Meeting Report
MicroRNA: Potential for Cancer Detection, Diagnosis, and Prognosis
James V. Tricoli and James W. Jacobson
Diagnostic Biomarkers and Technology Branch, Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, Rockville, Maryland
requirements, technology platforms, data normalization, the effect
Introduction of contaminating cells, and standardization of data reporting.
The Cancer Diagnosis Program of the National Cancer Institute [Cancer Res 2007;67(10):4553–5]
sponsored a workshop entitled ‘‘MicroRNA: Potential for Cancer
Detection, Diagnosis, and Prognosis’’ in Rockville, Maryland on
November 28 and 29, 2006. The purpose of this workshop was to bring miRNA Gene Structure and Function
together leaders in the microRNA (miRNA) field to discuss the The ability to apply miRNAs to the diagnostic and therapeutic
potential for the application of miRNAs to translational research, with arenas stems from our basic scientific knowledge of miRNA gene
the ultimate goal of applying them as markers for cancer diagnosis. regulation and function. The first two speakers, Dr. Michael
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miRNAs are small (22–25 nucleotides in length) noncoding RNAs that T. McManus (University of California San Francisco, San Francisco,
can effectively reduce the translation of target mRNAs by binding to CA) and Dr. Bryan R. Cullen (Duke University, Durham, NC),
their 3¶ untranslated region (UTR). This occurs through the assembly provided an overview of the generation and functional role of
of an RNA-induced silencing complex composed of a variety of miRNAs in the cell. Dr. McManus discussed the role of miRNAs in
proteins including Argonaute. If the homology between the miRNA gene regulation and development. He described how Dicer is
sequence and the target 3¶-UTR is incomplete, then this complex essential to proper miRNA processing and that Dicer mutations in
reduces expression by blocking translation. If, however, the homology mice are lethal at day 7.5 of development. He also pointed out that
is complete, then degradation of the target mRNA can result. To date, mutations in Dicer can result in effects other than the build-up
more than 300 distinct human miRNAs capable of targeting thousand of unprocessed miRNAs, including problems in chromosome
of genes have been identified. segregation. Dr. Cullen discussed viral miRNAs and RNA interfer-
Since their discovery, investigations into the function and role of ence. miRNAs have now been identified in several DNA viruses
miRNAs have revolutionized studies in molecular biology, partic- including every herpesvirus analyzed. Dr. Cullen explained that
ularly in the field of development. A variety of studies have shown transforming herpesviruses express numerous viral miRNAs in
the ability of individual miRNAs to regulate oncogene and tumor latently infected transformed cells including EBV (23 miRNAs) and
suppressor gene expression and others have shown that miRNA KSHV (12 miRNAs). He presented recent data showing that each of
gene loss or mutation can contribute to tumorigenesis. miRNA these miRNAs down-regulates a distinct and unique set of cellular
expression patterns (or signatures) are now known to characterize mRNA when expressed in human B cells. These results suggest that
the developmental origins of tumors more effectively than mRNA viral miRNAs likely play a key role in the ability of some oncogenic
expression signatures and may provide a useful tool for the viruses to transform human cells in vivo.
diagnosis and prognosis of human cancer. Several approaches have
been developed to block the function of miRNAs resulting in the miRNA Expression and Cancer Signatures
inhibition of their oncogenic effects. These accomplishments have The expression of individual miRNAs and miRNA signatures
revealed a potential for miRNA to be used as a clinical tool in have now been linked to the diagnosis and prognosis of a number
cancer diagnosis and as a target for therapy. To address these of human cancers. These include chronic lymphocytic leukemia,
issues, the workshop was divided into four sections featuring a chronic myelogenous leukemia, and prostate, testicular, lung,
total of 14 speakers. Dr. Paul Meltzer (National Cancer Institute, breast, ovarian, pancreatic, and gastric cancers. Dr. Carlo M. Croce
Bethesda, MD) served as chair for the workshop and began by (The Ohio State Comprehensive Cancer Center, Columbus, OH)
discussing the recent effect of miRNA on translational research. discussed miRNA signatures in B-cell lymphomas and provided
Dr. Meltzer highlighted several questions about miRNAs that need data illustrating that many miRNA genes are located at fragile sites
to be addressed, including how we will know when miRNA within the human genome. Two of these, mir-15a and mir-16-1, are
discovery is complete, how to verify miRNA tissue specificity, located at the fragile site 13q14.3 and, along with 11 other miRNAs,
the effect of redundancy between closely related miRNAs, and show an association with disease progression in human CLL.
the influence of polymorphisms in miRNAs and their targets. In Dr. Croce explained that homozygous deletion of mir-15a and mir-
addition, technical issues were brought up, including biospecimen 16-1 can lead to overexpression of the targeted mRNAs resulting in
tumor proliferation and invasion. Dr. Scott Hammond (University
of North Carolina at Chapel Hill, Chapel Hill, NC) discussed
miRNAs as oncogenes and tumor suppressors and described his
Note: The complete list of speakers is available as supplemental data at Cancer investigation of miRNA expression patterns in mammalian
Research Online (http://cancerres.aacrjournals.org/). development and in cancer. He provided evidence that many
Requests for reprints: James V. Tricoli, Diagnostic Biomarkers and Technology
Branch, Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis,
miRNAs are down-regulated in some tumor cell lines due to
National Cancer Institute, 6130 Executive Boulevard, Executive Plaza North, Suite reduced maturation during miRNA biogenesis. A number of miRNA
6035A, Rockville, MD 20852. Phone: 301-496-1591; Fax: 301-402-7819; E-mail: tricolij@ genes are overexpressed in tumor cell lines and primary tumors.
mail.nih.gov.
I2007 American Association for Cancer Research. Seven of these cancer-associated miRNAs are clustered in a single
doi:10.1158/0008-5472.CAN-07-0563 primary transcript termed the miR-17 cluster (OncomiR-1) that is
www.aacrjournals.org 4553 Cancer Res 2007; 67: (10). May 15, 2007
Cancer Research
located in a region amplified in lymphoma and several solid to be redundant, may have distinct functions due to the presence of
malignancies. Ectopic expression of these in a mouse model of cis-acting regulatory motifs. Dr. Jun S. Wei (National Cancer
lymphoma resulted in accelerated disease progression, reduced Institute, Bethesda, MD) discussed the diagnostic and treatment
apoptosis, and more tumor dissemination. This work establishes potential of miRNAs. Dr. Wei explored the role of miRNAs in
miRNAs as oncogenes in human cancers and thus candidates for pediatric malignancies, profiling miRNA expression in a panel of 58
cancer marker development and targets of therapy. Dr. Lin Zhang pediatric tumor xenografts and cell lines using in-house developed
(University of Pennsylvania School of Medicine, Philadelphia, PA) miRNA microarrays. The expression profiles of miRNAs showed
discussed miRNA gene copy number alterations in human distinct signatures for each tumor category. He presented data that
epithelial cancers. Dr. Zhang cited work by Dr. Croce, which showed consistency between microarray and real-time reverse
showed that 52% of miRNA genes reside in genomic regions that transcription-PCR results that confirmed the expression of these
are altered in cancer. Dr. Zhang presented data confirming this cancer-specific miRNAs in corresponding clinical tumor samples
result using an array-based comparative genomic hybridization for the neuroblastoma-specific and rhabdomyosarcoma-specific
study of 227 human tumors. Of the 283 known miRNA genes miRNAs. Dr. Wei described experiments to identify the targets of
analyzed using high-resolution array-based comparative genomic tumor-specific miRNAs by performing a parallel gene expression
hybridization, a large proportion exhibit DNA copy number profiling study on the same set of xenograft samples correlating
alterations in ovarian (37%) and breast (73%) cancers and expression of miRNAs and mRNAs. The resulting data matrix will
melanoma (86%). These findings support the notion that copy facilitate the identification of genes regulated by miRNA that may
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number alterations of miRNA genes are highly prevalent in cancer, be potential targets for miRNA-based cancer therapies. Dr. Thomas
may account partly for the frequent miRNA gene deregulation, and Tuschl (Rockefeller University, New York, NY) discussed miRNA
could be used as diagnostic cancer markers. Dr. Jun Lu (The Broad discovery and regulation using small RNA cloning approaches.
Institute of MIT and Harvard, Cambridge, MA) discussed miRNA Dr. Tuschl described the inhibition of miRNAs using antisense
profiles and the classification of human cancer. Dr. Lu described a RNAs called antagomirs that target specific miRNAs. Antagomirs
bead-based miRNA profiling method that allows good detection are stabilized by a 2¶-O-methyl modification of the ribose sugar
fidelity, linearity, specificity, and reproducibility. He also discussed, moiety that renders them highly resistant to nucleases and provide
using miRNA expression profiling to develop novel cancer a high level of thermodynamic duplex stability. Dr. Tuschl showed
diagnostics, understand cancer mechanisms, and assist in cancer that targeting miR-122 in the mouse liver using an anti-miR-122
therapeutic discovery. In a human cancer cohort, miRNA antagomir resulted in complete degradation of miR-122 that is
expression profiles distinguish the lineage of solid tumors, raising dose dependent. The antagomirs are highly specific and long
the prospect of using miRNAs to classify poorly differentiated lasting, with little or no short-term toxicity. Gene expression
cancer specimens. Dr. Lu presented data on changes in miRNA studies showed that the inhibition of miR-122 resulted in
profiles of MCF-7 cells in response to histone deacetylase inhibitors alterations in the level of numerous genes including those involved
and heat shock protein inhibitors and showed that the miRNA in cholesterol biosynthesis. These studies suggest that targeting
expression patterns are reflective of the mechanism of action of specific miRNAs for the purpose of achieving a therapeutic benefit
these drugs. may be possible using antagomirs. Dr. Thomas Schmittgen (Ohio
State University Comprehensive Cancer Center, Columbus, OH)
discussed PCR-based miRNA expression profiling in pancreatic
Diagnostic and Treatment Potential of miRNAs in cancer. Dr. Schmittgen used a unique real-time PCR method to
Cancer identify a large group of miRNAs that can distinguish pancreatic
Recent evidence from translational studies suggests that miRNA tumors from normal or benign pancreas. He provided evidence that
signatures may be useful in categorizing, detecting, and predicting real-time PCR is a superior method for miRNA expression analysis
the course of an increasing number of human cancers. In addition, due to the sensitivity and specificity of the PCR. The expression
mechanistic studies on the role of miRNAs in oncogenesis and signature correctly classified 28 of 28 pancreatic tumors, 11 of 15
tumor progression have suggested therapeutic strategic targeting adjacent benign tissues, and 6 of 6 normal pancreatic tissues.
of miRNAs and miRNA regulated pathways. Dr. Joshua Mendell Interestingly, the majority of the differentially expressed miRNAs
(Johns Hopkins University, Baltimore, MD) discussed c-Myc– were increased in the pancreatic tumors, suggesting that they may
regulated miRNAs in cellular transformation and tumorigenesis. be good therapeutic targets for antisense oligonucleotides or as
Dr. Mendell presented data showing that c-Myc activates markers to diagnose pancreatic and possibly other forms of cancer.
expression of a group of six miRNAs on human chromosome 13,
known as the miR-17 cluster (OncomiR-1). Chromatin immuno-
precipitation showed that c-Myc binds directly to this locus to Future Directions and Clinical Implications
activate transcription of these miRNAs. Dr. Mendell also showed There are many issues to consider when validating a cancer
that this group of miRNAs is widely overexpressed in human marker or signature for use in the clinic. It is essential that we
cancers and can promote tumorigenesis in animal models. He apply the lessons learned from gene expression and proteomic
presented new data showing that miRNA stability and subcellular profiling to the emerging studies using miRNAs so as not to repeat
localization can be regulated in a sequence-specified manner. Every previous errors in data analysis and signature validation. Dr. Cheryl
studied miRNA, including miR-29a, is predominantly cytoplasmic Willman (University of New Mexico, Albuquerque, NM) discussed
in cycling cells. Fractionation and in situ hybridization experiments the lessons learned from gene expression profiling studies.
show that miR-29b is partially localized to the nucleus and that a Dr. Willman identified a number of key issues that need to be
unique hexanucleotide terminal motif of miR-29b is a transferable addressed to achieve meaningful and reproducible results in
element that specifies nuclear import and rapid decay. Dr. miRNA gene expression array studies. These include a well-defined
Mendell’s findings suggest that related miRNAs, generally believed clinical question, a statistically valid experimental design, selection
Cancer Res 2007; 67: (10). May 15, 2007 4554 www.aacrjournals.org
MicroRNA in Cancer Detection, Diagnosis, and Prognosis
of highly characterized cases appropriate to the question and cross-hybridization of >3% was observed in only 4 of 56 potential
representative of the population, consideration of tumor hetero- cross-hybridization events. This probe design strategy has enabled
geneity, identification of normal controls, a robust platform, and the design of probes for all of the human miRNA sequences in the
robust statistical and computational analysis of diagnostics and Sanger database. The larger dynamic range and the specificity of the
predictors with independent validation. Dr. Willman pointed out assay offer a robust method for miRNA profiling. Dr. David Brown
that the results of any array study are dependent on case inclusion (Asuragen Corporation, Austin, TX) discussed methods and
and statistical design, and may or may not be intrinsically stable or applications for miRNA expression analysis. Dr. Brown described
readily generalized. In addition, gene selection can markedly a miRNA array platform that facilitates the quantitative analysis of
influence presentation of the data and has the potential to more than 15,000 validated and predicted miRNAs. The platform is
introduce bias. Finally, results must be fully validated on being used to identify miRNAs signatures that correlate with patient
independent data sets. Dr. Kevin Dobbin (National Cancer prognosis or response to therapy. This array platform can be used to
Institute, Bethesda, MD) discussed the statistical similarities and evaluate formalin-fixed paraffin-embedded samples from cancer
differences between mRNA and miRNA array studies. Dr. Dobbin patients. He stated that the expression levels of as few as two
pointed out that there are typically far fewer features represented miRNAs can be used to distinguish tumor, chronic pancreatitis, and
on miRNA arrays than on mRNA arrays. This makes array normal pancreatic tissue samples. The presence of miRNAs in
normalization more problematic. The normalization methods clinical samples such as formalin-fixed paraffin-embedded tissues,
commonly used for mRNA arrays, based on median centering or serum, plasma, urine, and saliva, as well as the strong correlation
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lowess normalization, may not be adequate for miRNA arrays. between miRNA expression and disease state and patient prognosis,
There are also drawbacks to alternative exogenous or endogenous suggests that miRNAs may ultimately prove to be valuable cancer
control-based methods used for normalization in miRNA studies. diagnostic analytes.
Methods for determining the number of arrays required for an
mRNA experiment have been developed and, with minor The Future of miRNAs in Cancer Diagnosis and
modifications, can be applied validly to miRNA experiments as Treatment
well. Dr. Dobbin provided an example of how preselecting
‘‘informative’’ genes to use in a cluster analysis produces bias that The usefulness of miRNAs for the detection, diagnosis,
obscures the true structure in the data. The resulting cluster prognosis, and possible treatment of human cancer will depend
dendrogram can display significant structure even when none on carefully designed translational studies. In addition, it will
exists. Finally, more general issues related to designing reproduc- require careful consideration of the best methods for sample
ible and relevant studies (patient selection and potential clinical collection, miRNA isolation, miRNA quantitation, and data
treatment decisions that may be affected) and what constitutes analysis. To facilitate this, we need to gain a better understanding
adequate study validation (internal versus external validation) were of specific miRNA characteristics, such as how targeting of multiple
discussed, including the risk of inadequate cross-validation and mRNAs by a single miRNA affects data interpretation in marker
how this risk can be reduced by using appropriate statistical studies and the effect of miRNA isoforms on diagnostic utility. In
software. Dr. Hui Wang (Agilent Technologies, Santa Clara, CA) the therapeutic arena, there is a requirement to target the correct
discussed direct miRNA profiling from low-input total RNA. miRNA sites without affecting miRNA targets of similar sequence.
Dr. Wang stated that accurate miRNA measurements are challeng- Whereas, as presented by Dr. Tuschl, early mouse studies using
ing due to the large dynamic range of miRNA expression, high antagomirs show promise, actual treatment implementation in
miRNA sequence homology, and the lack of consensus on humans will likely present many unforeseen challenges. The
normalization methods. She described studies of miRNA profiling miRNA workshop provided a timely opportunity to address these
directly from low-input total RNA (100 ng) without size-fraction- and other issues and to form a framework of understanding upon
ation or amplification using a novel and highly efficient RNA which future studies can be conducted.
labeling method and a unique probe design. To optimize the
sequence specificity of the probe-target interactions, all probes on Acknowledgments
the microarray are empirically selected to have matched probe- Received 2/22/2007; accepted 3/7/2007.
target melting temperature (Tm). The Tm-balanced probes are We thank Drs. Rebecca Huppi and Natasha Caplen for their assistance in
organizing the workshop, Dr. Paul Meltzer for serving as chair, Dr. Sheila Taube for her
generally capable of single-nucleotide discrimination. Dr. Wang support of the workshop, and the members of the miRNA Workshop Coordinating
provided an example from the human let-7 family of miRNAs where Committee for their helpful suggestions.
www.aacrjournals.org 4555 Cancer Res 2007; 67: (10). May 15, 2007
