BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Morphology and genomic hallmarks of breast tumours developed by ATM deleterious variant carriers.

BACKGROUND: The ataxia telangiectasia mutated (ATM) gene is a moderate-risk breast cancer susceptibility gene; germline loss-of-function variants are found in up to 3% of hereditary breast and ovarian cancer (HBOC) families who undergo genetic testing. So far, no clear histopathological and molecular features of breast tumours occurring in ATM deleterious variant carriers have been described, but identification of an ATM-associated tumour signature may help in patient management. METHODS: To characterise hallmarks of ATM-associated tumours, we performed systematic pathology review of tumours from 21 participants from ataxia-telangiectasia families and 18 participants from HBOC families, as well as copy number profiling on a subset of 23 tumours. Morphology of ATM-associated tumours was compared with that of 599 patients with no BRCA1 and BRCA2 mutations from a hospital-based series, as well as with data from The Cancer Genome Atlas. Absolute copy number and loss of heterozygosity (LOH) profiles were obtained from the OncoScan SNP array. In addition, we performed whole-genome sequencing on four tumours from ATM loss-of-function variant carriers with available frozen material. RESULTS: We found that ATM-associated tumours belong mostly to the luminal B subtype, are tetraploid and show LOH at the ATM locus at 11q22-23. Unlike tumours in which BRCA1 or BRCA2 is inactivated, tumours arising in ATM deleterious variant carriers are not associated with increased large-scale genomic instability as measured by the large-scale state transitions signature. Losses at 13q14.11-q14.3, 17p13.2-p12, 21p11.2-p11.1 and 22q11.23 were observed. Somatic alterations at these loci may therefore represent biomarkers for ATM testing and harbour driver mutations in potentially 'druggable' genes that would allow patients to be directed towards tailored therapeutic strategies. CONCLUSIONS: Although ATM is involved in the DNA damage response, ATM-associated tumours are distinct from BRCA1-associated tumours in terms of morphological characteristics and genomic alterations, and they are also distinguishable from sporadic breast tumours, thus opening up the possibility to identify ATM variant carriers outside the ataxia-telangiectasia disorder and direct them towards effective cancer risk management and therapeutic strategies.

Whole-genome profiling helps to classify phyllodes tumours of the breast.

AIMS: The aim of this study was to analyse a series of borderline and malignant phyllodes tumours (PTs) of the breast by whole-genome profiling to identify genomic markers that could help to recognise potentially malignant tumours within borderline tumours. METHODS: We evaluated the genetic imbalances of a series of 53 PTs (30 borderline, 23 malignant) using the Human CNV370 BeadChip microarray (Illumina), containing 370 000 SNP markers and correlate this alterations with clinicopathological features. RESULTS: Forty-five PTs (85%) showed chromosome copy number variations (CNVs). Twenty PTs (37%) showed five or more chromosomal imbalances (8/30 borderline (27%) and 12/23 malignant (52%)). The large-scale genetic changes associated with malignant were+7p (9/23), +1q (8/23), -10p (8/23), -13q14 (7/23), +8q (6/23) and +10q (6/23) and borderline were+1q (13/30), -13q14 (9/30), -6q (8/30) and -10p (8/30). Losses in 9p21.3, encompassing CDKN2A/B gene, were present in three tumours (malignant), whereas deletions of 13q, with a minimal region in 13q14.2 encompassing the RB1 gene, were found in 9/30 borderline and 7/28 malignant tumours. High-level amplifications were seen in eight tumours (seven malignant and one borderline): in 7p in three tumours (including EGFR in two), 7q31.2 (including TFEC and MET), 8q24.21 (including MYC) and 8q23.3 (including CSMD3) in one tumour each. CONCLUSIONS: Whole-genome profiling by SNP arrays in PTs leads to identify a high number of CNV, gains of 7p and 8q, losses of 13q and 10, losses in 9p21.3 (CDKN2A/B) and the presence of amplifications, especially involving EGFR, as markers of potentially malignant tumours.

Bioinformatics for precision medicine in oncology: principles and application to the SHIVA clinical trial.

Precision medicine (PM) requires the delivery of individually adapted medical care based on the genetic characteristics of each patient and his/her tumor. The last decade witnessed the development of high-throughput technologies such as microarrays and next-generation sequencing which paved the way to PM in the field of oncology. While the cost of these technologies decreases, we are facing an exponential increase in the amount of data produced. Our ability to use this information in daily practice relies strongly on the availability of an efficient bioinformatics system that assists in the translation of knowledge from the bench towards molecular targeting and diagnosis. Clinical trials and routine diagnoses constitute different approaches, both requiring a strong bioinformatics environment capable of (i) warranting the integration and the traceability of data, (ii) ensuring the correct processing and analyses of genomic data, and (iii) applying well-defined and reproducible procedures for workflow management and decision-making. To address the issues, a seamless information system was developed at Institut Curie which facilitates the data integration and tracks in real-time the processing of individual samples. Moreover, computational pipelines were developed to identify reliably genomic alterations and mutations from the molecular profiles of each patient. After a rigorous quality control, a meaningful report is delivered to the clinicians and biologists for the therapeutic decision. The complete bioinformatics environment and the key points of its implementation are presented in the context of the SHIVA clinical trial, a multicentric randomized phase II trial comparing targeted therapy based on tumor molecular profiling versus conventional therapy in patients with refractory cancer. The numerous challenges faced in practice during the setting up and the conduct of this trial are discussed as an illustration of PM application.

ITALICS: an algorithm for normalization and DNA copy number calling for Affymetrix SNP arrays.

MOTIVATION: Affymetrix SNP arrays can be used to determine the DNA copy number measurement of 11 000-500 000 SNPs along the genome. Their high density facilitates the precise localization of genomic alterations and makes them a powerful tool for studies of cancers and copy number polymorphism. Like other microarray technologies it is influenced by non-relevant sources of variation, requiring correction. Moreover, the amplitude of variation induced by non-relevant effects is similar or greater than the biologically relevant effect (i.e. true copy number), making it difficult to estimate non-relevant effects accurately without including the biologically relevant effect. RESULTS: We addressed this problem by developing ITALICS, a normalization method that estimates both biological and non-relevant effects in an alternate, iterative manner, accurately eliminating irrelevant effects. We compared our normalization method with other existing and available methods, and found that ITALICS outperformed these methods for several in-house datasets and one public dataset. These results were validated biologically by quantitative PCR. AVAILABILITY: The R package ITALICS (ITerative and Alternative normaLIzation and Copy number calling for affymetrix Snp arrays) has been submitted to Bioconductor.

VAMP: visualization and analysis of array-CGH, transcriptome and other molecular profiles.

MOTIVATION: Microarray-based CGH (Comparative Genomic Hybridization), transcriptome arrays and other large-scale genomic technologies are now routinely used to generate a vast amount of genomic profiles. Exploratory analysis of this data is crucial in helping to understand the data and to help form biological hypotheses. This step requires visualization of the data in a meaningful way to visualize the results and to perform first level analyses. RESULTS: We have developed a graphical user interface for visualization and first level analysis of molecular profiles. It is currently in use at the Institut Curie for cancer research projects involving CGH arrays, transcriptome arrays, SNP (single nucleotide polymorphism) arrays, loss of heterozygosity results (LOH), and Chromatin ImmunoPrecipitation arrays (ChIP chips). The interface offers the possibility of studying these different types of information in a consistent way. Several views are proposed, such as the classical CGH karyotype view or genome-wide multi-tumor comparison. Many functionalities for analyzing CGH data are provided by the interface, including looking for recurrent regions of alterations, confrontation to transcriptome data or clinical information, and clustering. Our tool consists of PHP scripts and of an applet written in Java. It can be run on public datasets at http://bioinfo.curie.fr/vamp AVAILABILITY: The VAMP software (Visualization and Analysis of array-CGH,transcriptome and other Molecular Profiles) is available upon request. It can be tested on public datasets at http://bioinfo.curie.fr/vamp. The documentation is available at http://bioinfo.curie.fr/vamp/doc.

CAPweb: a bioinformatics CGH array Analysis Platform.

Assessing variations in DNA copy number is crucial for understanding constitutional or somatic diseases, particularly cancers. The recently developed array-CGH (comparative genomic hybridization) technology allows this to be investigated at the genomic level. We report the availability of a web tool for analysing array-CGH data. CAPweb (CGH array Analysis Platform on the Web) is intended as a user-friendly tool enabling biologists to completely analyse CGH arrays from the raw data to the visualization and biological interpretation. The user typically performs the following bioinformatics steps of a CGH array project within CAPweb: the secure upload of the results of CGH array image analysis and of the array annotation (genomic position of the probes); first level analysis of each array, including automatic normalization of the data (for correcting experimental biases), breakpoint detection and status assignment (gain, loss or normal); validation or deletion of the analysis based on a summary report and quality criteria; visualization and biological analysis of the genomic profiles and results through a user-friendly interface. CAPweb is accessible at http://bioinfo.curie.fr/CAPweb.

ITTACA: a new database for integrated tumor transcriptome array and clinical data analysis.

Transcriptome microarrays have become one of the tools of choice for investigating the genes involved in tumorigenesis and tumor progression, as well as finding new biomarkers and gene expression signatures for the diagnosis and prognosis of cancer. Here, we describe a new database for Integrated Tumor Transcriptome Array and Clinical data Analysis (ITTACA). ITTACA centralizes public datasets containing both gene expression and clinical data. ITTACA currently focuses on the types of cancer that are of particular interest to research teams at Institut Curie: breast carcinoma, bladder carcinoma and uveal melanoma. A web interface allows users to carry out different class comparison analyses, including the comparison of expression distribution profiles, tests for differential expression and patient survival analyses. ITTACA is complementary to other databases, such as GEO and SMD, because it offers a better integration of clinical data and different functionalities. It also offers more options for class comparison analyses when compared with similar projects such as Oncomine. For example, users can define their own patient groups according to clinical data or gene expression levels. This added flexibility and the user-friendly web interface makes ITTACA especially useful for comparing personal results with the results in the existing literature. ITTACA is accessible online at http://bioinfo.curie.fr/ittaca.

Preferential occurrence of chromosome breakpoints within early replicating regions in neuroblastoma.

Neuroblastoma (NB) is a frequent paediatric extra cranial solid tumor characterized by the occurrence of unbalanced chromosome translocations, frequently, but not exclusively, involving chromosomes 1 and 17. We have used a 1 Mb resolution BAC array to further refine the mapping of breakpoints in NB cell lines. Replication timing profiles were evaluated in 7 NB cell lines, using DNAs from G1 and S phases flow sorted nuclei hybridised on the same array. Strikingly, these replication timing profiles were highly similar between the different NB cell lines. Furthermore, a significant level of similarity was also observed between NB cell lines and lymphoblastoid cells. A segmentation analysis using the Adaptative Weights Smoothing procedure was performed to determine regions of coordinate replication. More than 50% of the breakpoints mapped to early replicating regions, which account for 23.7% of the total genome. The breakpoints frequency per 10(8) bases was therefore 10.84 for early replicating regions, whereas it was only 2.94 for late replicating regions, these difference being highly significant (p < 10(-4)). This strong association was also observed when chromosomes 1 and 17, the two most frequent translocation partners in NB were excluded from the statistical analysis. These results unambiguously establish a link between unbalanced translocations, whose most likely mechanism of occurrence relies on break-induced replication, and early replication of the genome.