WO2008064519A1 - Méthodes et compositions permettant de diagnostiquer un cancer de l'oesophage, d'établir un pronostic et d'améliorer la survie des patients - Google Patents

Méthodes et compositions permettant de diagnostiquer un cancer de l'oesophage, d'établir un pronostic et d'améliorer la survie des patients Download PDF

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WO2008064519A1
WO2008064519A1 PCT/CN2006/003195 CN2006003195W WO2008064519A1 WO 2008064519 A1 WO2008064519 A1 WO 2008064519A1 CN 2006003195 W CN2006003195 W CN 2006003195W WO 2008064519 A1 WO2008064519 A1 WO 2008064519A1
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mirna
mir
level
hsa
individual
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Yong Guo
Zhaoli Chen
Jing Cheng
Jie He
Keith Mitchelson
Liang Zhang
Xin Meng
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Capitalbio Corporation
Tsinghua University
Cancer Hospital And Institute, Peking Union Medical College And Chinese Academy Of Medical Sciences
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Priority to PCT/CN2006/003195 priority Critical patent/WO2008064519A1/fr
Priority to JP2009537466A priority patent/JP2010510769A/ja
Priority to CN2006800198158A priority patent/CN101316935B/zh
Publication of WO2008064519A1 publication Critical patent/WO2008064519A1/fr

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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • TECHNICAL FIELD [0001] This application pertains to methods for diagnosis of disease (such as esophageal cancer) and prognosis and improvement of patient survival based on the levels of microRNAs.
  • Esophageal cancer is the third most common cancer of the digestive tract and the seventh leading cause of cancer-related deaths worldwide.
  • the most common types of esophageal cancer are squamous cell carcinoma and adenocarcinoma.
  • Squamous cell carcinoma develops in flat cells that line the esophagus.
  • Adenocarcinoma develops in the lining of the esophagus and is associated with a condition called Barrett's esophagus.
  • Esophageal squamous cell cancer has a well-defined progression from preinvasive dysplasia to invasive squamous cell carcinoma, with metastases frequently occur at later stages (Wen et al., Fam. Cancer.
  • microRNAs are small noncoding single-stranded RNAs of about 22 nucleotide long which, through partial or complete sequence homology, may interact with the 3' untranslated .region (3'-UTR) of target niRNA molecules (Bartel, Cell 2004, 116: 281-297). The interaction between miRNAs and their target mRNA block the translation of the mRNA into protein. In some instances when the target and the miRNA match exactly, degradation of the mRNA may also occur. miRNAs have been implicated in a broad range of cellular processes, such as cell differentiation, cell growth, and cell death (Cheng et al,
  • miRNA genes are often found associated with genomic sites that are subject to modification in cancers, regions such as fragile sites (FRAs), breakpoint sites and regions, minimal regions of genomic amplification as well as in minimal regions of loss of heterogeneity (Calin et al, Proc. Natl.
  • Changes in the expression of miRNAs have been identified in a number of cancers, such as chronic lymphoid leukemia (Calin et al., Proc Natl Acad Sci USA. 2004, 101:11755-11760), colon cancer (Bandres et al., MoI Cancer 2006, 5:29), glioblastoma (Ciafre et al., Biochem. Biophys. Res. Commun.
  • the present invention provides methods and compositions for cancer diagnosis and prognosis, particularly esophageal cancer diagnosis and prognosis, based on the levels of miRNAs or the gene status of corresponding miRNA genes. Also provided are uses of probes that detect miRNA or the gene status of corresponding genes for diagnosis and prognosis of cancer, particularly esophageal cancer.
  • the present invention provides methods of diagnosing cancer in an individual, comprising a) determining the level of at least one miRNA in a 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 cancer.
  • a method of diagnosing esophageal cancer in an individual comprising: a) determining the level of at least one miRNA (such as at least one miRNA shown in Figure 1 or their corresponding homologues) in an esophageal tissue sample of 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 esophageal cancer.
  • the cancer is esophageal squamous cell cancer.
  • the cancer is esophageal adenocarcinoma.
  • the miRNA is not miR-29b, miR-29a, miR-96, miR-182, miR-182a, miR-183, and miR- 129-1. In some embodiments, the miRNA is not miR-15 and miR-16.
  • the level of at least one (such as at least any of 2, 5, 10, 14) of the miRNAs of SEQ ID Nos. 1-14 or their corresponding homologues are determined, wherein a substantial increase in the levels of at least one of the measured miRNAs is indicative of esophageal cancer.
  • the levels of at least one (such as at least any of 2, 5, 10, 15, 20, 24) of the miRNAs of SEQ ID Nos. 15-38 or their corresponding homologies are determined, wherein a substantial decrease in the levels of at least one of the measured miRNAs is indicative of esophageal cancer.
  • the levels of at least one (such as at least any of 2, 5, 10, 14) of the miRNAs of SEQ ID Nos. 1-14 or their corresponding homologues and at least one (such as at least any of 2, 5, 10, 15, 20, 24) of the miRNAs of SEQ ID Nos. 15-38 or their corresponding homologues are determined, wherein a substantial increase in the levels of at least one of the miRNA from Nos. 1-14 of Figure 1 or their corresponding homologues and a substantial decrease in the levels of at least one of the miRNAs from Nos. 15-38 is or their corresponding homologues indicative of esophageal cancer.
  • the miRNA level is determined by a microarray analysis.
  • the miRNA level is determined based on the hybridization signal of the miRNA on the microarray. In some embodiments, the miRNA level is determined based on the ratio of the hybridization signal to the miRNA on the microarray to that of a reference sample. In some embodiments, the miRNA level is determined by any of Northern blot analysis, in situ hybridization, and quantitative reverse transcriptase polymerase chain reaction.
  • a method of diagnosing esophageal cancer in an individual comprising analyzing the gene status of at least one miRNA gene (such as at least one miRNA gene corresponding to an miRNA shown in Figure 1 or their corresponding homologues) in an esophageal 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 is indicative of esophageal cancer.
  • the change of the gene status is determined based on a deletion or amplification of the miRNA gene.
  • the change of the gene status is determined based on the change in gene copy number of the miRNA gene.
  • a system for determining the level of at least one miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA) in a sample
  • the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA) in an esophageal tissue sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • a system for diagnosis of esophageal cancer
  • the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 (or the gene status of the corresponding miRNA).
  • a system for diagnosing esophageal cancer comprising at least one (including for example at least any of 2, 5, 10, 15, 20, 25, 30, 35, and 40) probes (such as oligonucleotides), wherein each of the probes detects the level of a different miRNA shown in Figure 1 (or the gene status of the corresponding miRNA), and wherein a characteristic change of in the level of at least one of the miRNAs shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA) is indicative of esophageal cancer.
  • probes such as oligonucleotides
  • a system for diagnosing esophageal cancer
  • the system comprises a plurality of probes, wherein 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 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA), and wherein a characteristic change in the level of at least one of the miRNAs shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA) is indicative of esophageal cancer.
  • probes that detect miRNAs for the manufacture of systems described herein.
  • probes such as oligonucleotides
  • each probe is capable of determining the level of a miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • probes such as oligonucleotides
  • a system such as a microarray
  • each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probe are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • a method of classifying esophageal patients comprising determining the level of at least one miRNA (such as miRNA shown in Table 2 or their corresponding homologues) or the gene status of the corresponding miRNA gene in an esophageal cancer tissue of the individual, wherein the level of the miRNA (or the gene status of the corresponding miRNA gene) is used as a basis for classifying the esophageal patient.
  • miRNA such as miRNA shown in Table 2 or their corresponding homologues
  • the level of the miRNA or the gene status of the corresponding miRNA gene
  • a method for determining the level of differentiation of esophageal cancer in an individual comprising determining the level of an miRNA shown in Figure 2a or their corresponding homologues in an esophageal cancer tissue of the individual, wherein the level of the miRNA is used as a basis for determining the level of differentiation of esophageal cancer in the individual.
  • at least one miRNA is hsa-miR-335 or its corresponding homologue.
  • at least one miRNA is hsa-miR-25 or its corresponding homologue.
  • at least one miRNA is hsa-130b or its corresponding homologue.
  • at least one miRNA is hsa-miR-130a.
  • at least one miRNA is hsa-miR- 181 d or its corresponding homologue.
  • a system for determining the level of at least one miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene) in a sample
  • the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA gene) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a system for classifying esophageal cancer patients, wherein the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA gene) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a use of a system for classifying an individual having esophageal cancer wherein the system comprises one or more probes, and wherein each probe is capable of determining the level of an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • the system comprises one or more probes, wherein each of the probes is capable of detecting a different miRNA in the sample, and wherein at least about 50% of the probes are capable of detecting a miRNA shown in Table 2 or their corresponding homologues.
  • each probe is capable of determining the level of a miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • probes for the manufacture of a system for classifying an individual having esophageal cancer
  • each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA gene) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • the system is useful for determining the level of differentiation of esophageal cancer in an individual having esophageal cancer.
  • the system is useful for pathologically classifying an individual having esophageal squamous cell cancer. In some embodiments, the system is useful for determining the tumor stage of an individual having esophageal cancer. [0024]
  • the invention in another aspect provides methods of prognosing esophageal cancer patients, including for example methods of determining a prognosis for survival of an individual having esophageal cancer.
  • a method for determining a prognosis for survival for an individual having esophageal cancer comprising: (a) determining the level of at least one miRNA in an esophageal cancer tissue sample from the individual, and (b) comparing the level of the miRNA in said sample to a threshold level, wherein the level of the miRNA as compared to a threshold level correlates or reversely correlates with the survival of the individual.
  • a method of determining a prognosis for survival for an individual having esophageal cancer comprising analyzing the gene status of at least one miRNA genes (such as a miRNA gene corresponding to any of hsa-miR-103, hsa-miR-107, and hsa-miR-23b), wherein a change in gene status as compared to a that of a control sample indicates a high or low survival of the individual.
  • miRNA genes such as a miRNA gene corresponding to any of hsa-miR-103, hsa-miR-107, and hsa-miR-23b
  • At least one miRNA is hsa-miR-103, hsa-miR-107, or hsa- miR-23b or their corresponding homologues. In some embodiments, at least one miRNA is hsa-miR-103. In some embodiments, at least one miRNA is hsa-miR-107. In some embodiments, at least one miRNA is hsa-miR-23b. In some embodiments, the survival is overall survival. In some embodiments, the survival is disease-free survival. In some embodiments, the individual has an early stage of esophageal cancer. In some embodiments, the method further comprises determining a proper course of treatment for the individual.
  • probes that are capable of detecting the levels of the miRNAs (or the gene status of the corresponding miRNA gene) or systems comprising one or more probes for determining a prognosis for survival.
  • probes capable of detecting the levels of the miRNAs (or the gene status of the corresponding miRNA gene) or systems comprising one or more probes for determining a prognosis for survival.
  • the probe is capable of detecting an 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 said individual.
  • a use of one or more probes for determining prognosis of survival of an individual having esophageal cancer wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual, and wherein at least one miRNA is hsa-miR-103, hsa-miR- 107, or hsa-miR-23b or their corresponding homologues.
  • probes for the manufacture of an agent (or system) for determining a prognosis of survival of an individual having esophageal cancer, wherein the probe is capable of detecting an 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 said individual.
  • a use of one or more probe for the manufacture of an agent (or system) for determining prognosis of survival of an individual having esophageal cancer wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual, and wherein at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa- miR-23b or their corresponding homologues. In some embodiments, at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b.
  • a method of improving survival of an individual having esophageal cancer comprising administering to the individual an effective amount of an agent that decreases the level of an miRNA, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual.
  • an agent that decreases the level of an miRNA for the manufacture of a medicament for improving survival of an individual having esophageal cancer, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual.
  • a method of improving survival of an individual having esophageal cancer comprising 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-103, hsa-miR-107, hsa-miR-23b, and their corresponding homologues.
  • an agent for the manufacture of a medicament for improving survival of an individual having esophageal cancer wherein the agent decreases the level of a miRNA selected from the group consisting of hsa-miR-103, hsa-miR-107, hsa- miR-23b, and their corresponding homologues.
  • the agent(s) decrease the levels of at least two miRNAs selected from the group consisting of hsa-miR-103, hsa- miR-107, and hsa-miR-23b.
  • the agent(s) decrease the levels of hsa- miR-103, hsa-miR-107, and hsa-miR-23b.
  • a pharmaceutical composition comprising an agent that decreases the level of at least one miRNA and a pharmaceutically acceptable carrier, wherein at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b, or their corresponding homologues.
  • at least one miRNA is hsa-miR-103.
  • at least one miRNA is hsa-miR-107.
  • At least one miRNA is hsa-miR-23b.
  • the agent is a double-stranded RNA (such as short or small-interfering RNA or "siRNA"), an antisenses nucleic acid, or an enzymatic RNA molecules such as ribozyme.
  • siRNA short or small-interfering RNA or "siRNA”
  • the invention also provides kits for methods described herein.
  • Figure 1 provides miRNAs with an altered level in esophageal cancer patients.
  • Figure 2a provides miRNAs with altered levels in esophageal cancer patients at different levels of differentiation.
  • Figure 2b provides miRNAs with altered levels in fugating versus medullary forms of esophageal squamous cell cancer.
  • Figure 2c provides miRNAs with altered levels in different tumor stages (N0/N1) of esophageal cancer.
  • Figure 3 provides miRNAs whose levels correlate with esophageal cancer survival rate.
  • Figure 4 provides hierarchical clustering of the miRNA expression selected by
  • SAMs Significance Analysis of Microarrays
  • FIG. 1 provides a comparison of miRNA expression profiles of fresh tissues and frozen tissues. The miRNA profiles were validated by the 5 paired test samples of fresh tissue. The 10 test samples were validated by using SVM method, and 10 of them were correctly classified. The training samples labeled with # were 'wrongly classified',.
  • Figure 6 provides Kaplan-Meier survival curves for esophageal cancer patients.
  • the present invention is based, in part, on our studies on genome-wide miRNA expression profiling using a paired set of 31 frozen archival normal or cancerous esophageal tissue samples. Specifically, using DNA oligonucleotide microarrays, we have compared the expression of miRNAs of cancer samples with corresponding non-cancerous samples. We have identified 40 miRNAs that were either overexpressed or underexpressed in the cancer samples as compared to the corresponding non-cancerous samples. We have also identified certain miRNAs whose levels are altered in different disease states. In addition, we found that the levels of three miRNAs correlate with the overall and disease-free survival rates of esophageal cancer patients.
  • the invention provides methods and compositions for diagnosis and prognosis of diseases (such as cancer diagnosis and prognosis, particularly cancer diagnosis and prognosis), based on the levels of miRNAs or the gene status of corresponding miRNA genes.
  • diseases such as cancer diagnosis and prognosis, particularly cancer diagnosis and prognosis
  • methods and compositions for diagnosing a disease such as cancer, for example esophageal cancer
  • levels of certain miRNAs such as cancer, for example esophageal cancer
  • compositions for classifying cancer patients particularly esophageal cancer patients, based on pathological forms, levels of differentiation, and tumor stages based on the levels of certain miRNAs.
  • the invention further provides systems and kits useful for methods described 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, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. In some embodiments, the individual is human. In some embodiments, the individual is an animal model for the study of esophageal cancer. It is understood that, when the individual is not human, the miRNA would refer to the corresponding homologs or orthologs of the human miRNA identified herein. [0049] In some embodiments the individual is a male. In some embodiments, the individual is a female. In some embodiments, the individual does not show any pathological phenotypes of esophageal cancer.
  • the individual is has a family history of esophageal cancer.
  • An "esophageal tissue sample” described herein refers to a tissue sample from the esophagus.
  • the tissue sample is a fresh sample.
  • the tissue sample is a frozen sample.
  • the tissue sample is preserved.
  • the tissue sample is formalin preserved.
  • the tissue sample is paraffin embedded. As described below, and depending on the particular method, the tissue can be used whole or subject to various methods known in the art to disassociate the sample into small pieces, cell aggregates or individual cells.
  • Esophageal cancer includes, but is not limited to, esophageal squamous cell cancer or esophageal adenocarcinoma.
  • the present invention in one aspect provides a method of diagnosing a disease (such as cancer) in an individual, comprising: a) determining the level of at least one miRNA in a sample of 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 a disease (such as cancer).
  • Suitable diseases that can be diagnosed in the present invention include, but are not limited to, cancer, eg. 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, testis cancer, cardiovascular disease, eg. coronary heart disease, hypertension, atherosclerosis, age-related disease, eg. Parkinson's disease, Alzheimer's disease, diabetes.
  • cancer eg. 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, testis cancer, cardiovascular disease, eg. coronary heart disease, hypertension, atherosclerosis, age-related disease, eg. Parkinson's disease, Alzheimer's disease, diabetes.
  • cardiovascular disease e
  • the present invention in one aspect provides a method of diagnosing esophageal cancer in an individual, comprising: a) determining the level of at least one miRNA (such as at least one miRNA shown in Figure 1 or their corresponding homologues) in an esophageal tissue sample of 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 esophageal cancer.
  • a miRNA such as at least one miRNA shown in Figure 1 or their corresponding homologues
  • a method of diagnosing esophageal cancer in an individual comprising: a) comparing the level of at least one miRNA (such as at least one miRNA shown in Figure 1 or their corresponding homologues) in a esophageal tissue sample from the individual with a reference level, wherein the tissue is suspected of being cancerous, and b) determining whether the individual has esophageal cancer based on a characteristic change in the level of at least one miRNA.
  • the method further comprises the step of providing an esophageal tissue sample from the individual.
  • the method further comprises isolating miRNAs from the tissue sample.
  • a method of providing information for diagnosis of esophageal cancer in an individual comprising: a) determining the level of at least one miRNA shown in Figure 1 or their corresponding homologues in an esophageal tissue sample of the individual, wherein the tissue is suspected of being cancerous, and b) providing information about the level of the miRNA for diagnosis of esophageal cancer, wherein the level of the miRNA is used as basis for diagnosing esophageal cancer, and wherein a characteristic change in the level of the at least one miRNA is indicative of esophageal cancer.
  • the miRNA is not miR-29b, miR-29a, miR-96, miR-182*, miR-182a*, miR-183, and miR-129-1. In some embodiments, the miRNA is not miR-15 and miR-16.
  • the level of at least one (such as at least any of 2, 5, 10, 14) of the miRNAs of SEQ ID Nos. 1-14 are determined, wherein a substantial increase in the levels of at least one of the measured miRNAs is indicative of esophageal cancer.
  • the levels of at least one (such as at least any of 2, 5, 10, 15, 20, 24) of the miRNAs of SEQ ID Nos. 15-38 are determined, wherein a substantial decrease in the levels of at least one of the measured miRNAs is indicative of esophageal cancer.
  • the levels of all miRNAs shown in Figure 1 are determined, wherein a substantial increase in the levels of at least one of the miRNA of SEQ ID Nos. 1- 14 and a substantial decrease in the levels of at least one of the miRNAs of SEQ ID Nos. 15-
  • a substantial increase in the levels of at least two of the miRNAs of SEQ ID Nos. 1-14 and a substantial decrease in the levels of at least two of the miRNAs of SEQ ID Nos. 15-38 are indicative of esophageal cancer.
  • a substantial increase in the levels of miRNAs of SEQ ID NOs. 1-14 and a substantial decrease in the levels of the miRNAs of SEQ ID NOs. 15-38 are indicative of esophageal cancer.
  • Levels of miRNA in the tissue sample may also reflect changes of the gene status of the miRNAs. Gene status can be reflected, for example, by deletion or amplification of the miRNA gene or change in gene copy number of the miRNA gene.
  • a method of diagnosing esophageal cancer in an individual comprising analyzing the gene status of at least one miRNA gene (such as at least one miRNA gene corresponding to an miRNA shown in Figure 1) in an esophageal 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 is indicative of esophageal cancer.
  • the change of the gene status is determined based on a deletion or amplification of the miRNA gene.
  • the change of the gene status is determined based on the change in gene copy number of the miRNA gene.
  • a method of diagnosing esophageal cancer in an individual comprising analyzing at least one miRNA gene corresponding to at least one miRNA shown in Figure 1 in an esophageal tissue sample suspected of being cancerous from the individual for deletion or amplification, wherein a deletion or amplification of the miRNA gene relative to the corresponding miRNA gene in a control sample is indicative of esophageal cancer.
  • the method comprises analyzing at least one miRNA gene corresponding to at least one miRNA of SEQ ID Nos. 1-14 for amplification, wherein an amplification of the miRNA gene relative to the corresponding miRNA gene in a control sample is indicative of esophageal cancer.
  • the method comprises analyzing at least one miRNA gene corresponding to at least one miRNA of SEQ ID Nos. 15-38 for deletion, wherein a deletion of the miRNA gene relative to the corresponding miRNA gene in a control sample is indicative of esophageal cancer.
  • the method further comprises the step of providing an esophageal tissue sample suspected of being cancerous from the individual.
  • the method further comprises the step of isolating DNA from the esophageal tissue sample.
  • a method of diagnosing esophageal cancer in an individual comprising determining the gene copy number of at least one miRNA gene corresponding to at least one of the miRNAs shown in Figure 1 or their corresponding homologues in an esophageal tissue sample suspected of being cancerous from the individual, wherein a copy number other than two for miRNA genes located on a somatic chromosome or a sex chromosome for female, and other than one for miRNA genes located on a sex chromosome for male, is indicative of esophageal cancer.
  • the method comprises determining the gene copy number of at least one miRNA gene corresponding to at least one of the miRNAs of SEQ ID Nos. 1-14 in a sample from the individual, wherein a copy number more than two for miRNA genes located on a somatic chromosome or a sex chromosome for female, or more than one for miRNA genes located on a sex chromosome for male, is indicative of esophageal cancer.
  • the method comprises determining the gene copy number of at least one miRNA gene corresponding to at least one of the miRNAs of SEQ ID Nos.
  • the method further comprises the step of providing an esophageal tissue sample suspected of being cancerous from the individual. In some embodiments, the method further comprises the step of isolating DNA from the esophageal tissue sample.
  • the miRNAs described herein are also useful for one or more of the following: classifying esophageal cancer patients, predicting risk of developing esophageal cancer, monitoring tumor progression in esophageal cancer patients, and monitoring treatment in esophageal cancer patients, based on the levels of at least one or more miRNAs in an esophageal tissue sample, or the gene status of at least one or more miRNAs in an esophageal tissue sample of the individual.
  • systems such as microarrays
  • probes such as oligonucleotides
  • microRNAs such as microarrays
  • probes that are capable of determining the gene status of miRNA genes corresponding to microRNAs shown in Figure 1 or their corresponding homologues.
  • These systems are useful for determining levels of miRNAs shown in Figure 1 or their corresponding homologues and for diagnosing esophageal cancer. While some of the discussion below focus on systems for detecting miRNAs, it is 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.
  • a system for determining the level of at least one miRNA shown in Figure 1 (or the gene status of the corresponding miRNA) in a sample
  • the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA) in an esophageal tissue sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • a system for diagnosis of esophageal cancer
  • the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 (or the gene status of the corresponding miRNA).
  • a system for diagnosing esophageal cancer, comprising (including for example consisting essentially of) at least one (including for example at least any of 2, 5, 10, 15, 20, 25, 30, 35, and 40) probes, wherein each of the probes detects the level of a different miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • Systems (such as microarrays) provided herein are further described below in detail.
  • a system for diagnosing cancer comprising at least one (including for example at least any of 2, 5, 10, 15, 20, 25, 30, 35, and 40) probes (such as oligonucleotides), wherein each of the probes detects the level of a different miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA), and wherein a characteristic change of in the level of at least one of the miRNAs shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA) is indicative of esophageal cancer.
  • probes such as oligonucleotides
  • a system for diagnosing esophageal cancer
  • the system comprises a plurality of probes, wherein 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 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA), and wherein a characteristic change in the level of at least one of the miRNAs shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA) is indicative of esophagreal cancer.
  • probes that detect miRNAs for the manufacture of systems described herein.
  • probes such as oligonucleotides
  • each probe is capable of determining the level of an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • probes such as oligonucleotides
  • a system such as a microarray
  • each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probe are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • the present invention identifies 40 miRNAs whose levels correlate with esophageal cancer. These miRNAs are shown in Table 2 and Figure 1. Figure 1 also provides the name, sequence, and chromosomal location of the miRNAs. Information about miRNAs can be generally found at http://miRNA.sanger.ac.uk/. See Griffths-Jones et al., Nucleic Acids Research, 2006, Vol. 34, Database issue. Methods of diagnosing esophageal cancer can be based the levels or gene status of any of the miRNAs shown in Figure 1. Systems described herein can be used for determining the levels of one of more miRNAs shown in Figure 1 and diagnosing esophageal cancer based on the levels of one or more miRNAs shown in Figure 1.
  • the effectiveness (e.g., sensitivity and/or specificity) of the methods described herein are generally enhanced when at least two miRNAs are utilized.
  • at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 38 miRNAs shown in Figure 1 are utilized.
  • the levels or gene status of at least two (such as at least any of 3, 5, 10 or more) of miRNA of SEQ ID Nos. 1-14 are determined. In some embodiments, the levels or gene status of at least two (such as at least any of 3, 5, 10, 15, 20, or more) miRNAs of SEQ ID Nos. 15-38 are determined. In some embodiments, the levels or gene status of at least one of the miRNAs selected from the miRNAs of SEQ ID Nos. 1-14 and at least one of the miRNAs selected from the miRNAs of SEQ ID Nos. 15-38 are determined.
  • the levels or gene status of at least two (such as at least any of 3, 5, 10, or more) of the miRNAs selected from the miRNAs of SEQ ID Nos. 1-14 and the levels of at least two (such as at least any of 3, 5, 10, 15, 20, or more) miRNAs of SEQ ID Nos. 15- 38 are determined. In some embodiments, the levels or gene status of all miRNAs shown in Figure 1 are determined.
  • the levels of the corresponding homologues of the miRNA described herein are determined.
  • the "corresponding homologues" of miRNA described herein refers to miRNAs having at least about 50% sequence identity (including for example at least about any of 60%, 70%, 80%, 90%, 95%, 98%, or 99%) sequence identity to the corresponding miRNA described herein.
  • the corresponding homologue of a miRNA of SEQ ID NO:1 has at least about 50% sequence identity (including for example at least about any of 60%, 70%, 80%, 90%, 95%, 98%, or 99%) sequence identity to SEQ ID NO-.l.
  • a miRNA sequence that has at least about, for example, 95% identical to a reference sequence 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 nucleotide 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 method of diagnosing esophageal cancer is based on levels of the miRNAs.
  • level refers to the amount or rate of accumulation of an miRNA molecule or its precursor. The term can be used to refer to the absolute amount of an 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).
  • the control can be a different miRNA from the same sample whose level does not alter in an esophageal cancer tissue sample, or can be the same miRNA from a different sample (such as a noncancerous tissue sample from the same individual or a tissue sample from another individual not having esophageal cancer).
  • the "precursor" of an miRNA molecule or "miRNA precursor” refers to the unprocessed miRNA gene transcript, and typically comprises an RNA transcript of about 70 nucleotides in length.
  • the miRNA precursors are typically processed by digestion with an RNAase (such as Dicer, Argonaut, or RNAase III) into an active miRNA molecule, which are typically 19-25 nucleotide long.
  • a "level of miRNA in an esophageal tissue sample” refers to the miRNA level in the tissue sample. While in most cases the level of the miRNA in an esophageal tissue sample is determined based directly on measuring the miRNA level in an esophageal tissue sample, it is contemplated that the miRNA level in an esophageal tissue sample can also be reflected by (and thus based on) the level of miRNA in a lymph node sample (such as the proximal lymph nodes or lymph fluid), serum, blood, or other proximal biological fluid materials such as sputum.
  • a lymph node sample such as the proximal lymph nodes or lymph fluid
  • the miRNA level is determined based on the level of the miRNA in a lymph node sample (such as a lymph node section or needle aspirate). In some embodiments, the miRNA level is determined based on the level of the miRNA in the blood or serum. In some embodiments, the miRNA level is determined based on the level of the miRNA in an esophageal tissue swab. In some embodiments, the miRNA level is determined based on the level of the miRNA from a sample is obtained by endoscopic ultrasound-guided sampling procedures (for example by RT-PCR analysis).
  • Endoscopic ultrasound-guided fine-needle aspiration is a minimally invasive technique for the nonoperative sampling of mediastinal lymph nodes, which allows more detailed molecular marker analysis. Determination of miRNA levels in samples other than esophageal cancer tissues can be used along or in conjunction with each other. For example, the level of the miRNA can first be determined from the serum, then a follow-up analysis of miRNA in regional lymph nodes can be conducted. Such multi-step analysis could provide additional information and increase confidentiality of the diagnosis.
  • miRNA levels can be determined in various stages. For example, the miRNA level can be determined immediately prior to surgery, during surgery, after the surgery, prior to tumor treatment, during tumor treatmet, or after tumor treatment. In some embodiments, the miRNA level is determined based on the level of the miRNA in a tissue sample (particularly esophageal tissue sample) collected by swabbing.
  • miRNA levels can be determined by Northern blot, in situ hybridization, RT-PCR, and microarrays. Such methods are known in the art. See, e.g., Einat, Methods MoI. Biol. 2006, 342:139-157; Thompson et al. Genes Dev. 2006, 20:2202-2207.
  • total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agrose gels according to standard techniques, and transferred to nitrocellulose filters by, e.g., the so-called "Northern" blotting techniques. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question. Autoradiographic detection of probe hybridization to miRNA can be performed by exposing hybridized filters to photographic film.
  • RNA transcript level can be quantified by computerized imaging of the hybridization blot, for example with a phosphoimager.
  • the levels of RNA transcripts can be carried out according to the technique of 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 (such as cRNA probes).
  • the levels of the miRNAs can also be determined by reverse transcription of the miRNA transcripts, followed by amplification in a polymerase chain reaction (RT-PCR).
  • the levels of the miRNAs can be quantified in comparison with an internal standard, for example, levels of mRNA from a "housekeeping" gene present in the same sample.
  • a suitable "housekeeping" gene for use as an internal standard include myosin or glyceraldehydes-3 -phosphate dehydrogenase (G3PDH) or human U6.
  • G3PDH myosin or glyceraldehydes-3 -phosphate dehydrogenase
  • the 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.
  • qRT-PCR real-time quantitative PCR
  • the qRT-PCR for miRNA level determination provided herein may provide a sensitive and specific tool for the diagnosis, classification, and prognosis of esophageal cancer.
  • the present invention in one aspect provides methods of determining the miRNA level in a sample of an individual (such as an individual having a disease, for example cancer) by RT-PCR.
  • the level of the miRNA is determined by qRT-PCR.
  • the levels of miRNAs are determined by using a microarray, such as microarrays described herein.
  • Nucleic acid probes for one or more methods described above can be produced recombinantly or chemically synthesized using methods 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 various stringency conditions readily determined by one skilled in the art. Depending on the particular assay, one skilled in the art can readily vary the stringency conditions to optimize detection of a particular miRNA in a particular sample.
  • the stability of a hybrid is a function of the ion concentration and temperature.
  • 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.
  • the hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.
  • Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5xDenhart's solution, 5xSSPE, 0.2% SDS at 42°C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42°C.
  • High stringency conditions can be provided, for example, by 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 0 C.
  • Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5xDenhart's solution, 6xSSPE, 0.2% SDS at 22°C, followed by washing in IxSSPE, 0.2% SDS, at 37 0 C.
  • Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • 2OxSSPE sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA).
  • levels of the miRNA are obtained from an individual at more than one time point.
  • Such "serial" sampling is well suited for the aspects of the invention relating to monitoring progression of esophageal cancer in an individual having esophageal cancer.
  • Serial sampling can be performed on any desired timeline, such as semi- annually, annually, biennially, or less frequently. The comparison between the measured levels and the reference level may be carried out each time a new sample is measured, or the data relating to levels may be held for less frequent analysis.
  • the reference level is generally a level that is considered "normal" for the particular miRNA. In some embodiments, the reference level is based on the level of the miRNA in the non-cancerous esophageal tissue from same individual. In some embodiments, the reference level is based on the level of an individual not having esophageal cancer. In some embodiments, the reference level is based on an average of levels obtained from a population that is not having esophageal cancer. In some embodiments, the reference level is derived from a pool of samples including the sample being tested. The reference level can be predetermined or determined contemporaneously with the sample being tested.
  • 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 at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 38 miRNAs shown in Figure 1 or their corresponding homologues. The process of comparing the levels of an miRNA with a reference level can be carried out in any convenient manner appropriate to the type of measured values for the miRNAs at issue.
  • the levels may be compared qualitatively by visually comparing the intensities of the hybridization signals.
  • 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.
  • 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).
  • fold difference refers to a numerical representation of the magnitude difference between a measured value and a reference value for an miRNA.
  • a "characteristic change" in the levels of an miRNA can simply be a substantial decrease or an substantial increase in the miRNA levels in the individual as compared to a reference level.
  • “Substantial increase” used herein refers to an increase in miRNA level by at least about 5%, including for example at least any of 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more.
  • substantial decrease used herein refers to a decrease in miRNA level by at least about 5%, including for example at least any of 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or more.
  • Figure 1 provides a summary of changes of the listed miRNAs in one exemplary method.
  • a characteristic change of the levels of the miRNAs are used as a basis for diagnosing esophageal cancer. For example, in some embodiments when the level of at least one of the miRNAs of SEQ ID Nos. 1-14 are determined, a substantial increase in the levels of at least one of the measured miRNAs is indicative of esophageal cancer. In some embodiments when at least one of the miRNAs of SEQ ID Nos. 15-38 are determined, a substantial decrease in the levels of at least one of the measured miRNAs is indicative of esophageal cancer. In some embodiments when at least one of the miRNAs of SEQ ID Nos.
  • the levels of all miRNAs shown in Figure 1 are determined, and a substantial increase in the levels of at least one of the miRNA of SEQ ID Nos. 1-14 and a substantial decrease in the levels of at least one of the miRNAs of SEQ ID Nos. 15-38 are indicative of esophageal cancer.
  • a substantial increase in the levels of at least two of the miRNAs of SEQ ID Nos. 1-14 and a substantial decrease in the levels of at least two of the miRNAs of SEQ ID Nos. 15-38 are indicative of esophageal cancer.
  • the "majority" suggestion or indication may be considered the result of the assay.
  • the result may be considered as suggesting or indicating a diagnosis of esophageal cancer for the individual.
  • a diagnosis of esophageal cancer requires a characteristic change of at least one, or more, specific miRNAs. For example, in cases when one of the miRNAs is hsa-miR-16, a substantial increased level of hsa-miR-16 in some embodiments may be prerequisite for a diagnosis of esophageal cancer.
  • the gene status is 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 in a control sample is indicative of the presence of esophageal cancer in the individual.
  • a deletion or amplification in an miRNA gene can be detected by determining the structure or sequence of genes in cells from an esophageal tissue sample from an individual suspected of having esophageal cancer, and comparing this with the structure or sequence of these genes in cells from a control sample. Any techniques suitable for detecting alteration in the structure or sequence of genes can be used in the practice of the present method. 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 can also be used.
  • Deletions or amplifications of an miRNA gene can also be detected by amplifying a fragment of these genes 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 an miRNA gene can also be identified by detecting deletions of chromosomal markers that are closely linked to the miRNA gene.
  • the status of an miRNA gene in cells of an individual can also 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 miRNA genes on somatic chromosomes and sex chromosomes in a female, or other than one for miRNA genes on sex chromosomes in a male, is indicative of the presence of esophagreal cancer in the individual.
  • Any techniques suitable for detecting gene copy number can be used in the practice of the present method, including the Southern blot and PCR amplification techniques.
  • An alternative method of determining the miRNA gene copy number in an esophageal 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 an 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.
  • a "control sample” can be a tissue sample from an individual not having esophageal cancer.
  • the control sample can be a collection of tissue samples from a population of individuals.
  • Gene status can be determined for at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 38 miRNAs shown in Figure 1 or their corresponding homologues. In those embodiments when the gene status of more than one miRNAs are used but do not unanimously suggest or indicate a diagnosis of esophageal cancer, the "majority" suggestion or indication may be considered the result of the assay.
  • a diagnosis of esophageal cancer requires a characteristic change of at least one, or more, specific miRNA genes.
  • RNA genes can be determined using a variety of techniques. These 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.
  • a method of classifying esophageal patients comprising determining the level of at least one miRNA (such as miRNA shown in Table 2 or their corresponding homologues) or the gene status of the corresponding miRNA gene in an esophageal cancer tissue of the individual, wherein the level of the miRNA (or the gene status of the corresponding miRNA gene) is used as a basis for classifying the esophageal patient.
  • miRNA such as miRNA shown in Table 2 or their corresponding homologues
  • the level of the miRNA or the gene status of the corresponding miRNA gene
  • a method for determining the level of differentiation of esophageal cancer in an individual comprising determining the level of at least one miRNA shown in Figure 2a (or the gene status of the corresponding miRNA gene) in an esophageal cancer tissue of the individual, wherein the level of the miRNA (or the gene status of the corresponding miRNA gene) is used as a basis for determining the level of differentiation of esophageal cancer in the individual.
  • the individual can be determined to have high, middle, and low levels of differentiation based on the level of the miRNA tested.
  • the correlation between the miRNA level (or the gene status of the corresponding miRNA gene) and the level of differentiation can be determined, for example, by analyzing miRNA levels (or the gene status of the corresponding miRNA gene) of a population of esophageal cancer samples which have been classified into different levels of differentiation based on methods known in the art.
  • the miRNA is any one or more of hsa-miR-25, hsa-130b.
  • At least one miRNA is hsa-miR-335. In some embodiments, at least one miRNAs is hsa-miR-25. In some embodiments, at least one miRNAs is hsa-130b. In some embodiments, at least one miRNA is hsa-miR-130a. In some embodiments, at least one miRNAs is hsa-miR-181d.
  • a method for pathologically classifying an individual having esophageal squamous cell cancer comprising determining the level of at least one miRNA shown in Figure 2b or their corresponding homologues (or the gene status of the corresponding miRNA gene) in an esophageal cancer tissue of the individual, wherein the level of the miRNA (or the gene status of the corresponding miRNA gene) is used as a basis for pathologically classifying the individual.
  • the individual can be determined to have a fungating form of esophageal squamous cell cancer or medullary form of esophageal squamous cell cancer, depending on the level of the miRNA tested.
  • the correlation between the level of the miRNA (or the gene status of the corresponding miRNA gene) and the different pathological forms of the cancer can be established, for example, by analyzing miRNA levels (or the gene status of the corresponding miRNA gene) of a population of samples which have been pathologically classified by methods known in the art.
  • a method for determining the stage of a tumor in an individual having esophageal cancer comprising determining the level of at least one miRNA shown in Figure 2c or their corresponding homologues (or the gene status of the corresponding miRNA gene) in an esophageal cancer tissue of the individual, wherein the level of the miRNA (or the gene status of the corresponding miRNA gene) is used as a basis for determining the stage of the tumor in the individual.
  • the individual can be determined to have stage I, stage II, or stage III tumor based on the levels of the miRNAs (or the gene status of the corresponding miRNA gene).
  • the individual is determined to have stage Tl, T2, or T3 tumor based on the levels of the miRNAs (or the gene status of the corresponding miRNA genes). In some embodiments, the individual is determined to have stage NO and stage Nl based on the levels of the miRNAs (or the gene status of the corresponding miRNA genes).
  • the correlation between the level of the miRNA (or the gene status of the corresponding miRNA gene) and the different tumor stages can be established, for example, by analyzing miRNA levels (or the gene status of the corresponding miRNA genes) of a population of samples which have been classified into different tumor stages by methods known in the art.
  • Also provided herein are systems comprising probes that are capable of detecting miRNAs shown in Table 2 (including for example any of the miRNAs shown in Figure 2a, 2b, and 2c, or their corresponding homologues) and systems for determining the gene status of miRNA genes corresponding to miRNAs shown in Table 2 or their corresponding homologues. These systems are useful for determining levels of miRNAs (or gene status of corresponding miRNA genes) shown in Table 2 and classifying esophageal cancer patients.
  • a system for determining the level of at least one miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene) in a sample
  • the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA gene) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a system for classifying esophageal cancer patients, wherein the system comprises a plurality of probes, wherein each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA gene) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 (or the gene status of the corresponding miRNA gene).
  • a system for classifying an individual having esophageal cancer, comprising (including for example consisting essentially of or consisting of) at least about any of 2, 5, 10, 20, 30, or 40 probes, wherein each of the probes is capable of detecting an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a system for classifying an individual having esophageal cancer wherein the system comprises one or more probes, and wherein each probe is capable of determining the level of an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a use of a system for determining the level of differentiation of esophageal cancer in an individual having esophageal cancer wherein the system comprises one or more probes, and wherein each probe is capable of determining the level of an miRNA shown in Figure 2a or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a system for pathologically classifying an individual having esophageal squamous cell cancer wherein the system comprises one or more probes, and wherein each probe is capable of determining the level of an miRNA shown in Figure 2b or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a use of a system for determining the tumor stage of an individual having esophageal cancer wherein the system comprises one or more probes, and wherein each probe is capable of determining the level of an miRNA shown in Figure 2c or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • each probe is capable of determining the level of an miRNA shown in Figure 2c or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • probes that detect miRNAs (or determine the gene status of the corresponding miRNA gene) for the manufacture of systems described herein.
  • each probe is capable of determining the level of an miRNA Shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • probes for the manufacture of a system for classifying an individual having esophageal cancer
  • each of the probes is capable of detecting a different miRNA (or the gene status of the corresponding miRNA gene) in the sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 or their corresponding homologues (or the gene status of the corresponding miRNA).
  • a use of one or more probes for the manufacture of a system for determining the level of differentiation of esophageal cancer in an individual having esophageal cancer wherein each probe is capable of determining the level of an miRNA shown in Figure 2a or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • each probe is capable of determining the level of an miRNA shown in Figure 2a or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • a use of one or more probes for the manufacture of a system for pathologically classifying an individual having esophageal squamous cell cancer wherein each probe is capable of determining the level of an miRNA shown in Figure 2b or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • each probe for the manufacture of a system for determining the tumor stage of an individual having esophageal cancer, wherein each probe is capable of determining the level of an miRNA shown in Figure 2c or their corresponding homologues (or the gene status of the corresponding miRNA gene).
  • the invention in another aspect provides methods of prognosing esophageal cancer patients, including for example methods of determining a prognosis for survival of an individual having esophageal cancer.
  • the prognostic methods of the invention are useful for determining a proper course of treatment for an individual having esophageal 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.
  • prognosis can help determine whether agents for improving survival (such as agents described herein) are necessary and/or effective.
  • a method for determining a prognosis for survival for an individual having esophageal cancer comprising: (a) determining the level of at least one miRNA in an esophageal cancer tissue sample from the individual, and (b) comparing the level of the miRNA in said sample to a threshold level, wherein the level of the miRNA as compared to a threshold level correlates or reversely correlates with the survival of the individual.
  • “correlate” means that a low level of the miRNA as compared to the threshold level is indicative of a low survival of the individual having esophageal cancer, and vise versa.
  • At least one miRNA is an miRNA that regulates a target gene selected from the group consisting of: PPP6C, SATB2, CHSTI l, CRELDl, ESRRA, MTMR4, RNF125;SYNJ1, TAF5, YWHAH, ZYX, CHSTI l, KIAA 1033, TGFBR3, SNRK, RNF125, AXIN2, CAPZA2, SYNJl, DLLl, YWHAH, MTMR4, PPP6C, CAMKV, and TAF5, PPPlCB, POU4F2, MYHl, MYH2, TOP2B, STX17, GBAS 5 MYH4, CPSF4, EIF4EBP3, LHX4, CLK3, CAPN6, KIAA1622, AUH, PP
  • ZNF579 NRGN 5 CUL3, CIB2, ZBTB26, GPBPl, TMEM16D, HOXAl, CAMTAl, MCM3AP, MPPED2, HOOK2, PLAU, MCFD2, BLCAP, DHX15, FBNl, NCOA6, SNRPC, CCK, SFRS15, TMODl, GPRC5B, ZNF403, DCUN1D5, ZNF423, and GPR64.
  • At least one of the miRNAs is an miRNA that regulates a target gene selected from the group consisting of: PPP6C, SATB2, CHSTI l, CRELDl, ESRRA, MTMR4, RNF125;SYNJ1, TAF5, YWHAH, and ZYX.
  • at least one miRNAs is a miRNA that regulates a target gene selected from the group consisting of CHSTI l, KIAA1033, TGFBR3, SNRK, RNF125, AXIN2, CAPZA2, SYNJl, DLLl, YWHAH, MTMR4, PPP6C, CAMKV, and TAF5.
  • At least one miRNAs is an miRNA that regulates a target gene selected from the group consisting of: PPPlCB, POU4F2, MYHl, MYH2, TOP2B, STXl 7, GBAS, MYH4, CPSF4, EIF4EBP3, LHX4, CLK3, CAPN6, KIAA1622, AUH, PPIF, KCNK3, IL6R, CSNK2A2, ZNF579, NRGN, CUL3, CIB2, ZBTB26, GPBPl, TMEM16D, HOXAl, CAMTAl, MCM3AP, MPPED2, H00K2, PLAU, MCFD2, BLCAP, DHX15, FBNl, NCOA6, SNRPC, CCK, SFRS15, TMODl, GPRC5B, ZNF403, DCUN1D5, ZNF423, and GPR64.
  • a target gene selected from the group consisting of: PPPlCB, POU4F
  • the gene corresponding to the miRNA is located to any of Chromosome 5, Chromosome 10, and Chromosome 9.
  • the miRNA is any of the miRNAs shown in Figure 3 or their corresponding homologues.
  • at least one miRNA is hsa- miR-103.
  • at least one miRNA is hsa-miR-107.
  • at least one miRNA is hsa-miR-23b.
  • a method of determining a prognosis for survival for an individual having esophageal cancer comprising: (a) determining the level of at least one miRNA in an esophageal cancer tissue sample from the individual, and (b) comparing the level of the miRNA in said sample to a threshold level, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual, and wherein at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b or their corresponding homologues. In some embodiments, at least one miRNA is hsa-miR- 103.
  • At least one miRNA is hsa-miR-107. In some embodiments, at least one miRNA is hsa-miR-23b. [0112] Levels of the miRNAs described herein may also reflect changes in the gene status of the miRNAs (such as miRNAs described herein).
  • a method of determining a prognosis for survival for an individual having esophageal cancer comprising analyzing the gene status of at least one miRNA genes (such as a miRNA gene corresponding to any of hsa-miR-103, hsa-miR-107, and hsa-miR-23b or their corresponding homologues), wherein a change in gene status as compared to a that of a control sample indicates a high or low survival of the individual.
  • miRNA genes such as a miRNA gene corresponding to any of hsa-miR-103, hsa-miR-107, and hsa-miR-23b or their corresponding homologues
  • a method of determining a prognosis for survival for an individual having esophageal cancer comprising analyzing at least one miRNA genes corresponding to any of hsa-miR-103, hsa-miR-107, and hsa-miR-23b 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.
  • a method of determining a prognosis for survival of an individual having esophageal cancer comprising determining the gene copy number of at least one miRNA genes corresponding to any of hsa-miR-103, hsa-miR-107, and hsa-miR-23b, wherein a copy number of more than two indicates a low survival rate of the individual.
  • probes that are capable of detecting the levels of the miRNAs (or the gene status of the corresponding miRNA gene) or systems comprising one or more probes for determining a prognosis for survival.
  • a use of one or more probes (or system comprising one or more probes) for determining prognosis for survival of an individual having esophageal cancer wherein the probe is capable of detecting an 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 said individual.
  • a use of one or more probes for determining prognosis of survival of an individual having esophageal cancer wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual, and wherein at least one miRNA is hsa-miR-103, hsa-miR- 107, or hsa-miR-23b or their corresponding homologues.
  • probes for the manufacture of an agent (or system) for determining a prognosis of survival of an individual having esophageal cancer, wherein the probe is capable of detecting an 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 said individual.
  • a use of one or more probe for the manufacture of an agent (or system) for determining prognosis of survival of an individual having esophageal cancer wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual, and wherein at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa- miR-23b or their corresponding homologues.
  • the invention also provides methods of improving survival of individuals having esophageal cancer.
  • a method of improving survival of an individual having esophageal cancer comprising administering to the individual an effective amount of an agent that decreases the level of an miRNA, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual.
  • an agent that decreases the level of an miRNA for the manufacture of a medicament for improving survival of an individual having esophageal cancer, wherein the level of the miRNA as compared to the threshold level reversely correlates with the survival of said individual.
  • a method of improving survival of an individual having esophageal cancer comprising administering to the individual an effective amount of an agent that decreases the level of an miRNA selected from the group consisting of hsa-miR-103, hsa-miR-107, hsa-miR-23b, and their corresponding homologues.
  • an agent for the manufacture of a medicament for improving survival of an individual having esophageal cancer wherein the agent decreases the level of an miRNA selected from the group consisting of hsa-miR-103, hsa-miR-107, hsa-miR-23b, and their corresponding homologues.
  • the methods described herein may further comprises a step of determining for prognosis for survival of the individual (for example, by methods described herein) prior to the administration of the agents.
  • the levels of more than one miRNAs are decreased. This can be achieved, for example, by use of an agent that decreases the levels of two or more miRNAs. Alternatively, two or more agents are used for decreasing the levels of two or more miRNAs.
  • a method of improving survival of an individual having esophageal cancer comprising administering to the individual an effective amount of one or more agents that decreases the levels of at least two miRNAs selected from the group consisting of hsa-miR-103, hsa-miR-107, hsa-miR- 23b, and their corresponding homologues.
  • a method of improving survival of an individual having esophageal cancer comprising administering to the individual an effective amount of one or more agents that decreases the levels of hsa- miR-103, hsa-miR-107, and hsa-miR-23b.
  • a pharmaceutical composition comprising an agent that decreases the level of an miRNA and a pharmaceutically acceptable carrier, wherein at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b.
  • at least one miRNA is hsa-miR-103.
  • at least one miRNA is hsa-miR-107.
  • at least one miRNA is hsa-miR-23b.
  • the agent is a double-stranded RNA (such as short or small-interfering RNA or "siRNA”), an antisenses nucleic acid, or an enzymatic RNA molecules such as ribozyme.
  • siRNA short or small-interfering RNA or "siRNA”
  • an antisenses nucleic acid or an enzymatic RNA molecules such as ribozyme.
  • the survival described herein can be disease free survival or overall survival.
  • 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.
  • Certain methods and uses described herein involve determining a prognosis for survival based on miRNA levels relative to a threshold level.
  • the threshold level can be determined by a plurality of methods, provided that the resulting threshold level accurately provides a level of miRNA above which exist a first group of patient having a different survival rate than that of a second group of patients having an miRNA level below the threshold level.
  • the threshold level can be determined by, for example, the miRNA level of a noncancerous esophageal cancer tissue sample.
  • the threshold level can also be determined by analyzing the levels of an miRNA in a population of individuals having esophageal 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 determined by identification of subset populations of the cohort which have the same or similar levels of the miRNA. Determination of the threshold level can then be made based on an miRNA level which best distinguish these separate groups.
  • the 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.
  • the threshold level can be a single number that is equally applicable to every individual having esophageal cancer, or 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.
  • a threshold level can be a level determined for each individual. For example, the threshold level may be a certain ratio of an miRNA in the esophageal cancer tissue relative to the miRNA level in a non-cancerous tissue within the same individual.
  • Verification that the threshold level distinguishes the likelihood of survival in esophageal 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 determine the likelihood of a correlation between one or more variables and a given outcome. In the specific case, the methods will determine the likelihood of a correlation between an miRNA level and disease free or overall survival of 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-Meir method or the Cox proportional-hazards regression model. [0126] Population-based determination of threshold levels, for example, by histogram analysis can be carried out using a cohort of patients sufficient in size in order to determine two or more separate groups of patients having different miRNA levels. Typically, such a cohort comprises at least 25 patients, including for example at least about any of 50, 75, 100, 125, 150, or 200 patients.
  • verification of determined threshold levels can also comprises at least 25 patients, including for example at least about any of 50, 75, 100, 125, 150, or 200 patients.
  • a single threshold level can separate two groups of patients, several threshold values might exist which separate a plurality of populations. For example, two threshold values can separate a first group of patients with high levels of miRNA from a second group of patients with intermediate levels of 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 describes the likelihood of disease-free or overall survival in a patient as a function of the miRNA level in that patient.
  • Such a curve will 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.
  • the miRNA (such as miRNAs described herein) can be combined with each other in the methods of the invention 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 an miRNA can also be used in conjunction with another variable found to be statistically significant as indicators of the likelihood of disease-free or overall survival for esophageal cancer patient, such as pathological indicators (for example, age, tumor size, tumor histology, clinical stage, family history and the like).
  • pathological indicators for example, age, tumor size, tumor histology, clinical stage, family history and the like.
  • clinical stage of the cancer is a statistically significant indicator of disease-free or overall survival
  • the threshold level of an miRNA can vary as a function of another statistically significant indicator of disease-free or overall survival for esophageal cancer.
  • Kaplan-Meier analysis is used to determine the correlation between survival rate and the miKNA level.
  • the method comprises: (a) determining a level of at least one miRNA in an esophageal cancer tissue from the individual, (b) classifying the individual as belonging to either a first or second group of individuals having esophageal cancer, wherein the first group of individuals having a low levels of the miRNA is classified as having an increased likelihood of survival compared to the second group of individuals having high level of the miRNA, wherein at least one miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b.
  • the patient is then classified into a group having a certain likelihood of disease free or overall survival. Then the likelihood of disease-free or overall survival for the patient is assessed based on the likelihood of disease-free or overall survival for patients in that group.
  • a sample can be determined to have low levels of miRNA. This patient would then be classified into a group of patients having low levels of miRNA. Because it has been established that there is an increased likelihood of disease-free or overall survival for the group of patients expressing high levels of miRNA, the specific cancer patient would be considered to have an increased likelihood of disease free or overall survival.
  • the methods described herein may further comprise a step of determining the proper course of treatment for the individual.
  • the prognostic indicators of survival for cancer patients suffering from an early stage of cancer may be different from those for cancer patients suffering from a later stage of cancer.
  • prognosis for stage I cancer patient may be oriented toward the likelihood of continued growth and/or metastasis of the cancer
  • 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.
  • Methods and compositions for improving survival [0135] Also provided are methods of improving survival of an esophageal cancer patient using agents that decreases the levels of certain miRNAs, such as miRNAs shown in Figure 3.
  • agents that can decrease the level of miRNAs can be used in methods of the present invention.
  • Suitable agents for inhibiting miRNA gene expression include, but are not limited to, double-stranded RNA (such as short or small-interfering RNA or "siRNA"), antisense nucleic acids, enzymatic RNA molecules such as ribozyme, small molecule compounds, and proteins. These agents can be used alone or in combination with other agents (such as other agents described herein).
  • the agents can decrease the miRNA levels directly (e.g., by inhibiting the miRNA expression or function) or indirectly (e.g., by affecting the gene status of the corresponding miRNA gene).
  • expression of a given miRNA gene can be inhibited by inducing RNA interference of the miRNA gene with an isolated double-stranded RNA (“dsRNA") molecule which has at least 70%, including for example at least any of 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, sequence homology with at least a portion of the miRNA gene product.
  • dsRNA isolated double-stranded RNA
  • the dsRNA molecule is a "short or small interfering RNA" or "siRNA.”
  • siRNA useful in the present methods may comprise short double-stranded RNA of about 10-30 nucleotides, including for example about any of 12-28, 14-26, 16-24, or 18-22 nucleotides.
  • the siRNA comprises a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions.
  • the sense strand comprises a nucleic acid sequence which 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.
  • the siRNA can differ from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alteration can include addition of non-nucleotide material, such as to the end(s) of the siRNA or one or two internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxynucleotides.
  • one or both strands of the siKNA also comprise a 3' overhang.
  • the siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described further below.
  • an antisense nucleic acid refers to a nucleic acid molecule that binds to target KNA by means of RNA-RNA or RNA-DNA interactions, which alters the activity of the target RNA.
  • Antisense nucleic acids suitable for use in the present methods are single- stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA, and LNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miRNA.
  • the antisense nucleic acid comprises a nucleic acid sequence that is at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a contiguous nucleic acid sequence in an miRNA. In some embodiments, the antisense nucleic acid has about 10-30 nucleotides, including for example, about any of 12-28, 14-26, 16-24, or 18-22 nucleotides. [0141] Antisense nucleic acids can also contain modifications to the nucleic acid backbone or to the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule.
  • 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 can also be inhibited by an enzymatic nucleic acid.
  • an "enzymatic nucleic acid” refers to a nucleic acid comprising a substrate binding region that is complementary to a contiguous nucleic acid sequence of an miRNA, and which is able to specifically cleave the miRNA.
  • the enzymatic nucleic acid binding region is 50-100% complementary, including for example 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a miRNA.
  • the enzymatic nucleic acid can also comprise modifications at the base, sugar, and/or phosphate groups.
  • An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.
  • the enzymatic nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as further described below. [0145] 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 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, gramacidinS, artificial viral envelopes or other intracellular carriers such as TAT.
  • a nucleic acid agent can also be delivered into a mammalian cell in vitro or in vivo using suitable vectors known in the art.
  • Suitable vectors for delivering a nucleic acid to a mammalian cell include viral vectors and non-viral vectors such as plasmid vector. Such vectors are useful for providing therapeutic amounts of an agent such as antisense RNA or siRNA.
  • Viral based systems provide the advantage of being able 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 the vital vectors can also be modified by pseudotyping the vectors with envelope proteins or surface antigens from other viruses.
  • an AAV vector can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV) rabies, Ebola, Mokola, and the like.
  • VSV vesicular stomatitis virus
  • Any of a variety of inducible promoters or enhancers can also be included in a nucleic acid or vector of the invention to allow control of 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 short promoters inducible by temperature changes.
  • An agent is in an effective amount if the amount of the agent is enough to decrease the level of miRNA. In some embodiments, the agent decreases the level of the miRNA by about any of 10%, 20%, 30%, 40%, or 50% of the difference between the miRNA level and threshold level.
  • Exemplary amounts for the 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, 100-500 mg/kg body weight.
  • the amount of the agent is about 10-500 mg/gram tumor mass, such as any of 20-300 mg/gram tumor mass, 50-200 mg/gram tumor mass, and 100-150 mg/gram tumor mass.
  • One of ordinary skill in the art can readily determine an appropriate dosage regimen for the administration of the agents to an individual.
  • Exemplary dosing frequency for the agents includes, but is not limited to, at least about any of 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.
  • the interval between each administration is less than about a week, such as less than about any of 6, 5, 4, 3, 2, or 1 day(s).
  • the interval between each administration is constant.
  • the administration can be carried out daily, every two days, every three days, every four days, every five days, or weekly.
  • the administration can be carried out twice daily, three times daily, or more frequent.
  • the administration of the agent can be extended over an extended period of time, such as from about a month up to about three years.
  • the dosing regime can be extended over a period of any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, and 36 months.
  • the interval between each administration is no more than about a week.
  • composition described herein can be administered to an individual via any route in the art, including, but not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transdermal, transpleural, intraarterial, topical, inhalational (e.g., as mists of sprays), transmucosal (such as via nasal mucosa), subcutaneous, transdermal, gastrointestinal, intraarticular, intracisternal, intraventricular, rectal (i.e., via suppository), vaginal (i.e., via pessary), intracranial, intraurethral, intrahepatic, and intratumoral.
  • the composition is administered systemically.
  • the composition is administered locally.
  • compositions comprising an agent that decreases the level of an miRNA and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an agent that decreases the level of a miRNA selected from the group consisting of hsa-miR-103, hsa-miR-107, hsa-miR-23b and their corresponding homologues.
  • at least one miRNA is hsa- miR-103.
  • at least one miRNA is hsa-miR-107.
  • at least one miRNA is hsa-miR-23b.
  • the agent is a siRNA.
  • the agent is an antisense RNA.
  • the agent is a ribozyme.
  • the pharmaceutical compositions are sterile. In some embodiments, the pharmaceutical compositions are pyrogene-free.
  • Suitable pharmaceutically acceptable carriers include, for example, water, buffered water, normal saline, 0.4% saline, 0.3% glycine, and hyaluronic acid.
  • the pharmaceutical compositions may also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTP A-bisamide) or calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers e.g., tromethamine hydrochloride
  • chelants such as, for example, DTPA or DTPA-bisamide
  • calcium chelate complexes such as, for example, calcium DTPA, CaNaDTP A-bisamide
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • solid pharmaceutically acceptable carriers for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • the invention provides various systems for detecting miRNA levels that undergo characteristic changes in esophageal cancer patients. Also provided are systems for determining gene status of miRNAs. As described above, the systems can be used for various purposes, including for example diagnosing esophageal cancer, classifying esophageal cancer patients, and determining a prognosis for survival of esophageal cancer patients. [0158] The systems described herein comprise probes for detecting miRNAs and/or determining gene status of miRNAs.
  • a system comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1, Table 2, or Figure 3 or their corresponding homologues.
  • the system comprises (including for example consisting essentially of or consisting of) at least about any of 2, 5, 10, 20, 30, 40, or 50 probes, wherein each of the probes is capable of detecting an miRNA shown in Figure 1, Table 2, or Figure 3 or their corresponding homologues.
  • a system comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues.
  • the system comprises (including for example consisting essentially of or consisting of) at least about any of 2, 5, 10, 20, 30, or 40 probes, wherein each of the probes is capable of detecting an miRNA shown in Figure 1 or their corresponding homologues.
  • a system comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%,
  • the system comprises (including for example consisting essentially of or consisting of) at least about any of 2, 5, 10, 20, 30, or 40 probes, wherein each of the probes is capable of detecting an miRNA shown in Table 2 or their corresponding homologues (including for example any miRNA shown in Figures 2a, 2b, and 2c).
  • a system comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 3 or their corresponding homologues.
  • the system comprises (including for example consisting essentially of or consisting of) at least about any of 1, 2, 3, 6, 9, 12, 15, 18, or 21 probes, wherein each of the probes is capable of detecting an miRNA shown in Figure 3.
  • the systems described herein may comprise two or more probes that detect the same miRNA.
  • the probes when the system is a microarray, the probes may be present in multiple (such as any of 2, 3, 4, 5, 6, 7, or more) copies in the microarray.
  • the system comprises different probes that detect the same miRNA.
  • these probes may bind to different (overlapping or nonoverlapping) regions of the miRNA.
  • Any probes that are capable of determining the levels of miRNA can be used.
  • the probe is an oligonucleotide. It is understood that, for detection of miRNAs, certain sequence variations are acceptable. Thus, the sequence of the oligonucleotides (or their complementary sequences) may be slightly different from those of the miRNAs described herein.
  • sequence variations are understood by those of ordinary skill in the art to be variations in the sequence that do not significantly affect the ability of the oligonucleotide to determine miRNA levels.
  • homologs and variants of these oligonucleotide molecules possess a relatively high degree of sequence identity when aligned using standard methods.
  • Oligonucleotide sequences encompassed by the invention have at least 40%, including for example at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or more sequence identity to the sequence of the miRNAs described herein.
  • the oligonucleotide comprises a portion for detecting the miRNAs and another portion.
  • Such other portion may be used, for example, for attaching the oligonucleotides to a substrate.
  • the other portion comprises a non-specific sequence (such as polyT) for increasing the distance between the complementary sequence portion and the surface of the substrate.
  • the oligonucleotides for systems described herein include, for example, DNA, RNA, PNA, LNA, combinations thereof, and/or modified forms thereof. They may also include a modified oligonucleotide backbone.
  • the oligonucleotide comprises at least about any of 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more continuous oligonucleotides complementary or identical to all or part of a miRNA described herein.
  • a single oligonucleotide may comprise two or more such complementary sequences.
  • there is a reactive group (such as an amine) attached to the 5' or 3' end of the oligonucleotide for attaching the oligonuceotide to a substrate.
  • the system is a microarray of probes.
  • "Microarray” and “array,” as used interchangeably herein, comprise a surface with an array, preferably an ordered array, of putative binding (e.g., by hybridization) sites for a biochemical sample (target) which often have undetermined characteristics.
  • a microarray refers to an assembly of distinct oligonucleotide probes immobilized at defined positions on a substrate.
  • a microarray comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1, Table 2, or Figure 3 or their corresponding homologues.
  • a microarray comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 1 or their corresponding homologues.
  • a microarray comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Table 2 or their corresponding homologues.
  • a microarray comprising a plurality of probes, wherein each of the probes is capable of detecting a different miRNA in a sample, and wherein at least about 15% (including for example at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the probes are capable of detecting an miRNA shown in Figure 3 or their corresponding homologues.
  • microarrays for determining gene status of miRNAs genes corresponding to miRNAs disclosed herein are provided.
  • Microarrays for determining gene status are known in the art.
  • the system can comprise sequence-tagged molecular inversion probes for determining the gene status.
  • Arrays may be formed on substrates fabricated with materials such as paper, glass, plastic (e.g., polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose, silicon, optical fiber or any other suitable solid or semisolid support, and configured in a planar (e.g., glass plates, silicon chips) or three dimensional (e.g., pins, fibers, beads, particles, microtiter wells, capillaries) configuration.
  • plastic e.g., polypropylene, nylon, polystyrene
  • polyacrylamide nitrocellulose
  • silicon optical fiber or any other suitable solid or semisolid support
  • planar e.g., glass plates, silicon chips
  • three dimensional e.g., pins, fibers, beads, particles, microtiter wells, capillaries
  • the probes are oligonucleotides.
  • Oligonucleotides forming the array may be attached to the substrate by any number of ways including, but not limiting to, (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 may also be non-covalently immobilize on the substrate by hybridization to anchors, by means of magnetic beads, or in a fluid phase such as in microtiter wells or capillaries.
  • anchors by means of magnetic beads, or in a fluid phase such as in microtiter wells or capillaries.
  • a fluid phase such as in microtiter wells or capillaries.
  • the amplified product is then contacted with a solid substrate, such as a glass slide, which is coated with an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified product and become covalently attached to the glass slide.
  • a solid substrate such as a glass slide
  • an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified product and become covalently attached to the glass slide.
  • Microarrays comprising the amplified products can be fabricated using 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 processed according to published procedures (Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1995) 93:10614-10619).
  • Arrays can also be printed by robotics onto glass, nylon (Ramsay, G., Nature Biotechnol. (1998), 16:40-44), polypropylene (Matson, et al., Anal Biochem. (1995), 224(1): 110-6), and silicone slides (Marshall, A. and Hodgson, J., Nature Biotechnol. (1998), 16:27-31).
  • Other approaches to array assembly include fine micropipetting within electric fields (Marshall and Hodgson, supra), and spotting the polynucleotides directly onto positively coated plates. Methods such as those using amino propyl silicon surface chemistry are also known in the art, as disclosed at www.cmt.corning.com and http://cmgm.stanford.edu/pbrown/.
  • microarrays are by making high-density nucleotide arrays. Techniques are known for rapid deposition of polynucleotides (Blanchard et al., Biosensors & Bioelectronics, 11:687-690). Other methods for making microarrays, e.g., by masking (Maskos and Southern, Nucleic. Acids. Res. (1992), 20:1679-1684), may also be used. In principle, and as noted above, any type of array, for example, dot blots on a nylon hybridization membrane, could be used. However, as will be recognized by those skilled in the art, very small arrays will frequently be preferred because hybridization volumes will be smaller.
  • the invention also provides kits for various methods described herein.
  • kits for diagnosing esophageal cancer.
  • the kit may further comprise control sample(s) for determination of a reference level, and/or information about obtaining a reference level.
  • the kit may further comprise instructions on use of the kits for diagnosing esophageal cancer, as described herein.
  • kits containing a system (such as microarray) described herein for classifying individuals having esophageal cancer.
  • the kit may further comprise control samples for classifying the individual and/or information about control samples, and in some embodiments, instructions on use of the kit for classifying individuals.
  • kits for determining a prognosis for survival of an individual having esophageal cancer may comprise, for example, probes that detect an miRNA, such as an miRNA shown in Figure 3.
  • the kit further comprises a control sample for the determination of a threshold level and/or information about obtaining a threshold level.
  • the kit may further comprise instruction on use of the kit to determine prognosis of survival of an individual.
  • the kit may further comprise agents that decrease the levels of miRNA or pharmaceutical compositions comprising such agents for improvement of survival.
  • kits described herein may further comprise reagents, which include, but are not limited to, 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 of miRNA levels using statistical methods as described herein, and optionally a password and/or account number for assessing the compiled database.
  • reagents include, but are not limited to, 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.
  • a software package e.g., for analyzing miRNA levels and
  • kits comprising a pharmaceutical composition comprising an agent that decreases the level of an miRNA shown in Figure 3 and an instruction on use of the composition for improvement of survival in an individual having esophageal cancer.
  • the kit further comprises vectors or other agents for delivery of the composition.
  • the kit further comprises instructions on administration of the pharmaceutical composition.
  • This example shows preparation of samples of analysis of miRNA levels.
  • Peripheral portions of resected esophageal cancers were paraffin embedded, and sectioned and routine H&E stained. The tumor cell concentrations were evaluated and tumor histology was confirmed by a pathologist.
  • follow-up information was extracted from the follow-up registry of the Cancer Institute and Hospital, CAMS. For all the samples, clinico-pathological information (age, sex, pathology, differentiation, TNM classification, tumor stage, and survival time after surgery) was available. The study was approved by the medical-ethics committee of Cancer Institute and Hospital, CAMS.
  • All of the miRNA probe sequences were designed to be fully complementary to their cognate full-length mature miRNA.
  • the probe sequences were concatenated up to a length of 40 nt (3 '-end miRNA probe plus 5 '-end 19mer polyT) with C6 5'-amino-modif ⁇ er.
  • Oligonucleotide probes were synthesized at MWG Biotech. Company and dissolved in EasyArrayTM spotting solution (CapitalBio Corp.) at a concentration of 40 ⁇ M. Each probe was printed in triplicate using a SmartArrayTM microarrayer (CapitalBio Corp.).
  • T4 RNA ligase labeling method according to Thomson' protocol (Thomson et al., 2004).
  • 4 ⁇ g of low-molecular-weight RNA was labeled with 500 ng of 5'- phosphate-cytidyl-uridyl-cyS-S' (Dharmacon, Lafayette, CO) with 2 units T4 RNA ligase (New England Biolabs, Beijing, China).
  • the labeling reaction was performed at 4oC for 2 h.
  • Hybridization was performed under LifterSlipTM (Erie, Portsmouth, NH) in a hybridization chamber which was placed in a three-phase-tiling agitator BioMixerTM (CapitalBio) to provide continuous mixing of the hybridization buffer that results in more uniform hybridization across the entire slide surface and prevents edge effects, the efficiency of which has been demonstrated with our genome-wide mRNA expression profiling.
  • the hybridization was performed overnight in water-bath at 42oC. The array was then washed with two consecutive washing solutions of 0.2% SDS, 2xSSC at 42°C for 5 min, and 0.2% SSC for 5 min at room temperature. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0TM software
  • Average values of the replicate spots of each miRNA were background subtracted, normalized, and subjected to further analysis. Normalization was performed by using per chip median normalization method and the median array. Data were filtered to eliminate genes with expression signal lower than 800 in all samples. Differentially expressed miRNAs were identified by Significance Analysis of Microarrays (SAM) (available at www- stat.stanford.edu/ ⁇ tibs/SAM/index.html). SAM calculates a score for each gene on the basis of the change in expression relative to the standard deviation of all measurements. Hierarchical clustering was performed with average linkage and Pearson correlation. The Support Vector Machine tool was used for cross-validation and prediction of the test set.
  • SAM Significance Analysis of Microarrays
  • RNAs were subjected for qRT-PCR with microRNA specific primers.
  • Reverse transcriptase reactions contained 2.5 ng/ ⁇ l total RNA, 25 nM stem-loop RT primer, 1 x RT buffer, 0.25 mM each of dNTPs, 200 U M-MLV reverse transcriptase and 0.25 U/ml RNase inhibitor (Invitrogen).
  • the 7.5 ml reactions were incubated in an MJ Research PTC-225 Thermocycler in a for 30 min at 16°C, 30 min at 42 0 C 5 5 min at 85 0 C and then held at 4°C.
  • the real-time PCR employed a FastStart DNA Master SYBR green I kit and a LightCycler (both from Roche Diagnostics, Mannheim, Germany) following the manufacturer's protocols.
  • the 10 ⁇ l PCR included 1 ⁇ l RT product, 1 * PCR Master Mix, 15 nM forward primer and 15 nM reverse primer. The reactions were incubated at 95 0 C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60°C for 35 s and 72 0 C for 3 s. All qRT-PCR reactions, including no-template controls were performed in duplicate.
  • the relative expression ratios of miRNAs were determined with the crossing point (CP) as the cycle number.
  • Gene Expression Assays for human U6 were used as the endogenous controls. The results were analyzed using LightCycler software version 3.5 (Roche Diagnostics). The real-time PCR amplification product was analyzed by melting curve analysis and agarose gel electrophoresis confirmation. The primer sequences are listed in Table 1. Table 1.
  • EXAMPLE 2 [0190] This example shows altered miRNA expression in esophageal cancers tissue samples.
  • miRNA genes were identified by direct signal strength than by ratio, but significantly several miRNA genes were identified by both classification methods with 5 genes (hsa-miR-335, -18 Id, -25, -7, -495) for pathological classification (fungating vs medullary) and two genes (hsa-miR-25, -130b) identified for position of cancer in the eosophagus (up v.y middle vs down). Table 2. Comparative analysis of clinicopathological classifications
  • EXAMPLE 3 shows a correlation between miRNA levels and survival of esophageal cancer patients.
  • the initial training set of 31 patient samples were divided into two groups based on survival for a period shorter or longer than 20 months after initial diagnosis.
  • the intensity of each individual differentially expressed miRNA was tested by averaging the signal for each survival group. The mean of these two average intensity values was then used as the threshold signal intensity in Kaplan-Meier analysis.
  • Three miRNAs (hsa-miR-103, -107, - 23b) showed a strong correlation between low expression and high overall survival period up to 90 months post-diagnosis.
  • Figures 3 and 6 Considering that most miRNAs normally function to down-regulate the translation of their target mRNA into its protein, the correlation suggests these mRNA target(s) function as tumor suppressor(s).
  • hsa-miR-23b was also identified in association with Tumor Stage Classification I-III but only by direct signal intensity (Table 2).
  • Kaplan-Meier analysis of the disease-free survival of patients also identified two miRNAs (hsa-miR-103, -107), again with low levels of expression correlating with high disease-free survival periods.
  • Validation of microarray data by real-time PCR analysis were carried out. Both real-time analysis and microarray profiles were performed for hsa-m ⁇ R-23b, hsa-miR-103 and hsa-miR-107 to determine the mature miRNA abundance had a prognostic signature with esophageal cancer patients.

Abstract

La présente invention concerne des méthodes et des compositions permettant de diagnostiquer et de classer un cancer de l'oesophage, ainsi que d'établir un pronostic de survie pour des sujets atteints d'un cancer de l'oesophage, en fonction du niveau ou du statut génétique de certains micro-ARN. L'invention a également trait à des compositions contenant des agents qui réduisent le niveau de miARN et à des utilisations desdites compositions pour améliorer la survie.
PCT/CN2006/003195 2006-11-28 2006-11-28 Méthodes et compositions permettant de diagnostiquer un cancer de l'oesophage, d'établir un pronostic et d'améliorer la survie des patients WO2008064519A1 (fr)

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JP2009537466A JP2010510769A (ja) 2006-11-28 2006-11-28 食道癌の診断ならびに患者の生存の予後および改善のための方法および組成物
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