calling using whole-exome sequencing data with blood samples as controls. Cancer Res; 76(20); 5954–61. ©2016 AACR.

Immunotherapy is currently transforming cancer treatment. Notably, immune checkpoint blockers (ICB) have shown unprecedented therapeutic successes in numerous tumor types, including cancers that were traditionally considered as nonimmunogenic. However, a significant proportion of patients do not respond to these therapies. Thus, early selection of the most sensitive patients is key, and the development of predictive companion biomarkers constitutes one of the biggest challenges of ICB development. Recent publications have suggested that the tumor genomic landscape, mutational load, and tumor-specific neoantigens are potential determinants of the response to ICB and can influence patients' outcomes upon immunotherapy. Furthermore, defects in the DNA repair machinery have consistently been associated with improved survival and durable clinical benefit from ICB. Thus, closely reflecting the DNA damage repair capacity of tumor cells and their intrinsic genomic instability, the mutational load and its associated tumor-specific neoantigens appear as key predictive paths to anticipate potential clinical benefits of ICB. In the era of next-generation sequencing, while more and more patients are getting the full molecular portrait of their tumor, it is crucial to optimally exploit sequencing data for the benefit of patients. Therefore, sequencing technologies, analytic tools, and relevant criteria for mutational load and neoantigens prediction should be homogenized and combined in more integrative pipelines to fully optimize the measurement of such parameters, so that these biomarkers can ultimately reach the analytic validity and reproducibility required for a clinical implementation. Clin Cancer Res; 22(17); 4309–21. ©2016 AACR.

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.

Little is known about mutational landscape of rare breast cancer (BC) subtypes. The aim of the study was to apply next generation sequencing to three different subtypes of rare BCs in order to identify new genes related to cancer progression. We performed whole exome and targeted sequencing of 29 micropapillary, 23 metaplastic, and 27 pleomorphic lobular BCs. Micropapillary BCs exhibit a profile comparable to common BCs: PIK3CA, TP53, GATA3, and MAP2K4 were the most frequently mutated genes. Metaplastic BCs presented a high frequency of TP53 (78 %) and PIK3CA (48 %) mutations and were recurrently mutated on KDM6A (13 %), a gene involved in histone demethylation. Pleomorphic lobular carcinoma exhibited high mutation rate of PIK3CA (30 %), TP53 (22 %), and CDH1 (41 %) and also presented mutations in PYGM, a gene involved in glycogen metabolism, in 8 out of 27 samples (30 %). Further analyses of publicly available datasets showed that PYGM is dramatically underexpressed in common cancers as compared to normal tissues and that low expression in tumors is correlated with poor relapse-free survival. Immunohistochemical staining on formalin-fixed paraffin-embedded tissues available in our cohort of patients confirmed higher PYGM expression in normal breast tissue compared to equivalent tumoral zone. Next generation sequencing methods applied on rare cancer subtypes can serve as a useful tool in order to uncover new potential therapeutic targets. Sequencing of pleomorphic lobular carcinoma identified a high rate of alterations in PYGM. These findings emphasize the role of glycogen metabolism in cancer progression.

Purpose Focal and temporal tumour heterogeneity can represent a major challenge for biology-guided therapies. This study proposes to investigative molecular discrepancies between primary colorectal cancer (CRC) samples and matched metastases. Experimental design Surgical samples from primary and matched metastatic tissues from 13 CRC patients along with their adjacent normal tissue were evaluated. A mutational analysis was performed using a targeted Next Generation Sequencing assay (Foundation Medicine) with a focus on known recurrent somatic mutations as surrogate of key oncogenic events. Gene expression analysis was also performed to investigate transcriptional discrepancies. Results Among the 26 samples, 191 mutations were identified including mutations in APC (13 pts), TP53 (11 pts), and KRAS (7 pts). Global concordance rate for mutations was 78% between primary and metastatic tumours and raised to 90% for 12 known recurrent mutations in CRC. Differential gene expression analysis revealed a low number of significantly variant transcripts between primary and metastatic tumours once the tissue effect was taken into account. Only two pathways (ST_ADRENERGIC, PID_REELINPATHWAY) were differentially up-regulated in metastases among 17 variant pathways. A common profile in primary and metastatic tumours revealed conserved pathways mostly involved in cell cycle regulation. Only two pathways were significantly down regulated compared to normal control, including regulation of autophagy (KEGG_REGULATION_OF_AUTOPHAGY). Conclusion These results suggest that profiles of primary tumour can identify key alterations present in matched CRC metastases at first metastatic progression. Gene expression analysis identified mainly conserved pathways between primary tumour and matched liver metastases.

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.

Molecular characterization of metastatic castration-resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTCs) offers an attractive non invasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in 11 mCRPC patients. We examined single somatic nucleotide variant (sSNV) shared between matched metastatic tumor sample and CTCs and sSNV specific to CTCs. Blood samples were drawn from 11 patients enrolled in the clinical program MOSCATO (2011-A00841-40). CTC enrichment, detection and single cell isolation were performed using three methods to obtain pools of 1-10 CTCs. The first method used ISET filtration, immunofluorescent staining (CD45, pan-cytokeratin, EpCAM, Vimentin and Hoechst 33342) on filters and laser microdissection of single CTCs; the second combined CellSearch and the VyCap puncher system; the third used RosetteSep enrichment, immunofluorescent staining and isolation by cell sorting. Whole Genome Amplification (WGA) was performed using the Ampli1 kit. WGA quality was assessed by qPCR of 7 genes located on different regions of the genome. WES was performed by preparation of a genomic DNA bank, Agilent capture and sequencing on the Illumina HiSeq 2000 platform. Data were aligned to the human genome reference hg19. 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. 189 WGAs of CTC pools were performed. A first round of WES showed that at least 3 well amplified genes were required to obtain a coverage of at least 50% at 10X depth sequencing. 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 to 10 cells. 17/34 (50%) CTC samples (4 patients) 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 Cytokeratin-Vimentin- CTC. Shared sSNV in cancer genes between epithelial CTC pools, but not in the paired biopsy, were present in 2 patients. We report WES of CTC pools harboring distinct EMT marker phenotypes is possible with the use of 3 different approaches to enrich, detect and isolate CTCs. 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. Ongoing work examining if sSNV detected in phenotypically different CTCs converge to similar signaling pathways will be presented. Citation Format: Vincent Faugeroux, Céline Lefebvre, Emma Pailler, Valérie Pierron, Fanny Billiot, Charles Marcaillou, Philippe Vielh, Semih Dogan, Philippe Rameau, Maud Ngocamus, Jean Charles Soria, Karim Fizazi, Yohann Loriot, Sylvia Julien, Françoise Farace. Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer. [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 4953.

Both the PI3K → Akt → mTOR and mitogen-activated protein kinase (MAPK) signaling pathways are often deregulated in prostate tumors with poor prognosis. Here we describe a new genetically engineered mouse model of prostate cancer in which PI3K-Akt-mTOR signaling is activated by inducible disruption of PTEN, and extracellular signal-regulated kinase 1/2 (ERK1/2) MAPK signaling is activated by inducible expression of a BRAFV600E oncogene. These tissue-specific compound mutant mice develop lethal prostate tumors that are inherently resistant to castration. These tumors bypass cellular senescence and disseminate to lymph nodes, bone marrow, and lungs where they form overt metastases in approximately 30% of the cases. Activation of PI3K → Akt → mTOR and MAPK signaling pathways in these prostate tumors cooperate to upregulate c-Myc. Accordingly, therapeutic treatments with rapamycin and PD0325901 to target these pathways, respectively, attenuate c-Myc levels and reduce tumor and metastatic burden. Together, our findings suggest a generalized therapeutic approach to target c-Myc activation in prostate cancer by combinatorial targeting of the PI3K → Akt → mTOR and ERK1/2 MAPK signaling pathways. Cancer Res; 72(18); 4765–76. ©2012 AACR.

Although the prognosis for clinically localized prostate cancer is now favorable, there are still no curative treatments for castration-resistant prostate cancer (CRPC) and, therefore, it remains fatal. In this study, we investigate a new therapeutic approach for treatment of CRPC, which involves dual targeting of a major signaling pathway that is frequently deregulated in the disease. We found that dual targeting of the Akt and mTOR signaling pathways with their respective inhibitors, MK-2206 and ridaforolimus (MK-8669), is highly effective for inhibiting CRPC in preclinical studies in vivo using a refined genetically engineered mouse model of the disease. The efficacy of the combination treatment contrasts with their limited efficacy as single agents, since delivery of MK-2206 or MK-8669 individually had a modest impact in vivo on the overall tumor phenotype. In human prostate cancer cell lines, although not in the mouse model, the synergistic actions of MK-2206 and ridaforolimus (MK-8669) are due in part to limiting the mTORC2 feedback activation of Akt. Moreover, the effects of these drugs are mediated by inhibition of cellular proliferation via the retinoblastoma (Rb) pathway. Our findings suggest that dual targeting of the Akt and mTOR signaling pathways using MK-2206 and ridaforolimus (MK-8669) may be effective for treatment of CRPC, particularly for patients with deregulated Rb pathway activity. Cancer Res; 72(17); 4483–93. ©2012 AACR.

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

Backgroung : Cisplatin is part of standard therapy for a number of solid tumors including lung cancer. The initial response to this antineoplastic agent is in most cases only transient and tumors quickly become resistant to the drug. The mechanisms of resistance have been intensively explored, mostly by functional high throughput screening methods or by genomic and transcriptomic approaches. Our aim was to analyze the translational reprogramming following cisplatin treatment in order to identify candidates that mediate cisplatin resistance. Material and methods: Here we explored the response of a sensitive and of a matched resistant NSCLC cell line to cisplatin at the level of the nascent proteome by performing polysome profiling. Cellular mRNAs were resolved on a linear sucrose gradient. The heavily translated mRNAs that are associated with ribosome particles (the so-called polysomes) could be isolated and competitively hybridized on a cDNA microarray against the total mRNA fraction. This approach enabled us to identify the mRNAs whose translation is modified upon cisplatin treatment in the resistant cells. Results : Amongst the 200 candidate genes identified by this approach, we focused on the Ubiquitin-Specific Peptidase 1 (USP1) gene regulation. We found that while the transcription of the gene remains unaffected in both the sensitive and the resistant cell lines, the translation of USP1 is modulated by cisplatin in the sensitive cells. The immediate response (4h after treatment) results in a decrease of USP1 mRNA translation, followed by a stimulation of the translation at a later time point (16h). Importantly, this translational regulation of USP1 is lost in the resistant cells which instead show a constitutively high translation rate for USP1. Inhibition of cell growth by cisplatin was potentiated when USP 1 expression was suppressed by siRNA. Similarly, ML323, a specific inhibitor of the USP1-UAF1 deubiquitinase complex, dramatically increased cell sensitivity to cisplatin. Thus interfering with USP1 activity in resistant cells by both an siRNA approach and the use of a small molecule inhibitor re-sensitized the resistant cells to cisplatin. Conclusion: Our original approach led to the identification of USP1 as a potential determinant of cisplatin resistance of a lung cancer cell line. USP1 protein levels in tumor samples could potentially serve as a predictive marker of the response to cisplatin. We suggest that small molecule inhibitors of USP1 should be tested as cisplatin-sensitizers. The analysis of the ‘nascent proteome’ by polysome profiling could enable the identification of additional candidates mediating cisplatin-resistance. Citation Format: Carole Helissey, Tony Sourisseau, Hélène Mahieu, Céline Lefebvre, Stephan Vagner, Jean-Charles Soria, Ken Olaussen. Loss of USP1 translational control as a targetable cisplatin resistance mechanism in non-small cell lung cancer (NSCLC). [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 758. doi:10.1158/1538-7445.AM2015-758

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.

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.

Many anticancer targets with role in drug resistance are involved in key metabolic processes. In recent years, the interest in targeting cell metabolism and metabolic pathways connected to energy storage and redox potential has been renewed. Comparative analyses of paired samples of tumor cells and corresponding normal tissue can reveal the altered metabolite profiles and can increase the understanding of metabolic involvement in cancer progression. By applying whole exome sequencing on eight pairs of pleiomorphic lobular carcinoma frozen samples, an aggressive breast cancer subtype, and matched normal tissue, we recently identified recurrent somatic mutations in four genes. Three of these genes are already described to be implicated in breast cancer (PIK3CA, TP53 and CDH1), and one novel mutated gene was identified: PYGM which encodes for glycogen phosphorylase firstly isolated from muscular tissue. Targeted sequencing performed on 27 pleiomorphic lobular carcinoma samples confirmed PYGM as a frequently mutated gene in this pathology (30%). Subsequent analysis of the breast gene expression TCGA dataset uncovered that PYGM is significantly downregulated in breast cancer samples compared to normal breast tissue, irrespectively of the breast cancer molecular subtype. The same trend was observed in other cancer types, such as colon, bladder or head and neck carcinoma, after analysis of publicly available gene expression databases. PYGM is expressed in many other cell types and initiates the breakdown of glycogen by degradation in glucose-1-phosphate, which can be enzymatically converted to glucose-6-phosphate. This generates an alternative cellular energy source to immediate ATP. In muscle cells, various mutations impairing PYGM activity lead to McArdle disease (autosomic recessive glycogen storage disease type V) which is characterized by exercise intolerance, pain, muscle weakness, and cramps. In cancer cells, some few abnormalities of PYGM have been reported. We thus investigated the potential dysregulation of PYGM expression and/or function in breast cancer and its consequences on cell metabolism. We found that PYGM expression is downregulated in breast cancer tissue compared to paired normal tissue. In order to analyze the role of PYGM in preclinical models, we established new breast cancer cell lines overexpressing PYGM. Biochemical and molecular analyses are ongoing to investigate cell behavior according to PYGM expression level in various culture conditions and will inform us about a potential efficacy of PYGM targeting in new therapy development based on metabolic biomarkers applied to breast cancer treatment. Citation Format: Veronika SMUTNA, Maria Vittoria DIECI, Céline LEFEBVRE, Véronique SCOTT, Fabrice ANDRE, Olivia FROMIGUE. Is PYGM dysregulation involved in breast cancer cell metabolism. [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 435. doi:10.1158/1538-7445.AM2014-435

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: Rare cancers sometimes present clonal evolution. Genomic characterisation of these rare entities could lead to identify new genes involved in the progression of more frequent cancers. In the present study, we have performed whole exome sequencing of three rare and aggressive histological subtypes in order to identify new targets that could subsequently be validated in common breast cancers. Methods: Whole exome sequencing was applied in ten pleiomorphic lobular carcinoma, seven micropapillary BC and eight metaplastic BC. Each cancer sample was matched with normal DNA from the same patient. Overall 50 samples were therefore sequenced. Tumor samples were selected to present >30% cancer cells. Histological diagnosis was confirmed by a BC pathologist. Whole exome sequencing (WES) was performed by BGI (Bejing Genomic Institute, Hong-Kong, China) using the Illumina Hiseq2000 platform (90bp paired-end reads, depth ≥80x). Genes recurrently mutated (with somatic mutations in >2 cases) were selected for validation. The validation by IonTorrent target sequencing was performed on additional formalin-fixed paraffin-embedded (FFPE) tumor samples (>30% of tumor cells). Results: Complete results of both WES and targeted resequencing validation are available for pleiomorphic lobular carcinoma. Nine of the 10 DNA pairs were eligible for WES. Genes already implicated in BC were significantly mutated: PIK3CA (3/9), TP53 (3/9) and CDH1 (3/9). Novel significantly mutated genes were identified: PYGM (2/9), EMR1 (2/9), ALDH1A2 (2/9), and DCLK1 (2/9). The validation using target gene sequencing was performed on the same 9 cases and on 19 additional confirmed pleiomorphic lobular BC cases (n = 28). For 8 out of the 9 cases with WES data, the results were reproducible on FFPE samples. Validation set confirmed recurrent mutations on PYGM genes (25%). PYGM is involved in glycogen metabolism. Further analyses on gene expression array revealed that PYGM is downregulated in most of the common BC samples, as compared to normal tissue, suggesting a role for this gene in the metabolism of common BC. WES data are available for micropapillary and metaplastic BC. HSPA8 (coding for a Heat shock protein) was found mutated in 3/7 of micropapillary samples. Validation on additional FFPE samples is ongoing and will be presented. WES of metaplastic BC revealed a high frequency of TP53 and PIK3CA mutations (50% in both cases). Conclusions: A novel mutated gene was identified in PLBC. PYGM is a gene involved in glycogen metabolism and was found downregulated in most of the common breast cancer. WES of micropapillary cancers suggests mutations on HSPA8, a gene encoding for a chaperon protein. Validation is ongoing. Deeper sequencing is ongoing for metaplastic BC that may help identifying new mutated genes in this multiclonal disease. Results will be presented. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-4.

The study of cancer therapeutics in mouse models is impaired by our limited understanding of whether the regulatory mechanisms that mediate their activity are conserved in human patients. To address this limitation, we have constructed a mouse prostate cancer regulatory model (interactome) based on in vivo drug-based perturbations of 15 transgenic models of prostate cancer as well as a human interactome from a large-scale set of molecular profiles of patient derived tissue. For the mouse interactome, we performed molecular perturbations by treatment with small molecules, including inhibitors of the androgen receptor, tyrosine kinase receptors, MAPK, Akt/mTOR, JAK/STAT, and NF-KB signaling pathways as well as several chemotherapeutic agents. We then used a cross-species analysis to independently identify and match master regulators of cancer progression with the therapeutic compounds that optimally abrogate their activity both in the human and in the mouse regulatory models. Cross-species interactome interrogation represents a new paradigm for ‘human to mouse to human’ (H2M2H) analyses, aimed at maximizing translational potential. By interrogating our regulatory models using molecular profiles of cancer progression, we identified two genes, FOXM1 and CENP-F, that are synergistically required for the maintenance of aggressive, metastatic prostate tumors and are predictive of clinical outcome. Indeed, lentivirus mediated shRNA co-silencing of these master regulators dramatically decreases tumorigenesis in vivo. Interrogation of these interactomes using small molecule signatures further identified candidate combinations that successfully abrogate the activity of the FOXM1/CENPF axis in metastatic prostate cancer GEM models, and significantly reduce tumor and metastatic burden in vivo, thus improving survival significantly over standard of care chemotherapy. Our findings suggest that simultaneous targeting of FOXM1 and CENPF may be especially beneficial in patients who have failed conventional chemotherapy. Citation Format: Antonina Mitrofanova, Alvaro Aytes, Celine Lefebvre, Michael Shen, Cory Abate-Shen, Andrea Califano. Cross-species analysis of genome-wide regulatory models elucidates master regulators of prostate cancer progression and treatment response. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A26.

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.

Identification of master regulators driving advanced prostate cancer and treatment response through the assembly of mouse and human prostate cancer interactomes. Alvaro Aytes1,4, Antonina Mitrofanova2,4, Celine Lefebvre2,4, Carolyn W. Kinkade1,4, Michael M. Shen3,4, Andrea Califano2,4, and Cory Abate-Shen1,4. 1Departments of Urology, Pathology and Cell Biology, 2Biomedical Informatics and Center for Computational Biology and Bioinformatics, 3Medicine and Genetics & Development, 4Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032 We have developed new genetically-engineered mouse models (GEM) based on inducible prostate-specific deletion of Pten alone or together with activation of oncogenic Kras. These models recapitulate all stages of prostate tumorigenesis, including metastases. We have used these GEM models to develop unbiased molecular networks of transcriptional and post-translational interactions, namely interactomes. Importantly, interactomes are predicated on the availability of large gene expression profile (GEP) datasets representative of the phenotypic variability and genetic perturbations of prostate cancer. We have generated GEPs using a collection of prostate cancer mouse models from our laboratory, as well as from the mouse modeling community. Each model has been treated with a variety of drugs against members of the AR, MAPk, Akt/mTOR, JAK/STAT, NF-KB signaling pathways and other kinase inhibitors as well as chemotherapeutic agents. In parallel, a human prostate cancer interactome has been generated using publicly available datasets. Interrogation of these interactomes represents a new paradigm for ‘human to mouse to human’ analyses. In particular, we have used these interactomes to identify molecular mechanisms and biomarkers of response to combinatorial targeting of Akt/mTOR and MAPK pathways in our metastatic prostate cancer GEM model of Kras activation and Pten loss-of-function. Molecular profiles from these tumors parallel those of human prostate cancers, having poor prognosis, concomitant activation of Akt/mTOR and MAP kinase signaling and displaying robust phosphorylation (inactivation) of FoxO3a tumor suppressor. This combined therapy profoundly reduces tumor and metastatic burden, improves survival and decreases FoxO3a phosphorylation. Computational analyses show that combination therapy in mice shifts the malignant signature toward that of normal prostate, while cross-species analyses identify a two-gene signature downstream of FoxO3a, including FOXM1 and CENP-F that is expressed in human metastatic tumors and is predictive of clinical outcome. Our findings suggest that targeting the FoxO3a regulatory network may be particularly beneficial for patients who have failed conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-405. doi:1538-7445.AM2012-LB-405

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

Pluripotent embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) are an excellent model system to study the interface between malignancy, pluripotency, and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 germ cell tumor (GCT) biopsies comprising all described differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). We applied the ARACNE reverse engineering algorithm to this GEP to obtain the first de novo and context-specific transcriptional interactome (GCTNet) comprising 1,310 transcription factors (TFs). GCTnet recapitulated 830 regulatory targets together of POU5F1, NANOG, and SOX2, the core pluripotency TFs. ChIP-seq and ChIP-PCR analysis of NT2/D1 embryonal carcinoma and H9 ES cells provided significant validation of ARACNe-inferred targets, besides providing the first detailed mapping of DNA binding sites of these 3 genes in human cells. Functional validation of ARACNe-inferred targets by stable knockdown of the POU5F1, NANOG and SOX2 in NT2/D1 cells also showed a significant enrichment of ARACNe targets. Analysis of GEP in a time-course of SOX2 knockdown by principal component analysis (PCA) identified 3 orthogonal gene expression programs: (1) genes with monotonic change (PC1), (2) genes showing a strong and focal gene expression at day 5 (PC2), and (3) genes which show oscillatory pattern of expression (PC3). Ingenuity and GO annotation analysis of PCA provided new insights into the pathways regulated by this TF. Our results provide a novel, comprehensive, and accurate regulatory networks responsible for pluripotency and lineage differentiation in GCTs. 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 3410. doi:10.1158/1538-7445.AM2011-3410

SummaryUnsustained enzyme inhibition is a barrier to targeted therapy for cancer. Here, resistance to a class I PI3K inhibitor in a model of metastatic breast cancer driven by PI3K and MYC was associated with feedback activation of tyrosine kinase receptors (RTKs), AKT, mTOR, and MYC. Inhibitors of bromodomain and extra terminal domain (BET) proteins also failed to affect tumor growth. Interestingly, BET inhibitors lowered PI3K signaling and dissociated BRD4 from chromatin at regulatory regions of insulin receptor and EGFR family RTKs to reduce their expression. Combined PI3K and BET inhibition induced cell death, tumor regression, and clamped inhibition of PI3K signaling in a broad range of tumor cell lines to provide a strategy to overcome resistance to kinase inhibitor therapy.

SummaryTo identify regulatory drivers of prostate cancer malignancy, we have assembled genome-wide regulatory networks (interactomes) for human and mouse prostate cancer from expression profiles of human tumors and of genetically engineered mouse models, respectively. Cross-species computational analysis of these interactomes has identified FOXM1 and CENPF as synergistic master regulators of prostate cancer malignancy. Experimental validation shows that FOXM1 and CENPF function synergistically to promote tumor growth by coordinated regulation of target gene expression and activation of key signaling pathways associated with prostate cancer malignancy. Furthermore, co-expression of FOXM1 and CENPF is a robust prognostic indicator of poor survival and metastasis. Thus, genome-wide cross-species interrogation of regulatory networks represents a valuable strategy to identify causal mechanisms of human cancer.