A Signature of Genomic Instability Resulting from Deficient Replication Licensing.

Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea). In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats.

Treatment recommendations to cancer patients in the context of FDA guidance for next generation sequencing.

BACKGROUND: Regulatory approval of next generation sequencing (NGS) by the FDA is advancing the use of genomic-based precision medicine for the therapeutic management of cancer as standard care. Recent FDA guidance for the classification of genomic variants based on clinical evidence to aid clinicians in understanding the actionability of identified variants provided by comprehensive NGS panels has also been set forth. In this retrospective analysis, we interpreted and applied the FDA variant classification guidance to comprehensive NGS testing performed for advanced cancer patients and assessed oncologist agreement with NGS test treatment recommendations. METHODS: NGS comprehensive genomic profiling was performed in a CLIA certified lab (657 completed tests for 646 patients treated at Roswell Park Comprehensive Cancer Center) between June 2016 and June 2017. Physician treatment recommendations made within 120 days post-test were gathered from tested patients' medical records and classified as targeted therapy, precision medicine clinical trial, immunotherapy, hormonal therapy, chemotherapy/radiation, surgery, transplant, or non-therapeutic (hospice, surveillance, or palliative care). Agreement between NGS test report targeted therapy recommendations based on the FDA variant classification and physician targeted therapy treatment recommendations were evaluated. RESULTS: Excluding variants contraindicating targeted therapy (i.e., KRAS or NRAS mutations), at least one variant with FDA level 1 companion diagnostic supporting evidence as the most actionable was identified in 14% of tests, with physicians most frequently recommending targeted therapy (48%) for patients with these results. This stands in contrast to physicians recommending targeted therapy based on test results with FDA level 2 (practice guideline) or FDA level 3 (clinical trial or off label) evidence as the most actionable result (11 and 4%, respectively). CONCLUSIONS: We found an appropriate "dose-response" relationship between the strength of clinical evidence supporting biomarker-directed targeted therapy based on application of FDA guidance for NGS test variant classification, and subsequent treatment recommendations made by treating physicians. In view of recent changes at FDA, it is paramount to define regulatory grounds and medical policy coverage for NGS testing based on this guidance.

Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins.

Minichromosome maintenance (MCM) proteins are loaded onto chromatin during G1-phase and define potential locations of DNA replication initiation. MCM protein deficiency results in genome instability and high rates of cancer in mouse models. Here we develop a method of nascent strand capture and release and show that MCM2 deficiency reduces DNA replication initiation in gene-rich regions of the genome. DNA structural properties are shown to correlate with sequence motifs associated with replication origins and with locations that are preferentially affected by MCM2 deficiency. Reduced nascent strand density correlates with sites of recurrent focal CNVs in tumors arising in MCM2-deficient mice, consistent with a direct relationship between sites of reduced DNA replication initiation and genetic damage. Between 10% and 90% of human tumors, depending on type, carry heterozygous loss or mutation of one or more MCM2-7 genes, which is expected to compromise DNA replication origin licensing and result in elevated rates of genome damage at a subset of gene-rich locations.

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer.

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as "stitchers," to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication-licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.

cnvCurator: an interactive visualization and editing tool for somatic copy number variations.

BACKGROUND: One of the most important somatic aberrations, copy number variations (CNVs) in tumor genomes is believed to have a high probability of harboring oncotargets. Detection of somatic CNVs is an essential part of cancer genome sequencing analysis, but the accuracy is usually limited due to various factors. A post-processing procedure including manual review and refinement of CNV segments is often needed in practice to achieve better accuracy. RESULTS: cnvCurator is a user-friendly tool with functions specifically designed to facilitate the process of interactively visualizing and editing somatic CNV calling results. Different from other general genomics viewers, the index and display of CNV calling results in cnvCurator is segment central. It incorporates multiple CNV-specific information for concurrent, interactive display, as well as a number of relevant features allowing user to examine and curate the CNV calls. CONCLUSIONS: cnvCurator provides important and practical utilities to assist the manual review and edition of results from a chosen somatic CNV caller, such that curated CNV segments will be used for down-stream applications.

SCNVSim: somatic copy number variation and structure variation simulator.

BACKGROUND: Somatically acquired structure variations (SVs) and copy number variations (CNVs) can induce genetic changes that are directly related to tumor genesis. Somatic SV/CNV detection using next-generation sequencing (NGS) data still faces major challenges introduced by tumor sample characteristics, such as ploidy, heterogeneity, and purity. A simulated cancer genome with known SVs and CNVs can serve as a benchmark for evaluating the performance of existing somatic SV/CNV detection tools and developing new methods. RESULTS: SCNVSim is a tool for simulating somatic CNVs and structure variations SVs. Other than multiple types of SV and CNV events, the tool is capable of simulating important features related to tumor samples including aneuploidy, heterogeneity and purity. CONCLUSIONS: SCNVSim generates the genomes of a cancer cell population with detailed information of copy number status, loss of heterozygosity (LOH), and event break points, which is essential for developing and evaluating somatic CNV and SV detection methods in cancer genomics studies.

Nomogram to predict the likelihood of upgrade of atypical ductal hyperplasia diagnosed on a core needle biopsy in mammographically detected lesions.

AIMS: To estimate the likelihood of the upgrade for atypical ductal hyperplasia (ADH) diagnosed on a core needle biopsy of a mammographically detected lesion. METHODS AND RESULTS: A total of 203 consecutive ADH cases diagnosed on core biopsy in mammographically detected lesions and having subsequent surgical excision were reviewed. The pathological features of ADH were assessed with multivariable logistic regression to predict the likelihood of upgrade for these patients. A nomogram was created using statistically significant variables. A corresponding formula was created to calculate the risk of upgrade. This risk was divided further into low, intermediate and high. A total of 57 (28.1%) cases had upgrade. A nomogram was created that included age, menopausal status, hormone therapy status, personal history of breast cancer, number of involved cores, solid growth pattern, size of largest focus and mammographic mass versus calcifications. The nomogram had an area under the receiver operating characteristic curve of 0.775. CONCLUSIONS: We have developed a user-friendly nomogram that uses easily recognized variables to calculate the likelihood of upgrade for ADH. The nomogram could assist the treating surgeon in decision-making, particularly when the patient is at risk for surgical intervention.

FUSIM: a software tool for simulating fusion transcripts.

BACKGROUND: Gene fusions are the result of chromosomal aberrations and encode chimeric RNA (fusion transcripts) that play an important role in cancer genesis. Recent advances in high throughput transcriptome sequencing have given rise to computational methods for new fusion discovery. The ability to simulate fusion transcripts is essential for testing and improving those tools. RESULTS: To facilitate this need, we developed FUSIM (FUsion SIMulator), a software tool for simulating fusion transcripts. The simulation of events known to create fusion genes and their resulting chimeric proteins is supported, including inter-chromosome translocation, trans-splicing, complex chromosomal rearrangements, and transcriptional read through events. CONCLUSIONS: FUSIM provides the ability to assemble a dataset of fusion transcripts useful for testing and benchmarking applications in fusion gene discovery.

OSAT: a tool for sample-to-batch allocations in genomics experiments.

BACKGROUND: Batch effect is one type of variability that is not of primary interest but ubiquitous in sizable genomic experiments. To minimize the impact of batch effects, an ideal experiment design should ensure the even distribution of biological groups and confounding factors across batches. However, due to the practical complications, the availability of the final collection of samples in genomics study might be unbalanced and incomplete, which, without appropriate attention in sample-to-batch allocation, could lead to drastic batch effects. Therefore, it is necessary to develop effective and handy tool to assign collected samples across batches in an appropriate way in order to minimize the impact of batch effects. RESULTS: We describe OSAT (Optimal Sample Assignment Tool), a bioconductor package designed for automated sample-to-batch allocations in genomics experiments. CONCLUSIONS: OSAT is developed to facilitate the allocation of collected samples to different batches in genomics study. Through optimizing the even distribution of samples in groups of biological interest into different batches, it can reduce the confounding or correlation between batches and the biological variables of interest. It can also optimize the homogeneous distribution of confounding factors across batches. It can handle challenging instances where incomplete and unbalanced sample collections are involved as well as ideally balanced designs.

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development.

BACKGROUND: Changes in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease. RESULTS: In this study Methylated DNA ImmunoPrecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array). CONCLUSIONS: Our data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

Bioinformatic identification of novel putative photoreceptor specific cis-elements.

BACKGROUND: Cell specific gene expression is largely regulated by different combinations of transcription factors that bind cis-elements in the upstream promoter sequence. However, experimental detection of cis-elements is difficult, expensive, and time-consuming. This provides a motivation for developing bioinformatic methods to identify cis-elements that could prioritize future experimental studies. Here, we use motif discovery algorithms to predict transcription factor binding sites involved in regulating the differences between murine rod and cone photoreceptor populations. RESULTS: To identify highly conserved motifs enriched in promoters that drive expression in either rod or cone photoreceptors, we assembled a set of murine rod-specific, cone-specific, and non-photoreceptor background promoter sequences. These sets were used as input to a newly devised motif discovery algorithm called Iterative Alignment/Modular Motif Selection (IAMMS). Using IAMMS, we predicted 34 motifs that may contribute to rod-specific (19 motifs) or cone-specific (15 motifs) expression patterns. Of these, 16 rod- and 12 cone-specific motifs were found in clusters near the transcription start site. New findings include the observation that cone promoters tend to contain TATA boxes, while rod promoters tend to be TATA-less (exempting Rho and Cnga1). Additionally, we identify putative sites for IL-6 effectors (in rods) and RXR family members (in cones) that can explain experimental data showing changes to cell-fate by activating these signaling pathways during rod/cone development. Two of the predicted motifs (NRE and ROP2) have been confirmed experimentally to be involved in cell-specific expression patterns. We provide a full database of predictions as additional data that may contain further valuable information. IAMMS predictions are compared with existing motif discovery algorithms, DME and BioProspector. We find that over 60% of IAMMS predictions are confirmed by at least one other motif discovery algorithm. CONCLUSION: We predict novel, putative cis-elements enriched in the promoter of rod-specific or cone-specific genes. These are candidate binding sites for transcription factors involved in maintaining functional differences between rod and cone photoreceptor populations.

Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium.

BACKGROUND: Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been used successfully for optical mapping in cardiac cells and tissues. However, their utility for probing electrical activity deep inside the myocardial wall and in blood-perfused myocardium has been limited because of light scattering and high absorption by endogenous chromophores and hemoglobin at blue-green excitation wavelengths. OBJECTIVE: The purpose of this study was to characterize two new styryl dyes--di-4-ANBDQPQ (JPW-6003) and di-4-ANBDQBS (JPW-6033)--optimized for blood-perfused tissue and intramural optical mapping. METHODS: Voltage-dependent spectra were recorded in a model lipid bilayer. Optical mapping experiments were conducted in four species (mouse, rat, guinea pig, and pig). Hearts were Langendorff perfused using Tyrode's solution and blood (pig). Dyes were loaded via bolus injection into perfusate. Transillumination experiments were conducted in isolated coronary-perfused pig right ventricular wall preparations. RESULTS: The optimal excitation wavelength in cardiac tissues (650 nm) was >70 nm beyond the absorption maximum of hemoglobin. Voltage sensitivity of both dyes was approximately 10% to 20%. Signal decay half-life due to dye internalization was 80 to 210 minutes, which is 5 to 7 times slower than for di-4-ANEPPS. In transillumination mode, DeltaF/F was as high as 20%. In blood-perfused tissues, DeltaF/F reached 5.5% (1.8 times higher than for di-4-ANEPPS). CONCLUSION: We have synthesized and characterized two new near-infrared dyes with excitation/emission wavelengths shifted >100 nm to the red. They provide both high voltage sensitivity and 5 to 7 times slower internalization rate compared to conventional dyes. The dyes are optimized for deeper tissue probing and optical mapping of blood-perfused tissue, but they also can be used for conventional applications.

Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures.

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

Robust detection of immune transcripts in FFPE samples using targeted RNA sequencing.

Current criteria for identifying cancer patients suitable for immunotherapy with immune checkpoint blockers (ICBs) are subjective and prone to misinterpretation, as they mainly rely on the visual assessment of CD274 (best known as PD-L1) expression levels by immunohistochemistry (IHC). To address this issue, we developed a RNA sequencing (RNAseq)-based approach that specifically measures the abundance of immune transcripts in formalin-fixed paraffin embedded (FFPE) specimens. Besides exhibiting superior sensitivity as compared to whole transcriptome RNAseq, our assay requires little starting material, implying that it is compatible with RNA degradation normally caused by formalin. Here, we demonstrate that a targeted RNAseq panel reliably profiles mRNA expression levels in FFPE samples from a cohort of ovarian carcinoma patients. The expression profile of immune transcripts as measured by targeted RNAseq in FFPE versus freshly frozen (FF) samples from the same tumor was highly concordant, in spite of the RNA quality issues associated with formalin fixation. Moreover, the results of targeted RNAseq on FFPE specimens exhibited a robust correlation with mRNA expression levels as measured on the same samples by quantitative RT-PCR, as well as with protein abundance as determined by IHC. These findings demonstrate that RNAseq profiling on archival FFPE tissues can be used reliably in studies assessing the efficacy of cancer immunotherapy.

Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing.

BACKGROUND: The rapid adoption of next-generation sequencing provides an efficient system for detecting somatic alterations in neoplasms. The detection of such alterations requires a matched non-neoplastic sample for adequate filtering of non-somatic events such as germline polymorphisms. Non-neoplastic tissue adjacent to the excised neoplasm is often used for this purpose as it is simultaneously collected and generally contains the same tissue type as the neoplasm. Following NGS analysis, we and others have frequently observed low-level somatic mutations in these non-neoplastic tissues, which may impose additional challenges to somatic mutation detection as it complicates germline variant filtering. METHODS: We hypothesized that the low-level somatic mutation observed in non-neoplastic tissues may be entirely or partially caused by inadvertent contamination by neoplastic cells during the surgical pathology gross assessment or tissue procurement process. To test this hypothesis, we applied a systematic protocol designed to collect multiple grossly non-neoplastic tissues using different methods surrounding each single neoplasm. The procedure was applied in two breast cancer lumpectomy specimens. In each case, all samples were first sequenced by whole-exome sequencing to identify somatic mutations in the neoplasm and determine their presence in the adjacent non-neoplastic tissues. We then generated ultra-deep coverage using targeted sequencing to assess the levels of contamination in non-neoplastic tissue samples collected under different conditions. RESULTS: Contamination levels in non-neoplastic tissues ranged up to 3.5 and 20.9 % respectively in the two cases tested, with consistent pattern correlated with the manner of grossing and procurement. By carefully controlling the conditions of various steps during this process, we were able to eliminate any detectable contamination in both patients. CONCLUSION: The results demonstrated that the process of tissue procurement contributes to the level of contamination in non-neoplastic tissue, and contamination can be reduced to below detectable levels by using a carefully designed collection method. A standard protocol dedicated for acquiring adjacent non-neoplastic tissue that minimizes neoplasm contamination should be implemented for all somatic mutation detection studies.

Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR).

Nascent strand capture and release (NSCR) is a method for isolation of short nascent strands to identify origins of DNA replication. The protocol provided involves isolation of total DNA, denaturation, size fractionation on a sucrose gradient, 5'-biotinylation of the appropriate size nucleic acids, binding to a streptavidin coated magnetic beads, intensive washing, and specific release of only the RNA-containing chimeric nascent strand DNA using ribonuclease I (RNase I). The method has been applied to mammalian cells derived from proliferative tissues and cell culture but could be used for any system where DNA replication is primed by a small RNA resulting in chimeric RNA-DNA molecules.

Structural variation discovery in the cancer genome using next generation sequencing: computational solutions and perspectives.

Somatic Structural Variations (SVs) are a complex collection of chromosomal mutations that could directly contribute to carcinogenesis. Next Generation Sequencing (NGS) technology has emerged as the primary means of interrogating the SVs of the cancer genome in recent investigations. Sophisticated computational methods are required to accurately identify the SV events and delineate their breakpoints from the massive amounts of reads generated by a NGS experiment. In this review, we provide an overview of current analytic tools used for SV detection in NGS-based cancer studies. We summarize the features of common SV groups and the primary types of NGS signatures that can be used in SV detection methods. We discuss the principles and key similarities and differences of existing computational programs and comment on unresolved issues related to this research field. The aim of this article is to provide a practical guide of relevant concepts, computational methods, software tools and important factors for analyzing and interpreting NGS data for the detection of SVs in the cancer genome.

Whole-genome sequencing of a malignant granular cell tumor with metabolic response to pazopanib.

Granular cell tumors are an uncommon soft tissue neoplasm. Malignant granular cell tumors comprise <2% of all granular cell tumors, are associated with aggressive behavior and poor clinical outcome, and are poorly understood in terms of tumor etiology and systematic treatment. Because of its rarity, the genetic basis of malignant granular cell tumor remains unknown. We performed whole-genome sequencing of one malignant granular cell tumor with metabolic response to pazopanib. This tumor exhibited a very low mutation rate and an overall stable genome with local complex rearrangements. The mutation signature was dominated by C>T transitions, particularly when immediately preceded by a 5' G. A loss-of-function mutation was detected in a newly recognized tumor suppressor candidate, BRD7. No mutations were found in known targets of pazopanib. However, we identified a receptor tyrosine kinase pathway mutation in GFRA2 that warrants further evaluation. To the best of our knowledge, this is only the second reported case of a malignant granular cell tumor exhibiting a response to pazopanib, and the first whole-genome sequencing of this uncommon tumor type. The findings provide insight into the genetic basis of malignant granular cell tumors and identify potential targets for further investigation.

Effects of preanalytic variables on circulating microRNAs in whole blood.

Research in the last decade suggests the clinical potential of circulating microRNAs in whole blood as biomarkers for cancer detection. However, before applying the identified circulating microRNAs clinically, biospecimen-focused research has to be performed to identify possible preanalytic variables that may significantly affect the levels of circulating microRNAs. In this study, using a unique resource of the Data Bank and BioRepository (DBBR) at Roswell Park Cancer Institute, we conducted a two-step analysis to identify internal control circulating microRNAs in whole blood and then to study how selected major preanalytic variables (namely, processing delay, storage condition, storage time, and freeze/thaw cycles) might affect the detection of circulating microRNAs. In the discovery phase of the first step, we identified three microRNAs, including miR346, miR134, and miR934, whose levels exhibited the smallest variation between the case-control groups, as well as within each group interindividually. In the further validation analysis, the consistency was validated for miR346 and miR134 but not for miR934. At the second step, using miR346 and miR134 as internal controls, we observed that as the numbers of freeze/thaw cycles increased, levels of both miR346 and miR134 were significantly decreased (Ptrend < 0.0001); varying other processing and storage conditions did not affect miRNA levels. In the paralleled analysis in plasma samples, levels of miR16 were significantly decreased by increasing processing delay and increasing numbers of freeze/thaw cycles but not affected by storage condition and duration. The results from this study highlight the necessity of biospecimen-focused research on circulating microRNAs before clinical utilization. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology."

Circulating miR-148b and miR-133a as biomarkers for breast cancer detection.

Circulating microRNAs have drawn a great deal of attention as promising novel biomarkers for breast cancer. However, to date, the results are mixed. Here, we performed a three-stage microRNA analysis using plasma samples from breast cancer patients and healthy controls, with efforts taken to address several pitfalls in detection techniques and study design observed in previous studies. In the discovery phase with 122 Caucasian study subjects, we identified 43 microRNAs differentially expressed between breast cancer cases and healthy controls. When those microRNAs were compared with published data from other studies, we identified three microRNAs, including miR-148b, miR-133a and miR-409-3p, whose plasma levels were significantly higher in breast cancer cases than healthy controls and were also significant in previous independent studies. In the validation phase with 50 breast cancer cases and 50 healthy controls, we validated the associations with breast cancer detection for miR-148b and miR-133a (P = 1.5×10-6 and 1.3×10-10, respectively). In the in-vitro study phase, we found that both miR-148b and miR-133a were secreted from breast cancer cell lines, showing their secretory potential and possible tumor origin. Thus, our data suggest that both miR-148b and miR-133a have potential use as biomarkers for breast cancer detection.