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Metastatic relapse after treatment is the leading cause of cancer mortality, and known resistance mechanisms are missing for most treatments administered to patients. To bridge this gap, we analyze a pan-cancer cohort (META-PRISM) of 1,031 refractory metastatic tumors profiled via whole-exome and transcriptome sequencing. META-PRISM tumors, particularly prostate, bladder, and pancreatic types, displayed the most transformed genomes compared with primary untreated tumors. Standard-of-care resistance biomarkers were identified only in lung and colon cancers—9.6% of META-PRISM tumors, indicating that too few resistance mechanisms have received clinical validation. In contrast, we verified the enrichment of multiple investigational and hypothetical resistance mechanisms in treated compared with nontreated patients, thereby confirming their putative role in treatment resistance. Additionally, we demonstrated that molecular markers improve 6-month survival prediction, particularly in patients with advanced breast cancer. Our analysis establishes the utility of the META-PRISM cohort for investigating resistance mechanisms and performing predictive analyses in cancer.Significance:This study highlights the paucity of standard-of-care markers that explain treatment resistance and the promise of investigational and hypothetical markers awaiting further validation. It also demonstrates the utility of molecular profiling in advanced-stage cancers, particularly breast cancer, to improve the survival prediction and assess eligibility to phase I clinical trials.This article is highlighted in the In This Issue feature, p. 1027

These figures provide complementary information for the figures presented in the main text and illustrations of the methods section.

These tables present clinical data, sample data, whole-exome and transcriptome sequencing results, literature references, and external data sources that were used for the analyses presented in the main text.

PDF file - 272K, Supplemental figure S1: Correlation analysis between proliferation marker MIK67 and the spliceosome-related genes Supplemental figure S2: Expression of the indicated spliceosomal components in SNRPE or SNRPD1-depleted cells. Supplemental figure S3: Effect of siRNA-mediated depletion of SNRPE or SNRPD1 on cell viability in additional breast, melanoma and lung tumoral cell lines Supplemental figure S4: Correlation analysis between the proliferation rate of all tested cell lines tested and their sensitivity to SNRPE or SNRPD1 depletion Supplemental figure S5: Apoptosis detection in SKBr-3 cell line following SNRPD1 depletion Supplemental figure S6: Genes whose initial exon expression is modulated by SNRPE. Supplemental figure S7: Expression of GUSB and RPLPO control genes in SNRPE-depleted cells treated or not with cycloheximide

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PDF file - 43K, Supplemental Table S1: SNRPD1 and SNRPE specific siRNA sequences used for transfection

Metastatic relapse after treatment is the leading cause of cancer mortality, and known resistance mechanisms are missing for most treatments administered to patients. To bridge this gap, we analyze a pan-cancer cohort (META-PRISM) of 1,031 refractory metastatic tumors profiled via whole-exome and transcriptome sequencing. META-PRISM tumors, particularly prostate, bladder, and pancreatic types, displayed the most transformed genomes compared with primary untreated tumors. Standard-of-care resistance biomarkers were identified only in lung and colon cancers—9.6% of META-PRISM tumors, indicating that too few resistance mechanisms have received clinical validation. In contrast, we verified the enrichment of multiple investigational and hypothetical resistance mechanisms in treated compared with nontreated patients, thereby confirming their putative role in treatment resistance. Additionally, we demonstrated that molecular markers improve 6-month survival prediction, particularly in patients with advanced breast cancer. Our analysis establishes the utility of the META-PRISM cohort for investigating resistance mechanisms and performing predictive analyses in cancer. Significance: This study highlights the paucity of standard-of-care markers that explain treatment resistance and the promise of investigational and hypothetical markers awaiting further validation. It also demonstrates the utility of molecular profiling in advanced-stage cancers, particularly breast cancer, to improve the survival prediction and assess eligibility to phase I clinical trials. This article is highlighted in the In This Issue feature, p. 1027

Metastatic relapse after treatment is the primary cause of cancer morbidity and mortality. While genetic mechanisms of primary tumors and, to a lesser extent, metastatic cancers have been studied in large cohorts, refractory metastatic tumors are not yet sufficiently characterized. Markers of aggressiveness and resistance that molecular profiling can extract from these tumors have yet to be identified and incorporated into clinical care. In this study, we present a pan-cancer cohort of 1,031 metastatic tumors (which we refer to as META-PRISM) that are resistant to at least one systemic therapy or with no approved treatment options. We retrieved the complete clinical history of patients and performed whole-exome (n=571) and transcriptome sequencing (n=947) for this cohort. The prevalence of detected cancer biomarkers was assessed and compared to an external cohort of primary tumors. In the META-PRISM cohort, we observed an increase in (i) whole-genome duplication frequency, (ii) tumor mutational burden, (iii) germline cancer-predisposing variants, and (iv) somatic alterations in cancer genes, including KRAS, EGFR, CCND1, MYC, and TP53, as compared to the tumor type-matched primary tumors. The most extensive increase in genomic variation at metastatic stage was observed in prostate bladder, and pancreatic cancer types. We also identified enrichment of standard-of-care resistance biomarkers in most cancer types. However, only 9.3% of tumors harbored at least one such biomarker, indicating that the current understanding of resistance mechanisms remains insufficient. Our cohort demonstrated a significantly improved 6-month survival prediction from models incorporating molecular markers over models with only clinical markers for breast cancer patients and to a lesser extent for other studied tumor types. Overall, our data establish a unique resource for investigating treatment resistance mechanisms and performing predictive analyses in cancer. Citation Format: Yoann Pradat, Julien Viot, Konstantin Gunbin, Andrey Yurchenko, Luigi Cerbone, Marc Deloger, Guillaume Grisay, Loic Verlingue, Véronique Scott, Ismael Padioleau, Leonardo Panunzi, Stefan Michiels, Antoine Hollebecque, Gerome Jules-Clément, Laura Mezquita, Antoine Lainé, Yohann Loriot, Benjamin Besse, Luc Friboulet, Fabrice André, Paul-Henry Cournède, Daniel Gautheret, Sergey Nikolaev. Integrative pan-cancer genomic and transcriptomic analyses of refractory metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6570.

Rapalogs have become standard-of-care in patients with metastatic breast, kidney, and neuroendocrine cancers. Nevertheless, tumor escape occurs after several months in most patients, highlighting the need to understand mechanisms of resistance. Using a panel of cancer cell lines, we show that rapalogs downregulate the putative protein kinase TRIB3 (tribbles pseudokinase 3). Blood samples of a small cohort of patients with cancer treated with rapalogs confirmed downregulation of TRIB3. Downregulation of TRIB3 was mediated by LRRFIP1 independently of mTOR and disrupted its interaction with the spliceosome, where it participated in rapalog-induced deregulation of RNA splicing. Conversely, overexpression of TRIB3 in a panel of cancer cell lines abolished the cytotoxic effects of rapalogs. These findings identify TRIB3 as a key component of the spliceosome, whose repression contributes significantly to the mechanism of resistance to rapalog therapy. Significance: Independent of mTOR signaling, rapalogs induce cytoxicity by dysregulating spliceosome function via repression of TRIB3, the loss of which may, in the long term, contribute to therapeutic resistance.

The cyclin-dependent kinase 4 (CDK4)/6 inhibitor Palbociclib is a new standard treatment in hormone-receptor positive breast cancer patients. No predictive biomarkers have been identified and no pharmacodynamics has properly been described so far.Patients with early-breast cancer were randomized 3 : 1 to oral palbociclib 125 mg daily for 14 days until the day before the surgery versus no treatment. Primary objective was antiproliferative response defined as a natural logarithm of Ki67 expression at day 15 below 1. Secondary end points were subgroups analyses and safety. Exploratory analyses included search for predictive biomarkers. Immunostainings (Ki67, RB, pRB, p16, pAKT, pER, pCDK2, CyclinD1), FISH (CCND1) and gene expression (GE) arrays were carried out at baseline and at surgery. In addition, activating PIK3CA and AKT1 mutations were assessed at baseline.74 patients were allocated to palbociclib and 26 to control. Most patients (93%) were hormone-receptor (HR)-positive, whereas 8% were HER2-positive. Palbociclib led to significantly more antiproliferative responses when compared with control (58% versus 12%, P 0.001), and to a significantly higher Ki67 decrease (P 0.001). In the HR-positive/HER2-negative subgroup, this antiproliferative effect was even more marked in the palbociclib arm when compared with control (70% versus 9%, P 0.001). Palbociclib treatment led also to a significantly higher decrease from baseline in phospho-Rb when compared with control (P 0.001). Among treated patients, changes in Ki67 correlated with changes in phospho-Rb (Spearman rank r = 0.41, P 0.0001). GE analyses confirmed a major effect on proliferation and cell cycle genes. Among treated patients, CCNE2 expression was significantly more decreased in antiproliferative responders versus nonresponders (P = 0.006).Short-term preoperative palbociclib decreases Ki67 in early-breast cancer patients. Early decrease of Rb phosphorylation correlates with drug's effect on cell proliferation and could potentially identify patients with primary resistance.NCT02008734.

Background While deregulation of the cyclin D1-CDK4/6-retinoblastoma pathway is common in hormone receptor positive (HR+) breast cancer, Rb is usually intact in HR+ breast cancer, and targeted CDK 4/6 inhibitors that act upstream of Rb, are routinely being utilized in clinical practice. However, factors that can lead to clinical resistance to CDK 4/6 inhibitors are not known. Patients and methods We identified patients who had pre- and post-genotyping in tissue and peripheral blood samples after receiving CDK 4/6 inhibitors. Genotyping was carried out in tumor tissue or blood collected before start of CDK 4/6 inhibitor and after disease progression on CDK 4/6 inhibitor, covering more than 90% of the coding region in RB1. Results We identified detectable acquired RB1 mutations in circulating tumor DNA (ctDNA) after exposure to CDK4/6 inhibitor (palbociclib, palbociclib, ribociclib) for 5, 8, and 13 months, respectively, in three patients. The RB1 mutations included substitution in donor splicing site of exon 8 of the RB1 gene in patient #1; substitution in donor splicing site of exon 22 of RB1 gene, exon 19 deletion, exon 3 insertion in patient #2; and RB1 exon 16 H483Y mutation in patient #3. None of these RB1 mutations were present in the pre-CDK 4/6 specimen highlighting these molecular alterations, which lead to functional loss of Rb1, likely emerged under selective pressure from the CDK4/6 inhibitor potentially confering therapeutic resistance. Conclusion This is the first clinical report to describe the emergence of somatic RB1 mutations after exposure to palbociclib or ribociclib, in patients with metastatic breast cancer. Further research is needed to validate these findings, identify how these mutations temporally emerge under selective pressure of CDK 4/6 inhibitor, and develop rational therapeutic strategies.

Purpose: There is a need to identify which patients present a high risk of relapse after optimal adjuvant therapies, in order to better define which population of early breast cancer patients is eligible to new drugs. Previous studies have shown that gene copy numbers drive breast cancer progression. In the BIG1-98 trial, FGFR1 (8p11) and ZNF217 (20q13) amplifications were associated with poor outcome in patients with HR+/Her2- early breast cancers. In the present study, we aim to validate these findings using retrospective analysis of samples prospectively collected in a randomized trial (PACS04). Patients and methods: Tumor DNA was extracted from FFPE samples obtained from patients included in PACS04 trial. This trial compared FEC to ED75 in patients with node positive, early breast cancer and included 3000 patients between 2001 and 2004. The biomarker study focuses on HR+/Her2- breast cancer. 500 samples were collected and hybridization was performed on 390. DNA copy numbers were assessed on 900 genes using Oncoscan FFPE assay kit. Prognostic value of FGFR1 (8p11) and ZNF217 (20q13) copy numbers was assessed in a Cox model that included prognostic parameters. Prognostic value was assessed using CNA as continuous variable, and with a predefined cut-off (CN>=3). Metastases-free survival (MFS) was chosen as endpoint for this biomarker study. Results: Tumor copy numbers alterations were obtained in 390 patients with HR+/Her2-, node positive, early breast cancer. 31% of the patients presented more than 3 lymph node involved, and 25% a poorly differenciated tumor. The median tumor size was 20 mm. 120 (30%) and 112 (28%) patients presented FGFR1 and/or ZNF217 gene gain with a cut-off for amplification predefined at 3 copies. When assessed as continuous variable, FGFR1 (HR: 1.02, 95%CI:1.007-1.04, p=0.0045) and ZNF217 (HR: 1.011, 95%CI:1.003-1.01, p=0.006) copy numbers were associated with MFS. The 10 years MFS rates were 68% (95%CI: 59-78%) and 85% (95%CI: 81-91%) in patients with FGFR1-gained and FGFR1-normal tumors respectively (HR: 2.51, 95%CI:1.56-4.01, p=0.0001). The 10 years MFS rates were 72% (95%CI:65%-83%) and 83% (95%CI: 77-87%) in patients with ZNF217-gained and ZNF217-normal tumors (HR: 1.79, 95%CI:1.12-2.86, p=0.013). In a multivariate analysis, FGFR1-gain (HR: 2.45, 95%CI:1.42-4.22, p=0.0012) and ZNF217-gain (HR:1.78, 95%CI:1.00-3.17, p=0.049) were associated with a higher likelihood of metastases and/or death. The 10 MFS rate was 88% (95%CI: 83-94%) for patients who presented FGFR1- and ZNF217-normal tumor (n=191), and 71% (95%CI: 66-76%) in patients presenting FGFR1 and/or ZNF217 gene gain. Conclusion: Using samples prospectively collected in a randomized trial, this study validates the prognostic value of FGFR1- and ZNF217- copy numbers in patients who present HR+/Her2- early breast cancer. Exploratory analyses on copy number alterations, together with targeted sequencing are ongoing and will be presented. Citation Format: Andre F, Penault Llorca F, Carene D, Roche H, Delaloge S, Lacroix L, Scott V, Richon C, Lacroix-Ticrit M, Lemonnier J, Michiels S. FGFR1 / ZNF217 copy numbers and outcome in patients with node positive, HR+/Her2- early breast cancer: A genomic analysis of PACS04 trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-10.

Neoadjuvant systemic therapy (NAC) is currently used in the treatment of stage II/III breast cancer. Pathological complete response as a surrogate endpoint for clinical outcomes is not completely validated for all subgroups of breast cancers. Therefore, there is a need for reliable predictive tests of the most effective treatment.We used a combination of predictive clinical, pathological, and gene expression-based markers of response to NAC in a prospective phase II multicentre randomized clinical trial in breast cancer patients, with a long follow-up (8 years). This study concerned the subpopulation of 188 patients with similar levels of pathological response rates to sequential epirubicin/cyclophosphamide and docetaxel to determine predictive marker of pCR and DFS. We used a set of 45 genes selected from high throughput analysis and a standardized RT-qPCR. We analyzed the predictive markers of pathological complete response (pCR) and DFS in the overall population and DFS the subpopulation of 159 patients with no pCR.In the overall population, combining both clinical and genomic variables, large tumor size, low TFF1, and MYBL2 overexpression were significantly associated with pCR. T4 Stage, lymphovascular invasion, negative PR status, histological type, and high values of CCNB1 were associated with DFS. In the no pCR population, only lymphovascular invasion and high values of BIRC5 were associated with DFS.We confirm the importance of ER-related and proliferation genes in the prediction of pCR in NAC-treated breast cancer patients. Furthermore, we identified BIRC5 (survivin) as a main pejorative prognostic factor in patients with breast cancers with no pCR. These results also open perspective for predictive markers of new targeted therapies.

International audience; Purpose: The identification of molecular biomarkers for classification of breast cancer is needed to better stratify the patients and guide therapeutic decisions. The aim of this study was to investigate the value of MAPRE1 gene encoding microtubule-end binding proteins EB1 as a biomarker in breast cancer and evaluate whether combinatorial expression of MAPRE1 and MTUS1 gene encoding EB1-negative regulator ATIP3 may improve breast cancer diagnosis and prognosis.Methods: Probeset intensities for MAPRE1 and MTUS1 genes were retrieved from Exonhit splice array analyses of 45 benign and 120 malignant breast tumors for diagnostic purposes. Transcriptomic analyses (U133 Affymetrix array) of one exploratory cohort of 150 invasive breast cancer patients and two independent series of 130 and 155 samples were compared with clinical data of the patients for prognostic studies. A tissue microarray from an independent cohort of 212 invasive breast tumors was immunostained with anti-EB1 and anti-ATIP3 antibodies.Results: We show that MAPRE1 gene is a diagnostic and prognostic biomarker in breast cancer. High MAPRE1 levels correlate with tumor malignancy, high histological grade and poor clinical outcome. Combination of high-MAPRE1 and low-MTUS1 levels in tumors is significantly associated with tumor aggressiveness and reduced patient survival. IHC studies of combined EB1/ATIP3 protein expression confirmed these results.Conclusions: These studies emphasize the importance of studying combinatorial expression of EB1 and ATIP3 genes and proteins rather than each biomarker alone. A population of highly aggressive breast tumors expressing high-EB1/low-ATIP3 may be considered for the development of new molecular therapies.

BACKGROUND The prognostic and predictive role of cyclo-oxygenase-2 (COX2) in breast cancer is still debated, and in particular, its role as a target of COX2 inhibitor (celecoxib) in neoadjuvant setting. MATERIALS AND METHODS We analyzed a series of 156 breast cancer samples from patients of the COX2 inhibitor-treated arm included in the REMAGUS-02 randomized phase II trial. COX2 gene expression was assessed by reverse transcription and quantitative polymerase chain reaction using ribonucleic acid from frozen biopsies. Pathological complete response (pCR) was the surrogate end-point. RESULTS Significantly higher rates of grade 3, and estrogen and progesterone receptor negativity were observed in tumors with the highest expression of COX2. pCR rates were significantly higher in COX2-overexpressing tumors in patients receiving celecoxib. The test for interaction between COX2 gene expression and the celecoxib effect was statistically significant (p

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 goal of this study was CGH array profiling of breast cancer from Malian women in order to define differences with those from USA. CGH array was performed in 28 samples, 17 with a triple negative phenotype. The profiles were compared to those of 106 tumors from USA. 6 chromosomal regions (6p21, 9q34, 11q13, 12q24, 17q25 and 22q12.1-22q13.1) were identified with a significant higher rate of copy number alterations. These regions contain several genes of interest including BCR. FISH and IHC confirmed that BCR was amplified and overexpressed particularly in triple negative tumors. Finally, 5 regions presented a high level of amplification in two or more samples, including 2 regions located between 9p22.3-9p23 and 9p23-9p24.1. This study confirms that breast cancers from African women present biological differences with those from USA. Larger studies are needed to go further in the identification of therapeutic targets that would be specific to African women.

The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology. Cancer Res; 73(7); 2247–58. ©2013 AACR.

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.

Breast cancer includes high number of molecular entities targetable by specific agents. In this study, array CGH and PIK3CA/AKT1 mutations were used to drive patients into targeted therapy. A prospective molecular analysis was offered to metastatic breast cancer patients for whom samples were collected prospectively or retrospectively either from frozen or paraffin-embedded tissue. Analyses were performed using array CGH (Agilent platform) and PIK3CA (exon 10 and 21) and AKT1 mutations were explored by standard Sanger sequencing. One hundred and eight patients were included. Good quality CGH was obtained in 79% cases and was better for frozen samples. Genomic alterations were identified in 50% of patients including 11 PIK3CA and 8 AKT1 mutations. Eighteen treatments (17 patients) were administered according to their molecular profile with evidence of activity in nine. Reasons for not providing a genomic-driven treatment included absence of progressive disease (38%), investigator's choice (9%), rapid PD (19%), and no drug access (21%). Array CGH correctly identified Her2 status in 97% cases; failures were related to low % of tumour cells. Our study showed that array CGH is feasible in the context of daily practice and, in combination with PIK3CA/AKT1 mutations, identifies a significant number of actionable molecular alterations that allow driving patients into specific targeted agents.

Background The aim of neoadjuvant chemotherapy is to increase the likelihood of successful breast conservation surgery (BCS). Accurate identification of BCS candidates is a diagnostic challenge. Breast Cancer Index (BCI) predicts recurrence risk in estrogen receptor + lymph node - breast cancer. Performance of BCI to predict chemosensitivity based on pathological complete response (pCR) and BCS was assessed. Methods Real-time RT-PCR BCI assay was conducted using tumor samples from 150 breast cancer patients treated with neoadjuvant chemotherapy. Logistical regression and c-index were used to assess predictive strength and additive accuracy of BCI beyond clinicopathologic factors. Results BCI classified 42 % of patients as low, 35 % as intermediate and 23 % as high risk. Low BCI risk group had 98.4 % negative predictive value (NPV) for pCR and 86 % NPV for BCS. High versus low BCI group had a 34 and 5.8 greater likelihood of achieving pCR and BCS, respectively (P = 0.0055; P = 0.0022). BCI increased c-index for pCR (0.875–0.924; P = 0.017) and BCS prediction (0.788–0.843; P = 0.027) beyond clinicopathologic factors. Conclusions BCI significantly predicted pCR and BCS beyond clinicopathologic factors. High NPVs indicate that BCI could be a useful tool to identify breast cancer patients who are not eligible for neoadjuvant chemotherapy. These results suggest that BCI could be used to assess both chemosensitivity and eligibility for BCS.

In addition to their major interest in refining disease classifications and improving diagnostic and prognostic accuracy by means of molecular signatures, microarrays have been proposed in breast cancer as an alternative to routine immunohistochemistry (IHC) determination of biomarkers such as estrogen and progesterone receptors (ER, PR) and HER2, that are crucial for treatment decisions in breast cancer. IHC is currently the standard technique for determination of ER, PR, and HER2 status, with additional in situ hybridization assays to clarify equivocal HER2 IHC results. Despite its long-term use and efforts to achieve interlaboratory standardization and quality control programs, marked variability in the determination of these variables has been reported between pathology laboratories with relatively high false-positive (for HER2) and falsenegative rates (for ER and PR) [1]. The historical ligandbinding assay reported ER and PR protein content on a continuous scale with a large dynamic range. ER and PR IHC data are now used as a dichotomous variable, and information has been lost compared to the results obtained with continuous variables (high ER values correlated with better response to hormone therapy) [2, 3]. Semi quantitative IHC approaches have been proposed, but implementation of such systems in different laboratories gives results that may differ from one laboratory to another Electronic supplementary material The online version of this article (doi:10.1007/s10549-012-2042-4) contains supplementary material, which is available to authorized users.

Introduction: Neoadjuvant chemotherapy increases the likelihood that breast conservation therapy for breast cancer patients will be successful. Breast Cancer Index (BCI), a gene expression assay combining HoxB13/IL17BR ratio and Molecular Grade Index (MGI), is prognostic for the risk of distant recurrence and overall survival in tamoxifen-treated and untreated breast cancer patients. It was previously reported that high risk patients, as determined by BCI, had a 10-fold greater probability of pathologic complete response (pCR) with neoadjuvant chemotherapy than low risk patients. The aim of the current study was to examine the relationship between BCI score and the use of breast conservation surgery (BCS) following treatment with neoadjuvant chemotherapy. Material and Methods: A total of 145 women (tumor size T1, T2 and T3) were treated with neoadjuvant cheomtherapy for stage I-III breast cancer. RNA was extracted from FFPE tumor samples and a real-time RT-PCR assay was completed to generate a BCI score and risk group categorization as previously described (Jerevall et al. Br J Cancer 2011). The relationship between BCS, BCI and clinicopathological factors was examined using univariate and multivariate logistic regression. Results: Of the 145 patients (67% ER+, 54% PR+, 57% >50 y old), 48 (33.1%) underwent BCS. BCI categorized 62 (43%) of patients as low, 50 (34%) as intermediate and 33 (23%) as high risk. The rate of BCS for the three BCI risk categories was 15% (low risk), 48% (intermediate risk) and 45% (high risk). In the low risk group, the rate of BCS was 15% corresponding to a NPV of 85%. This is consistent with previous data from the same cohort, where the NPV of BCI for pCR was 98.4% with only one patient in the low risk group achieving pCR. In univariate analysis, pathological tumor size (pT), ER, PR, grade and BCI were predictors of BCS. A higher BCI score was associated with higher likelihood of BCS (odds ratio of 3.90; CI: 1.45−10.49; p=0.0069). In multivariate analysis, pT and BCI remained significantly associated with BCS, while ER status was not (p=0.23). Results were similar in the subset of patients with T1 and T2 tumors (N=97). In this subset, BCI categorized 42% of patients as low, 37% as intermediate and 21% as high risk and the rate of BCS was 22%, 64% and 60%, respectively. In multivariate analysis of this subset, only BCI was significantly associated with BCS. In all patients, the concordance index based on a model with pT alone was 0.695. When BCI was incorporated into the model with pT, the concordance index increased to 0.801 (p= 0.0002). Conclusion: In this study, we have shown that patients with higher BCI scores were associated with a higher likelihood of receiving BCS after neoadjuvant chemotherapy. Addition of BCI to tumor size increased accuracy in predicting likelihood of BCS. BCI along with standard pathological factors may improve estimation of individual probability of BCS after neoadjuvant chemotherapy. This study gives rise to the hypothesis that patients with low BCI should not be eligible for neoadjuvant chemotherapy since the likelihood of breast conservation is low. Further large confirmatory studies are necessary. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-08.

Background: Mechanistic Target of Rapamycin (mTOR), a key component of the PI3K/Akt signaling pathway, coordinates various cellular processes such as metabolism, growth, cell proliferation and cell cycle progression. Dysregulations affecting mTOR frequently occur in cancer, and mTOR inhibitors/rapalogs are used as monotherapy or a part of combination therapy in breast or kidney metastatic cancer. Despite this, response rate is modest. Several mechanisms of resistance have been reported, but do not fully characterize insensitive tumor cells. Purpose: This study investigated new target genes of rapamycin/rapalogs. Methods: Expression levels of target genes were evaluated in a panel of cancer cell lines by transcriptomic analyses, real-time qPCR and western blot. Transcriptional regulation was investigated by in silico analyses of the promoter region, gene reporter assays using site-directed mutagenesis, and ChIP. Stably silenced cell lines were established by lentiviral transduction of shRNA sequences. Results: We identified one gene: TRIB3 (Tribbles Pseudokinase 3), whose expression is downregulated in the presence of rapamycin in a panel of cancer cell lines and in PBMCs isolated from cancer patients. This gene encodes for a pseudokinase involved in a glucose-induced insulin resistance in Type 2 Diabetes by inhibiting the activation of Akt1/2. We observed that silencing mTOR did not mimic rapamycin downregulation of TRIB3, suggesting an mTOR-independent effect of rapamycin. We identified a binding site for the transcriptional repressor GCF2/LRRFIP1 in the promoter region of TRIB3, and confirmed that the presence and the binding of GCF2 to TRIB3 promoter are required for TRIB3 downregulation by rapamycin. Finally, we identified FKBP3 (FK506-Binding Protein 3), a protein that can bind rapamycin with high affinity, as a GCF2 partner, confirming the direct effect of rapamycin on TRIB3 promoter. Conclusions: Taken together, our investigations identified a novel transcriptional target gene of rapamycin: TRIB3. We also characterized the molecular mechanism of regulation by rapamycin through a FKBP3/GCF2-dependent repression of TRIB3 promoter activity, and independently of mTOR signaling. Such variations in TRIB3 expression level in cancer cells could be linked to rapamycin sensitivity, and may represent a valuable therapeutic target. Citation Format: Bojana Stefanovska, Cécile Vicier, Véronique Scott, Guillaume Meurice, Fabrice André, Olivia Fromigué. A new mTOR-independent effect of rapamycin: Transcriptional regulation of TRIB3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2524.

Background: There is a need to develop blood-based bioassays for breast cancer (BC) screening. In this study, differential gene expression between BC samples and benign tumours was used to identify candidate biomarkers for blood-based screening. Methods: We identified two proteins (Fibronectin 1 and CXCL9) from a gene expression data set that included 120 BC samples and 45 benign lesions. These proteins fulfil the following criteria: differential gene expression between cancer and benign lesion, protein released in the extracellular medium and stable in the serum, commercially available ELISA kit, ELISA accuracy in a feasibility study. Protein concentrations were determined by ELISA. Blood samples were from normal volunteers (n=119) and early BC patients (n=133). Results: Seventy-three per cent of patients had cT1-T2 tumour. Patients had higher CXCL9 and Fibronectin 1 concentrations than volunteers. CXCL9 mean concentration was 851 and 635 pg ml−1 for patients and volunteers respectively (P=0.013). CXCL9 concentration was significantly higher in patients with estrogen receptor (ER)-negative compared with volunteers (P=0.003), data consistent with gene expression profile. Fibronectin 1 mean concentration was 190 μg ml−1 for patients and 125 μg ml−1 for volunteers (P

BACKGROUND: With the widespread development of genomic analysis, an accurate and quick method for obtaining high-quality nucleic acids is needed. The objective of the current study was to evaluate the quality and potential use of material obtained from fine-needle aspiration cytology (FNAC). METHODS: Ultrasound- or palpation-guided FNAC was performed in 124 consecutive patients who had nodular breast lesions. The authors evaluated the amount of messenger RNA (mRNA) obtained, its quality through the RNA Integrity (RIN) score, and the factors that influenced both. For malignant lesions, the authors attempted to correlate estrogen receptor 1 (ESR1) and HER-2 (c-erb–B2) mRNA expression measured by real-time quantitative polymerase chain reaction with estrogen receptor and HER-2 detection obtained by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH) on the surgical specimen. RESULTS: The amount of mRNA obtained was >1 μg in 89.5% of 124 samples (43 benign lesions and 81 adenocarcinomas). Overall, 59.3% of samples yielded >1 μg RNA with a RIN score >6. The most significant predictors of quality and quantity of mRNA were the cytopathologist who sampled the tumors and a diagnosis of cancer versus benign lesion. The median ESR1 expression level, which was expressed as the polymerase chain reaction cycle threshold (CT) level minus the average 18S value (dct), was 17.7 dct in patients with estrogen receptor-negative tumors and 11.1 dct in patients with estrogen receptor-positive tumors,. The median HER-2 expression level was 15.1 dct in patients with HER-2-negative tumors and 10.7 dct in patients with HER-2-positive tumors. mRNA expression was concordant with the IHC/FISH evaluation in 90.3% of patients for estrogen receptor status and in 98.5% of patients for HER-2 status. CONCLUSIONS: In 70% of cases, FNAC of breast lesions in well trained hands allowed the extraction of mRNA suitable for gene expression analysis. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.

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.

Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation has been suggested to negatively influence response to anti-HER2 therapy in breast cancer patients. The present study focused on mutations of the PIK3CA gene, encoding one of the two PI3K subunits. Methods: PIK3CA mutations were assessed by direct sequencing in 80 HER2-positive patients treated with 1 year of trastuzumab. All patients preoperatively received four cycles of anthracycline-based chemotherapy, followed by four cycles of docetaxel and 1 year of trastuzumab, starting either before surgery with the first cycle of docetaxel and continuing after surgery (neoadjuvant trastuzumab arm, n=43), or only after surgery (adjuvant trastuzumab arm, n=37). Results: PIK3CA mutations were found in 17 tumours (21.3%). Better disease-free survival (DFS) was observed in patients with PIK3CA wild-type compared with mutated tumours (P=0.0063). By combining PIK3CA status and treatment arms, four separate prognostic groups with significantly different DFS (P=0.0013) were identified. Conclusion: These results confirm that the outcome of HER2-positive patients treated with trastuzumab is significantly worse in patients with PIK3CA-mutated compared with wild-type tumours.

Starting from a clinical observation, a small subset of breast cancer patients presenting retinoic acid receptor alpha (RARA) amplification and overexpression was identified. Retinoic acid reduced cell viability of RARAamplified cell lines through interferon regulatory factor (IRF)-1 and caspase (CASP)-1 activation and RARAnonamplified cells were most likely resistant to all-trans-retinoic acid (ATRA). This study suggests that RARA-amplified breast cancers could be sensitive to retinoic acid. Background: Molecular segmentation of breast cancer allows identification of small groups of patients who present high sensitivity to targeted agents. A patient, with chemo- and trastuzumab-resistant HER2-overexpressing breast cancer, who presented concomitant acute promyelocytic leukemia, showed a response in her breast lesions to retinoic acid, arsenic, and aracytin. We therefore investigated whether RARA gene amplification could be associated with sensitivity to retinoic acid derivatives in breast cancers. Materials and Methods: Array comparative genomic hybridization and gene expression arrays were used to characterize RARA amplifications and expression in 103 breast cancer samples. In vitro activity of ATRA was characterized in T47D, SKBR3, and BT474 cell lines. Results: Retinoic acid receptor alpha was gained or amplified in 27% of HER2-positive and 13% of HER2-negative breast cancer samples. Retinoic acid receptor alpha can be coamplified with HER2. Retinoic acid receptor alpha copy number changes could be correlated with messenger RNA expression. All-trans-retinoic acid reduced cell viability of RARAamplified, but not RARA-normal, cell lines through apoptosis. Gene expression arrays showed that ATRA-induced apoptosis in RARA-amplified cell lines was related to an increase in CASP1 and IRF1. Conclusion: The results of this study suggest that breast cancers exhibiting RARA amplifications could be sensitive to retinoic acid. A phase II trial will evaluate this hypothesis in the clinical setting.

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: In this monocentric randomized trial we aimed at identifying if short-term preoperative palbociclib treatment is associated with decreased proliferation and early biomarker changes in patients with early breast cancer (EBC) Methods: Untreated EBC patients were randomized 3:1 to oral palbociclib 125mg daily for 14 days until the day before the surgery vs no treatment. FFPE and frozen samples were extracted at baseline and at surgery. Primary objective was antiproliferative response defined as a natural logarithm of Ki67 expression at day 15 below 1. Immunostaining (Ki67, RB, pRB, p16, pAKT, pER), FISH (CCND1) and gene expression (GE) arrays were performed pre- and post-treatment. PIK3CA and AKT1 mutations were assessed pretreatment. Results: 74 patients were randomized in the palbociclib arm and 26 in the control arm. 93% tumors were HR-positive; 8% HER2-positive. Palbociclib treatment led to significantly more patients with antiproliferative response as compared to control (58% vs 10%, p = 0.0003). In addition, mean decrease from baseline in ln(Ki67) was significantly higher at day 15 in the palbociclib arm (p Citation Format: Monica Arnedos, Bianca Cheaib, Mohamed Amine Bayar, Stefan Michiels, Veronique Scott, Julien Adam, Valerie Leroux-Kozal, Virginie Marty, Chafika Mazouni, Benjamin Sarfati, Ivan Bieche, David Gentien, Suzette Delaloge, Magali Lacroix-Triki, Fabrice Andre. Anti-proliferative response and predictive biomarkers to palbociclib in early breast cancer: The Preoperative Palbociclib (POP) randomized trial. [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 CT041.

This study was designed to identify predictive signatures of pathological complete response (pCR) in breast cancer treated by taxane-based regimen, using clinicopathological variables and transcriptomic data (Affymetrix Hgu133 Plus 2.0 devices). The REMAGUS 02 trial ( n = 153,training set) and the publicly available M.D. Anderson data set ( n = 133, validation set) were used. A re-sampling method was applied. All predictive models were defined using logistic regression and their classification performances were tested through Area Under the Curve (AUC) estimation. A stable set of 42 probesets (31 genes) differentiate pCR or no pCR samples. Single-or 2-probesets signatures, mainly related to ER pathway, were equally predictive of pCR with AUC greater then 0.80. Models including probesets associated with ESR1, MAPT, CA12 or PIGH presented good classification performances. When clinical variables were entered into the model, only CA12 and PIGH, remained informative ( p = 0.05 and p = 0.005) showing that a combination of a few genes provided robust and reliable prediction of pCR.

La cytoponction a l’aiguille fine est une procedure simple, rapide et peu onereuse qui a montre son utilite dans le diagnostic du cancer mammaire [1]. Celle-ci est cependant associee a une frequence plus elevee de resultats indetermines ou de faux negatifs par rapport a la biopsie, ce qui limite son utilisation a des centres tres experimentes [2]. L’Institut de Cancerologie Gustave Roussy (IGR) a identifie une signature moleculaire transcriptomique (EHT Dx14), basee sur l’utilisation de la technologie de biopuces d’ExonHit SA [3], et ayant la capacite de differencier une tumeur mammaire maligne de lesions benignes, a partir d’echantillons obtenus par cytoponction [4]. Dans cette etude, 68 des 71 echantillons ont ete correctement identifies par la signature moleculaire (96 %, 88 – 99 %) avec une sensibilite de 96 % (86 – 100 %) et une specificite de 95 % (76 – 100 %). Les resultats de la cytologie ont egalement ete compares a ceux de la signature moleculaire. L’analyse cytologique n’a pas permis d’etablir un diagnostic definitif de la nature maligne ou benigne de la tumeur mammaire dans 5 des 71 echantillons analyses. Le recours a la signature moleculaire dans ces echantillons a permis une classification correcte dans 4 des 5 cas.

Background Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial. Methods This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA. Results Thirty seven samples were excluded because i) they contained less than 30% malignant cells, or ii) they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy. Conclusions Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications.

Gene-expression arrays have generated molecular predictors of relapse and drug sensitivity in breast cancer. We aimed to identify exons differently expressed in malignant and benign breast lesions and to generate a molecular classifier for breast-cancer diagnosis.165 breast samples were obtained by fine-needle aspiration. Complementary DNA was hybridised on splice array. A nearest centroid prediction rule was developed to classify lesions as malignant or benign on a training set, and its performance was assessed on an independent validation set. A two-way ANOVA model identified probe sets with differential expression in malignant and benign lesions while adjusting for scan dates.120 breast cancers and 45 benign lesions were included in the study. A molecular classifier for breast-cancer diagnosis with 1228 probe sets was generated from the training set (n=94). This signature accurately classified all samples (100% accuracy, 95% CI 96-100%). In the validation set (n=71), the molecular predictor accurately classified 68 of 71 tumours (96%, 88-99%). When the 165 samples were taken into account, 37 858 exon probe sets (5.4%) and 3733 genes (7.0%) were differently expressed in malignant and benign lesions (threshold: adjusted p0.05). Genes involved in spliceosome assembly were significantly overexpressed in malignant disease (permutation p=0.002). In the same population of 165 samples, 956 exon probe sets presented both higher intensity and higher splice index in breast cancer than in benign lesions, although located on unchanged genes.Many exons are differently expressed by breast cancer and benign lesions, and alternative transcripts contribute to the molecular characteristics of breast malignancy. Development of molecular classifiers for breast-cancer diagnosis with fine-needle aspiration should be possible.

BACKGROUND. Children with high-risk neuroblastomas (NB) potentially may benefit from treatment with recombinant human erythropoietin (Epo). Epo is a stimulator of erythropoiesis, acting through its receptor (EpoR). The objective of the current study was to evaluate expression levels of Epo and EpoR in NB and in normal tissues and their effects on the proliferation of tumor cells. METHODS. A tissue microarray study was performed with 101 primary tumors, 39 paired metastases, 56 paired control tissues, and 6 human NB cell lines. Immunohistochemical staining was performed using antibodies against Epo and EpoR. Immunostaining intensity was evaluated by using a semiquantitative score based on the percentage of positive cells. An in vitro analysis of cell proliferation and cell cycle in the presence of recombinant Epo was performed in the 6 cell lines. RESULTS. The expression of EpoR was found to be significantly higher in tumors than in paired control tissues (P < .0001) compared with the expression of Epo (P = .06), and the expression of EpoR was significantly higher in lymph node metastases than in paired primary tumors (P = .02) compared with Epo (P = .99). Survival analysis demonstrated that the patients who had tumors with the highest expression of EpoR had a significantly better overall survival (P = .03). In the in vitro study, recombinant Epo did not modify proliferation and cell cycle in the cell lines regardless of the EpoR expression level. CONCLUSIONS. Epo and EpoR were expressed in NB but did not modify tumor cell proliferation. The results of the current study suggested that Epo may be used safely for supportive care in children with NB. Cancer 2007. © 2007 American Cancer Society.

Cells expressing the human immunodeficiency virus (HIV-1) envelope glycoprotein complex (Env) can fuse with CD4+ cells. When the apoptotic pathway is initiated in Env+ cells (`donor cells'), co-culture with a healthy CD4+ fusion partner (`acceptor cells') results in apoptosis of the syncytium and thus is `contagious'. The cell-to-cell transmission of the lethal signal was only observed when the nuclei from donor cells exhibited pre-apoptotic chromatin condensation (PACC), correlating with comet assay-detectable DNA strand breaks, which precede caspase activation, as well as the loss of the mitochondrial transmembrane potential. Transmission of the lethal signal resulted into mitochondrial alterations, and caspase-dependent nuclear pyknosis with chromatinolysis affecting both the donor and the acceptor nuclei. In the presence of caspase inhibitors, all nuclei of the syncytium formed by fusion of the pre-apoptotic and the healthy cell manifested PACC, exhibited DNA lesions and lost transcriptional activity. Transmission of the lethal signal did not require donor cells to contain a nucleus or mitochondrial DNA, yet was inhibited when two mitochondrion-stabilizing proteins, Bcl-2 or vMIA, were overexpressed. Contagious apoptosis could be induced in primary human T cells, as well as in vivo, in T cells exposed to dying Env-expressing cells. Altogether, these data point to a novel mechanism through which HIV-1 can induce bystander killing.

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

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.

Background Elucidation of promising cancer biomarkers from gene expression data can provide important insight into the relationship between signaling networks and cancer. SORBS2, sorbin and SH3 domain containing 2, is a multi-adapter protein involved in signal transduction associated to the cytoskeleton and was reported to be strongly repressed in pancreatic and cervical cancers. Methods: With the purpose of identifying genes involved in metastatic process, we compared gene expression profiling of 19 invasive ovarian cancers and 24 borderline tumors. Prognostic value of the selected genes was then tested in a gene expression array database that includes 1659 patients with early breast cancer (Gyorffy B et al. 2010). Upon isolation of SORBS2 as a predictor, its involvement in cell migration and tumor progression was investigated in vitro. Small interfering RNA targeting SORBS2 was used to downregulate its expression in T47D and Hela, two cell lines overexpressing SORBS2. Functional effect of siRNA-induced knockdown of SORBS2 on cell viability was determined by WST-1 assay and Trypan Blue exclusion test. Effect on cell migration was evaluated by wound-healing and transwell assays. Western blot analyses were also performed to examine the expressions of proteins involved in cell survival, death and migration. Results: High-throughput analyses of genes that are differentially expressed between borderline ovarian tumors and invasive carcinoma demonstrated that SORBS2 is significantly downregulated in invasive carcinoma (FDR Conclusion: This study is the first to provide evidence for an antiproliferative activity of SORBS2 with no effect on cell migration in breast cancer cells. Our clinical and in vitro data suggest that SORBS2 is a candidate marker to predict relapse in patients with early breast cancer. Molecular mechanisms mediating the antiproliferative effect of SORBS2 are currently being investigated. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-07.

Background: Rapid advancements in genomics paired with significant growth in the availability of targeted therapies offers clinicians expanding opportunities to provide increasingly effective cancer treatment. Currently, individual gene sequencing (e.g. EGFR) from formalin-fixed paraffinembedded (FFPE) tissue is widely used in cancer diagnosis. Shifting this paradigm towards NGS-based, comprehensive mutation testing in routinely collected FFPE cancer specimens will enable more complete and accurate characterization of patients’ cancers for individualized targeted therapy selection. Materials and Methods: DNA was extracted from 2×20micron sections of 83 specimens consisting of colorectal cancer, non-small cell lung cancer and melanoma. Hybridization-capture of 2574 exons across 176 oncogenes, tumour suppressor genes and ADME-related genes was performed to produce libraries appropriate for paired-end sequence analysis on the Illumina HiSeq2000 platform. Results: In-depth sequence analysis of 176 genes in 50 CRC, 29 NSCLC, and 4 melanoma specimens with median coverage averaging 213-fold (range 8 to 461) detected a per-sample average of 2 previously-described mutations, 7 novel mutations and 2 CNAs in the colon specimens, including frequent alterations in TP53 (33), APC (27), KRAS (12) and BRAF (6). The lung specimens averaged 1 previously described mutation, 8 novel mutations and 1 CNA per sample, most frequently KRAS (10), TP53 (7), JAK2 (3), EGFR (2) and BRAF (2). The melanoma cases exhibited on average 1 previously described mutation, 7 novel mutations and 3 CNAs including TP53 (4) and BRAF (2). In addition to validated clinically actionable mutations in EGFR, KRAS, and BRAF, and multiple alterations in well-known cancer genes such as TP53, STK11, APC,MLH1, BRCA2, and SMAD4, we detected many other mutations that are plausibly clinically actionable. These included activating mutations in the PI3 kinase subunit gene PIK3CA, as well as mutations in MET , KIT , ERBB2 and CDKN2A. Conclusions: It is feasible to perform highly sensitive and specific sequence analysis of hundreds of genes from routinely collected FFPE tissues. This approach detects not only the “hot spot” mutations commonly tested for in CRC, NSCLC and melanoma but also many additional mutations that could plausibly inform therapeutic decision-making. We suggest that clinical-grade next-generation sequencing should become a routine part of all clinical trials, and increasingly, of clinical care.

573 Background: Breast Cancer Index (BCI), a gene expression assay combining HoxB13/IL17BR ratio and Molecular Grade Index (MGI), is prognostic for the risk of distant recurrence and overall surviv...

646 Background: To describe microRNA expression in breast cancer and identifies whether they could contribute to prognosis assessment. Methods: MicroRNA expression was assessed in 178 patients with...