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Molecular tests predicting the risk of distant recurrence (DR) can be used to assist therapy decision-making in oestrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer patients after considerations of standard clinical markers. The Oncotype DX Recurrence Score (RS) is a widespread tool used for this purpose. Here, we compared the RS with the StemPrintER Risk Score (SPRS), a novel genomic predictor with a unique biological basis in its ability to measure the expression of cancer stemness genes.We benchmarked the SPRS vs. RS, alone or in combination with clinicopathological variables expressed by the Clinical Treatment Score (CTS), for the prognostication of DR in a retrospective cohort of 776 postmenopausal patients with ER+/HER2-breast cancer enrolled in the translational arm of the randomised Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. Likelihood ratio (LR) with χIn all patients, the SPRS provided significantly more prognostic information than the RS for ten-year DR prognostication (C-index = 0.688, LR-χIn postmenopausal ER+/HER2- breast cancer patients, SPRS provided more prognostic information than RS for DR when used alone or in combination with the CTS. The SPRS could therefore potentially identify high-risk patients, who might benefit from aggressive treatments, from low-risk patients who might safely avoid adjuvant chemotherapy or prolongation of endocrine therapy.

Motivation Breast cancer is the most commonly diagnosed malignancy in women and the second cause of cancer death in developed countries. While advancements in early detection and therapeutic options have led to a significant decrease in mortality, response to treatment is affected by the genetic heterogeneity of the disease. Recent genome-wide DNA mutation analyses revealed the existence of hundreds of low-frequency mutated genes, in addition to known cancer drivers: a finding that is prompting research into the impact of these genes on the pathogenesis of the disease. Results Herein, we describe a strategy towards the characterization of the role of low-frequency mutated genes in breast cancer. Through the combined analyses of publicly available gene expression and mutational datasets, we identified several Cancer Gene Modules (CMs) that we re-organized in Gene Regulatory Networks (GRN) enriched in low-frequency mutated genes. Importantly, these low-frequency mutated genes were mutually exclusive with known cancer drivers. Finally, we provide evidence that gene expression analysis of these mutated GRNs can predict resistance/sensitivity to chemotherapeutic drugs for breast cancer treatment. Availability and implementation Datasets are available at https://www.ncbi.nlm.nih.gov/geo/ and at https://www.ebi.ac.uk/ega/datasets/. Molecular signatures and GSEA software are available at http://www.gsea-msigdb.org/gsea/index.jsp. Source codes are available at https://github.com/EleonoraLusito/Reverse_Engineering_BC_GRNs. Supplementary information Supplementary data are available at Bioinformatics online.

1020 Background: Accurate prediction of distant metastasis (DM) in early stage ER+/HER2- breast cancer (BC) patients is vital to avoid over/under-treatment with adjuvant chemotherapy (CT). The OncotypeDX Recurrence Score (RS) is a widely used tool to assist clinical decision-making for CT. The StemPrintER Risk Score (SPRS) is an alternative genomic predictor based on the biology of cancer stem cells that predicts recurrence risk in ER+/HER2- BCs (Pece S. et al., EBioMedicine 2019). Here, we analyze the prognostic value of SPRS in the TransATAC cohort of post-menopausal ER+/HER2- BC patients, and compare the prognostic information provided by SPRS and RS for 10-year risk of DM. Methods: The likelihood χ2 (LRχ2) and Kaplan-Meier survival analyses were used to assess prognostic information provided by SPRS, RS and the clinical treatment score (CTS) in 818 TransATAC patients treated with anastrozole or tamoxifen for 5 years. Comparative analyses were made for DM risk over the 10-year follow-up, as well as in the early (0-5 years) or late (5-10 years) interval, according to nodal status. Results: Used as a continuous variable, SPRS was an independent predictor of DM in years 0-10 among all patients when adjusted for clinical parameters as expressed by the CTS [HR=1.43 (1.18-1.73), PHIGH vs. LOW=2.96 (1.85-4.73); P2=33.4; RS: HR=1.52, P2=22.1), with even greater differences in late DM prediction in N0 patients. SPRS also provided more prognostic information than RS to CTS (ΔLRχ2: SPRS+CTS vs. CTS= 14.9; RS+CTS vs. CTS= 9.7). Conclusions: In ER+/HER2- TransATAC BC patients, SPRS was highly prognostic for DM and was superior to RS in providing additional prognostic information to conventional clinicopathological parameters.

Summary Cancer genetic heterogeneity offers a wide repertoire of molecular determinants to be screened as therapeutic targets. Here, we identify potential anticancer targets by exploiting negative genetic interactions between genes with driver loss-of-function mutations (recessive cancer genes) and their functionally redundant paralogs. We identify recessive genes with additional copies and experimentally test our predictions on three paralogous pairs. We confirm digenic negative interactions between two cancer genes ( SMARCA4 and CDH1 ) and their corresponding paralogs ( SMARCA2 and CDH3 ). Furthermore, we identify a trigenic negative interaction between the cancer gene DNMT3A , its functional paralog DNMT3B , and a third gene, DNMT1 , which encodes the only other human DNA-methylase domain. Although our study does not exclude other causes of synthetic lethality, it suggests that functionally redundant paralogs of cancer genes could be targets in anticancer therapy.

The phosphatase Cdc14 exerts negative feedback on its upstream regulators to limit its release from the nucleolus to once per cell cycle., In budding yeast, the phosphatase Cdc14 orchestrates progress through anaphase and mitotic exit, thereby resetting the cell cycle for a new round of cell division. Two consecutive pathways, Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN), contribute to the progressive activation of Cdc14 by regulating its release from the nucleolus, where it is kept inactive by Cfi1. In this study, we show that Cdc14 activation requires the polo-like kinase Cdc5 together with either Clb–cyclin-dependent kinase (Cdk) or the MEN kinase Dbf2. Once active, Cdc14 triggers a negative feedback loop that, in the presence of stable levels of mitotic cyclins, generates periodic cycles of Cdc14 release and sequestration. Similar phenotypes have been described for yeast bud formation and centrosome duplication. A common theme emerges where events that must happen only once per cycle, although intrinsically capable of oscillations, are limited to one occurrence by the cyclin–Cdk cell cycle engine.

BACKGROUND The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting. METHODS We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score). RESULTS We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors). CONCLUSIONS We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection.

Lung cancer is the leading cause of cancer death worldwide. Low-dose computed tomography screening (LDCT) was recently shown to anticipate the time of diagnosis, thus reducing lung cancer mortality. However, concerns persist about the feasibility and costs of large-scale LDCT programs. Such concerns may be addressed by clearly defining the target "high-risk" population that needs to be screened by LDCT. We recently identified a serum microRNA signature (the miR-Test) that could identify the optimal target population. Here, we performed a large-scale validation study of the miR-Test in high-risk individuals (n = 1115) enrolled in the Continuous Observation of Smoking Subjects (COSMOS) lung cancer screening program. The overall accuracy, sensitivity, and specificity of the miR-Test are 74.9% (95% confidence interval [CI] = 72.2% to 77.6%), 77.8% (95% CI = 64.2% to 91.4%), and 74.8% (95% CI = 72.1% to 77.5%), respectively; the area under the curve is 0.85 (95% CI = 0.78 to 0.92). These results argue that the miR-Test might represent a useful tool for lung cancer screening in high-risk individuals.

Summary Cells entering mitosis become rounded, lose attachment to the substrate, and increase their cortical rigidity. Pivotal to these events is the dismantling of focal adhesions (FAs). How mitotic reshaping is linked to commitment to divide is unclear. Here, we show that DEPDC1B, a protein that accumulates in G2, coordinates de-adhesion events and cell-cycle progression at mitosis. DEPDC1B functions as an inhibitor of a RhoA-based signaling complex, which assembles on the FA-associated protein tyrosine phosphatase, receptor type, F (PTPRF) and mediates the integrity of FAs. By competing with RhoA for the interaction with PTPRF, DEPDC1B promotes the dismantling of FAs, which is necessary for the morphological changes preceding mitosis. The circuitry is relevant in whole organisms, as shown by the control exerted by the DEPDC1B/RhoA/PTPRF axis on mitotic dynamics during zebrafish development. Our results uncover an adhesion-dependent signaling mechanism that coordinates adhesion events with the control of cell-cycle progression., Graphical Abstract, Highlights • DEPDC1B is a cell-cycle gene involved in the transition from G2 phase to mitosis • Persistent adhesion at G2 phase delays CycB/CDK1 activation and G2/M transition • DEPDC1B controls RhoA/ROCK-dependent adhesion dynamics at G2 phase • DEPDC1B inhibits RhoA activation by displacing it from the PTPRF/GEF-H1 complex, During mitosis, cells become rounded and lose attachment to the substrate. Marchesi et al. show that DEPDC1B, a cell-cycle-regulated protein, binds to the focal-adhesion-associated receptor PTPRF, thus inhibiting RhoA activation and leading to dismantling of focal adhesions upon mitotic entry. DEPDC1B thus links mitotic progression to de-adhesion.

Following the identification of cyclin-dependent kinases in the 1980s, kinases were hailed as the directors of mitosis. Although the action of kinases must necessarily be reversible, only recently has the involvement of specific phosphatases in mitosis become appreciated. Studies are now revealing how the timely execution of mitotic events depends on the delicate interplay between kinases and phosphatases. To date, the best-characterized mitotic phosphatases are Cdc25, that is required for entry into mitosis and Cdc14, that controls exit from mitosis in budding yeast. Recent work has now exposed the conserved serine-threonine phosphatases PP1 and PP2A as key regulators of various mitotic processes.

Lung cancer is leading cause of cancer death worldwide. Being lung cancer asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease. Therefore, it is vital that screening programs and novel diagnostic tools are developed to increase lung cancer early detection. The development of a minimally invasive blood-based diagnostic tool would be ideal as a first-line screening procedure. An increasing number of studies are demonstrating that fluctuations of circulating miRNAs are associated to lung cancer. Recently, we described a serum circulating miRNA signature (miR-Test) diagnostic for asymptomatic, early stage, lung cancer, that was validated in a large cohort of individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects). However, the transfer to the clinic of a blood test based on circ-miRNAs requires the establishment of standardized operating procedures (SOPs), working instructions and guidelines for all pre-analytical and analytical procedures. We identified possible sources of variability affecting circulating miRNAs, analyzed their impact on the miR-Test performance, and defined a standardized protocol to optimize miR-Test application. Analysis of all possible technical and biological variation affecting circ-miRNAs level, revealed two main sources of variability: one related to analytical procedures for miRNAs extraction and quantification, and the other due to pre-analytical conditions, on how samples are prepared. The extraction causes the main source of analytical imprecision. In conclusion, we identified an optimal protocol for the application of miR-Test for lung cancer early diagnosis. Citation Format: Francesca Montani, Matteo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Serum circulating miR–Test application: Standardized protocol for miR–Test clinical application. [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 1575. doi:10.1158/1538-7445.AM2015-1575

Background: Lung cancer is the leading cause of cancer death worldwide. As lung cancer is asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease, when the tumor is unresectable. Consequently, the survival rate is very low: 15% at 5 years. It is vital, therefore, that screening programs and novel diagnostic tools are developed, which will increase the detection of lung cancer in its early stages (stage I-II), when the tumor is still curable, to reduce lung cancer mortality. Recently, we described a serum microRNA signature diagnostic for asymptomatic, early stage, lung cancer. The availability of reliable biomarkers to identify high-risk individuals might help to reduce the size of the target population for LDCT-based programs, thereby reducing costs and probably increasing compliance Methods: We performed a large-scale validation study of a miRNA blood test based on our signature (the miR-Test) in a population of high-risk individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects), and other 74 lung cancer patients diagnosed outside of screening. Results: The miR-Test showed overall accuracy, specificity and sensitivity of 75%, 78%, and 75%, respectively, with an AUC of 0.85. The test appears to have a dual origin: the first from epithelial cells (the epithelial-like component); the second from cells of hematopoietic origin (the inflammatory-like component). Of note, we found that both components are needed to maintain a good performance of the miR-Test. Conclusions: The relatively high sensitivity of the miR-Test in detecting asymptomatic lung cancer and its high negative predictive value (NPV > 99%), confirm the effectiveness of the test, both interms of its ability to identify asymptomatic lung cancer patients and to reduce significantly unnecessary CTs on healthy individuals. Citation Format: Francesca Montani, Matteo Jacopo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giuseppina Bonizzi, Raffaella Bertolotti, Massimo Bellomi, Cristiano Rampinelli, Patrick Maisonneuve, Lorenzo Spaggiari, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. miR-Test: a blood test for lung cancer early detection. [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 1573. doi:10.1158/1538-7445.AM2015-1573

Background. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, highlighting the existence of numerous species of non-coding RNAs (ncRNAs) that play a significant regulatory role in complex organisms, impacting on both physiology and disease. In particular, microRNAs (miRNAs) have been proposed as “next-generation” markers for their ability to unambiguously distinguish cellular states, including SCs, progenitors and differentiated cells, as well as tumour types, even among closely related cancers or in different biological fluids. However, a comprehensive approach for the identification and characterization of small non-coding RNAs from pathological samples (blood, FFPE, primary cultures) has not been established nor compared with current available methodologies (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for the analysis of small non-coding RNAs from pathological samples, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of cancer associated small ncRNAs. Results: An optimized protocol to analyse small ncRNAs from different pathological samples has been developed and compared with high-throughput QPCR. Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of cancer associated non-coding RNAs. Conclusion: We propose that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of ncRNAs, expanding our ability to fish out robust and efficient molecular markers for diagnostic (early detection), prognostic or therapeutic use. Note: This abstract was not presented at the meeting. Citation Format: Francesca Montani, Matteo Marzi, Rose Mary Carletti, Pier Paolo Di Fiore, Francesco Nicassio. Revealing the complexity of cancer associated small non-coding RNAs by next generation sequencing (NGS) and low-density array. [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 522. doi:10.1158/1538-7445.AM2014-522

Background. High incidence and mortality rate tumours present a more favourable prognosis if diagnosed in early stages (Jemal A. et al, 2011). Screening programs based on effective radiological techniques (e.g. mammography or low dose spiral CT) promise a mortality reduction, but their application on large scale suffers of disadvantages such as the use of ionizing radiation, overdiagnosis risk and high costs. Thus, the identification of molecular markers for cancer early diagnosis, especially if detectable through a simple non-invasive blood test, would provide powerful tools for the improvement of screening programs. Recently, a novel class of small non-coding RNAs, namely microRNAs, has been identified. MiRNA are extremely stable in biological fluids and their levels are different in malignant vs. control sera, making them a promising class of biological markers for early diagnosis. Indeed, studies from our and other labs led to the identification of “miRNA signatures” able to predict the presence of tumours even within a population of asymptomatic high-risk individuals (Bianchi F. et al., 2011; Boeri M. et al., 2011). However, methods used in these studies rely on QPCR approaches, which require previous knowledge on the molecules to be investigated. Indeed, the real complexity of circulating RNAs is still obscure, comprising other classes of molecules not yet investigated that could behave as novel biomarkers. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, but a complete protocol for the identification of circulating RNAs has not been established nor compared with current available approaches (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for serum miRNA analysis, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of circulating small-RNAs. Methods: Total RNA has been isolated from the serum of healthy donors and analysed simultaneously by low-density QPCR (TaqMan Low Density Array, Life Technologies) and by NGS (Illumina). Results: A step-wise protocol to simultaneously analyze non-coding RNAs using NGS technology (Illumina) and high-throughput QPCR has been developed. The protocol has been optimized to allow both analyses using even limited amount of serum (up to 1mL). Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of circulating non-coding RNAs. Conclusion: We proposed that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of circulating RNAs, expanding our ability to fish out robust and efficient molecular markers for early detection of cancer. Citation Format: Francesca Montani, Rose Mary Carletti, Matteo J. Marzi, Gabriele Bucci, Francesco Nicassio, Pier Paolo Di Fiore. Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2013-5280