WO2010055488A2 - Compositions et procédés pour le profilage de l’expression des microarn du cancer hépatocellulaire - Google Patents

Compositions et procédés pour le profilage de l’expression des microarn du cancer hépatocellulaire Download PDF

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WO2010055488A2
WO2010055488A2 PCT/IB2009/055058 IB2009055058W WO2010055488A2 WO 2010055488 A2 WO2010055488 A2 WO 2010055488A2 IB 2009055058 W IB2009055058 W IB 2009055058W WO 2010055488 A2 WO2010055488 A2 WO 2010055488A2
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mir
hsa
nucleic acid
expression
acid molecules
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PCT/IB2009/055058
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WO2010055488A3 (fr
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Ying Wu
Hongguang Zhu
Lei Wang
Shuyang Wang
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Koninklijke Philips Electronics N.V.
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Priority to CN200980150551.3A priority Critical patent/CN102985558B/zh
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Publication of WO2010055488A3 publication Critical patent/WO2010055488A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to compositions and methods for microRNA expression profiling of hepatocellular cancer.
  • Hepatocellular cancer also referred to as hepatocellular carcinoma, HCC
  • HCC hepatocellular carcinoma
  • Hepatocellular cancer thus represents a type of an extremely poor prognostic cancer.
  • the dismal outcome has been attributed to the major hallmarks of hepatocellular cancer, namely intra-hepatic metastases or postsurgical recurrence. New tumor colonies frequently invade into the major branches of the portal vein and possibly other parts of the liver (reviewed, for example, in Tang, Z. Y. (2001) World J
  • Hepatocellular tumors comprise diverse benign and malignant neoplasms. Although the pheno types can be broadly distinguished histologically, these tumors can vary widely in their clinical behavior and prognosis. Therefore, early detection of such tumors would be desirable in order to discriminate these different types of tumor and to enable a reliably risk assessment of patients exhibiting or being supposed to have a (benign) pre-cancerous state whether or not the pre-cancerous state will progress in a carcinoma.
  • the prediction of cancer progression may be used to guide the therapy decision in patients exhibiting a type of hepatocellular cancer and thus can markedly help to improve long-term survival.
  • AFP alpha- fetoprotein
  • bio markers would be of utmost clinical importance, particularly if these markers enable a diagnosis at an early stage of tumor progression in order to allow early stage treatment of carcinomas while avoiding unnecessary surgical intervention.
  • such markers should enable the identification of a carcinoma at a stage where the presence of malignant cells is not yet detectable by in situ techniques or microscopic analysis of biopsy or resection material.
  • Many diagnostic assays are also hampered by the fact that they are typically based on the analysis of only a single molecular marker, which might affect detection reliability and/or accuracy.
  • a single marker normally does not enable detailed predictions concerning latency stages, tumor progression, and the like.
  • miRNAs small regulatory RNA molecules
  • nt nucleotides
  • MiRNAs are produced from primary transcripts that are processed to stem-loop structured precursors (pre-miRNAs) by the RNase III Drosha. After transport to the cytoplasm, another RNase III termed Dicer cleaves of the loop of the pre-miRNA hairpin to form a short double-stranded (ds) RNA, one strand of which is incorporated as mature miRNA into a miRNA-protein (miRNP).
  • ds short double-stranded
  • miRNA-protein miRNA-protein
  • the miRNA guides the miRNPs to their target mRNAs where they exert their function (reviewed, e.g. in Bartel, D.P.
  • miRNAs can guide different regulatory processes.
  • Target mRNAs that are highly complementary to miRNAs are specifically cleaved by mechanisms identical to RNA interference (RNAi).
  • RNAi RNA interference
  • the miRNAs function as short interfering RNAs (siRNAs).
  • Target mRNAs with less complementarity to miRNAs are either directed to cellular degradation pathways or are translationally repressed without affecting the mRNA level.
  • the mechanism of how miRNAs repress translation of their target mRNAs is still a matter of controversy .
  • miRNAs are shown to map to a genetic locus that is deleted in chronic lymphatic leukemia (CLL) and it was found that in about 70% of the CLL patients, both miRNA genes are deleted or down-regulated.
  • CLL chronic lymphatic leukemia
  • down-regulation of miR-143 and miR-145 was observed in colorectal neoplasia, whereas expression of the miRNA let-7 is frequently reduced in lung cancers (Michael, M.Z. et al. (2003) MoI. Cancer Res. 1, 882-891; Mayr, C. et al.
  • miRNAs may provide insights into cellular processes involved in malignant transformation.
  • these studies focused on tumor tissues without separating the malignant cells. Therefore, 40% of the reported HCC-associated miRNAs showed different (partially even opposing) regulatory patterns across the different studies.
  • no data are available on a correlation between miRNA expression and early stages of HCC.
  • miRNA microRNA
  • nucleic acid expression signatures and/or compositions for one or more mammalian target cells exhibiting or having the predepostion to develop hepatocelluar cancer and/or discriminating different types of hepatocellular cancer and/or different stages of tumor progression, i.e. hepatocellular carcinoma and carcinoma embolus.
  • the present invention relates to a diagnostic kit of molecular markers for identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of or the predisposition to develop hepatocellular cancer.
  • the nucleic acid expression signature may comprise at least five nucleic acid molecules, preferably at least eight nucleic acid molecules, and particularly preferably at least twelve nucleic acid molecules.
  • the nucleic acid expression signature may comprise at least six nucleic acid molecules, preferably at least twelve nucleic acid molecules, and particularly preferably at least twenty nucleic acid molecules.
  • the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target cells compared to the one or more control cells and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in the one or more target cells compared to the one or more control cells.
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-mir-221, hsa- mir-324-5p, hsa-mir-96, hsa-mir-18a, hsa-mir-18b, hsa-mir-30d, hsa-mir-331-3p, hsa- mir-183, hsa-mir-551b, hsa-mir-139-5p, hsa-mir-144, hsa-mir-144*, and hsa-mir-450a.
  • the expression of any one or more of the nucleic acid molecules encoding hsa-mir-221, hsa-mir-324-5p, hsa-mir-96, hsa-mir-18a, hsa- mir-18b, hsa-mir-30d, hsa-mir-331-3p, hsa-mir-183, and hsa-mir-551b is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-mir-139-5p, hsa-mir-144, hsa-mir-144*, and hsa-mir-450a is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • the diagnostic kit is for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the hepatocellular cancer is hepatocellular carcinoma
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-224, hsa-miR-532-3p, hsa-miR-663, hsa- miR-99b, hsa-miR-362-5p, hsa-miR-652, hsa-miR-222, hsa-miR-501-3p, hsa-miR-375, hsa-miR-451, and hsa-miR-335.
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-224, hsa-miR-532-3p, hsa-miR-663, hsa-miR-99b, hsa-miR-362-5p, hsa-miR-652, hsa-miR-222, and hsa-miR-501-3p is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-375, hsa-miR- 451, and hsa-miR-335 is down-regulated in the in the one or more target cells compared to the one or more control cells
  • the hepatocellular cancer is carcinoma embolus
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-199b-5p, hsa-miR-21, hsa-miR-19b, hsa- miR-103, hsa-miR-23a, hsa-miR-125b-2*, hsa-miR-30c-2*, hsa-miR-505*, and hsa- miR-203.
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-199b-5p, hsa-miR-21, hsa-miR-19b, hsa-miR-103, and hsa-miR-23a is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-125b-2*, hsa-miR-30c-2*, hsa-miR-505*, and hsa-miR-203 is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • the nucleic acid expression signature may comprise at least six nucleic acid molecules, preferably at least twelve nucleic acid molecules, and particularly preferably at least twenty nucleic acid molecules.
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa-miR-139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*.
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa-miR-139-5p, hsa-miR- 122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa- miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*.
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa- miR-139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR- 144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa- miR-194*, hsa-miR-215, hsa-miR-331-3p, hsa-miR-335, hsa-miR-451, hsa-miR-486- 5p, hsa-miR-505*, h
  • the diagnostic kit is for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the hepatocellular cancer is carcinoma embolus
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa-miR-139-5p, hsa-miR-122*, hsa- miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa-miR-194*, hsa-miR-215, hsa-miR- 331-3p, hsa-miR-335, hsa-miR-451, hsa-miR-486-5p,
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-331-3p, and hsa-miR-551b is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-505, hsa-miR- 139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa-miR- 194*, hsa-miR-215, hsa-miR-335, hsa-miR-451,
  • the present invention relates to a method for identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer, the method comprising: (a) determining in the one or more target cells the expression levels of a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence; (b) determining the expression levels of the plurality of nucleic acid molecules in one or more control cells; and (c) identifying from the plurality of nucleic acid molecules one or more nucleic acid molecules that are differentially expressed in the target and control cells by comparing the respective expression levels obtained in steps (a) and (b), wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature, as defined herein, that is indicative for the presence of or the predisposition to develop hepatocellular cancer.
  • the method is for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the present invention relates to a method for preventing or treating hepatocellular cancer in one or more mammalian target cells, the method comprising: (a) identifying in one or more target cells a nucleic acid expression signature by using a method, as defined herein; and (b) modifying in the one or more cells the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression is up-regulated in the one or more target cells is down-regulated and the expression of a nucleic acid molecule whose expression is down-regulated in the one or more target cells is up-regulated.
  • the present invention relates to a pharmaceutical composition for the prevention and/or treatment of hepatocellular cancer in one or more mammalian target cells, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence that is at least partially complementary to a microRNA sequence encoded by a nucleic acid molecule whose expression is up- regulated in the one or more target cells, as defined herein, and/or that corresponds to a microRNA sequence encoded by a nucleic acid molecule whose expression is down- regulated in the one or more target cells, as defined herein.
  • the present invention relates to the use of said pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of hepatocellular cancer.
  • Figure 1 illustrates the human miRNAs comprised in particularly preferred expression signatures according to the present invention for identifying one or more target cells exhibiting or having a predisposition to develop hepatocellular cancer (i.e. hepatocellular carcinoma and carcinoma embolus) as well as for discriminating between hepatocellular carcinoma and carcinoma embolus, respectively. Also indicated are the expression levels ("REG” for "regulation”) of these miRNAs in cancerous tissue as compared to (healthy) control tissue (i.e. an up-regulation or a down-regulation) .
  • Figure 2 depicts a flow chart schematically illustrating the essential method steps for determining an expression signature according to the present invention for identifying one or more target cells exhibiting or having a predisposition to develop hepatocellular cancer.
  • Figure 3 displays the expression levels of the identified miRNAs in the progression of hepatocelluar carcinoma (HCC) and carcinoma embolus (CE).
  • HCC hepatocelluar carcinoma
  • CE carcinoma embolus
  • Figure 4 illustrates an unsupervised hierarchical clustering of the differentially expressed miRNAs and shows that the clustering placed 31/31 normal-cirrhosis samples in one group and 29/32 HCC-CE samples in another group.
  • Figure 5 show the PAM scores for individual miRNAs correspond to their predictive power in the prediction analysis of microarray.
  • a minimal set of 2 miRNAs hsa-miR-139-5p and hsa-miR-101
  • the hsa-miR-139-5p is another identified miRNA in hepatocellular carcinoma.
  • the present invention is based on the unexpected finding that cells exhibiting or having a predisposition to develop hepatocellular cancer (HCC) can be reliably identified and different types of HCC discriminated based on particular miRNA expression signatures both with high accuracy, sensitivity and specificity, wherein the expression signatures as defined herein typically comprises both up- and down- regulated human miRNAs. More specifically, said miRNA expression signatures - by analyzing the overall miRNA expression pattern and/or the respective individual miRNA expression level(s) - allow the detection of HCC at an early disease state as well as an evaluation of the risk that a benign pre-cancerous state transforms into a malignant carcinoma.
  • HCC hepatocellular cancer
  • the present invention relates to a diagnostic kit of molecular markers for identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of or the predisposition to develop hepatocellular cancer.
  • cancer also referred to as “carcinoma”
  • cancer generally denotes any type of malignant neoplasm, that is, any morphological and/or physiological alterations (based on genetic re-programming) of target cells exhibiting or having a predisposition to develop characteristics of a carcinoma as compared to unaffected (healthy) wild-type control cells.
  • alterations may relate inter alia to cell size and shape (enlargement or reduction), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death) or cell survival.
  • hepatocellular (or “hepatic”), as used herein, relates to the liver.
  • hepatocellular cancer refers to cancerous growths in the liver.
  • the term "having a predisposition to develop cancer”, as used herein, denotes any cellular phenotype being indicative for a pre-cancerous state, i.e. an intermediate state in the transformation of a normal cell into a tumor cell. In other words, the term denotes a state of risk of developing cancer.
  • hepatocellular carcinoma also referred to as “hepatoma” and commonly abbreviated as "HCC”
  • hepatocellular carcinoma denotes a primary malignancy of the liver.
  • Most cases of HCC are secondary to either a viral hepatitide infection (hepatitis B or C) or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis).
  • hepatitis B or C hepatitis B or C
  • cirrhosis cirrhosis
  • Treatment options of HCC and prognosis are dependent on many factors but especially on tumor size and staging. Tumor grade is also important. High-grade tumors will have a poor prognosis, while low-grade tumors may go unnoticed for many years. The usual outcome is poor, because only 10% to 20% of hepatocellular carcinomas can be removed completely using surgery. If the cancer cannot be completely removed, the disease is usually deadly within 3 to 6 months.
  • Hepatocellular carcinoma like any other cancer, develops when there is a mutation to the cellular machinery that causes the cell to replicate at a higher rate and/or results in the cell avoiding apoptosis.
  • chronic viral infections of hepatitis B and/or C can aid the development of hepatocellular carcinoma by repeatedly causing the body's own immune system to attack the liver cells, some of which are infected by the virus, others merely bystanders. While this constant cycle of damage followed by repair can lead to mistakes during repair which in turn lead to carcinogenesis, this hypothesis is more applicable, at present, to hepatitis C.
  • hepatitis B the integration of the viral genome into infected cells is the most consistently associated factor in malignancy.
  • hepatitis B and/or C infections or hepatic cirrhosis are not merely to be considered as risk factors for tumor etiology but as early/intermediate stages in tumor progression (i.e. "pre-cancerous states") that are associated with hyper-proliferative tissue growth resulting in (often benign) noninvasive neoplasm which, in turn, may progress to malignant tumors such as HCC.
  • malignant tumors invade other tissues and often metastasize given enough time to do so.
  • Malignant cells are often characterized by progressive and uncontrolled growth. Macroscopically, HCC appears as a nodular or infiltrative tumor.
  • the nodular type may be solitary (having a large mass) or multiple (when developed as a complication of cirrhosis). Tumor nodules are round to oval, well circumscribed but not encapsulated. The diffuse type is poorly circumscribed and infiltrates the portal veins, rarely the hepatic veins.
  • tumor tissue may form an embolus, that is, an object migrating from one part of the body (through circulation) and causes a blockage (occlusion) of a blood vessel in another part of the body.
  • embolus Such secondary hepatocellular tumors resulting from tumor embolism are referred herein as "carcinoma embolus”.
  • the mammalian target cells employed in the present invention may be of human or non-human origin. However, the invention is typically performed with human cells.
  • the term "one or more cells”, as used herein, is to be understood not only to include individual cells but also tissues, organs, and organisms.
  • the term "target cell”, as used herein, refers to a cell being at least supposed to exhibit or to have a predisposition to develop hepatocellular cancer, whereas the term “control cell” typically denotes a (healthy) wild-type cell not having characteristics of such a cancerous phenotype. However, in some applications, for example, when comparing cells exhibiting different cancerous or pre-cancerous states, the cells having the less severe disease characteristics are typically considered the "control cells".
  • the target and control cells used are derived from biological samples collected from the subjects to be diagnosed for the presence or the predisposition to develop hepatocellular cancer. Furthermore, in order to corroborate the data obtained "comparative samples" may also be collected from subjects having a given known disease state.
  • the biological samples may include body tissues and fluids, such as blood, sputum, and urine.
  • the biological sample may contain a cell extract derived from or a cell population including a hepatic cell, preferably a cancerous hepatic cell or a hepatic cell derived from tissue suspected to be cancerous. Even more preferably the biological sample comprises a cell population derived from a hepatic tissue.
  • the cell may be purified from the obtained body tissues and fluids if necessary, and then used as the biological sample.
  • the expression level of the nucleic acid markers of the present invention is determined in the subject-derived biological sample(s).
  • the sample used for detection in the in vitro methods of the present invention should generally be collected in a clinically acceptable manner, preferably in a way that nucleic acids (in particular RNA) or proteins are preserved.
  • the samples to be analyzed are typically hepatic biopsies or resections. Furthermore, hepatocellular carcinoma cells may migrate into other tissues. Hence, blood and other types of sample can be used as well.
  • a biopsy or resection may contain only a minority of (pre-) carcinoma cells. To increase the signal/background ratio, a resection can be divided into different sub- samples prior to analysis (for example, by laser-capture microdissection).
  • At least one of the sub-samples may contain an increased ratio of (pre-)cancerous versus non-cancerous cells.
  • Samples, in particular after initial processing may be pooled. However, also non- pooled samples may be used.
  • microRNA (or “miRNA”), as used herein, is given its ordinary meaning in the art (reviewed, e.g. in Bartel, D.P. (2004) Cell 23, 281-292; He, L. and Hannon, G.J. (2004) Nat. Rev. Genet. 5, 522-531). Accordingly, a "microRNA” denotes a RNA molecule derived from a genomic locus that is processed from transcripts that can form local RNA precursor miRNA structures.
  • the mature miRNA is usually 20, 21, 22, 23, 24, or 25 nucleotides in length, although other numbers of nucleotides may be present as well, for example 18, 19, 26 or 27 nucleotides.
  • the miRNA encoding sequence has the potential to pair with flanking genomic sequences, placing the mature miRNA within an imperfect RNA duplex (herein also referred to as stem-loop or hairpin structure or as pre-miRNA), which serves as an intermediate for miRNA processing from a longer precursor transcript.
  • This processing typically occurs through the consecutive action of two specific endonucleases termed Drosha and Dicer, respectively.
  • Drosha generates from the primary transcript (herein also denoted "pri-miRNA”) a miRNA precursor (herein also denoted "pre-miRNA”) that typically folds into a hairpin or stem-loop structure.
  • miRNA precursor (or “precursor miRNA” or “pre-miRNA”), as used herein, refers to the portion of a miRNA primary transcript from which the mature miRNA is processed. Typically, the pre-miRNA folds into a stable hairpin (i.e.
  • the hairpin structures typically range from 50 to 80 nucleotides in length, preferably from 60 to 70 nucleotides (counting the miRNA residues, those pairing to the miRNA, and any intervening segment(s) but excluding more distal sequences).
  • nucleic acid molecule encoding a microRNA sequence denotes any nucleic acid molecule coding for a microRNA (miRNA). Thus, the term does not only refer to mature miRNAs but also to the respective precursor miRNAs and primary miRNA transcripts as defined above. Furthermore, the present invention is not restricted to RNA molecules but also includes corresponding DNA molecules encoding a microRNA, e.g. DNA molecules generated by reverse transcribing a miRNA sequence.
  • a nucleic acid molecule encoding a microRNA sequence according to the invention typically encodes a single miRNA sequence (i.e. an individual miRNA). However, it is also possible that such nucleic acid molecule encodes two or more miRNA sequences (i.e.
  • nucleic acid molecule encoding a microRNA sequence is also to be understood to include "sense nucleic acid molecules” (i.e. molecules whose nucleic acid sequence (5' ⁇ 3') matches or corresponds to the encoded miRNA (5' ⁇ 3') sequence) and "anti-sense nucleic acid molecules” (i.e. molecules whose nucleic acid sequence is complementary to the encoded miRNA (5'— > 3') sequence or, in other words, matches the reverse complement (3' ⁇ 5') of the encoded miRNA sequence).
  • sense nucleic acid molecules i.e. molecules whose nucleic acid sequence (5' ⁇ 3') matches or corresponds to the encoded miRNA (5' ⁇ 3') sequence
  • anti-sense nucleic acid molecules i.e. molecules whose nucleic acid sequence is complementary to the encoded miRNA (5'— > 3') sequence or, in other words, matches the reverse complement (3' ⁇ 5') of the encoded miRNA sequence).
  • complementary refers to the capability of an "anti-sense” nucleic acid molecule sequence of forming base pairs, preferably Watson-Crick base pairs, with the corresponding "sense" nucleic acid molecule sequence (having a sequence complementary to the anti-sense sequence).
  • an "anti-sense” nucleic acid molecule sequence of forming base pairs preferably Watson-Crick base pairs
  • the corresponding "sense” nucleic acid molecule sequence having a sequence complementary to the anti-sense sequence.
  • two nucleic acid molecules i.e. the
  • the "sense” and the “anti-sense” molecule) may be perfectly complementary, that is, they do not contain any base mismatches and/or additional or missing nucleotides.
  • the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions).
  • the "complementary" nucleic acid molecule comprises at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in corresponding "sense" nucleic acid molecule.
  • the plurality of nucleic acid molecules encoding a miRNA sequence that are comprised in a diagnostic kit of the present invention may include one or more "sense nucleic acid molecules" and/or one or more "anti-sense nucleic acid molecules".
  • the diagnostic kit includes one or more "sense nucleic acid molecules” (i.e. the miRNA sequences as such), said molecules are to be considered to constitute the totality or at least a subset of differentially expressed miRNAs (i.e. molecular markers) being indicative for the presence of or the disposition to develop a particular condition, here hepatocellular cancer.
  • a diagnostic kit includes one or more "anti-sense nucleic acid molecules” (i.e.
  • said molecules may comprise inter alia probe molecules (for performing hybridization assays) and/or oligonucleotide primers (e.g., for reverse transcription or PCR applications) that are suitable for detecting and/or quantifying one or more particular (complementary) miRNA sequences in a given sample.
  • a plurality of nucleic acid molecules as defined within the present invention may comprise at least two, at least ten, at least 50, at least 100, at least 200, at least 500, at least 1.000, at least 10.000 or at least 100.000 nucleic acid molecules, each molecule encoding a miRNA sequence.
  • the term “differentially expressed”, as used herein, denotes an altered expression level of a particular miRNA in the target cells as compared to the healthy control cells, which may be an up-regulation (i.e. an increased miRNA concentration in the target cells) or a down-regulation (i.e. a reduced or abolished miRNA concentration in the target cells).
  • the nucleic acid molecule is activated to a higher or lower level in the target cells than in the control cells.
  • a nucleic acid molecule is to considered differentially expressed if the respective expression levels of this nucleic acid molecule in target cells and control cells typically differ by at least 5% or at least 10%, preferably by at least 20% or at least 25%, and most preferably by at least 30% or at least 50%.
  • the latter values correspond to an at least 1.3-fold or at least 1.5-fold up-regulation of the expression level of a given nucleic acid molecule in the target cells compared to the wild-type control cells or vice versa an at least 0.7-fold or at least 0.5- fold down-regulation of the expression level in the target cells, respectively.
  • expression level refers to extent to which a particular miRNA sequence is transcribed from its genomic locus, that is, the concentration of a miRNA in the one or more cells to be analyzed.
  • control cell typically denotes a (healthy) wild- type cell not having characteristics of a HCC phenotype. However, in some applications, for example, when comparing cells exhibiting different cancerous or precancerous states, the cells having the less severe disease characteristics are typically considered the "control cells”.
  • RNA level for example by Northern blot analysis using miRNA-specific probes, or at the DNA level following reverse transcription (and cloning) of the RNA population, for example by quantitative PCR or real-time PCR techniques.
  • determining includes the analysis of any nucleic acid molecules encoding a microRNA sequence as described above. However, due to the short half- life of pri-miRNAs and pre-mRNAs typically the concentration of only the mature miRNA is measured.
  • the standard value of the expression levels obtained in several independent measurements of a given sample for example, two, three, five or ten measurements
  • the standard value may be obtained by any method known in the art. For example, a range of mean ⁇ 2 SD (standard deviation) or mean ⁇ 3 SD may be used as standard value.
  • control nucleic acids e.g. housekeeping genes whose expression levels are known not to differ depending on the disease states of the cell.
  • housekeeping genes include inter alia ⁇ -actin, glycerinaldehyde 3 -phosphate dehydrogenase, and ribosomal protein Pl.
  • the control nucleic acid is another miRNA known to be stably expressed during the various non-cancerous and (pre-)cancerous states of the cell.
  • the expression levels for one or more control cells it may also be possible to define based on experimental evidence and/or prior art data on or more cut-off values for a particular cell phenotype (i.e. a disease state).
  • the respective expression levels for the one or more target cells can be determined by using a stably expressed control miRNA for normalization. If the "normalized” expression levels calculated are higher than the respective cutoff value defined, then this finding would be indicative for an up- regulation of gene expression. Vice versa, if the "normalized” expression levels calculated are lower than the respective cutoff value defined, then this finding would be indicative for a down-regulation of gene expression.
  • the term "identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer” is intended to also encompass predictions and likelihood analysis (in the sense of "diagnosing”).
  • the compositions and methods disclosed herein are intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for the disease.
  • an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease.
  • the invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information for diagnosis.
  • the invention may also be used to discriminate between different types of hepatocellular tumors.
  • one or more differentially expressed nucleic acid molecules identified together represent a nucleic acid expression signature that is indicative for the presence of or the predisposition to develop hepatocellular cancer in the target cells.
  • expression signature denotes a set of nucleic acid molecules (e.g., miRNAs), wherein the expression level of the individual nucleic acid molecules differs between the (cancerous) target cells and the (non-cancerous) control cells.
  • a nucleic acid expression signature is also referred to as a set of markers and represents a minimum number of (different) nucleic acid molecules, each encoding a miRNA sequence that is capable for identifying a phenotypic state of a target cell.
  • the nucleic acid expression signature comprises at least five nucleic acid molecules, each encoding a (different) miRNA sequence.
  • the nucleic acid expression signature comprises at least eight or at least ten (different) nucleic acid molecules.
  • the nucleic acid signature comprises at least twelve or at least fifteen (different) nucleic acid molecules.
  • the nucleic acid molecules comprised in the nucleic acid expression signature are human sequences (hereinafter designated “hsa” (Homo sapiens)).
  • the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a miRNA sequence whose expression is up-regulated (i.e. its concentration is increased) in the one or more target cells compared to the one or more control cells and at least one nucleic acid molecule encoding a miRNA sequence whose expression is down-regulated (i.e. its concentration is reduced) in the one or more target cells compared to the one or more control cells.
  • the nucleic acid expression signature of the diagnostic kit comprises any one or more human target cell-derived nucleic acid molecules encoding microRNA sequences selected from the group consisting of hsa-mir-221 (SEQ ID NO:1), hsa-mir-324-5p (SEQ ID NO:2), hsa-mir-96 (SEQ ID NO: 3), hsa-mir-18a (SEQ ID NO:4), hsa-mir-18b (SEQ ID NO:5), hsa-mir- 3Od (SEQ ID NO:6), hsa-mir-331-3p (SEQ ID NO:7), hsa-mir-183 (SEQ ID NO:8), hsa-mir-551b (SEQ ID NO:9), hsa-mir-139-5p (SEQ ID NO: 10), hsa-mir-144 (SEQ ID NO: 11), hsa-mir-144* (SEQ ID NO: 12), and hsa
  • the expression of any one or more of the nucleic acid molecules encoding hsa-mir-221, hsa-mir-324-5p, hsa-mir-96, hsa-mir-18a, hsa- mir-18b, hsa-mir-30d, hsa-mir-331-3p, hsa-mir-183, and hsa-mir-551b is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-mir-139-5p, hsa-mir-144, hsa-mir-144*, and hsa-mir-450a is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • one or more of the plurality of nucleic acid molecules and "any one or more human target cell-derived nucleic acid molecules”, as used herein, may relate to any subgroup of the plurality of nucleic acid molecules, e.g., any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, and so forth nucleic acid molecules, each encoding a microRNA sequence that are comprised in the nucleic acid expression signature, as defined herein.
  • the nucleic acid expression signature includes at least any one or more nucleic acid molecules encoding the miRNAs specified above and also contains one or more additional nucleic acid molecules encoding any further miRNA sequences that are differentially expressed in the target cells and in one or more control cells analyzed, particularly any one or more human target cell-derived nucleic acid molecules encoding microRNA sequences selected from the group consisting of hsa-mir-100 (SEQ ID NO: 14), hsa-mir-101 (SEQ ID NO: 15), hsa-mir-lOa (SEQ ID NO: 16), hsa-mir-139-3p (SEQ ID NO: 17), hsa-mir- 148a (SEQ ID NO:18), hsa-mir-30c (SEQ ID NO:19), hsa-mir-122* (SEQ ID NO:20), hsa-mir-152 (SEQ ID NO:21), hsa-mir-24-1* (SEQ ID NO:
  • the expression of any of these nucleic acid molecules is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • the above-mentioned miRNAs (SEQ ID NO: 1 to SEQ ID NO: 36) were found to differentially expressed in several types of hepatocellular cancer tested as compared to control cells, particularly in hepatocellular carcinoma (i.e. a primary liver malignancy) and in carcinoma embolus (i.e. a secondary malignancy).
  • hepatocellular carcinoma i.e. a primary liver malignancy
  • carcinoma embolus i.e. a secondary malignancy
  • the diagnostic kit is for the further use of discriminating different types of hepatocellular cancer, particularly for discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the hepatocellular cancer is hepatocellular carcinoma
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-224 (SEQ ID NO:37), hsa-miR-532-3p (SE ID NO:38), hsa-miR-663 (SEQ ID NO:39), hsa-miR-99b (SEQ ID NO:40), hsa-miR- 362-5p (SEQ ID NO: 41), hsa-miR-652 (SEQ ID NO:42), hsa-miR-222 (SEQ ID NO:43), hsa-miR-501-3p (SEQ ID NO:44), hsa-miR-375 (SEQ ID NO:45), hsa-miR- 451 (SEQ ID NO:46), and hsa-miR-335 (SEQ ID NO: 47).
  • the expression signature for discriminating hepatocellular carcinoma may comprise any one or more of the nucleic acid molecules identified as SEQ ID NO:37 to SEQ ID NO:47 but none of the before- mentioned nucleic acid molecules identified as SEQ ID NO:1 to SEQ ID NO:36.
  • the expression signature for discriminating hepatocellular carcinoma may comprise any one or more of the nucleic acid molecules identified as SEQ ID NO:37 to SEQ ID NO:47 and in addition any one or more of the before-mentioned nucleic acid molecules identified as SEQ ID NO:1 to SEQ ID NO:36.
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-224, hsa-miR-532-3p, hsa-miR-663, hsa-miR-99b, hsa-miR-362-5p, hsa-miR-652, hsa-miR-222, and hsa-miR-501-3p is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-375, hsa-miR- 451, and hsa-miR-335 is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • the nucleic acid expression signature for discriminating hepatocellular carcinoma includes at least any one or more nucleic acid molecules encoding the miRNAs specified above (SEQ ID NO:37 to SEQ ID NO:47) and also contains one or more additional nucleic acid molecules encoding any further miRNA sequences that are differentially expressed in the target cells and in one or more control cells analyzed, particularly any one or more human target cell- derived nucleic acid molecules encoding microRNA sequences selected from the group consisting of hsa-miR-130b (SEQ ID NO:48), hsa-miR-132 (SEQ ID NO:49), hsa-miR- 106b (SEQ ID NO:50), hsa-miR-125a-5p (SEQ ID NO:51), hsa-miR-151-3p (SEQ ID NO:52), hsa-miR-17 (SEQ ID NO:53), hsa-miR-21 * (
  • the hepatocellular cancer is carcinoma embolus
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-199b-5p (SEQ ID NO:71), hsa-miR-21 (SEQ ID NO:72), hsa-miR-19b (SEQ ID NO:73), hsa-miR-103 (SEQ ID NO:74), hsa-miR- 23a (SEQ ID NO:75), hsa-miR-125b-2* (SEQ ID NO:76), hsa-miR-30c-2* (SEQ ID NO:77), hsa-miR-505* (SEQ ID NO:78), and hsa-miR-203 (SEQ ID NO:79).
  • nucleic acid sequences of the above-referenced miRNAs are listed in Table 3.
  • the expression signature for discriminating carcinoma embolus may comprise any one or more of the nucleic acid molecules identified as SEQ ID NO:71 to SEQ ID NO:79 but none of the before- mentioned nucleic acid molecules identified as SEQ ID NO:1 to SEQ ID NO:36.
  • the expression signature for discriminating carcinoma embolus may comprise any one or more of the nucleic acid molecules identified as SEQ ID NO:71 to SEQ ID NO:79 and in addition any one or more of the before-mentioned nucleic acid molecules identified as SEQ ID NO:1 to SEQ ID NO:36.
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-199b-5p, hsa-miR-21, hsa-miR-19b, hsa-miR-103, and hsa-miR-23a is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-125b-2*, hsa-miR-30c-2*, hsa-miR-505*, and hsa-miR-203 is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • the nucleic acid expression signature for discriminating carcinoma embolus includes at least any one or more nucleic acid molecules encoding the miRNAs specified above (SEQ ID NO:71 to SEQ ID NO: 79) and also contains one or more additional nucleic acid molecules encoding any further miRNA sequences that are differentially expressed in the target cells and in one or more control cells analyzed, particularly any one or more human target cell- derived nucleic acid molecules encoding microRNA sequences selected from the group consisting of hsa-let-7c (SEQ ID NO: 80), hsa-miR-122 (SEQ ID NO:81), hsa-miR- 125b (SEQ ID NO:82), hsa-miR-130a (SEQ ID NO:83), hsa-miR-195 (SEQ ID NO:84), hsa-miR-30a (SEQ ID NO:85), hsa-miR-126* (SEQ ID NO:
  • the nucleic acid expression signature of the diagnostic kit comprises any one or more human target cell-derived nucleic acid molecules encoding microRNA sequences selected from the group consisting of hsa-miR-505 (SEQ ID NO:32), hsa-miR-139-5p (SEQ ID NO: 10), hsa- miR-122* (SEQ ID NO:20), hsa-miR-30a* (SEQ ID NO: 90), hsa-miR-30e* (SEQ ID NO:26), hsa-miR-378* (SEQ ID NO:28), hsa-miR-144* (SEQ ID NO: 12), hsa-miR- 378 (SEQ ID NO:27), hsa-miR-100 (SEQ ID NO: 14), hsa-miR-lOa (SEQ ID NO: 16), hsa-miR-192* (SEQ ID NO:
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa-miR-139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*.
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa-miR-139-5p, hsa-miR- 122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa- miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*.
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505, hsa- miR-139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR- 144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa- miR-194*, hsa-miR-215, hsa-miR-331-3p, hsa-miR-335, hsa-miR-451, hsa-miR-486- 5p, hsa-miR-505*, h
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-331-3p, and hsa-miR-55 Ib is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-505, hsa-miR- 139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa-miR- 194*, hsa-miR-215, hsa-miR-335, hsa-miR-451
  • a diagnostic kit is for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the hepatocellular cancer is carcinoma embolus
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-505 (SEQ ID NO:32), hsa-miR-139-5p (SEQ ID NO: 10), hsa-miR-122* (SEQ ID NO:20), hsa-miR-30a* (SEQ ID NO: 90), hsa- miR-30e* (SEQ ID NO:26), hsa-miR-378* (SEQ ID NO:28), hsa-miR-144* (SEQ ID NO:12), hsa-miR-378 (SEQ ID NO:27), hsa-miR-100 (SEQ ID NO:14), hssa-miR-100
  • the expression of any one or more of the nucleic acid molecules encoding hsa-miR-331-3p, and hsa-miR-55 Ib is up-regulated and the expression of any one or more of the nucleic acid molecules hsa-miR-505, hsa-miR- 139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa-miR- 194*, hsa-miR-215, hsa-miR-335, hsa-miR-451
  • the expression signature for identifying one or more target cells exhibiting or having a predisposition to develop hepatocellular cancer i.e. hepatocellular carcinoma and carcinoma embolus
  • the expression signature for identifying one or more target cells exhibiting or having a predisposition to develop hepatocellular cancer may comprise any one or more of the nucleic acid molecules identified as in Table 4.
  • the expression signature for identifying one or more target cells exhibiting or having a predisposition to develop hepatocellular cancer (i.e. hepatocellular carcinoma and carcinoma embolus) as well as for discriminating between hepatocellular carcinoma and carcinoma embolus may comprise any one or more of the nucleic acid molecules identified as in Tables 1 to 3, respectively, of the present invention.
  • the present invention relates to a method for identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer, the method comprising:
  • the method is for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the method of the present invention comprises determining and comparing the expression levels of a plurality of nucleic acid molecules encoding a microRNA sequence both in one or more target cells supposed to exhibit or to have a predisposition to develop hepatocellular cancer and in one or more control cells, i.e. typically wild- type cells not showing the characteristics of such a cancerous phenotype (cf. also the discussion above).
  • the invention relates to a method for preventing or treating hepatocellular cancer in one or more mammalian target cells, the method comprising:
  • modifying the expression of a nucleic acid molecule encoding a miRNA sequence denotes any manipulation of a particular nucleic acid molecule resulting in an altered expression level of said molecule, that is, the production of a different amount of corresponding miRNA as compared to the expression of the "wild-type" (i.e. the unmodified control).
  • the term "different amount”, as used herein, includes both a higher amount and a lower amount than determined in the unmodified control.
  • a manipulation, as defined herein may either up-regulate (i.e. activate) or down-regulate (i.e. inhibit) the expression (i.e. particularly transcription) of a nucleic acid molecule.
  • expression of one or more nucleic acid molecules encoding a microRNA sequence comprised in the nucleic acid expression signature is modified in such way that the expression of a nucleic acid molecule whose expression is up-regulated in the one or more target cells is down-regulated and the expression of a nucleic acid molecule whose expression is down-regulated in the one or more target cells is up-regulated.
  • the modification of expression of a particular nucleic acid molecule encoding a miRNA sequence occurs in an anti-cyclical pattern to the regulation of said molecule in the one or more cancerous target cells in order to interfere with the "excess activity" of an up-regulated molecule and/or to restore the "deficient activity" of a down-regulated molecule in the one or more target cells.
  • down-regulating the expression of a nucleic acid molecule comprises introducing into the one or more target cells a nucleic acid molecule encoding a sequence that is complementary to the microRNA sequence encoded by nucleic acid molecule to be down-regulated.
  • introducing into a cell refers to any manipulation allowing the transfer of one or more nucleic acid molecules into a cell. Examples of such techniques include inter alia transfection or transduction techniques all of them well established in the art (cf, for example, Sambrook, J. et al. (1989) Molecular, Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F.M. et al. (2001) Current Protocols in Molecular Biology, Wiley & Sons, Hoboken, NJ).
  • complementary sequence is to be understood that the "complementary" nucleic acid molecule (herein also referred to as an "anti-sense nucleic acid molecule”) introduced into the one or more cells is capable of forming base pairs, preferably Watson-Crick base pairs, with the up-regulated endogenous "sense" nucleic acid molecule.
  • nucleic acid molecules may be perfectly complementary, that is, they do not contain any base mismatches and/or additional or missing nucleotides.
  • the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions).
  • the "complementary" nucleic acid molecule comprises a stretch of at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in the up-regulated "sense" nucleic acid molecule.
  • the "complementary" nucleic acid molecule i.e.
  • nucleic acid molecule encoding a nucleic acid sequence that is complementary to the microRNA sequence encoded by nucleic acid molecule to be down-regulated may be a naturally occurring DNA- or RNA molecule or a synthetic nucleic acid molecule comprising in its sequence one or more modified nucleotides which may be of the same type or of one or more different types.
  • nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit.
  • the nucleic acid molecule may contain one or more modifications of the RNA backbone into 2'-0-methyl group or 2'-0-methoxyethyl group (also referred to as "2'-0-methylation”), which prevented nuclease degradation in the culture media and, importantly, also prevented endonucleo lytic cleavage by the RNA- induced silencing complex nuclease, leading to irreversible inhibition of the miRNA.
  • LNAs locked nucleic acids
  • RNA-mimicking sugar conformation cf, e.g., Oram, U.A. et al. (2006) Gene 372, 137-141.
  • Another class of silencers of miRNA expression was recently developed. These chemically engineered oligonucleotides, named “antagomirs”, represent single- stranded 23-nucleotide RNA molecules conjugated to cholesterol (Krutzfeldt, J. et al. (2005) Nature 438, 685-689).
  • microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes, were generated as well. Termed “microRNA sponges", these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest (Ebert, M.S. et al. (2007) Nat. Methods 4, 721- 726).
  • the one or more nucleic acid molecules whose expression is to be down-regulated encode microRNA sequences selected from:
  • the one or more nucleic acid molecules whose expression is to be down-regulated encode microRNA sequences selected from the group consisting of hsa-miR-505, hsa-miR- 139-5p, hsa-miR-122*, hsa-miR-30a*, hsa-miR-30e*, hsa-miR-378*, hsa-miR-144*, hsa-miR-378, hsa-miR-100, hsa-miR-lOa, hsa-miR-192*, hsa-miR-29c*, hsa-miR- 194*, hsa-miR-215, hsa-miR-331-3p, hsa-miR-335, hsa-miR-451, hsa-miR-486-5p
  • up-regulating the expression of a nucleic acid molecule comprises introducing into the one or more target cells a nucleic acid molecule encoding the microRNA sequence encoded by nucleic acid molecule to be up-regulated.
  • the up-regulation of the expression of a nucleic acid molecule encoding a miRNA sequence is accomplished by introducing into the one or more cells another copy of said miRNA sequence (i.e. an additional "sense" nucleic acid molecule).
  • the "sense" nucleic acid molecule to be introduced into the one or more target cells may comprise the same modification as the "anti-sense" nucleic acid molecules described above.
  • the one or more nucleic acid molecules whose expression is to be up-regulated encode microRNA sequences selected from: (a) the group consisting of hsa-miR-139-5p, hsa-miR-144, hsa-miR-144*, and hsa-miR-450a with respect to the "general" expression signature, presumably indicative for tumor invasiveness as defined above; and/or (b) the group consisting of hsa-miR-375, hsa-miR-451, and hsa-miR-335 with respect to the expression signature for discriminating hepatocellular carcinoma as defined above; and/or .
  • the "sense” and/or the “anti-sense” nucleic acid molecules to be introduced into the one or more target cells in order to modify the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature may be operably linked to a regulatory sequence in order to allow expression of the nucleotide sequence.
  • a nucleic acid molecule is referred to as "capable of expressing a nucleic acid molecule" or capable “to allow expression of a nucleotide sequence” if it comprises sequence elements which contain information regarding to transcriptional and/or translational regulation, and such sequences are “operably linked” to the nucleotide sequence encoding the polypeptide.
  • An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed (and/or the sequences to be expressed among each other) are connected in a way that enables gene expression.
  • the precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e.
  • promoter regions normally include 5' non- coding sequences involved in initiation of transcription and translation, such as the -35/- 10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.
  • the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.
  • nucleic molecules may also be influenced by the presence, e.g., of modified nucleotides (cf. the discussion above).
  • modified nucleotides cf. the discussion above.
  • LNA locked nucleic acid
  • a nucleic acid molecule of the invention to be introduced into the one or more cells provided may include a regulatory sequence, preferably a promoter sequence, and optionally also a transcriptional termination sequence.
  • the promoters may allow for either a constitutive or an inducible gene expression.
  • Suitable promoters include inter alia the E. coli / ⁇ cUV5 and tet (tetracycline-responsive) promoters, the T7 promoter as well as the SV40 promoter or the CMV promoter.
  • the nucleic acid molecules of the invention may also be comprised in a vector or other cloning vehicles, such as plasmids, phagemids, phages, cosmids or artificial chromosomes.
  • the nucleic acid molecule is comprised in a vector, particularly in an expression vector.
  • Such an expression vector can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a genetic construct as defined in the invention, replication and control sequences derived from a species compatible with the host that is used for expression as well as selection markers conferring a selectable phenotype on transfected cells. Large numbers of suitable vectors such as pSUPER and pSUPERIOR are known in the art, and are commercially available.
  • the invention relates to a pharmaceutical composition for the prevention and/or treatment of hepatocellular cancer, preferably manifested as an adenocarcinoma, in one or more mammalian target cells, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence that is at least partially complementary to a microRNA sequence encoded by a nucleic acid molecule whose expression is up-regulated in the one or more target cells, as defined herein, and/or that corresponds to a microRNA sequence encoded by a nucleic acid molecule whose expression is down-regulated in the one or more target cells, as defined herein.
  • the invention is directed to the use of such a pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of hepatocellular cancer, preferably manifested as an adenocarcinoma.
  • suitable pharmaceutical compositions include inter alia those compositions suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), peritoneal and parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Administration may be local or systemic. Preferably, administration is accomplished via the oral or intravenous routes.
  • the formulations may also be packaged in discrete dosage units.
  • compositions according to the present invention include any pharmaceutical dosage forms established in the art, such as inter alia capsules, microcapsules, cachets, pills, tablets, powders, pellets, multi-particulate formulations (e.g., beads, granules or crystals), aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions such as ointments, and oil- in water emulsions such as creams, lotions, and balms.
  • pharmaceutical dosage forms established in the art, such as inter alia capsules, microcapsules, cachets, pills, tablets, powders, pellets, multi-particulate formulations (e.g., beads, granules or crystals), aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions such as oint
  • compositions in order to prepare the pharmaceutical compositions, pharmaceutically inert inorganic or organic excipients (i.e. carriers) can be used.
  • excipients for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils may be used.
  • Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols, and suitable mixtures thereof as well as vegetable oils.
  • the pharmaceutical composition may also contain additives, such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsif ⁇ ers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsif ⁇ ers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsif ⁇ ers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • the nucleic acid molecules may be incorporated into slow or sustained release or targeted delivery systems, such as liposomes, nanoparticles, and microcapsules.
  • One approach involves covalently coupling the passenger strand (miRNA* strand) of the miRNA to cholesterol or derivatives/conjugates thereof to facilitate uptake through ubiquitously expressed cell-surface LDL receptors (Soutschek, J. et al.
  • LNA-antimiR locked- nucleic-acid-modified oligonucleotides
  • lipidoids synthesis scheme based upon the conjugate addition of alkylacrylates or alkyl-acrylamides to primary or secondary amines
  • RNAi therapeutics Akinc, A. et al. (2008) Nat. Biotechnol 26, 561-569.
  • a further cell-specific targeting strategy involves the mixing of miRNAs with a fusion protein composed of a targeting antibody fragment linked to protamine, the basic protein that nucleates DNA in sperm and binds miRNAs by charge (Song, E. et al. (2005) Nat. Biotechnol. 23, 709-717).
  • a fusion protein composed of a targeting antibody fragment linked to protamine, the basic protein that nucleates DNA in sperm and binds miRNAs by charge
  • a fusion protein composed of a targeting antibody fragment linked to protamine, the basic protein that nucleates DNA in sperm and binds miRNAs by charge
  • Example 1 Sample collection and preparation
  • Fig. 2 The principal method steps for identifying one or more target cells in a patient's sample exhibiting or having a predisposition to develop colorectal cancer are shown in Fig. 2.
  • Biopsies were taken during surgery (e.g., liver resection), if abnormal areas were found. Biopsy samples are usually about 0.5 cm in diameter. Surgical specimens were snap-frozen in liquid nitrogen at or immediately after collection. Samples may also be stored at -80 0 C.
  • Patient data (age, sex, imaging data, therapy, other medical conditions, family history, and the like) were derived from the hospital databases for matching the various samples collected.
  • Pathologic follow-up (for example, histological analysis via hematoxylin and eosin (H&E) staining) was used for evidently determining the disease state (i.e. control, pre-cancerous stage (e.g., hepatic cirrhosis), primary malignancy (e.g., hepatocellular carcinoma) or secondary/metastatic malignancy (e.g., carcinoma embolus)) of a given sample as well as to ensure a consistent classification of the specimens.
  • H&E histological analysis via hematoxylin and eosin
  • Laser-capture micro-dissection was optionally performed for each cancerous sample in order to specifically isolate tumor cell populations (about 200.000 cells).
  • a transparent transfer film is applied to the surface of a tissue section or specimen. Under a microscope, the thin tissue section is viewed through the glass slide on which it is mounted and clusters of cells are identified for isolation.
  • a near IR laser diode integral with the microscope optics is activated.
  • the pulsed laser beam activates a spot on the transfer film, fusing the film with the underlying cells of choice.
  • the transfer film with the bonded cells is then lifted off the thin tissue section (reviewed, e.g., in Emmert-Buck, M.R. et al. (1996).
  • RNA purification was performed according to the guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159) using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions.
  • the population of miRNAs was isolated via the mzVVanaTM miRNA Isolation Kit (Ambion, Inc., Austin, TX, USA) according to the manufacturer's instruction.
  • a qualitative analysis of the miRNAs (differentially) expressed in a particular sample may optionally be performed using the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions.
  • the raw data obtained for single-color (CY3) hybridization were normalized by applying a Quantile method and using the R software known in the art.
  • a synthesized heterogenous miRNA may be added ("spiked-in") to the samples at certain ratio with respect to the total RNA concentration as an internal positive control for quantitative analysis.
  • a "spike-in” miRNA may be a plant miRNA, for example, ath-miR168a, ath-miR162a, ppt- miR898b, or smo-miRl 100, that has a low homology to human gene or transcript sequences.
  • the "spike-in” miRNA may be any sequence 18 nt. to 30 nt. in length that is less than 70% homo log to human gene or transcript sequences.
  • the miRNA expression data obtained an established real-time quantitative RT-PCR employing a TaqMan MicroRNA assay (Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's instructions. For assessing whether a particular miRNA is differentially expressed in cancerogenous target cells as compared to control cells the following criteria were used:
  • the miRNA was considered to be differentially expressed in the target and control cells, respectively.
  • the miRNA expression level determined represents the mean value of the respective individual data obtained.
  • Table 4 lists the miRNAs exhibiting a differential expression both in hepatocellular carcinoma (HCC) and carcinoma embolus (CE).
  • Table 5 summarizes the miRNAs exhibiting a differential expression only in hepatocellular carcinoma
  • Table 6 lists the miRNAs exhibiting a differential expression only in carcinoma embolus.
  • ID the abbreviation "k” denotes a known miRNA in hepatocellular cancer
  • n denotes a newly identified miRNA.
  • the expression levels and the degree of differential expression are listed in the columns “geometric mean”.
  • SENS denotes the sensitivity of detection, that is, the occurrence of the differentially expressed miRNA in HCC or CE samples.
  • Particularly preferred miRNAs are shown in bold.
  • hepatocellular carcinoma and carcinoma embolus The principal method steps for identifying one or more target cells in a patient's sample exhibiting or having a predisposition to develop hepatocellular cancer (i.e. hepatocellular carcinoma and carcinoma embolus) are shown in Fig. 2.
  • liver tissue specimens 13 normal tissues, 18 hepatic cirrhosis, 24 HCC without embolus and 8 carcinoma embolus
  • Surgical specimens were snap-frozen in liquid nitrogen at or immediately after collection. Samples may also be stored at -80 0 C.
  • Patient data (age, sex, imaging data, therapy, other medical conditions, family history, and the like) were derived from the hospital databases for matching the various samples collected.
  • Pathologic follow-up for example, histological analysis via hematoxylin and eosin (H&E) staining was used for evidently determining the disease state (i.e.
  • pre-cancerous stage e.g., hepatic cirrhosis
  • primary malignancy e.g., hepatocellular carcinoma
  • secondary/metastatic malignancy e.g., carcinoma embolus
  • Laser-capture micro-dissection was optionally performed for each cancerous sample in order to specifically isolate tumor cell populations (about 200.000 cells).
  • a transparent transfer film is applied to the surface of a tissue section or specimen. Under a microscope, the thin tissue section is viewed through the glass slide on which it is mounted and clusters of cells are identified for isolation.
  • a near IR laser diode integral with the microscope optics is activated.
  • the pulsed laser beam activates a spot on the transfer film, fusing the film with the underlying cells of choice.
  • the transfer film with the bonded cells is then lifted off the thin tissue section (reviewed, e.g., in Emmert-Buck, M. R. et al. (1996).
  • Example 4 Analysis of the miRNA expression profile in the samples
  • a qualitative analysis of the miRNAs (differentially) expressed in a particular sample may optionally be performed using the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA).
  • the microarray contains probes for 723 human miRNAs from the Sanger database v.10.1. Total RNA (100 ng) derived from each of 63 LCM-selected liver samples were used as inputs for labeling via Cy3 incorporation.
  • Microarray slides were scanned by XDR Scan (PMTlOO, PMT5). The labeling and hybridization were performed according to the protocols in the Agilent miRNA microarray system.
  • the raw data obtained for single-color (CY3) hybridization were normalized by applying a Quantile method and using GeneSpring GXlO software (Agilent Technologies, Santa Clara, CA, USA) known in the art.
  • the miRNA expression data obtained an established real-time quantitative RT-PCR employing a TaqMan MicroRNA assay (Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's instructions.
  • the miRNA was considered to be differentially expressed in the target and control cells, respectively.
  • the miRNA expression level determined represents the mean value of the respective individual data obtained.
  • Table 7 lists preferable miRNAs exhibiting a differential expression in hepatocellualar carcinoma (HCC) and carcinoma embolus (CE). Particularly preferred miRNAs with
  • the results obtained demonstrate a global highly specific regulation of miRNA expression in hepatocellular cancer.
  • the respective subsets of miRNAs specified herein represent unique miRNA expression signatures for expression profiling of hepatocellular cancer that do not only allow the identification of a cancerogenous state as such but also enables the discrimination between different types of tumors, namely hepatocellular carcinoma (a primary malignancy) and carcinoma embolus (a secondary malignancy).
  • the miRNA expression signatures defined herein do not run out in the mere identification of hepatocellular cancer (HCC) but also enable a reliably risk assessment of patients exhibiting or being supposed to have a pre-cancerous state (e.g., a hepatic cirrhosis) whether or not the pre-cancerous state will progress in a carcinoma.
  • the miRNA expression signatures as defined herein enable a prediction of disease progression for patients having a predisposition to develop HCC.
  • the identification of the miRNA expression signatures of the present invention provides a unique molecular marker that allows the detection of HCC at an early disease stage (that is, at a stage where the presence of malignant cells is not yet detectable by in situ techniques or microscopic analysis of biopsy or resection material), where HCC still can be efficiently treated markedly. Furthermore, the prediction of cancer progression may be used to guide the therapy decision in patients exhibiting a pre-cancerous state of HCC.
  • Diagnostic kit of molecular markers for identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of or the predisposition to develop hepatocellular cancer.
  • nucleic acid expression signature comprises at least five nucleic acid molecules, preferably at least eight nucleic acid molecules, and particularly preferably at least twelve nucleic acid molecules.
  • nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target cells compared to the one or more control cells and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in the one or more target cells compared to the one or more control cells.
  • nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-mir- 221, hsa-mir-324-5p, hsa-mir-96, hsa-mir-18a, hsa-mir-18b, hsa-mir-30d, hsa-mir-331- 3p, hsa-mir-183, hsa-mir-551b, hsa-mir-139-5p, hsa-mir-144, hsa-mir-144*, and hsa- mir-450a.
  • kit of 4 wherein the expression of any one or more of the nucleic acid molecules encoding hsa-mir-221, hsa-mir-324-5p, hsa-mir-96, hsa-mir- 18a, hsa-mir-18b, hsa-mir-30d, hsa-mir-331-3p, hsa-mir-183, and hsa-mir-551b is up- regulated and the expression of any one or more of the nucleic acid molecules hsa-mir- 139-5p, hsa-mir-144, hsa-mir-144*, and hsa-mir-450a is down-regulated in the in the one or more target cells compared to the one or more control cells.
  • kit of any of 1 to 5, for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus.
  • the hepatocellular cancer is hepatocellular carcinoma
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-224, hsa-miR-532-3p, hsa-miR-663, hsa-miR-99b, hsa-miR-362-5p, hsa-miR-652, hsa-miR-222, hsa-miR-501-3p, hsa-miR- 375, hsa-miR-451, and hsa-miR-335
  • the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding hsa-miR-199b-5p, hsa-miR-21, hsa-miR-19b, hsa-miR-103, hsa-miR-23a, hsa-miR-125b-2*, hsa-miR-30c-2*, hsa-miR-505*, and hsa-miR-203.
  • Method for identifying one or more mammalian target cells exhibiting or having a predisposition to develop hepatocellular cancer comprising:
  • step (c) identifying from the plurality of nucleic acid molecules one or more nucleic acid molecules that are differentially expressed in the target and control cells by comparing the respective expression levels obtained in steps (a) and (b), wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature, as defined in any of 1 to 10, that is indicative for the presence of or the predisposition to develop hepatocellular cancer.
  • the method of 11 for the further use of discriminating hepatocellular cancer selected from the group consisting of hepatocellular carcinoma and carcinoma embolus. 13.
  • Method for preventing or treating hepatocellular cancer in one or more mammalian target cells comprising:
  • compositions for the prevention and/or treatment of hepatocellular cancer in one or more mammalian target cells comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence that is at least partially complementary to a microRNA sequence encoded by a nucleic acid molecule whose expression is up-regulated in the one or more target cells, as defined in any of 1 to 10, and/or that corresponds to a microRNA sequence encoded by a nucleic acid molecule whose expression is down-regulated in the one or more target cells, as defined in any of 1 to 10.

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Abstract

La présente invention porte sur des compositions et procédés pour le profilage de l’expression des microARN (ARNmi) du cancer hépatocellulaire. L’invention concerne en particulier un kit de diagnostic de marqueurs moléculaires permettant d’identifier une ou plusieurs cellules mammaliennes cibles présentant ou possédant une prédisposition à développer un cancer hépatocellulaire, ledit kit comprenant une pluralité de molécules d’acide nucléique, chaque molécule d’acide nucléique codant pour une séquence de microARN, une ou plusieurs molécules appartenant à ladite pluralité de molécules d’acide nucléique étant exprimées de façon différentielle dans les cellules cibles et dans une ou plusieurs cellules témoins, et lesdites une ou plusieurs molécules d’acide nucléique exprimées de façon différentielle représentant, ensemble, une signature d’expression d’acide nucléique indicatrice de la présence d’un cancer hépatocellulaire ou d’une prédisposition à développer un cancer hépatocellulaire. L’invention concerne également des procédés correspondants utilisant de telles signatures d’expression d’acide nucléique pour identifier une ou plusieurs cellules mammaliennes cibles présentant ou possédant une prédisposition à développer un cancer hépatocellulaire, ainsi que pour prévenir ou traiter un tel état pathologique. L’invention porte enfin sur une composition pharmaceutique pour la prévention et/ou le traitement du cancer hépatocellulaire.
PCT/IB2009/055058 2008-11-13 2009-11-13 Compositions et procédés pour le profilage de l’expression des microarn du cancer hépatocellulaire WO2010055488A2 (fr)

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WO2012151736A1 (fr) * 2011-05-06 2012-11-15 Zhongshan Hospital Fudan University Marqueur constitué d'un microarn du plasma et nouvelle méthode de diagnostic d'un carcinome hépatocellulaire
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WO2018231851A1 (fr) * 2017-06-13 2018-12-20 Gilead Sciences, Inc. Méthodes de traitement de la fibrose hépatique
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