A phase II study of weekly irinotecan in patients with locally advanced or metastatic HER2- negative breast cancer and increased copy numbers of the topoisomerase 1 (TOP1) gene: a study protocol

BACKGROUND: About 20% of patients with primary breast cancer develop metastatic disease during the course of the disease. At this point the disease is considered incurable and thus treatment is aimed at palliation and life prolongation. As many patients will have received both an anthracycline and a taxane in the adjuvant setting, treatment options for metastatic breast cancer are limited. Furthermore response rates for the most commonly used drugs range from around 30% to 12% . Thus new treatment options are needed and preferably coupled to biomarkers predictive of response. Irinotecan is a topoisomerase 1 inhibitor used for decades for the treatment of colorectal cancer. Four studies have investigated the efficacy of irinotecan monotherapy in breast cancer and all have included non-biomarker selected patients. In these studies response rates for irinotecan ranged from 5%-23% and are thus comparable to response rates obtained with drugs commonly used in the metastatic setting. If a predictive biomarker could be identified for irinotecan, response rates might be even higher. METHODS/DESIGN: This multi-centre phase II single arm trial was designed to investigate if patients with metastatic breast cancer and increased expression of the topoisomerase 1 gene have a high likelihood of obtaining a clinical benefit from treatment with irinotecan. Trial recruitment is two-staged as 19 patients are planned to participate in the first part. If less than 7 patients have clinical benefit the trial stops, if more than 7 patients have clinical benefit a total of 40 patients will be included. DISCUSSION: This ongoing trial is the first to prospectively test copy number of the topoisomerase I gene as a predictive biomarker of response to irinotecan. TRIAL REGISTRATION: EudraCT number 2012-002348-26

MicroRNAs, epigenetics and disease

Epigenetics is defined as the heritable chances that affect gene expression without changing the DNA sequence. Epigenetic regulation of gene expression can be through different mechanisms such as DNA methylation, histone modifications and nucleosome positioning. MicroRNAs are short RNA molecules which do not code for a protein but have a role in post-transcriptional silencing of multiple target genes by binding to their 3′ UTRs (untranslated regions). Both epigenetic mechanisms, such as DNA methylation and histone modifications, and the microRNAs are crucial for normal differentiation, development and maintenance of tissue-specific gene expression. These mechanisms also explain how cells with the same DNA content can differentiate into cells with different functions. Changes in epigenetic processes can lead to changes in gene function, cancer formation and progression, as well as other diseases. In the present chapter we will mainly focus on microRNAs and methylation and their implications in human disease, mainly in cancer.</jats:p

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer

BACKGROUND: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. RESULTS: We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of ‘DNA methylation entropy’ to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples. CONCLUSION: Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1552-y) contains supplementary material, which is available to authorized users

Integrative analysis of miRNA and gene expression reveals regulatory networks in tamoxifen-resistant breast cancer

Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer, however, tamoxifen resistance is frequently observed. To elucidate the underlying molecular mechanisms of tamoxifen resistance, we performed a systematic analysis of miRNA-mediated gene regulation in three clinically-relevant tamoxifen-resistant breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive cell line. Alterations in the expression of 131 miRNAs in tamoxifen-resistant vs. parental cell lines were identified, 22 of which were common to all TamRs using both sequencing and LNA-based quantitative PCR technologies. Although the target genes affected by the altered miRNA in the three TamRs differed, good agreement in terms of affected molecular pathways was observed. Moreover, we found evidence of miRNA-mediated regulation of ESR1, PGR1, FOXM1 and 14-3-3 family genes. Integrating the inferred miRNA-target relationships, we investigated the functional importance of 2 central genes, SNAI2 and FYN, which showed increased expression in TamR cells, while their corresponding regulatory miRNA were downregulated. Using specific chemical inhibitors and siRNA-mediated gene knockdown, we showed that both SNAI2 and FYN significantly affect the growth of TamR cell lines. Finally, we show that a combination of 2 miRNAs (miR-190b and miR-516a-5p) exhibiting altered expression in TamR cell lines were predictive of treatment outcome in a cohort of ER+ breast cancer patients receiving adjuvant tamoxifen mono-therapy. Our results provide new insight into the molecular mechanisms of tamoxifen resistance and may form the basis for future medical intervention for the large number of women with tamoxifen-resistant ER+ breast cancer

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines

Background: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. Methods: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. Results: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. Conclusions: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan

Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan- or oxaliplatin-specific resistance profiles, with non-reciprocal cross-resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance-associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models

Inhibition of microRNA function by antimiR oligonucleotides

MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Indeed, disease-associated miRNAs represent a new class of targets for the development of miRNA-based therapeutic modalities, which may yield patient benefits unobtainable by other therapeutic approaches. The recent explosion in miRNA research has accelerated the development of several computational and experimental approaches for probing miRNA functions in cell culture and in vivo. In this review, we focus on the use of antisense oligonucleotides (antimiRs) in miRNA inhibition for loss-of-function studies. We provide an overview of the currently employed antisense chemistries and their utility in designing antimiR oligonucleotides. Furthermore, we describe the most commonly used in vivo delivery strategies and discuss different approaches for assessment of miRNA inhibition and potential off-target effects. Finally, we summarize recent progress in antimiR mediated pharmacological inhibition of disease-associated miRNAs, which shows great promise in the development of novel miRNA-based therapeutics

Procalcitonin, lipopolysaccharide-binding protein, interleukin-6 and C-reactive protein in community-acquired infections and sepsis: a prospective study

INTRODUCTION: Clinicians are in need of better diagnostic markers in diagnosing infections and sepsis. We studied the ability of procalcitonin, lipopolysaccharide-binding protein, IL-6 and C-reactive protein to identify patients with infection and sepsis. METHODS: Plasma and serum samples were obtained on admission from patients with suspected community-acquired infections and sepsis. Procalcitonin was measured with a time-resolved amplified cryptate emission technology assay. Lipopolysaccharide-binding protein and IL-6 were measured with a chemiluminescent immunometric assay. RESULTS: Of 194 included patients, 106 had either infection without systemic inflammatory response syndrome or sepsis. Infected patients had significantly elevated levels of procalcitonin, lipopolysaccharide-binding protein, C-reactive protein and IL-6 compared with noninfected patients (P < 0.001). In a receiver-operating characteristic curve analysis, C-reactive protein and IL-6 performed best in distinguishing between noninfected and infected patients, with an area under the curve larger than 0.82 (P < 0.05). IL-6, lipopolysaccharide-binding protein and C-reactive protein performed best in distinguishing between systemic inflammatory response syndrome and sepsis, with an area under the curve larger than 0.84 (P < 0.01). Procalcitonin performed best in distinguishing between sepsis and severe sepsis, with an area under the curve of 0.74 (P < 0.01). CONCLUSION: C-reactive protein, IL-6 and lipopolysaccharide-binding protein appear to be superior to procalcitonin as diagnostic markers for infection and sepsis in patients admitted to a Department of Internal Medicine. Procalcitonin appears to be superior as a severity marker