METHODS AND COMPOSITIONS DIAGNOSING LUNG CANCER, DETERMINING PROGNOSIS, AND IMPROVING PATIENT SURVIVAL

TECHNICAL FIELD This document pertains to methods for diagnosing disease (such as lung cancer), determining prognosis, and improving patient survival, based on levels of microRNAs (miRNAs).

BACKGROUND Lung cancer is the leading cause of cancer death world- wide. Patients with similar lung cancer may experience different clinical outcomes, and it is difficult to predict patient's prognosis.

Although understanding of the molecular biology of lung cancer has increased in recent years, more detailed knowledge about the molecular mechanisms underlying progression remain elusive. There is a need for accurate prognostic indicators to identify high-risk patients, so that optimal treatments can be designed.

The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

SUMMARY

This document is based in part on the discovery that particular miRNAs have increased or decreased levels in cancer tissue samples. Thus, provided herein are methods and compositions for diagnosing cancer and determining prognosis of cancer patients (e.g., lung cancer), based on levels of miRNAs arid/or on the status of the corresponding miRNA genes. Also provided herein are methods for using probes that detect miRNA or the gene status of corresponding miRNA genes for diagnosing and determining prognosis of cancer, such as lung cancer.

In one aspect, this document features a method for identifying an individual as having lung cancer, comprising: a) determining the level of at least one miRNA in a lung tissue sample from the individual, wherein the tissue is suspected of being cancerous, b) comparing the level of the at least one miRNA with a reference level, and c) classifying the individual as having or being likely to have lung cancer if the sample exhibits a characteristic change in the level of the at least one miRNA. The lung cancer can be small cell lung cancer, lung adenocarcinoma, or lung squamous cell cancer. The at least one miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, or a corresponding homologue. The method can include comparing the levels of at least three miRNAs in the individual with reference levels, wherein the at least three miRNAs are selected from the group consisting of hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, and their corresponding homologues. The method can include determining the levels of hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, and hsa-miR-140-3p, and classifying the individual as having or being likely to have lung cancer if the sample exhibits a substantial increase in the levels of hsa-miR-210, hsa-miR-30a, and hsa-miR-182 in combination with a substantial decrease in the levels of hsa-miR-486-5p and hsa-miR-140-3p. The miRNA level can be determined by a microarray analysis (e.g., based on the hybridization signal of the miRNA on the microarray, or based on the ratio of the hybridization signal to the miRNA on the microarray to that of a reference sample). The miRNA level can be determined by Northern blot analysis, in situ hybridization, or quantitative reverse transcriptase polymerase chain reaction. The miRNA level can be determined based on the miRNA level from any of a lymph node sample, blood, serum, or a lung tissue swab.

In another aspect, this document features a method for identifying an individual as having lung cancer, comprising analyzing the gene status of at least one miRNA gene in a lung tissue sample from the individual, wherein the lung tissue is suspected of being cancerous, and classifying the individual as having or being likely to have lung cancer if the sample exhibits a characteristic change in the gene status relative to the corresponding miRNA gene in a control sample. The change in gene status can be determined based on a deletion or amplification of the miRNA gene, or based on a change in the copy number of the miRNA gene.

In another aspect, this document features a system for detecting lung cancer, comprising at least one pair of primers or a plurality of probes, wherein each pair of primers or each probe is capable of detecting a different miRNA in a sample, and wherein at least about 50 percent of the primer pairs and probes are capable of detecting a miRNA selected from the group consisting of hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140- 3p, and their corresponding homologues. In another aspect, this document features the use of a system for detecting lung cancer, wherein the system comprises at least one pair of primers or a plurality of probes, wherein each pair of primers or each of the probes is capable of detecting a different miRNA, or the gene status of the corresponding miRNA, in the sample, wherein at least about 50 percent of the primer pairs or probes are capable of detecting a miRNA selected from the group consisting of hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3ρ, and their corresponding homologues, and wherein a characteristic change in the level of at least one of the miRNAs or corresponding homologues is indicative of lung cancer. In still another aspect, this document features the use of at least one pair of primers or a plurality of probes for the manufacture of a system for detecting lung cancer, wherein each pair of primers or each of the probes is capable of detecting a different miRNA in the sample, and wherein at least about 50% of the primer pairs or probes are capable of detecting a miRNA selected from the group consisting of hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486- 5p, hsa-miR- 140-3p, and their corresponding homologues.

This document also features a method of classifying a lung cancer patient, comprising determining the level of at least one miRNA or the gene status of the corresponding miRNA gene in a lung cancer tissue sample from the individual, and classifying the lung cancer patient based on the level of the miRNA or the gene status of the corresponding miRNA gene. The method can include determining the level of a miRNA listed in Table 1 or Table 2 or a corresponding homologue, and determining the level of differentiation of lung cancer in the individual based on the level of the miRNA.

In another aspect, this document features a method for determining a prognosis for survival for an individual having lung squamous cell cancer, comprising: a) determining the level of at least one miRNA in a lung cancer tissue sample from the individual, b) comparing the level of the miRNA in the sample to a threshold level, and c) directly or inversely correlating the level of the miRNA as compared to a threshold level with the prognosis for survival of the individual. The at least one miRNA can be hsa-miR-31 or a corresponding homologue. In another aspect, this document features a method for determining a prognosis for survival for an individual having lung squamous cell cancer, comprising analyzing the gene status of at least one miRNA gene in a lung squamous cell cancer sample from the individual, and classifying the individual as having a high or low chance of survival based on whether there is a change in status of the at least one miRNA gene in the lung cancer sample as compared to the gene status in a control sample. The at least one miRNA gene can correspond to the gene encoding for hsa-miR-31 or a corresponding homologue. The method can further comprise determining a proper course of treatment for the individual. The miRNA level can be determined by Northern blot analysis, in situ hybridization, or quantitative real time polymerase chain reaction. The miRNA level can be determined based on the miRNA level in a lymph node sample, serum, blood, or a lung tissue.

In yet another aspect, this document features the use of one or more primer pairs or probes for determining prognosis for survival of an individual having lung squamous cell cancer, wherein the one or more primer pairs or probes are capable of being used to detect a miRNA in the sample, and wherein the level of the miRNA as compared to a threshold level correlates or reversely correlates with the survival of the individual. The at least one miRNA can be hsa-miR-31 or a corresponding homologue.

In another aspect, this document features the use of one or more primer pairs or probes for the manufacture of an agent or system for determining a prognosis of survival of an individual having lung squamous cell cancer, wherein the one or more primer pairs or probes are capable of being used to detect a miRNA in the sample, and wherein the level of the miRNA as compared to the threshold level correlates or reversely correlates with the survival of the individual. The at least one miRNA can be hsa-miR-31 or a corresponding homologue. In another aspect, this document features a method for improving survival of an individual having lung cancer, comprising administering to the individual an effective amount of an agent that decreases the level of a miRNA, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of the individual. The miRNA can be hsa-miR-31 or a corresponding homologue. The miRNA can be hsa-miR-210, hsa-miR-3 Oa, hsa-miR-182, or a corresponding homologue. The agent can be an antisense RNA or a siRNA. The method can include administering to the individual an effective amount of one or more agents that decrease the levels of at least two miRNAs, wherein the at least two miRNAs are selected from the group consisting of hsa-miR-31, hsa-miR-210, hsa-miR-3 Oa, hsa-miR-182, and their corresponding homologues. The method can include administering to the individual an effective amount of one or more agents that decrease the levels of at least three miRNAs, wherein the at least three miRNAs are selected from the group consisting of hsa-miR-31, hsa- miR-210, hsa-miR-3 Oa, hsa-miR-182, and their corresponding homologues. The method can include administering to the individual an effective amount of one or more agents that decrease the levels of hsa-miR-31, hsa-miR-210, hsa-miR-3 Oa, and hsa-miR-182, or corresponding homologues of hsa-miR-31 , hsa-miR-210, hsa-miR-3 Oa ₅ and hsa-miR- 182.

This document also features the use of an agent that decreases the level of a miRNA for the manufacture of a medicament for improving survival of an individual having lung cancer, wherein the level of the miRNA as compared to the threshold level inversely correlates with the survival of the individual. The at least one miRNA can be hsa-miR-31 or a corresponding homologue. The at least one miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, or a corresponding homologue. The agent can be an antisense RNA or a siRNA.

In another aspect, this document features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent that decreases the level of a miRNA, wherein at least one miRNA is hsa-miR-31 or a corresponding homologue. This document also features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent that decreases the level of a miRNA, wherein at least one miRNA is hsa-miR-210, hsa- miR-30a, hsa-miR-182, or a corresponding homologue. The agent can be an antisense RNA or a siRNA.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figures IA through IF are a series of graphs showing that a classifier can be used to distinguish malignant lung lesions from normal tissues. Principal Component Analysis (PCA) and Support Vector Machine (SVM) analysis were used to classify cancerous tissue and adjacent normal tissue. Dots represent cancerous tissue, and crosses represent adjacent normal tissue from lung cancer patients. For all pathological subtypes of lung cancer, 98.2% (127/132) were correctly classified in the training cohort (Fig. IA) and 92% (92/100) were correctly classified in the test cohort (Fig. IB), including 60 squamous cell cancer (SCC), 43 adenocarcinoma and 13 paired sets of small cell lung cancer (SCLC) tissues. For SCC, 93.3% (56/60) were correctly classified in the training cohort (Fig. 1C) and 96.7% (58/60) were correctly classified in the test cohort (Fig. ID), including 60 paired sets of SCC tissue. For adenocarcinoma, 97.8% (45/46) were correctly classified in the training cohort (Fig. IE) and 90% (36/40) were correctly classified in the test cohort (Fig. IF), including 43 paired sets of SCC tissues.

Figure 2 is a depiction of the results of unsupervised hierarchical clustering of miRNA expression in SCC, adenocarcinoma, and SCLC. Differentially expressed miRNAs in each histological subtype used for unsupervised hierarchical clustering were selected by Significance Analysis of Microarrays (S AM), based on the miRNA ratio of cancerous tissues versus corresponding adjacent normal tissues (left panel), and the miRNA signal of cancerous tissues (right panel). The 60 SCC, 43 adenocarcinoma, and 13 SCLC samples were analyzed using unsupervised hierarchical clustering with the differentially expressed miRNAs. The three pathological subtypes of lung cancers in this study could not be clearly divided into three groups by their miRNA expression profiling, and it is notable the SCLC samples tended to cluster into one group.

Figures 3 A and 3B are a pair of Kaplan-Meier survival curves for SCC patients. The survival curves are shown for patient samples from a training cohort (60 SCC cases; Fig. 3A) and test cohort (20 SCC cases; Fig. 3B). High levels of hsa-miR-31 expression correlated with a lower survival rate, while low hsa-miR-31 expression correlated with a high survival rate. The p value was 0.007 in the training cohort and 0.001 in the independent test cohort.

DETAILED DESCRIPTION

This document is based, in part, on studies of genome- wide miRNA expression profiling using a paired set of frozen archival normal or cancerous lung tissue samples. Specifically, using DNA oligonucleotide microarrays, the expression of miRNAs in cancer samples were compared with miRNA expression in corresponding non-cancerous samples. Five miRNAs were identified as being either over expressed or under expressed in the cancer samples as compared to the corresponding non-cancerous samples. Certain miRNAs also were identified as having altered levels in different disease states. In addition, the levels of one miRNA were found to correlate with overall and disease-free survival rates of lung cancer patients. Accordingly, this document provides methods and compositions for assessing diagnosis and prognosis of disease (e.g., cancer, such as lung cancer), based on the levels of miRNAs or on the status of the corresponding miRNA genes. In one aspect, there are provided methods and compositions for identifying an individual as having a disease (e.g., lung cancer), based on the levels of certain miRNAs. In another aspect, there are provided methods and compositions for classifying cancer patients (e.g., lung cancer patients) by pathological forms, levels of differentiation, and tumor stages, based on the levels of certain miRNAs. In another aspect, there are provided methods and compositions for determining a prognosis for survival of an individual having cancer (e.g., lung cancer), based on the levels of particular miRNAs.

Methods for improving survival of lung cancer patients and pharmaceutical compositions for use in such methods also are also provided, as are systems and kits that can be useful in the methods described herein.

As used herein, "a," "an," and "the" can mean singular or plural (i.e., can mean one or more) unless indicated otherwise.

An "individual" as used herein refers to a vertebrate (e.g., a mammal such as a human). Mammals include, without limitation, humans, non-human primates, cows, sheep, pigs, horses, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, mice, and rats. In some embodiments, an individual can be a human. In some embodiments, an individual can be an animal model for the study of a disease such as lung cancer. It is to be understood that, when the individual is not human, the miRNAs can be corresponding homologs or orthologs of the human miRNAs identified herein.

An individual can be male or female. An individual may or may not show any pathological phenotypes of lung cancer. In some embodiments, an individual can have a family history of lung cancer.

The term "lung tissue sample" refers to a tissue sample from a lung. A tissue sample can be, for example, a fresh sample, a frozen sample, or a preserved sample, (e.g., a formalin preserved sample or a paraffin embedded sample). As described below, and depending on the particular method, a tissue can be used whole, or can be subjected to one or more methods known in the art to dissociate the sample into small pieces, cell aggregates, or individual cells.

Lung cancer includes, but is not limited to, lung SCC, small cell lung cancer (SCLC), and lung adenocarcinoma.

miRNAs MicroRNAs (miRNAs) are small, noncoding, single-stranded RNAs that typically are about 22 (e.g., 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. Through partial or complete sequence homology, miRNAs can interact with the 3' untranslated region (3'-UTR) of target mRNA molecules (Bartel, Cell 2004, 116: 281-297). The interaction between miRNAs and their target mRNAs can block translation of the mRNAs into protein. In some instances, when the target and the miRNA match exactly, degradation of the mRNA also can occur.

Information about miRNAs generally can be found online at http://miRNA.sanger.ac.uk/. See, also, Griffiths- Jones et al., Nucleic Acids Research, 2006, Vol. 34, Database issue. miRNAs have been implicated in a broad range of cellular processes, such as cell differentiation, cell growth, and cell death (Cheng et al, Nucleic Acids Res 2005, 33:1290-1297; John et al, PLoS Biol. 2004, 2: e363). There may be as many as 1,000 miRNAs in the human genome (Zamore and Haley, Science 2005, 309:1519-1524), and as many as one- third of human mRNAs are predicted to be potential miRNA targets (Lewis et al, Cell 2005, 115:787-798). The discovery of RNA editing of human miRNAs suggested an even larger repertoire of regulatory targets (Blow et al, Genome Biology 2006, 7:R27).

This document discloses miRNAs having levels that correlate (e.g., directly or inversely) with lung cancer. Examples of such miRNAs are listed in Table 1 , which also provides the name, sequence, and chromosomal location of the miRNAs, as well as an indication of whether each is up regulated or down regulated in lung cancer. Methods of diagnosing diseases such as lung cancer can be based, for example, on the level or gene status of any of the miRNAs listed in Table 1. Systems described herein can be used to determine the levels of one of more miRNAs listed in Table 1, and/or for diagnosing lung cancer based on the level of one or more miRNAs listed in Table 1. Although acceptable levels of sensitivity and specificity with a single miRNA can be achieved for practice of the methods described herein, the effectiveness (e.g., sensitivity and/or specificity) of the methods described herein can be enhanced when at least two miRNAs are utilized. For example, in some embodiments, at least two, three, four, or five of the miRNAs listed in Table 1 can be utilized in a method for identifying an individual as having lung cancer. In some embodiments, the levels or gene status of at least two (e.g., at least two, three, four, or five) of the miRNAs set forth in SEQ ID NOS: 1-5 can be determined. For example, the levels or gene status of the miRNAs set forth in SEQ ID NOS: 1-3 can be determined, or the levels or gene status of at least two (e.g., two or three) miRNAs set forth in SEQ ID NOS :4 and 5 can be determined. In some embodiments, the levels or gene status of at least one of the miRNAs selected from the miRNAs of SEQ ID NOS : 1 -3 and at least one of the miRNAs selected from the miRNAs of SEQ ID NOS :4 and 5 can be determined. In some cases, the levels or gene status of at least two (e.g., two or three) miRNAs of SEQ ID NOS:l-3 and the levels of SEQ ID NOS :4 and 5 can be determined. In some embodiments, the levels or gene status of all miRNAs listed in Table 1 can be determined.

In some embodiments, the levels of one or more corresponding homologues of the miRNA described herein can be determined. A "corresponding homologue" of a miRNA as described herein refers to a miRNA from a non-human vertebrate that corresponds to a human miRNA. Corresponding homologues typically have at least about 50% sequence identity (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity to the corresponding miRNA described herein. For example, the corresponding homologue of a miRNA of SEQ ID NO:1 can have at least about 50% sequence identity (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity) to SEQ ID NO:1.

When a miRNA sequence is stated to have at least about, for example, 95% identity to a reference sequence (e.g., SEQ ID NO:1), it is intended that the miRNA sequence is identical to the reference sequence except that the miRNA sequence may include up to five point alterations per each 100 nucleotides of the reference sequence. These up to five point alterations may be deletions, substitutions, additions, and may occur anywhere in the sequence, interspersed either individually among nucleotides in the reference sequence or in one or more continuous groups within the reference sequence.

The percent sequence identity between a particular nucleic acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained on the World Wide Web at the United States government's National Center for Biotechnology Information web site (ncbi.nlm.nih.gov), for example. Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seql .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO: 1), or by an articulated length (e.g., 20 consecutive nucleotides from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 20 matches when aligned with the sequence set forth in SEQ ID NO:1 is 90.9 percent identical to the sequence set forth in SEQ ID NO: 1 (i.e., 20÷22* 100=90.9). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11 , 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It is also noted that the length value will always be an integer.

Methods for evaluating miRNA levels

The methods disclosed herein can be based on levels of one or more miRNAs. As used herein, the term "level" refers to the amount or rate of accumulation of a miRNA molecule or its precursor. The term can be used to refer to the absolute amount of a miRNA in a sample (as represented by the intensity of a hybridization signal, for example), or the ratio of the amount of the miRNA to that of a control (as represented by the ratio of the hybridization signal of the sample to that of a control, for example). A control can be a different miRNA from the same sample whose level does not alter in a lung cancer tissue sample, or can be the same miRNA from a different sample (e.g., a noncancerous tissue sample from the same individual, or a tissue sample from another individual not having lung cancer).

A "precursor" of a miRNA molecule, or "miRNA precursor," refers to the unprocessed miRNA gene transcript, and typically comprises an RNA transcript of about 70 nucleotides in length. miRNA precursors typically are processed by digestion with an RNAase (e.g., Dicer, Argonaut, or RNAase III) into an active miRNA molecule, which typically is about 19-25 (e.g., 19, 20, 21, 22, 23, 24, or 25) nucleotides in length.

A "level of miRNA in a lung tissue sample" refers to the miRNA level in the tissue sample. While in most cases the level of a miRNA in a lung tissue sample can be determined based directly on measuring the miRNA level in a lung tissue sample, it is contemplated that the miRNA level in a lung tissue sample also can be reflected by (and thus based on) the level of miRNA in a lymph node sample (e.g., the proximal lymph nodes or lymph fluid), serum, blood, or other proximal biological fluid materials such as sputum. In some embodiments, a miRNA level can be determined based on the level of the miRNA in a lymph node sample (e.g., a lymph node section or needle aspirate), based on the level of the miRNA in blood or serum, or based on the level of the miRNA in a lung tissue swab. In some embodiments, a miRNA level can be determined based on the level of the miRNA from a sample obtained by endoscopic ultrasound-guided sampling procedures (for example by RT-PCR analysis). Endoscopic ultrasound-guided fine-needle aspiration (FNA) is a minimally invasive technique for the non-surgical sampling of mediastinal lymph nodes, which can allow for more detailed molecular marker analysis. Determination of miRNA levels in samples other than lung cancer tissues can be used along or in conjunction with each other. For example, the level of a miRNA can first be determined in a serum sample, and a follow-up analysis of the miRNA in regional lymph nodes can be conducted. Such multi-step analysis may provide additional information and increase confidence in the diagnosis. miRNA levels can be determined in various stages. For example, a miRNA level can be determined immediately prior to surgery, during surgery, after surgery, prior to tumor treatment, during tumor treatment, and/or after tumor treatment. In some embodiments, a miRNA level can be determined based on the level of the miRNA in a tissue sample (e.g., a lung tissue sample) collected by swabbing.

Methods of determining levels of miRNAs include those known in the art. For example, miRNA levels can be determined by Northern blotting, in situ hybridization, RT-PCR, and/or microarrays analysis. See, e.g., Einat, Methods MoI. Biol. 2006, 342:139-157; and Thompson et al, Genes Dev. 2006, 20:2202-2207.

According to an exemplary method, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acid can be precipitated, and DNA can be removed by treatment with DNase and precipitation. RNA molecules then can be separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters by, e.g., Northern blotting techniques. The RNA then can be immobilized on the filters by heating. Detection and quantification of specific RNA can be accomplished using appropriately labeled DNA or RNA probes that are complementary to the RNA in question. Autoradiographic detection of probe hybridization to miRNAs can be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic film exposed to the hybridized filters can provide an accurate measurement of RNA transcript levels. Alternatively, RNA transcript levels can be quantified by computerized imaging of the hybridization blot, for example with a phosphorimager.

In addition to Northern and other RNA blotting hybridization techniques, levels of RNA transcripts can be measured by in situ hybridization. This technique involves depositing whole cells or tissues onto a microscopic cover slip and probing the nucleic acid content of the cell or tissue with a solution containing radioactive or otherwise labeled probes (e.g., cRNA probes).

The levels of miRNAs also can be determined by reverse transcription of miRNA transcripts, followed by amplification in a polymerase chain reaction (RT-PCR). The levels of miRNAs can be quantified in comparison to an internal standard (e.g., levels of mRNA from a "housekeeping" gene present in the same sample). Suitable "housekeeping" genes for use as internal standards include myosin, glyceraldehyde-3 -phosphate dehydrogenase (G3PDH), and human U6. Methods for quantitative RT-PCR and variations thereof are well known to those of ordinary skill in the art. Exemplary primers for RT-PCR experiments are provided in Table 1. In certain instances, real-time quantitative PCR (qRT-PCR) analysis of miRNAs may be more sensitive than classical tissue sectioning and staining for detecting miRNAs in some early-stage cancers. The qRT-PCR for miRNA level determination disclosed herein may provide a sensitive and specific tool for the diagnosis, classification, and prognosis of lung cancer. In one aspect, this document provides methods for determining miRNA level in a sample of an individual (e.g., an individual having a disease such as cancer) by RT-PCR. In some embodiments, the level of the miRNA is determined by qRT-PCR. In some cases, the levels of miRNAs are determined by using a microarray.

Nucleic acid probes for use in the methods described herein can be produced recombinantly, or can be chemically synthesized using methods that are well known in the art. Additionally, hybridization probes can be labeled with a variety of detectable labels, including, for example, radioisotopes, fluorescent tags, reporter enzymes, biotin, and other ligands. Such detectable labels can additionally be coupled with, for example, calorimetric or photometric indicator substance for spectrophotometric detection. Methods for labeling and detecting such probes are known in the art. Nucleic acid probes useful for detecting miRNAs in a sample can be hybridized under stringency conditions that can be readily determined by one skilled in the art. Depending on the particular assay, the stringency conditions can be varied to optimize detection of a particular miRNA in a particular sample.

In general, the stability of a hybrid is a function of ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. Hybridized nucleic acid molecules generally have at least 60% identity (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% identity). Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5xDenhart's solution, 6xSSPE, 0.2% SDS at 22 ⁰ C, followed by washing in IxSSPE, 0.2% SDS, at 37 ⁰ C. Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5xDenhart's solution, 5xSSPE, 0.2% SDS at 42 ⁰ C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 ⁰ C. High stringency conditions are provided by conditions equivalent to hybridization in 50% formamide, 5xDenhart's solution, 5xSSPE, 0.2% SDS at 42 ⁰ C, followed by washing in 0. IxSSPE, and 0.1% SDS at 65°C. Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al., supra, 1999). Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 2OxSSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3 M sodium chloride,

0.2M sodium phosphate, and 0.025 M (EDTA). In some embodiments, levels of one or more miRNAs can be obtained from an individual at more than one time point. Such "serial" sampling can be well suited for methods relating to monitoring progression of lung cancer in an individual. Serial sampling can be performed on any desired timeline, such as semi-annually, annually, biennially, or more or less frequently. The comparison between the measured level of a miRNA and the reference level can be carried out each time a new sample is measured, or data relating to levels may be held for less frequent analysis.

A reference level generally is a level that is considered "normal" for a particular miRNA. In some embodiments, a reference level can be based on the level of the miRNA in non-cancerous lung tissue from the same individual. In some embodiments, a reference level can be based on the level of the miRNA in an individual not having lung cancer. In some embodiments, a reference level can be based on an average of levels obtained from a population identified as not having lung cancer. In some cases, a reference level can be derived from a pool of samples including the sample being tested, and can be predetermined or determined contemporaneously with the sample being tested.

As used herein, a "reference" value can be an absolute value, a relative value, a value that has an upper or lower limit, a range of values, an average value, a median value, a mean value, or a value as compared to a particular control or baseline value.

A comparison to a reference value may be performed for any (e.g., one, two, three, four, or five) of the miRNAs listed in Table 1 or their corresponding homologues. The process of comparing the levels of a miRNA with a reference level can be carried out in any manner appropriate to the type of measured values for the miRNAs of interest. For example, when hybridization signals are used as a measure of miRNA levels, the levels can be compared qualitatively by visually comparing the intensities of the hybridization signals. For quantitative measures, the comparison can be made by inspecting the numerical data, inspecting representations of the data (e.g., inspecting graphical representations such as bar or line graphs). The process of comparing may be manual (such as visual inspection by the practitioner of the methods) or it may be automated.

In some embodiments, the comparison is performed by determining the magnitude of the difference between the measured and reference levels (e.g., comparing the "fold" or percentage difference between the measured and reference levels). As used herein, the phrase "fold difference" refers to a numerical representation of the magnitude difference between a measured value and a reference value for a miRNA. A "characteristic change" in the level of a miRNA can be a substantial decrease or a substantial increase in the miRNA level in an individual as compared to a reference level. As used herein, the phrase "substantial increase" refers to an increase in miRNA level by at least about 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more than 50%). Similarly, the phrase "substantial decrease" as used herein refers to a decrease in miRNA level by at least about 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more than 50%).

Table 1 provides a list of characteristic changes for particular miRNAs in one exemplary method. A characteristic change in the level of one or more miRNAs can be used as a basis for diagnosing lung cancer. For example, when the level of at least one of the miRNAs of SEQ ID NOS: 1-3 is determined, a substantial increase in the level of at least one of the measured miRNAs can be indicative of lung cancer. In some embodiments, when the level of at least one of the miRNAs of SEQ ID NOS :4 and 5 is determined, a substantial decrease in the level of at least one of the measured miRNAs can be indicative of lung cancer. In some embodiments, when the levels of at least one of the miRNAs of SEQ ID NOS:l-3 and at least one of the miRNAs of SEQ ID NOS :4 and 5 are determined, a substantial increase in the level of at least one of the miRNAs of SEQ ID NOS: 1-3 and a substantial decrease in the levels of at least one of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer.

In some embodiments, the levels of all miRNAs listed in Table 1 can be determined, and a substantial increase in the level of at least one of the miRNAs of SEQ ID NOS: 1-3, in combination with a substantial decrease in the levels of at least one of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer. In some embodiments, a substantial increase in the levels of at least two of the miRNAs of SEQ ID NOS: 1-3, in combination with a substantial decrease in the levels of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer. In embodiments where more than one miRNAs are used but the levels of the miRNAs do not unanimously suggest or indicate a diagnosis of lung cancer, a "majority" suggestion or indication may be considered the result of the assay. For example, when the method utilizes five miRNAs, three of which indicate lung cancer, the result may be considered as suggesting or indicating a diagnosis of lung cancer for the individual. In some embodiments, however, a diagnosis of lung cancer can require a characteristic change of at least one or more particular miRNAs. For example, in cases when one of the miRNAs is hsa-miR-210, a substantial increase in the level of hsa-miR-210 may be a prerequisite for a diagnosis of lung cancer. Methods for evaluating miRNA gene status

Also provided herein are methods for diagnosing lung cancer based on the gene status of at least one of the miRNAs listed in Table 1 (or their corresponding homologue(s)) in a sample from an individual. In some embodiments, gene status can be evaluated by analyzing at least one miRNA gene in the sample for deletion or amplification, wherein the detection of a deletion or amplification in the miRNA gene relative to the miRNA gene in a control sample can indicate the presence of lung cancer in the individual.

A deletion or amplification in a miRNA gene can be detected by determining the structure or sequence of the gene in a lung tissue sample from an individual suspected of having lung cancer, and comparing the structure or sequence with that of the gene in a control sample. Any techniques suitable for detecting alterations in gene structure or sequence can be used in the present methods. For example, the presence of miRNA gene deletions and amplifications can be detected by Southern blot hybridization of the genomic DNA from a subject, using nucleic acid probes specific for miRNA sequences. Sequence analyses and single strand conformational polymorphism analyses also can be used.

Deletions or amplifications of a miRNA gene also can be detected by amplifying a fragment of the gene by polymerase chain reaction (PCR), and analyzing the amplified fragment by sequencing or electrophoresis to determine if the sequence or length of the amplified fragment from the individual's DNA sample is different from that of a control DNA sample. Deletion of a miRNA gene also can be identified by detecting deletions of chromosomal markers that are closely linked to the miRNA gene.

The status of a miRNA gene in cells from an individual also can be evaluated by measuring the copy number of at least one miRNA gene in the sample, wherein a gene copy number other than two for a miRNA gene on somatic chromosomes in an individual of either gender and sex chromosomes in a female, or other than one for miRNA genes on sex chromosomes in a male, can indicate the presence of lung cancer in the individual.

Any techniques suitable for detecting gene copy number can be used, including Southern blotting and PCR amplification techniques. An alternative method for determining miRNA gene copy number in a lung tissue sample relies on the fact that many miRNAs or gene clusters are closely linked to chromosomal markers or other genes. The loss of a copy of a miRNA gene in an individual who is heterozygous at a marker or gene closely linked to the miRNA gene can be inferred from the loss of heterozygosity in the closely linked marker or gene. Methods for determining loss of heterozygosity of chromosomal markers are within the skill in the art.

Other techniques that can be used to determine the status of miRNA genes include, for example, allele-specific primer extension on microarrays, PCR/LDR universal arrays, microsphere-based single base chain extension, sequence-tagged molecular inversion probes, and combinatorial sequence-by-hybridization.

In some embodiments, a "control sample" can be a tissue sample from an individual not having lung cancer. Alternatively, a control sample can be a collection of tissue samples from a population of individuals (e.g., individuals known not to have lung cancer). Gene status can be determined for at least one (e.g., one, two, three, four or five) of the rm ^' RNAs listed in Table 1 or their corresponding homologues. In embodiments where the gene statuses of more than one miRNAs are used but do not unanimously suggest or indicate a diagnosis of lung cancer, the "majority" suggestion or indication may be considered the result of the assay. For example, when the method utilizes the gene status of five miRNAs, three of which suggest lung cancer, the result may be considered as suggesting or indicating a diagnosis of lung cancer for the individual. However in some embodiments, a diagnosis of lung cancer can require a change in the gene status of one or more specific miRNA genes.

Methods for diagnosing disease The present document provides methods for identifying an individual as having a disease (e.g., cancer), comprising: a) determining the level of at least one miRNA in a biological sample from the individual, and b) comparing the level of the miRNA with a reference level, wherein a characteristic change in the level of the miRNA is indicative of the disease. Diseases that can be diagnosed include, but are not limited to, cancer (e.g., lung cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, colorectal cancer, pancreas cancer, leukemia, lymphoma, kidney cancer, urinary bladder cancer, cervical cancer, endometrial cancer, ovary cancer, and testicular cancer), cardiovascular disease (e.g., coronary heart disease, hypertension, and atherosclerosis), and age-related disease (e.g., Parkinson's disease, Alzheimer's disease, and diabetes). For example, this document provides a method of diagnosing lung cancer in an individual, comprising: a) determining the level of at least one miRNA (e.g., at least one miRNA listed in Table 1, or a corresponding homologue) in a lung tissue sample from the individual, wherein the tissue is suspected of being cancerous, and b) comparing the level of the miRNA with a reference level, wherein a characteristic change in the level of the miRNA is indicative of lung cancer.

In some embodiments, a method for diagnosing lung cancer in an individual can comprise: a) comparing a reference level of at least one miRNA (e.g., at least one miRNA listed in Table 1, or a corresponding homologue) with the level of at least one miRNA in a lung tissue sample from the individual, wherein the tissue is suspected of being cancerous, and b) determining whether the individual has lung cancer based on a characteristic change in the level of the at least one miRNA. In some embodiments, the method can further comprise providing a lung tissue sample from the individual, and/or isolating miRNAs from the tissue sample.

This document also contemplates a method of providing information for diagnosis of lung cancer in an individual, comprising: a) determining the level of at least one miRNA listed in Table 1, or a corresponding homologue, in a lung tissue sample from the individual, wherein the tissue is suspected of being cancerous, and b) providing information about the level of the miRNA for diagnosis of lung cancer, wherein the level of the miRNA is used as basis for diagnosing lung cancer, and wherein a characteristic change in the level of the at least one miRNA is indicative of lung cancer.

In some embodiments, the level of at least one (e.g., one, two, or three) of the miRNAs of SEQ ID NOS: 1-3 can be determined, and a substantial increase in the level of at least one of the measured miRNAs can be indicative of lung cancer. In some embodiments, the level of at least one (e.g., one or two) of the miRNAs of SEQ ID NOS:4 and 5 can be determined, and a substantial decrease in the level of at least one of the measured miRNAs can be indicative of lung cancer. In some embodiments, the level of at least one (e.g., one, two, or three) of the miRNAs of SEQ ID NOS: 1-3 and at least one (e.g., one or two) of the miRNAs of SEQ ID NOS :4 and 5 can be determined, and a substantial increase in the level of at least one of the miRNAs of SEQ ID NOS: 1-3 and a substantial decrease in the level of at least one of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer.

In some embodiments, the levels of all miRNAs listed in Table 1 can be determined, and a substantial increase in the level of at least one of the miRNAs of SEQ ID NOS: 1-3 in combination with a substantial decrease in the level of at least one of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer. In some cases, a substantial increase in the levels of at least two (e.g., two or three) of the miRNAs of SEQ ID NOS: 1-3 in combination with a substantial decrease in the levels of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer. In some cases, a substantial increase in the levels of the miRNAs of SEQ ID NOS:l-3in combination with a substantial decrease in the levels of the miRNAs of SEQ ID NOS :4 and 5 can be indicative of lung cancer.

Altered levels of a miRNA in a tissue sample also may reflect changes in the status of the miRNA gene. Gene status can be reflected, for example, by deletion or amplification of the miRNA gene, or by a change in copy number of the miRNA gene.

Accordingly, in some embodiments, there is provided a method of diagnosing lung cancer in an individual, comprising analyzing the gene status of at least one miRNA gene (e.g., at least one gene encoding a miRNA listed in Table 1) in a lung tissue sample suspected of being cancerous in an individual, wherein a characteristic change in the gene status relative to the corresponding miRNA gene in a control sample can be indicative of lung cancer. In some embodiments, a change in gene status can be determined based on a deletion or amplification of a miRNA gene. In some cases, a change in gene status can be determined based on a change in copy number of a miRNA gene. This document also provides a method for diagnosing lung cancer in an individual, comprising analyzing for deletion or amplification a miRNA gene encoding a miRNA listed in Table I ₅ wherein the miRNA gene is analyzed in a lung tissue sample that is suspected of being cancerous. A deletion or amplification of the miRNA gene relative to the corresponding miRNA gene in a control sample can be indicative of lung cancer. For example, in some embodiments, a method can include analyzing at least one miRNA gene corresponding to a miRNA having a nucleotide sequence set forth in SEQ ID NO:1. SEQ ID NO:2, or SEQ ID NO: 3 for amplification, wherein amplification of the miRNA gene relative to the corresponding miRNA gene in a control sample can be indicative of lung cancer. In some embodiments, a method can include analyzing at least one miRNA gene corresponding to at least one miRNA having a nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:5 for deletion, wherein a deletion of the miRNA gene relative to the corresponding miRNA gene in a control sample can be indicative of lung cancer. In some cases, a method can further comprise providing a lung tissue sample suspected of being cancerous from the individual, and/or isolating DNA from the lung tissue sample. This document also provides a method for diagnosing lung cancer in an individual, comprising determining the gene copy number of at least one miRNA gene corresponding to a miRNA listed in Table 1 (or a corresponding homologue) in a lung tissue sample that is from the individual and is suspected of being cancerous. A copy number other than two for miRNA genes located on a somatic chromosome for either gender or on a sex chromosome for female, and other than one for miRNA genes located on a sex chromosome for male, can be indicative of lung cancer. For example, a method can include determining the gene copy number of at least one miRNA gene corresponding to at least one of the miRNAs set forth in SEQ ID NOS: 1-3 in a sample from the individual, and a copy number more than two for a miRNA gene located on a somatic chromosome for either gender or a sex chromosome for female, or more than one for a miRNA gene located on a sex chromosome for male, can be indicative of lung cancer. In some embodiments, a method can include determining the gene copy number of at least one miRNA gene corresponding to at least one of the miRNAs set forth in SEQ ID NOS :4 and 5 in a sample from the individual, and a copy number less than two for a miRNA gene located on a somatic chromosome for either gender or a sex chromosome for female, or less than one for a miRNA gene located on a sex chromosome for male, can be indicative of lung cancer. In some embodiments, a method can further comprise providing a lung tissue sample suspected of being cancerous from the individual, and/or isolating DNA from the lung tissue sample.

The miRNAs described herein also can be useful for one or more of the following: classifying lung cancer patients, predicting risk of developing lung cancer, monitoring tumor progression in lung cancer patients, and monitoring treatment in lung cancer patients, based on the levels of one or more miRNAs in a lung tissue sample, or based on the gene status of one or more miRNAs in a lung tissue sample.

Any of the methods disclosed herein can further include recording or causing to be recorded (e.g., via handwriting, computer, or audio means) information regarding the status of the individual who is assessed. For example, a method can include recording information related to whether or not an individual is identified as having lung cancer, the prognosis for survival of the individual, or the classification of the individual into a particular lung cancer group. Such steps are discussed in greater detail below.

Materials and methods for determining prognosis of lung cancer patients

This document provides methods for determining prognosis for lung cancer patients, including, for example, methods of determining a prognosis for survival of an individual having lung cancer. The prognostic methods can be useful for determining a proper course of treatment for an individual having lung cancer. For example, a determination of the likelihood of survival can assist in determining whether a more conservative or more radical approach to therapy should be taken, or whether treatment modalities should be combined. In addition, such prognosis can help determine whether agents for improving survival (such as agents described herein) may be necessary and/or effective.

In some embodiments, a method for determining a prognosis for survival for an individual having lung cancer can include: (a) determining the level of at least one miRNA in a lung cancer tissue sample from the individual, and (b) comparing the level of the miRNA in the sample to a threshold level, wherein the level of the miRNA as compared to the threshold level correlates or inversely correlates with the expected survival of the individual. As used herein, "correlate" means that a low level of the miRNA as compared to the threshold level is indicative of a low chance of survival for an individual having lung cancer, and vice versa. As used herein, "inversely correlate" means that a high level of the miRNA as compared to the threshold level is indicative of a low survival rate, and vice versa.

Certain methods and uses described herein can include determining a prognosis for survival based on miRNA levels relative to a threshold level. A threshold level can be determined by any of a plurality of methods, provided that the resulting threshold level provides a level of miRNA above which exists a first group of patients having a survival rate that differs from the survival rate of a second group of patients having a miRNA level below the threshold level.

A threshold level can be determined by, for example, measuring the miRNA level in one or more non-cancerous lung cancer tissue samples. A threshold level also can be determined by analyzing the levels of a miRNA in a population of individuals having lung cancer. This can be accomplished, for example, by histogram analysis, in which an entire cohort of tested individuals are graphically presented, wherein a first axis represents the level of the miRNA, and a second axis represents the survival rate of the individual. Two or more separate groups of individuals can be evaluated by identification of subset populations of the cohort that have the same or similar levels of a particular miRNA. Determination of a threshold level then can be made based on the miRNA level that best distinguishes these separate groups, or the miRNA levels that best distinguish the separate groups. For example, a threshold level can be based on the mean value of the average miRNA level of a group with high survival rate and the average miRNA level of a group with low survival rate. A threshold level also can represent the levels of two or more miRNAs. Two or more miRNAs can be represented, for example, by a ratio of values for levels of each miRNA. A threshold level can be a single number that is equally applicable to every individual having lung cancer, or can vary according to a specific subpopulation of individuals. For example, older men might have a different threshold level than younger men, and woman might have a different threshold level than men. Further, a threshold level can be a level determined for each individual. For example, a threshold level can be a certain ratio of a miRNA in lung cancer tissue relative to the miRNA level in non-cancerous tissue within the same individual.

Verification that the threshold level distinguishes the likelihood of survival in lung cancer patients expressing below threshold level versus patients expressing above threshold level can be carried out using single variable or multi-variable analysis. These methods can be used to determine the likelihood of a correlation between one or more variables and a given outcome. In the instant case, the methods can determine the likelihood of a correlation between a miRNA level and disease-free or overall survival of a cancer patient. Any one of a plurality of methods well known to those of ordinary skill in the art for carrying out these analyses can be used. Examples of single variable analysis are the Kaplan-Meier method and the Cox proportional-hazards regression model.

Population-based determination of threshold levels (e.g., by histogram analysis) can be carried out using a cohort of patients that is sufficient in size to determine two or more separate groups of patients having different miRNA levels. Typically, such a cohort comprises at least 25 patients (e.g., at least about 25, 30, 40, 50, 60, 75, 100, 125, 150, 200, or more than 200 patients). Similarly, a cohort for verification of determined threshold levels can also include at least 25 patients (e.g., at least about 25, 30, 40, 50, 60, 75, 100, 125, 150, 200, or more than 200 patients).

Further, while a single threshold level can separate two groups of patients, several threshold values might exist that can separate a plurality of populations. For example, two threshold values can separate a first group of patients with high levels of a miRNA from a second group of patients with intermediate levels of the miRNA, and from a third group of patients with low levels of the miRNA. The number of different threshold levels can be sufficient to proscribe a curve, such as a continuous line, which can describe the likelihood of disease-free or overall survival in a patient as a function of the miRNA level in that patient. Such a curve can constitute a "continuous" miRNA level, where the likelihood of disease free or overall survival in a patient is proportional to the miRNA level in that patient. Two or more miRNA levels can be represented by such a curve. In some embodiments, miRNAs can be combined with each other in the methods provided herein for determining prognosis for survival of a cancer patient. The use of a combination of two or more miRNAs can provide increased prognostic significance or confidence in a prognostic determination. The level of a miRNA can also be used in conjunction with another variable found to be statistically significant as an indicator of the likelihood of disease-free or overall survival for lung cancer patient, including pathological indicators (e.g., age, tumor size, tumor histology, clinical stage, family history, and the like). For example, the clinical stage of a cancer can be a statistically significant indicator of disease-free or overall survival, and the threshold level can vary according to the clinical stage of the cancer. Hence, the threshold level of a miRNA can vary as a function of another statistically significant indicator of disease-free or overall survival for lung cancer.

In some cases, Kaplan-Meier analysis can be used to determine the correlation between survival rate and miRNA levels. In some embodiments, a method can include: (a) determining a level of at least one miRNA in a lung cancer tissue from an individual, (b) classifying the individual as belonging to either a first or second group of individuals having lung cancer, wherein the first group of individuals having a low level of the at least one miRNA is classified as having an increased ^• likelihood of survival compared to the second group of individuals having a high level of the at least one miRNA. In some cases, the at least one miRNA can be hsa-miR-31.

After the levels of one or more miRNAs in patient sample have been determined and compared to a threshold level, the patient can then be classified into a group having a certain likelihood of disease free or overall survival. The likelihood of disease-free or overall survival for the patient can be assessed based on the likelihood of disease-free or overall survival for patients in that group.

For example, a sample from a patient may be determined to have low levels of a particular miRNA. The patient then can be classified into a group having low levels of miRNA.

If it had been established that there is an increased likelihood of disease-free or overall survival for the group of patients expressing low levels of the miRNA, the specific cancer patient would be considered to have an increased likelihood of disease free or overall survival.

The methods described herein can further comprise a step of determining the proper course of treatment for an individual. Those of skill in the art will appreciate that prognostic indicators of survival for cancer patients suffering from an early stage of cancer may be different from prognostic indicators for cancer patients suffering from a later stage of cancer. For example, the prognosis for a stage I cancer patient may be oriented toward the likelihood of continued growth and/or metastasis of the cancer, whereas prognosis for stage IV cancer patient may be oriented toward the likely effectiveness of therapeutic methods for treating the cancer. The determination of proper course of treatment will therefore take these variables into account.

In some cases, a method of determining prognosis for survival of an individual having lung cancer can include: (a) determining the level of at least one miRNA in a lung cancer tissue sample from the individual, and (b) comparing the level of the miRNA in the sample to a threshold level, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of the individual. The at least one miRNA can be hsa-miR-31 or a corresponding homologue.

Levels of the miRNAs described herein also may reflect changes in the gene status of the miRNAs (e.g., the miRNAs listed in Tables 1 and 2). In some embodiments, there is provided a method of determining a prognosis for survival for an individual having lung cancer, comprising analyzing the gene status of at least one miRNA gene (e.g., a miRNA gene encoding hsa-miR-31 or a corresponding homologue), wherein a change in gene status as compared to a that of a control sample indicates a relatively high or low chance of survival for the individual. For example, in some embodiments, there is provided a method for determining a prognosis for survival for an individual having lung cancer, comprising analyzing a miRNA gene corresponding to hsa-miR-31 for amplification, wherein an amplification of the miRNA gene relative to the corresponding miRNA gene in a control sample correlate with a low survival rate of the individual. In some embodiments, there is provided a method of determining a prognosis for survival of an individual having lung cancer, comprising determining the gene copy number of a miRNA gene corresponding to hsa-miR-31 , wherein a copy number of more than two indicates a low survival rate of the individual.

Also provided herein are uses of probes that are capable of detecting the levels of miRNAs (or the status of corresponding miRNA genes), and the use of systems comprising one or more probes for determining a prognosis for survival. For example, this document provides for the use of one or more probes (or a system comprising one or more probes) for determining prognosis for survival of an individual having lung cancer, wherein the probe is capable of detecting a miRNA in the sample, and wherein the level of the miRNA as compared to a threshold level correlates or reversely correlates with the projected survival of the individual. In some embodiments, one or more probes can be used for determining prognosis of survival for an individual having lung cancer, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of the individual, and wherein at least one miRNA is hsa-miR-31 or a corresponding homologue. In some embodiments, there is provided a use of one or more probes for the manufacture of an agent or system for determining a prognosis for survival of an individual having lung cancer, wherein the probe is capable of detecting a miRNA in the sample, and wherein the level of the miRNA as compared to the threshold level correlates or reversely correlates with the survival of the individual. In some embodiments, there is provided a use of one or more probe for the manufacture of an agent or system for determining prognosis of survival of an individual having lung cancer, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of the individual, and wherein at least one of hsa-miR-31 or a corresponding homologue.

Materials and methods for assisting medical and research professionals

This document also provides methods and materials to assist medical or research professionals in determining whether or not an individual has lung cancer. Medical professionals can be, for example, doctors, nurses, medical laboratory technologists, and pharmacists. Research professionals can be, for example, principle investigators, research technicians, postdoctoral trainees, and graduate students. A professional can be assisted by (1) determining the level of a miRNA in a lung tissue sample from an individual, and (2) communicating information about the level to that professional.

After the level of a miRNA (or the status of a corresponding miRNA gene) is reported, a medical professional can take one or more actions that can affect patient care. For example, a medical professional can record the level of a miRNA in a patient's medical record. In some cases, a medical professional can record a diagnosis of lung cancer, or otherwise transform the patient's medical record, to reflect the patient's medical condition. In some cases, a medical professional can review and evaluate a patient's entire medical record, and assess multiple treatment strategies, for clinical intervention of a patient's condition. A medical professional can initiate or modify treatment for lung cancer after receiving information regarding a patient's miRNA levels. In some cases, a medical professional can compare previous reports of miRNA levels with the recently communicated miRNA levels, and recommend a change in therapy. In some cases, a medical professional can enroll a patient in a clinical trial for novel therapeutic intervention of lung cancer. In some cases, a medical professional can elect waiting to begin therapy until the patient's symptoms require clinical intervention.

A medical professional can communicate the levels of a miRNA (or the status of a corresponding miRNA gene) to a patient or a patient's family. In some cases, a medical professional can provide a patient and/or a patient's family with information regarding lung cancer, including treatment options, prognosis, and referrals to specialists, e.g., oncologists. In some cases, a medical professional can provide a copy of a patient's medical records to communicate the levels of a miRNA, or the status of a corresponding miRNA gene, to a specialist.

A research professional can apply information regarding a subject's miRNA levels and/or miRNA gene status to advance lung cancer research. For example, a researcher can compile data on miRNA levels and/or miRNA gene status with information regarding the efficacy of a drug for treatment of lung cancer symptoms to identify an effective treatment. In some cases, a research professional can obtain a subject's miRNA levels to evaluate a subject's enrollment, or continued participation in a research study or clinical trial. In some cases, a research professional can classify the severity of a subject's condition, based on the levels of one or more particular miRNAs. In some cases, a research professional can communicate information regarding a subject's miRNA levels and/or miRNA gene status to a medical professional. In some cases, a research professional can refer a subject to a medical professional for clinical assessment of lung cancer, and treatment of lung cancer symptoms. Any appropriate method can be used to communicate information to another person (e.g., a professional). For example, information can be given directly or indirectly to a professional. For example, a laboratory technician can input miRNA levels into a computer- based record. In some cases, information is communicated by making an physical alteration to medical or research records. For example, a medical professional can make a permanent notation or flag a medical record for communicating a diagnosis to other medical professionals reviewing the record. In addition, any type of communication can be used to communicate the information. For example, mail, e-mail, telephone, and face-to-face interactions can be used. The information also can be communicated to a professional by making that information electronically available to the professional. For example, the information can be communicated to a professional by placing the information on a computer database such that the professional can access the information. In addition, the information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.

Materials and methods for improving survival of lung cancer patients Also provided are methods for improving the survival of lung cancer patients using agents that alter (e.g., increase or decrease) the levels of certain miRNAs, including the miRNAs listed in Tables 1 and 2. Any agents that can increase or decrease the level of miRNAs can be used in the methods provided herein. For example, agents that can be used to inhibit miRNA gene expression include, but are not limited to, double-stranded RNA (e.g., short or small-interfering RNA or "siRNA"), antisense nucleic acids, enzymatic RNA molecules such as ribozymes, small molecule compounds, and proteins. These agents can be used alone or in combination with other agents (e.g., other agents described herein). The agents can decrease the miRNA levels directly (e.g., by inhibiting miRNA expression or function) or indirectly (e.g., by affecting the status of the corresponding miRNA gene). A method for improving survival of an individual having lung cancer can include, for example, administering to the individual an effective amount of an agent that decreases the level of a miRNA, wherein the level of the miRNA as compared to a threshold level inversely correlates with the survival of the individual. In some embodiments, there is provided a use of an agent that decreases the level of a miRNA for the manufacture of a medicament for improving survival of an individual having lung cancer, wherein the level of the miRNA as compared to a threshold level inversely correlates with the survival of the individual.

In some embodiments, a method for improving survival of an individual having lung cancer can include administering to the individual an effective amount of an agent that decreases the level of a miRNA selected from the group consisting of hsa-miR-31, hsa-miR- 210, hsa-miR-30a, hsa-miR-182, and their corresponding homologues. In some embodiments, there is provided use of an agent for the manufacture of a medicament for improving survival of an individual having lung cancer, wherein the agent decreases the level of a miRNA selected from the group consisting of hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and their corresponding homologues. Agents that decrease the level of hsa-miR-31, or that decrease the level of hsa-miR-31 in combination with one or more of hsa-miR-210, hsa-miR-30a, and hsa- miR-182 can be particularly useful. The methods described herein can further include a step of determining prognosis for survival of an individual (e.g., by methods described herein) prior to administration of the agents.

In some embodiments, the levels of more than one miRNAs can be decreased to improve the survival of a lung cancer patient. This can be achieved, for example, with an agent that decreases the levels of two or more miRNAs. Alternatively, two or more agents can be used to decrease the levels of two or more miRNAs. For example, there is provided a method for improving survival of an individual having lung cancer, comprising administering to the individual an effective amount of one or more agents that decrease the levels of at least two miRNAs selected from the group consisting of hsa-miR-31 , hsa-miR-210, hsa-miR-3 Oa, hsa- miR-182, and their corresponding homologues. In some embodiments, there is provided use of one or more agents for the manufacture of a medicament for improving survival of an individual having lung cancer, wherein the agent decreases the levels of at least two miRNAs selected from the group consisting of hsa-miR-31, hsa-miR-210, hsa-miR-3 Oa, hsa-miR-182, and their corresponding homologues.

Also provided is a method for improving survival of an individual having lung cancer, comprising administering to the individual an effective amount of one or more agents that decrease the levels of at least three miRNAs selected from the group consisting of hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and their corresponding homologues. In some ' embodiments, there is provided use of one or more agents for the manufacture of a medicament for improving survival of an individual having lung cancer, wherein the agent decreases the levels of at least three miRNAs selected from the group consisting of hsa-miR-31, hsa-miR- 210, hsa-miR-30a, hsa-miR-182, and their corresponding homologues.

In some embodiments, there is provided a method of improving survival of an individual having lung cancer, comprising administering to the individual an effective amount of one or more agents that decrease the levels of hsa-miR-31, hsa-miR-210, hsa-miR-3 Oa, and hsa-miR-182. In some cases, there is provided use of one or more agents for the manufacture of a medicament for improving survival of an individual having lung cancer, wherein the agent decreases the levels of hsa-miR-31, hsa-miR-210, hsa-miR-30a, and hsa-miR-182. In some embodiments, there is provided a pharmaceutical composition comprising an agent that decreases the level of a miRNA and a pharmaceutically acceptable carrier, wherein at least one miRNA is hsa-miR-31, hsa-miR-210, hsa-miR-3 Oa, or hsa-miR-182.. In some cases, at least one miRNA can be hsa-miR-31. In some cases, at least one miRNA can be hsa- miR-210. In some cases, at least one miRNA can be hsa-miR~30a. In some cases, at least one miRNA can be hsa-miR-182. In some embodiments, the agent can be a double-stranded RNA (e.g., a short or small-interfering RNA or "siRNA"), an antisense nucleic acid, or an enzymatic RNA molecules such as a ribozyme. Methods and compositions for improving survival are further described herein in more detail.

"Survival" can be disease free survival or overall survival. As used herein, the term "disease-free survival" refers to the lack of tumor recurrence and/or spread and the fate of an individual after diagnosis, for example, an individual who is alive without tumor recurrence. The phase "overall survival" refers to the fate of the individual after diagnosis, regardless of whether the individual has a recurrence of the tumor.

In some embodiments, expression of a particular miRNA gene can be inhibited by inducing RNA interference of the miRNA gene with an isolated double-stranded RNA ("dsRNA") molecule having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) sequence homology with at least a portion of the miRNA gene product. In some embodiments, the dsRNA molecule can be a "short or small interfering RNA" or "siRNA." siRNAs that can be useful in the present methods can be, for example, short double- stranded RNAs of about 10-30 nucleotides (e.g., about 12-28, 14-26, 16-24, or 18-22 nucleotides). The siRNA can have a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions. The sense strand can contain a nucleic acid sequence that is substantially identical to a nucleic acid sequence contained within the target miRNA. The sense and antisense strands of the siRNA can comprises two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area.

A siRNA can differ from naturally-occurring RNA by addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non- nucleotide material (e.g., to the end(s) of the siRNA, or internal to the siRNA), modifications that make the siRNA resistant to nuclease digestion, or substitution of one or more nucleotides in the siRNA with deoxynucleotides. In some embodiments, one or both strands of a siRNA can comprise a 3' overhang. A siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described further below. Expression of a given miRNA can also be inhibited by an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid molecule that binds to a target RNA by means of RNA-RNA or RNA-DNA interactions, altering the activity of the target RNA. Antisense nucleic acids suitable for use in the present methods can be single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA, and LNA) that generally include a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miRNA. In some embodiments, an antisense nucleic acid can comprise a nucleic acid sequence that is at least about 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%) complementary to a contiguous nucleic acid sequence in a miRNA. In some embodiments, an antisense nucleic acid can be about 10-30 nucleotides (e.g., about 12-28, 14-26, 16-24, or 18- 22 nucleotides) in length.

Antisense nucleic acids also can contain modifications to the nucleic acid backbone or to the sugar and base moieties (or their equivalent) to, for example, enhance target specificity, nuclease resistance, delivery or other properties related to efficacy. Such modifications can include cholesterol moieties, duplex intercalators such as acridine, or the inclusion of one or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as further described below.

Expression of a given miRNA gene also can be inhibited by an enzymatic nucleic acid. As used herein, an "enzymatic nucleic acid" refers to a nucleic acid comprising a substrate binding region that is complementary to a contiguous nucleic acid sequence of a miRNA, and which is able to specifically cleave the miRNA. In some embodiments, an enzymatic nucleic acid binding region can be 50 to 100% complementary (e.g., 75-100% complementary or 95- 100% complementary) to a contiguous nucleic acid sequence in a miRNA. An enzymatic nucleic acid also can having modifications at the base, sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.

Enzymatic nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as further described below.

A variety of methods are known in the art for introducing a nucleic acid molecule into a cell, including a cancer cell. Such methods include, for example, microinjection, electroporation, lipofection, calcium-phosphate mediated transfection, DEAE-Dextran- mediated transfection, microparticle bombardment, delivery by a colloidal dispersion system (such as macromolecular complexes, beads, oil-in-water emulsions, micelles, mixed micelles, and liposomes), and conjugation to an antibody, gramacidin, artificial viral envelopes or other intracellular carriers such as TAT.

A nucleic acid agent also can be delivered into a mammalian cell in vitro or in vivo using vectors such as those known in the art. Suitable vectors for delivering a nucleic acid to a mammalian cell include, for example, viral vectors and non-viral vectors such as plasmid vectors. Such vectors can be useful for providing therapeutic amounts of an agent such as antisense RNA or siRNA.

Viral based systems can provide the advantageous ability to introduce relatively high levels of the heterologous nucleic acid into a variety of cells. Suitable viral vectors for introducing a nucleic acid include, for example, Herpes simplex virus vectors, vaccinia virus vectors, cytomegalovirus vectors, Moloney murine leukemia virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors. The tropism of vital vectors can also be modified by pseudotyping the vectors with envelope proteins or surface antigens from other viruses. For example, an AAV vector can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV) rabies, Ebola, Mokola, and the like.

Any of a variety of inducible promoters or enhancers can also be included in a nucleic acid or vector to control expression of the antisense RNAs or siRNAs by added stimuli or molecules. Such inducible systems include, for example, tetracycline inducible systems, metalothionein promoter induced by heavy metals, insect steroid hormone responsive to ecdysone or related steroids such as muristerone, mouse mammary tumor virus (MMTV) induced by steroids such as glucocorticoid and estrogen, and heat shock promoters inducible by temperature changes.

An agent can be said to be present in an effective amount if the amount of the agent is enough to alter (e.g., decrease) the level of the miRNA to which it is targeted. In some embodiments, an agent can decrease the level of the target miRNA by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, or more than 50%) of the difference between the miRNA level and threshold level. Exemplary amounts for the agents provide herein (e.g., nucleic acid agents) include, but are not limited to, 0.1-3000 mg/kg body weight, 10-2000 mg/kg body weight, 50-1000 mg/kg body weight, and 100-500 mg/kg body weight. In some embodiments, the amount of an agent (e.g., a nucleic acid agent) is about 10-500 mg/g tumor mass (e.g., about 20-300 mg/g tumor mass, about 50-200 mg/g tumor mass, or about 100-150 .. mg/g tumor mass). One of ordinary skill in the art can readily determine an appropriate dosage regimen for administration of one or more agents to an individual. Exemplary dosing frequencies include, without limitation, at least once ever month, at least about once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily, or more often. In some embodiments, the interval between each administrations can be less than about a week (e.g., less than about every six, five, four, three, two, or one day(s)). In some embodiments, the interval between each administration can be constant. For example, administration can be carried out daily, every two days, every three days, every four days, every five days, or weekly. In some embodiments, administration can be carried out twice daily, three times daily, or more frequently.

Administration of an agent can occur over an extended period of time, such as from about a month up to about three years. For example, a dosing regime can be extended over a period of any of about two, three, four, five, six, seven, eight, nine, ten, 11, 12, 18, 24, 30, or 36 months. In some embodiments, there may be no break in the dosing schedule. In some embodiments, the interval between each administration can be no more than about a week.

The compositions described herein can be administered to an individual via any route in the art, including, without limitation, intravenous^ intraperitoneal, intraocular, intra-arterial, intrapulmonary, oral, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transdermal, transpleural, intraarterial, topical, inhalational (e.g., as mists or sprays), transmucosal (e.g., via nasal mucosa), subcutaneous, transdermal, gastrointestinal, intraarticular, intracisternal, intraventricular, rectal (e.g., via suppository), vaginal (e.g., via pessary), intracranial, intraurethral, intrahepatic, and intratumoral routes. In some embodiments, a composition can be administered systemically. In some cases, a composition can be administered locally. Also provided herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an agent that alters (e.g., decreases) the level of a miRNA. In some embodiments, a pharmaceutical composition can include an agent that decreases the level of a miRNA selected from the group consisting of hsa-miR-210, hsa-miR-30a, and their corresponding homologues. The agent can be, for example, a siRNA, an antisense RNA, or a ribozyme.

In some embodiments, pharmaceutical compositions can be sterile. In some cases, pharmaceutical compositions can be pyrogen-free. Suitable pharmaceutically acceptable carriers include, for example, water, buffered water, normal saline, 0.4% saline, 0.3% glycine, and hyaluronic acid. Pharmaceutical compositions also can contain conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include, for example, stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include, without limitation, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), chelants (e.g., DTPA and DTPA-bisamide) and calcium chelate complexes (e.g., calcium DTPA and CaNaDTP A-bisamide), can calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, and calcium lactate). Pharmaceutical compositions can be packaged for use in liquid form, or can be lyophilized.

For solid pharmaceutical compositions, conventional nontoxic solid pharmaceutically acceptable carriers can be used. Solid pharmaceutically acceptable carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Systems for detecting miRNA levels and/or determining status ofmiRNA genes Also provided herein are systems (e.g., primer sets for quantitative real time PCR

(qPCR), in situ hybridization, or microarrays) comprising primers and/or probes (e.g., oligonucleotides) that can be used to detect miRNAs such as those listed in Tables 1 and 2. Systems (e.g., primer sets for qPCR, in situ hybridization, and microarrays) comprising primers and/or probes that are capable of determining the status ofmiRNA genes corresponding to miRNAs such as those listed in Tables 1 and 2, or to their corresponding homologues. These systems can be useful for determining levels of miRNAs listed in Tables 1 and 2 or their corresponding homologues, and for assessing lung cancer, for example. While some of the discussion herein focuses on systems for detecting miRNAs, it will be readily understood by a person of ordinary skill in the art that the description is equally applicable to systems for gene status of the miRNA genes.

Systems provided herein can include primers and/or probes for detecting miRNAs and/or determining gene status of miRNAs. While this discussion is focused on systems that are capable of detecting miRNAs, it will be readily understood by a person of skill in the art that certain aspects of the description are equally applicable to systems comprising primers and/or probes that can be used to assess gene deletion, amplification, and/or change in gene copy number for miRNA genes (collectively referred to herein as gene status of the miRNA genes). A system (e.g., a plurality of primers) for determining the level of at least one miRNA listed in Table 1 or Table 2 (or the status of a corresponding miRNA gene) in a sample can include, for example, one or more primer pairs, wherein each primer pair can be used to amplify a particular miRNA in a sample (e.g., a biological sample), provided that the particular miRNA is present in the sample.

A system (e.g., a plurality of primer pairs for qPCR, a plurality of probes for in situ hybridization, or a microarray) for determining the level of at least one miRNA listed in Table 1 or Table 2 (or the status of a corresponding miRNA gene) in a sample can include, for example, a plurality of primer pairs and/or probes, wherein each of the primer pairs and/or probes is capable of detecting a miRNA (or the status of the corresponding miRNA gene) in a lung tissue sample, and wherein at least about 15% (e.g., at least about 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) of the primer pairs and/or probes are capable of detecting a miRNA listed in Table 1 or Table 2 or their corresponding homologues (or determining the status of a corresponding miRNA gene). In some cases, each of the primer pairs and/or probes can be capable of detecting a different miRNA (or the status of the corresponding miRNA gene) in the lung tissue sample.

In some embodiments, a system (e.g., a microarray) for use in methods of diagnosing lung cancer in an individual can include a plurality of primer pairs and/or probes, wherein each primer pair or probe can be used to detect a miRNA (or the status of the corresponding miRNA gene) in a sample from the individual, and wherein at least about 15% (e.g., at least about 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) of the probes are capable of detecting a miRNA listed in Table 1 or Table 2 (or the status of a corresponding miRNA gene). In some cases, a system for diagnosing lung cancer can comprise (or consist essentially of) at least one (e.g., one, two, three, four, or five) pair of primers, wherein each pair can be used to detect the level of a miRNA listed in Table 1 or Table 2, or a corresponding homologue, or can be used to determine the status of a corresponding miRNA gene. In some cases, a system (e.g., a microarray) for diagnosing lung cancer can comprise (or consist essentially of) at least one (e.g., one, two, three, four, or five) probes, wherein each probe can detect the level of a miRNA listed in Table 1 or Table 2, or a corresponding homologue, or can determine the status of a corresponding miRNA gene. In some embodiments, each primer pair or probe in the system can be used to detect a different miRNA or determine the status of a different miRNA gene (e.g., a different miRNA listed in Table 1 or Table 2, or a corresponding homologue, or their corresponding genes). Systems (e.g., microarrays) provided herein are further described below in greater detail.

Also provided herein are uses of systems for diagnosing lung cancer. For example, there is provided a use of a system for diagnosing lung cancer, wherein the system comprises at least one (e.g., one, two, three, four, or five) primer pairs and/or probes (e.g., oligonucleotides), wherein each of the primer pairs and/or probes can be used to detect the level of a miRNA listed in Table 1 or Table 2, or a corresponding homologue, or to determine the status of a corresponding miRNA gene. In some cases, each primer pair or probe can be used to detect the level of a different miRNA listed in Table 1 or Table 2, or a corresponding homolog, or to detect the level of a different corresponding miRNA gene. A characteristic change of in the level of at least one of the miRNAs listed in Tables 1 and 2, or a corresponding homologue, or in the status of the corresponding miRNA gene, can be indicative of lung cancer.

Also provided herein is the use of a system (e.g., one or more primer pairs for qPCR, a plurality of probes for in situ hybridization, or a microarray) for diagnosing lung cancer, wherein the system comprises a plurality of primer pairs and/or probes, each of which can be capable of detecting a different miRNA (or the status of the corresponding miRNA genes) in the sample, wherein at least about 15% (e.g., at least about 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) of the probes are capable of detecting a miRNA listed in Table 1 or Table 2, or a corresponding homologue, or the status of a corresponding miRNA gene. A characteristic change in the level of at least one of the miRNAs or their corresponding homologues, or in the status of a corresponding miRNA gene, can be indicative of lung cancer.

Also provided are uses of primers and probes that can be used to detect miRNAs for the manufacture of systems as described herein. In some embodiments, there is provided a use of one or more primer pairs or probes (e.g., oligonucleotides) for manufacturing a system for diagnosis of lung cancer in an individual, wherein each primer pair or probe can be used to determine the level of a miRNA listed in Table 1 or Table 2 or a corresponding homologue, or to detect a corresponding miRNA gene. In some embodiments, there is provided a use of one or more primer pairs or probes (e.g., oligonucleotides) for the manufacture of a system (e.g., a microarray) for diagnosing lung cancer, wherein each primer pair or probe can be used to detect a different miRNA or to determine the status of the corresponding miRNA gene in the sample, and wherein at least about 15% (e.g., at least about 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) of the probes are capable of detecting a miRNA listed in Table 1 or Table 2, or a corresponding homologue, or a corresponding miRNA gene.

The systems provided herein can include two or more probes that detect the same miRNA. For example, in some embodiments (e.g., when the system is a microarray), the probes can be present in multiple (e.g., two, three, four, five, six, seven, or more) copies in the microarray. In some embodiments, a system can include different probes that detect the same miRNA. For example, two or more probes may bind to different (overlapping or nonoverlapping) regions of the same miRNA.

Any primers and/or probes that are capable of assessing the levels of miRNA can be used. In some embodiments, a primer or probe can be an oligonucleotide. It is to be understood that, for detection of miRNAs, certain sequence variations are acceptable. Thus, the sequences of oligonucleotides (or their complementary sequences) may be slightly different from those of the miRNAs described herein. Such sequence variations will be understood by those of skill in the art to be variations that do not significantly affect the ability of the oligonucleotide to determine miRNA levels. For example, homologs and variants of oligonucleotide molecules can possess a relatively high degree of sequence identity when aligned using the method set forth above. Oligonucleotide sequences encompassed herein can have at least 40% (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) sequence identity to the sequence of the miRNAs described herein. In some embodiments, an oligonucleotide can include a first portion for detecting a miRNA, as well as a second portion that can be, for example, for attaching the oligonucleotide to a substrate. In some embodiments, the second portion can comprise a non-specific sequence (e.g., polyT) for increasing the distance between the complementary sequence portion and the surface of the substrate.

Oligonucleotides for systems described herein can include, for example, DNA, RNA, PNA, LNA, combinations thereof, and/or modified forms thereof. Oligonucleotides also can include a modified backbone. In some embodiments, an oligonucleotide can comprise at least about nine, ten, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 continuous nucleotide segments that are complementary or identical to all or part of a miRNA described herein. A single oligonucleotide can include two or more such complementary sequences. In some embodiments, there can be a reactive group (e.g., an amine) attached to the 5' or 3' end of an oligonucleotide for attaching the oligonuceotide to a substrate.

In some embodiments, a system can include one or more pairs of primers for qPCR (also referred to as real time PCR (RT-PCR) or kinetic polymerase chain reaction). qPCR permits both detection and quantification of specific DNA sequence in a sample, as an absolute number of copies or as a relative amount when normalized to DNA input or other normalizing genes). A key feature of qPCR is that the amplified DNA product is quantified in real time as it accumulates in the reaction after each amplification cycle. Methods of quantification include the use of fluorescent dyes that intercalate with double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. Methods for qPCR are known in the art.

In some cases, a system can include one or more probes that can be used to detect one or more miRNAs in a tissue by in situ hybridization. For in situ hybridization, a labeled probe is used to localize a specific DNA or RNA sequence in a portion or section of tissue. Sample cells and tissues can be treated to fix the target nucleic acids in place, and to increase access of the probe. A probe can be a labeled complementary DNA or a complementary RNA (riboprobe). The probe can hybridize to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe). Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-specifically bound probe. The probe can be labeled (e.g., with radio-, fluorescent- or antigen-labeled bases such as digoxigenin) can be localized and quantitated in the tissue using autoradiography, fluorescence microscopy or immunohistochemistry, for example. In situ hybridization also can include the use of two or more labeled probes to simultaneously detect two or more transcripts.

In some embodiments, a system can be a microarray of probes. The terms "microarray" and "array" are used interchangeably herein, and refer to a surface comprising an array (e.g., an ordered array) of putative binding (e.g., by hybridization) sites for a biochemical sample (target) that may have undetermined characteristics. In some embodiments, a microarray can refers to an assembly of distinct oligonucleotide probes immobilized at defined positions on a substrate.

For example, in some embodiments, a microarray can comprise a plurality of probes, wherein each probe is capable of detecting a different miRNA in a sample, and wherein at least about 15% (at least about 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) of the probes are capable of detecting a miRNA listed in Table 1, or a corresponding homologue.

In some embodiments, microarrays for determining gene status of miRNAs genes corresponding to miRNAs disclosed herein are provided. Microarrays for determining gene status include, for example, those known in the art. For example, a system can include sequence-tagged molecular inversion probes for determining gene status.

Arrays can be formed on substrates fabricated with materials such as paper, glass, plastic (e.g., polypropylene, nylon, or polystyrene), polyacrylamide, nitrocellulose, silicon, optical fiber, or any other suitable solid or semisolid support, and can be configured in a planar (e.g., glass plates, silicon chips) or three dimensional (e.g., pins, fibers, beads, particles, microtiter wells, capillaries) configuration.

In some embodiments, probes can be oligonucleotides. Oligonucleotides forming an array may be attached to a substrate by any number of techniques, including, without limitation, (i) in situ synthesis (e.g., high-density oligonucleotide arrays) using photolithographic techniques; (ii) spotting/printing at medium to low density on glass, nylon or nitrocellulose; (iii) by masking, and (iv) by dot-blotting on a nylon or nitrocellulose hybridization membrane. Oligonucleotides also can be non-covalently immobilized on a substrate by hybridization to anchors, by means of magnetic beads, or in a fluid phase such as in microtiter wells or capillaries.

Several techniques are well known in the art for attaching nucleic acids to a solid substrate such as a glass slide. For example, one method is to incorporate into the amplified nucleic acids modified bases or analogs that contain a moiety capable of attachment to a solid substrate, such as an amine group, a derivative of an amine group or another group with a positive charge. The amplified product then can be contacted with a solid substrate (e.g., a glass slide) coated with an aldehyde or another reactive group that will form a covalent link with the reactive group on the amplified product, such that the amplified product becomes covalently attached to the glass slide. Microarrays containing amplified products can be fabricated using, for example, a Biodot (BioDot, Inc., Irvine, CA) spotting apparatus and aldehyde-coated glass slides (CEL Associates, Houston, TX). Amplification products can be spotted onto the aldehyde-coated slides, and can be processed according to published procedures (Schena et al., Proc. Natl. Acad. ScL U.S.A. (1995) 93:10614-10619). Arrays also can be printed by robotics onto glass, nylon (Ramsay, Nature Biotechnol. (1998), 16:40-44), polypropylene (Matson et al., Anal. Biochem. (1995), 224(1):110-6), and silicone slides (Marshall and Hodgson, Nature Biotechnol. (1998), 16:27-31). Other approaches to array assembly include fine micropipetting within electric fields (Marshall and Hodgson, supra), and spotting polynucleotides directly onto positively coated plates. Methods such as those using amino propyl silicon surface chemistry also are known in the art, as disclosed, for example, on the World Wide Web at cmt.corning.com and cmgm.stanford.edu/pbrown/.

One method for making microarrays is by making high-density nucleotide arrays. Techniques are known for rapid deposition of polynucleotides (Blanchard et al., Biosenesors & Bioelectronics, 11 :687-690). Other methods for making microarrays, e.g., by masking (Maskos and Southern, Nucleic. Acids. Res. (1992), 20:1679-1684), also can be used. In principle, and as noted above, any type of array (e.g., dot blots on a nylon hybridization membrane) can be used. As will be recognized by those skilled in the art, however, very small arrays can be particularly useful, as hybridization volumes will be smaller.

Kits

This document also provides kits for use in the methods described herein. In some embodiments, a kit can contain a system (e.g., one or more primer pairs for qPCR, one or more probes for in situ hybridization, or a microarray) for measuring miRNA levels as described herein. In some embodiments, a kit can further include additional reagents for carrying out the assays. A kit can further include an instruction or user manual detailing methods for performing the methods disclosed herein, and/or a reference to a site on the Internet where such instructions may be obtained.

In some embodiments, a kit can contain a system (e.g., one or more primer pairs for qPCR, one or more probes for in situ hybridization, or a microarray) as described herein for diagnosing lung cancer. A kit can further include one or more control samples for determination of a reference level, and/or information about obtaining a reference level. In some embodiments, a kit can further include instructions on use of the kits for diagnosing lung cancer, as described herein. In some embodiments, a kit can contain a system (e.g., one or more primer pairs for qPCR, one or more probes for in situ hybridization, or a microarray) as described herein for classifying individuals having lung cancer. A kit can further include one or more control ^• samples for classifying individuals, and/or information about control samples, and in some embodiments, instructions for use of the kit for classifying individuals. In some embodiments, there is provided herein a kit for determining a prognosis for survival of an individual having lung cancer. Such a kit can include, for example, primers and/or probes that detect one or more miRNAs (e.g., one or more miRNAs listed in Tables 1 and 2). In some embodiments, a kit can further comprise a control sample for determination of a threshold level, and/or information about obtaining a threshold level. In some embodiments, a kit can further comprise instructions for use of the kit to determine prognosis for survival of an individual. In some embodiments, a kit can further include one or more agents that alter (e.g., increase or decrease) the levels of one or more miRNAs, or pharmaceutical compositions comprising such agents for improvement of survival.

The kits described herein can further include reagents such as, without limitation, substrates, labels, primers, reagents for labeling miRNAs, reagents for isolating miRNA, negative or positive controls for hybridization and detection, tubes and/or other accessories, reagents for collecting tissue sample, buffers, hybridization chambers, cover slips, etc., and may also contain a software package (e.g., for analyzing miRNA levels and/or characteristic changes in miRNA levels using statistical methods as described herein), and optionally a password and/or account number for assessing the compiled database.

In some embodiments, a kit can include a pharmaceutical composition comprising an agent that alters (e.g., decreases) the level of a miRNA listed in Table 1, and instructions for using the composition for improvement of survival in an individual having lung cancer. In some embodiments, a kit can further include one or more vectors or other agents for delivery of the composition. In some embodiments, a kit can further include instructions for administration of the pharmaceutical composition.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLE 1 - Preparation of samples for analysis of miRNA levels. Patients and samples: One hundred and sixteen (116) pair of primary lung cancer tissues and matching non-cancerous lung tissues were randomly selected. Of these 116 specimens, 60 were SCC, 43 were adenocarcinomas and 13 were SCLC. These specimens were obtained from untreated patients undergoing surgery. Samples were snap-frozen in liquid nitrogen immediately after resection and stored (for a minimum of 5 years) at -80 °C until RNA extraction. Samples from a further 20 cases of primary lung cancer and corresponding non-cancerous lung tissues with follow-up information (minimum storage of 5 years) were used for an independent validation of survival analysis. Peripheral portions of the resected lung samples were paraffin embedded, sectioned, and hematoxylin and eosin (H&E) stained using routine methods. Tumor cell concentrations were evaluated, and tumor histology was independently confirmed by two pathologists. Follow-up information was extracted from a follow-up registry. For all samples, clinicopathological information (smoking, age, sex, pathological subtype, TNM classification, tumor stage, lymph node stage, differentiation status, and duration of survival after surgery) was available.

Fabrication ofmiRNA microarray: Altogether, 509 mature miRNA sequences were assembled and integrated into the miRNA microarray design. These included 435 human mature miRNAs (including a further 122 predicted miRNA sequences from published references (Xie et al., 2005)), 196 rat mature miRNAs, and 261 mouse mature miRNAs from the miRNA Registry at the Wellcome Trust Sanger Institute, Cambridge, UK (online at http://microrna.sanger.ac.uk). In addition, eight short oligonucleotides were designed that possessed no homology to any known RNA sequence, and their corresponding synthetic miRNAs were produced by in vitro transcription using Ambion's miRNA Probe Construction kit (Cat. No.1550; Ambion, Inc., Austin, TX). Various amounts of these synthetic miRNAs were added to the human miRNA samples prior to analysis, as internal controls.

All of the miRNA probe sequences were designed to be fully complementary to their cognate full-length mature miRNA. To facilitate probe immobilization onto the aldehyde" modified-surface of the glass slides (CapitalBio Corp., Beijing, China), probe sequences were concatenated up to a length of 40 nt (3'-end miRNA probe plus 5'-end 19mer polyT) with C6 5'-amino-modifier. Oligonucleotide probes were synthesized at MWG Biotech (Ebersberg, Germany), and were dissolved in Easy Array™ spotting solution (CapitalBio Corp.) at a concentration of 40 μM. Each probe was printed in triplicate using a SmartArray™ microarrayer (CapitalBio Corp.).

Labeling of target RNAs: Total RNA was extracted with TRIZOL reagent (Invitrogen; Carlsbad, CA) and the low-molecular-weight RNA was isolated by using an Ambion miRNA Isolation Kit. The T4 RNA ligase labeling method was used according to the Thomson protocol (Thomson et al., 2004). In brief, 4 μg of low-molecular- weight RNA was labeled with 500 ng of 5'-phosphate-cytidyl-uridyl-cy3-3' (Dharmacon; Lafayette, CO) with 2 units T4 RNA ligase (New England Biolabs, Beijing, China). The labeling reaction was performed at 4 ⁰ C for 2 hours. Labeled RNA was precipitated with 0.3 M sodium acetate and 2.5 volumes ethanol, and after washing with ethanol and drying, the labeled RNA was resuspended in 15 μl of hybridization buffer containing 3 ^χ SSC, 0.2% SDS and 15% formamide.

Slide Hybridization: Hybridization was performed under LIFTERSLIP ^{^} (Erie; Portsmouth, NH) in a hybridization chamber placed in a three-phase-tilting agitator

BIOMIXER-' (CapitalBio Corp.) to provide continuous mixing of the hybridization buffer, which resulted in more uniform hybridization across the entire slide surface and prevented edge effects. Hybridization was performed overnight in a water-bath at 42°C. The array was then washed with two consecutive washing solutions of 0.2% SDS, 2 ^χ SSC at 42°C for 5 minutes, and 0.2% SSC for 5 minutes at room temperature. Arrays were scanned with a confocal LUXSCAN-" scanner, and the images obtained were then analyzed using LUXSCAN 3.0 ^{^} software (both from CapitalBio Corp.).

Computational Analysis: For all samples, the average background values were subtracted from the replicate spots of each miRNA. Faint spots were filtered out if the expression signal was <1500. Signals were median-center normalized. Differentially expressed miRNAs were identified by SAM (Significance Analysis of Microarrays; available on the World Wide Web at stat.stanford.edu/~tibs/SAM/index.html. The miRNA sufficient minimum marker set to classify cancerous and adjacent normal tissues was analyzed by a PCA (Principal Component Analysis) and SVM (Support Vector Machine) method. The possible gene function of the most significant predicted miRNA targets were analyzed using four publicly available algorithms, i.e., miRBase (available online at microrna.sanger.ac.uk/sequences/), MIRANDA (available on the World Wide Web at microrna.org/), TARGETSCAN (available on the World Wide Web at targetscan.org/), and PICTAR (available online at pictar.bio.nyu.edu/). To reduce the number of false positives, only putative target genes predicted by at least three of the programs were accepted. Patient survival curves were estimated by the Kaplan-Meier method. The joint effect of covariables was examined using the Cox Proportional Hazard Regression Model.

Quantitative Reverse Transcription Polymerase Chain Reaction Analysis: For verification of miRNA expression profiles, total cellular RNAs were subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) with miRNA specific primers. The extraction of total RNA, reverse transcription, and PCR were performed according to the procedures described previously.

EXAMPLE 2 - miRNA expression can distinguish malignant tissues from adjacent normal tissues in lung cancers Studies were conducted to investigate whether miRNA expression can distinguish malignant tissues from adjacent normal tissues in lung cancers and SCC and adenocarcinoma, using 116 pairs of primary lung cancers (60 SCC, 43 adenocarcinoma and 13 SCLC), 60 pairs of SCC, and 43 pairs of adenocarcinoma, with corresponding adjacent normal lung tissues collected a minimum of 5 cm from the tumor. Tissues were initially snap-frozen in liquid nitrogen, and were stored frozen at -80 ⁰ C for a minimum of 5 years until analysis. The 116 pairs of primary lung cancers, 60 pairs of SCC, and 43 pairs of adenocarcinoma were randomly divided into training cohorts (66 pairs of lung cancer, 30 pairs of SCC, 23 pairs of adenocarcinoma) and test cohorts (50 pairs of lung cancer, 30 pairs of SCC, 20 pairs of adenocarcinoma). Faint spots were filtered out if the expression signal was <1500, and 29 miRNAs were chosen from the training data by SAM with q value = 0 and fold change > 2. PCA-SVM strategy was used to establish a classifier and to get the highest scored values with a set of five miRNAs (hsa-miR-486-5p, hsa-miR-210, hsa-miR-30a, hsa-miR-140-3p, and hsa- miR-182). These analyses revealed an accuracy of 98.2% for lung cancer, 93.3% for SCC, and 97.8% for adenocarcinoma in the original training set (Figures IA, 1C, and IE). Among these five miRNAs, three miRNAs (hsa-miR-210, hsa-miR-30a, and hsa-miR-182) showed up- regulation in cancer versus adjacent normal tissue, and two miRNAs (hsa-miR-486-5p and hsa- miR-140-3p) showed reduction in cancer versus adjacent normal tissue (Table 1). After establishing the classifier, the test cohort was assessed to confirm the modeling strategy. The accuracy was 92% in the test set for lung cancer, 96.7% for SCC, and 90% for adenocarcinoma (Figures IB, ID, and IF). Overall, the results showed that the classifier could efficiently distinguish malignant tissues from normal tissues in lung cancers, SCC, and adenocarcinoma with as few as five miRNA markers.

EXAMPLE 3 - miRNA expression does not distinguish different pathological subtypes of lung cancers

Studies were conducted to determine whether miRNA expression could distinguish the three pathological subtypes of lung cancer: SCC, adenocarcinoma, and SCLC. Differentially expressed miRNAs in each cancer were selected by SAM, based on the miRNA ratio of cancerous (C) tissues versus corresponding adjacent normal (N) tissues, and on the miRNA signal of cancerous tissues. Sixty-eight (68) miRNAs were differentially expressed among the three subtypes based on the C:N ratio, and 70 miRNAs were differentially expressed based on the strength of miRNA signals in cancerous tissues. The 60 SCC, 43 adenocarcinoma, and 13 SCLC samples were analyzed using unsupervised hierarchical clustering with the differentially expressed miRNAs. The three pathological subtypes of lung cancers in this study could not be clearly divided into three groups by their miRNA expression profiles, although it was notable that the SCLC samples tended to cluster into one group (Figure T). Importantly, this analysis suggested that the SCC miRNA expression profile is not significantly divergent from the profile of adenocarcinoma. In contrast, despite the small sample set, the analysis also suggested that the expression pattern of miRNAs in SCLC might be distinguishable from NSCLC.

EXAMPLE 4 — miRNA expression is associated with pathological and clinical features of lung cancer

Further studies were conducted to examine whether the microarray data revealed specific miRNA signatures for subsets of lung cancer that differed in their clinico-pathological status. These studies included comparison of miRNA expression in several group pairs, including smokers/non-smokers, different age groups, genders, pathological classifications, differentiation classifications, TNM classifications, tumor stage, lymph node stage, and tumor stage classifications, as listed in Table 2. SAM tools were used for the analyses, based on the miRNA ratio of C:N and the miRNA signal of cancerous tissues, respectively. Several miRNAs (Table 3) were identified by both direct signal strength and by ratio. Notably, three miRNAs (hsa-miR-205, hsa-miR-203, and hsa-miR-18b) had consistently low expression in SCC compared to adenocarcinoma. Two miRNAs (hsa-miR-205 and hsa-miR-181a) were differentially expressed between female and male patients with SCC. Two miRNAs (hsa-miR- 205 and hsa-miR-31) were differentially expressed among high versus middle versus low differentiation classification in SCC. One miRNA (hsa-miR-205) was differentially expressed in patients with a smoking index <400 per year versus a smoking index >400 per year in NSCLC. Four clinicopatholigical indications (histological subtype, gender, differentiation and smoking index) were not found to have significant statistical associations. Interestingly, hsa- miR-205 expression was prominently related to four different clinicopathological indications in lung cancer. No particular miRNA correlated with the TNM or individual T stages in the data set.

EXAMPLE 5 - Correlation between hsa-miR-31 expression and prognosis in SCC The correlation of miRNA expression profiles with patient survival was investigated. Thirty-seven (37) miRNAs were differentially expressed between SCC and corresponding adjacent normal tissues, 22 miRNAs were differentially expressed between adenocarcinoma and corresponding adjacent normal tissues, and 29 miRNAs were differentially expressed between non-small cell lung cancer (NSCLC) and corresponding adjacent normal tissues. AU of the differentially expressed miRNAs were used for Kaplan-Meier survival analysis. The median miRNA C:N values for the initial sets of 60 SCC and 43 adenocarcinomas, and the combined set of 103 NSCLC patient samples, were used as cut-points. Kaplan-Meier survival analysis showed that a high C:N expression ratio for hsa-miR-31 was associated with poor survival in SCC (p = 0.007; log-rank test, training cohort with 60 patients sample, Figure 3A). Further univariate and multivariate Cox analysis confirmed that high hsa-mir-31 expression was correlated with lower SCC survival rates than low hsa-mir-31 expression (Table 2).

Univariate Cox analysis with hsa-miR-31 and clinicopathological factors (smoking index, age, gender, differentiation, TNM classification) revealed that hsa-miR-31 expression level (p=0.011) and TNM (p=0.013) had prognostic significance in the training cohort (Table 4). Subsequently, a multivariate Cox proportional hazard regression analysis using these clinicopathological and molecular factors indicated that high hsa-miR-31 expression was a significantly unfavorable prognostic factor independent of other clinicopathological factors (p =0.021; risk ratio 3.05; 95% confidence interval [CI], 1.187-7.838), whereas TNM was not strongly associated with a poor patient outcome (p=0.179) (Table 5). The correlation of miRNAs with survival of adenocarcinoma and NSCLC patients was further investigated, and no miRNAs were observed to be correlated with survival.

The correlation of hsa-miR-31 with the prognosis of SCC patients was analyzed with an independent set of 20 SCC tissues. miRNA expression levels were analyzed by qRT-PCR. 'Kaplan-Meier survival analysis (Figure 3B) confirmed a significant decrease in survival for patients with high hsa-miR-31 expression (p=0.001 ; log-rank test, test cohort with 20 paired patients tissues). Univariate (p=0.007) and multivariate (p=0.029) Cox proportional hazard regression analyses also suggested that high hsa-miR-31 expression is an independent predictor of poor prognosis for SCC (Table 5).

Table 2. Comparison of clinicopathological classifications

Classifications (number) Total No. of No. of No. of FDR miRNAs miRNAs miRNAs (Intensity) (Ratio) (same * )

Phenotypic Classification

NSCLC (103) vs. corresponding (103) 206 29 0

SCC (60) vs. corresponding (60) 120 37 0

Adeno (43) vs. corresponding (43) 86 22 0

Histological Classification

SCC (60) vs. Adeno (43) 103 5 10 3 0

Age classification (median age years)

NSCLC; age<58 (49) vs. age>58 (54) 103 0 5 0 0

SCC; age<58 (28) vs. age>58 (32) 60 0 0 0 0

Adeno; age<58 (21) vs. age>58 (22) 43 0 0 0 0

Sex classification

NSCLC; male (70) vs. female (33) 103 4 4 0 0

SCC; male (50) vs. female (10) 60 3 2 2 0

Adeno; male (19) vs. female (24) 43 1 0 0 0

Stage (TNM) classification

NSCLC

I (34) vs. II (22) vs. Ill and IV (47) 103 0 0 0 0

SCC

I (21) vs. II (17) vs. Ill and IV (22) 60 0 0 0 0

Adeno

I (13) vs. II (6) vs. Ill and IV (24) 43 0 0 0 0

Stage (T) classification

NCSLC; 1 (5) vs. 2 (80) vs. 3 (18) 103 0 0 0 0

SCC; 1 (2) vs. 2 (45) vs. 3 (13) 60 0 0 0 0

Adeno; 1 (3) vs. 2 (35) vs. 3 (5) 43 0 0 0 0

Stage (N) classification

NSCLC; 0 (39) vs. 1 (23) vs. 2 (41) 103 3 0 0 0

SCC; 0 (26) vs. 1 (15) vs. 2 (19) 60 0 0 0 0

Adeno; 0 (13) vs. 1 (8) vs. 2 (22) 43 0 0 0 0

Differentiation classification

NSCLC high (10) vs. middle (54) vs. low (39) 103

SCC high (5) vs. middle (33) vs. low (22) 60 27

Adeno high (5) vs. middle (21) vs. low (17) 43

Smoke classification (Smoking Index 400 per year)

NSCLC

<400 per year (51 ) vs. >400 per year(52) 103

SCC

<400 per year (17) vs. >400 per year(43) 60

Adeno

<400 per year (34) vs. >400 per year (9) 43 3 0 0

NOTE: All miRNAs were selected by SAM analysis.

Intensity: Each miRNA was selected based on miRNA signal strength in cancer tissues.

Ratio: Each miRNA was selected based on the ratio of the miRNA signal in cancer tissues versus the signal in paired adjacent normal tissues.

Same * : No. of miRNAs are same between Intensity and Ratio.

Abbreviations: Adeno, adenocarcinoma; NSCLC, non-small cell lung cancer; SCC, squamous cell carcinoma.

Table 4: Post-operative survival of patients with SCC in relation to clinicopathological characteristics and miRNA expression analyzed by the Cox proportional hazard regression model in a training cohort of 60 cases. Univariate analysis Multivariate analysis

Hazard ratio (95% CI) P Hazard ratio (95% CI) P

Smoking Index >400 1.014 (0.385-2.668) 0.978 1.351 (0. 477-3.831) 0. 571

Age >59 1.111 (0.528-2.338) 0.781 1.053(0. 478-2.324) 0. 897

Sex MZF 1.265 (0.481-3.331) 0.634 1.710 (0. 595-4.915) 0. 319

TNM I/II/III 1.794 (1.131-2.845) 0.013 1.394 (0. 816-2.379) 0. 224

Differentiation high/middle/low 1.283 (0.656-2.508) 0.466 1.687 (0. 734-3.879) 0. 283 miKNA-31 high/low 2.827 (1.274-6.275) 0.011 3.278 (1.262-8.517) 0. 015

Abbreviations: 95% CI, 95% confidence interval.

Table 5: Post-operative survival of patients with SCC in relation to clinicopathological characteristics and miRNA expression analyzed by the Cox proportional hazard regression model in an independent test cohort of 20 cases

Univariate analysis Multivariate analysis

Hazard ratio (95% CI) P Hazard ratio (95% CI) P

Smoking Index >400 2,668 (0.583-12.198) 0.206 7.129 (0.888-57.233) 0.065

Age >60 0.559 (0.175-1.784) 0.326 1.082 (0.233-5.013) 0.920

TNM I/II/III 3.093 (1.296-7.382) 0.530 2.910 (0.772-10.961) 0.114

Differentiation High/middle/low 2.310 (0.773-6.901) 0.134 15.156 (1.759-130.605) 0.013 miRNA-31 High/low 8.067 (1.754-37.014) 0.007 10.320 (1.277-83.417) 0.029

Abbreviation: 95% CI, 95% confidence interval.

While this document contains many specifics, these should not be construed as limitations on the scope of the invention or of what may be claimed, but rather as descriptions of features specific to particular embodiments of the invention. Certain features that are described in this document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination. Only a few embodiments axe disclosed. Variations and enhancements of the disclosed embodiments and other embodiments can be made based on what is described and illustrated in this document.