WO2018054960A1 - Methods for predicting and treating resistance to chemotherapy in npm-alk(+) alcl - Google Patents

Methods for predicting and treating resistance to chemotherapy in npm-alk(+) alcl Download PDF

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WO2018054960A1
WO2018054960A1 PCT/EP2017/073748 EP2017073748W WO2018054960A1 WO 2018054960 A1 WO2018054960 A1 WO 2018054960A1 EP 2017073748 W EP2017073748 W EP 2017073748W WO 2018054960 A1 WO2018054960 A1 WO 2018054960A1
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alk
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Fabienne MEGGETTO
Coralie HOAREAU-AVEILLA
Annabelle CONGRAS
Nouritza TOROSSIAN
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paul Sabatier Toulouse Iii
Centre Hospitalier Universitaire De Toulouse
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Abstract

The present invention relates to methods for predicting and treating resistance to chemotherapy in NPM-ALK(+) ALCL. The inventors screened miR-125b expression in NPM- ALK(+) human and murine lymphoma models. They showed that NPM-ALK activity is responsible for miR-125b silencing in all NPM-ALK(+) models tested. Increasing miR-125b levels resulted in downregulation of the pro-apoptotic protein BAK-1, a bona fide miR-125b target, involved in the resistance to chemotherapy drug in cancers solid cells. The inventors also revealed that elevated miR-125b lead to resistance against doxorubicin. In particular, the present invention relates to a method for predicting whether a patient suffering from NPM- ALK(+) anaplastic large cell lymphoma (ALCL) will achieve a response with chemotherapy comprising determining the expression level of miR-125b in a tumor sample obtained from the patient.

Description

METHODS FOR PREDICTING AND TREATING RESISTANCE TO

CHEMOTHERAPY IN NPM-ALK(+) ALCL

FIELD OF THE INVENTION:

The present invention relates to methods for predicting and treating resistance to chemotherapy in NPM-ALK(+) ALCL.

BACKGROUND OF THE INVENTION:

Anaplastic large cell lymphoma (ALCL) is a biologic and clinically heterogeneous subtype of T-cell lymphoma with large lymphoid cells expressing the Ki-1 (CD30) molecule. Clinically, ALCL may present as localized (primary) cutaneous disease or widespread systemic disease (1-6). According to the 2008 WHO classification there are 2 forms of systemic ALCL based on the presence or absence of the aberrant expression of onco-protein NPM-ALK (1-5). NPM-ALK is a fusion protein containing the amino terminal region of NPM (nucleophosmin) juxtaposed to the entire intracytoplasmic domain of ALK. NPM-ALK is produced as a consequence of a reciprocal t(2;5) chromosomal translocation implicating the NPM gene on 5q35 and the ALK gene on 2p23 (7). NPM-ALK(+) ALCL usually affects children and young adults and accounts for about 3 percent of adult non-Hodgkin' s lymphoma and 10-15 percent of childhood lymphoma (1-5).

The anthracycline doxorubicin also known as hydroxydaunorubicin (Adriamycini®) is one of the most typical anticancer drugs commonly employed in the clinic (8). It is well accepted that the antitumor activity of doxorubicin is mainly due to its capacity to intercale into DNA and to impair the topoisomerase-II-mediated DNA repair resulting in apoptosis (9). Doxorubicin is indicated in the treatment of a broad spectrum of solid tumors and in the treatment of leukemias, as well as lymphoma including NPM-ALK(+) ALCL (10). Doxorubicin-containing polychemotherapy, typically CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), is the standard first-line treatment for NPM-ALK(+) ALCL (11, 12). Although conventional CHOP polychemotherapy is able to achieve high rate of remission, relapse and resistance occur in more than 40% of the patients, and prognosis of these patients remains invariably poor (11, 12). New treatments for ALCL are being researched all the time. For the treatment of patients with relapsed or refractory NPM-ALK(+) ALCL there are several new drugs now in clinical trials that are showing promising results, including Alisertib (MLN8237, aurora A kinase inhibitor), Bortezomib (Velcade®, proteasome inhibitor), Combination of brentuximab vedotin (Adcetris®, antibody-drug conjugate directed to the CD30) and chemotherapy and crizotinib (Xalkori®).

The cell-transforming potential of NPM-ALK chimeric protein in tumors largely depends on its deregulated tyrosine kinase activity that results from spontaneous dimerization through the NPM amino terminal domain (7). NPM-ALK interacts with molecules involved in the regulation of cell proliferation, survival, motility and cytoskeletal rearrangements such as JAK/STAT, PI3K/AKT, Ras/MAPK/ERK, Shp2, pl30Cas, PLCy and Src (13). In addition, we and other have highlighted the role of specific microRNAs in oncogenic ALK signaling in NPM-ALK(+) ALCL (14-22). MicroRNAs (miRNAs) are 18-24 bases in length and are a class of endogenously expressed small non-coding ribonucleic acids that are able to regulate gene expression at the post-transcriptional level through binding with the complementary sequences of the target mRNA. Numerous studies have provided certain evidence on the close correlation between aberrant expression of miRNAs and tumoral development. Some miRNAs are considered to be oncogenes or tumor suppressors and are aberrantly expressed in tumors including hematological malignancies (23, 24). However the action of certain miRNAs is dependent upon cellular or environmental context and results in both tumor-suppressive and - promoting roles (25, 26). The cellular concentration of miRNAs can be controlled at the transcriptional level by epigenetic modifications. Promoter methylation is a common mechanism for silencing miRNAs in cancer (27). Our laboratories showed for the first time that NPM-ALK(+) ALCL cell lines and biopsy specimens express low levels of two miRNAs, miR- 29a and miR-150 by DNA methylation (15, 22). Thus, miRNAs are likely to be useful as diagnostic and prognostic biomarkers and for cancer therapy (28, 29).

DNA methylation occurs in mammalian genome principally at cytosine residue of the CpG motif. This covalent epigenetic modification is catalysed by a family of enzymes named DNMTs (DNA methyltranferases) comprising 3 members, DNMT1, DNMT3A and DNMT3B. DNA hypermethylation by these DNMTs helps to regulate gene expression. Malignant cells tend to present an aberrant DNA hypermethylation on tumor suppressor genes that promotes tumorigenesis (30, 31). DNA methylation changes are reversible, in contrast to gene mutations, and there is a potential to reverse gene silencing using molecules, such as the decitabine (also referred as 5-Aza-2'-deoxycytidine) which inhibits DNMTs (32, 33). A multiple number of studies suggest a direct role of epigenetic mechanisms in cancer chemoresistance, normally due to deregulation of genes involved in DNA damage response, cell-cycle control, apoptosis, and DNA repair pathways. Moreover, the acquisition of resistance in cancer cells is associated with profound deregulation of cellular epigenetic landscape as characterized by global DNA hypomethylation (34). Furthermore, it is proposed that chemotherapy itself can exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance (35). Besides being the predominant substrate of DNA methylation (36), the CpG doublet is also a target of numerous DNA damaging agents, including doxorubicin (37). In addition, many TopoII inhibitors, such as etoposide and doxorubicin, suppress the DNMT1 -dependent DNA methylation (38). Thus, doxorubicin could promote transcriptional upregulation of microRNAs and promotes resistance of cancer cells to anthracyclins (39). Hence, identifying the methylation changes of microRNAs related to drug resistance might provide a diagnostic clue. MicroRNAs have recently been shown to play important roles in the development of chemoresistance and resistance to doxorubicin is a common and representative barrier for successful treatment of ALCL NPM-ALK(+). To investigate the potential role of methylated microRNAs in the doxorubicin-response of NPM- ALK(+) ALCLs, we focused our attention on miR- 125b.

MiR-125, which is a highly conserved miRNA throughout diverse species from nematode to humans, consists of three homologs hsa-miR-125a, hsa-miR- 125b- l and hsa-miR- 125b-2. Mature miR-125b, the human orthologue of lin-4, one of the very first miRNA identified in C. elegans, is transcribed from two loci located on chromosomes l lq23 (MIR- 125B-1) and 21q21 (MIR-125B-2) (40-42). Previous studies have shown that miR-125b have an ubiquitous expression in a variety of organs, including stomach, liver, lung, rectum, mammary gland, prostate, ovarian and hematopoietic cells. An increase number of studies confirmed that when tumor occurred in these organs miR- 125b generally exhibits aberrant expression, which could either act as tumor suppressor by down-regulating oncogene expression or tumor promoter through inhibition of tumor suppressor genes expression (43-49). With regard to drug resistance, miR- 125b has been shown to increase cell resistance to anthracyclins (daunorubicin and doxorubicin), in leukemia and Ewing sarcoma by suppressing the expression of apoptotic mediators, such as p53 and/or BAK1 (Bcl2 antagonist killer 1) (50, 51). High BAK1 expression correlates with drug sensitivity in malignant lymphohematopoietic cells whereas low BAK1 levels correlate with resistance and relapse (52). Thus aberrant expression of miR- 125b is closely related to proliferation, apoptosis, invasion, metastasis and immune response. Although deregulation of miR-125b plays a role in tumor development, the regulation of miR-125b expression is not well understood. Recent studies showed that in solid tumors, the methylation state of CpG-rich regions is correlated with the expression pattern of miR-125b and hypermethylation highlight miR- 125b as a potential biomarker for clinical outcome (53-55). Merkel and coworkers reported that miR-125a is downregulated in both NPM-ALK(+) and NPM-ALK(-) ALCL human cell lines and primary tissues while miR-125b expression are low only in mouse and various human NPM-ALK(+) lymphoma models suggesting that this miRNA may play important roles in the pathogenesis of NPM-ALK(+) ALCL (18). However, the impact of miR-125b deregulation on the oncogenic potency of NPM-ALK-expressing cells has not yet been investigated.

SUMMARY OF THE INVENTION:

The present invention relates to methods for predicting resistance to chemotherapy in NPM-ALK(+) ALCL. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors hypothesized that reversal of miR125b DNA methylation silencing by doxorubicin in NPM-ALK(+)-cells could displayed a resistance to doxorubicin. To test this hypothesis they screened for miR-125b expression in NPM-ALK(+) human and murine lymphoma models (human NPM-ALK[+] cell lines and primary tissues, and NPM-ALK conditional transgenic mice). The inventors show that NPM-ALK activity is responsible for miR-125b silencing in all NPM-ALK(+) models tested. This repression is likely mediated by the epigenetic silencing of miR-125b because there is a potential to reverse gene silencing using DNA methyltransferase (DNMT) inhibitors, such as the decitabine or doxorubicin treatment which could induced hypomethylation of gene promoters. Increasing miR-125b levels resulted in downregulation of the pro-apoptotic protein BAK-1, a bona fide miR-125b target, involved in the resistance to chemotherapy drug in cancers solid cells. Interestingly, the inventors revealed that elevated miR-125b lead to resistance against doxorubicin. Thus, the data from NPM-ALK(+) patients strongly suggest that miR-125b alteration might be linking to recurrence of NPM-ALK(+) after doxorubicin containing polychemotherapy. The inventors thus propose that miR- 125b as potential theranostic tool with potential clinical applications for doxorubicin- containing polychemotherapy.

Accordingly, the first object of the present invention relates to a method for predicting whether a patient suffering from NPM-ALK(+) anaplastic large cell lymphoma (ALCL) will achieve a response with chemotherapy comprising i) determining the expression level of miR- 125b in a tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value, and iii) concluding that the patient will achieve a response when the expression level of miR- 125b determined at step i) is higher than the predetermined reference value or concluding that the patient will not achieve a response when the level of miR- 125b is lower than the predetermined reference value. As used herein, the expression "NPM-ALK(+) anaplastic large cell lymphoma (ALCL)" has its general meaning in the art and refers to an ALCL characterized by the presence absence of the aberrant expression of onco fusion protein NPM-ALK.

As used herein, the term "chemotherapy" refers to the treatment which includes doxorubicin. In some embodiments, the chemotherapy is a polychemotherapy. The term "polychemothrapy" indicates chemotherapy which

As used herein, the "doxorubicin" has its general meaning in the art and refers to a compound having the structure:

Figure imgf000006_0001

or a pharmaceutically acceptable salt, solvate and/or prodrug thereof. Acceptable salts include for example hydrochloride and citrate salts.

The term "predicting whether a patient will achieve a response", as used herein refers to the determination of the likelihood that the patient will respond either favorably or unfavorably to the treatment. Especially, the term "prediction", as used herein, relates to an individual assessment of any parameter that can be useful in determining the evolution of a patient. As will be understood by those skilled in the art, the prediction of the clinical response to the treatment, although preferred to be, need not be correct for 100% of the patients to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of patients can be identified as having an increased probability of having a positive response. Whether a patient is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%. The p-values are, preferably, 0.2, 0.1 or 0.05. The method is thus particularly suitable for discriminating responder from non responder. As used herein the term "responder" in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or improved. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after chemotherapy. A non- responder includes patients for whom the cancer does not show reduction or improvement after chemotherapy. According to the invention the term "non responder" also includes patients having a stabilized cancer. The term "non-responder" also includes patient having a relapse. As used herein, the term "relapse" refers to the return of cancer after a period of improvement in which no cancer could be detected. Thus, the method of the present invention is particularly useful to prevent relapse after putatively successful treatment with chemotherapy. Typically, the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a patient who is known to be a responder or non responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media. When it is concluded that the patient is a non responder, the physician could take the decision to stop the immune checkpoint therapy to avoid any further adverse sides effects.

As used herein, the term "tumor sample" refers to any sample that include lymphoma cells and typically includes tissue samples obtained from a biopsy (e.g. lymph node or skin) or blood samples obtained from the patients. The lymphoma cells can be detected by any method well known in the art.

As used herein, the term "miR" or 'miRNA" has its general meaning in the art and refers to the miRNA sequence publicly available from the data base http://microrna.sanger.ac.uk/sequences/ under the miRBase Accession number. miR-125b pertaining to the invention is thus known per se.

According to the invention, measuring the expression level of miR- 125b in the sample obtained from the subject can be performed by a variety of techniques. For example the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. Conventional methods and reagents for isolating RNA from a sample comprise High Pure miRNA Isolation Kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction, PureLink™ miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, Pure Yield™ RNA Midiprep (Pro mega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG- RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNA™ Kit (Ambion), Poly(A)Purist™ Kit (Ambion) and any other methods, commercially available or not, known to the skilled person. The expression level of miR-125b in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of miRNA in a sample may be used. Generally, miRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms). Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy.

In some embodiments, RNA is converted to DNA (cDNA) prior to analysis. cDNA can be generated by reverse transcription of isolated miRNA using conventional techniques. miRNA reverse transcription kits are known and commercially available. Universal primers, or specific primers, including miRNA- specific stem-loop primers, are known and commercially available, for example, from Applied Biosystems. In some embodiments, miRNA is amplified prior to measurement. In some embodiments, the expression level of miRNA is measured during the amplification process. In some embodiments, the expression level of miRNA is not amplified prior to measurement. Some exemplary methods suitable for determining the expression level of miRNA in a sample are described in greater hereinafter. These methods are provided by way of illustration only, and it will be apparent to a skilled person that other suitable methods may likewise be used.

Many amplification-based methods exist for detecting the expression level of miRNA nucleic acid sequences, including, but not limited to, PCR, RT-PCR, qPCR, and rolling circle amplification. Other amplification-based techniques include, for example, ligase chain reaction (LCR), multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. A typical PCR reaction includes multiple steps, or cycles, that selectively amplify target nucleic acid species: a denaturing step, in which a target nucleic acid is denatured; an annealing step, in which a set of PCR primers (i.e., forward and reverse primers) anneal to complementary DNA strands, and an elongation step, in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target sequence. Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. A reverse transcription reaction (which produces a cDNA sequence having complementarity to a miRNA) may be performed prior to PCR amplification. Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer. Kits for quantitative real time PCR of miRNA are known, and are commercially available. Examples of suitable kits include, but are not limited to, the TaqMan® miRNA Assay (Applied Biosystems) and the mirVana™ qRT-PCR miRNA detection kit (Ambion). The miRNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence. In some embodiments, custom qRT-PCR assays can be developed for determination of miRNA levels. Custom qRT-PCR assays to measure miRNAs in a sample can be developed using, for example, methods that involve an extended reverse transcription primer and locked nucleic acid modified PCR. Custom miRNA assays can be tested by running the assay on a dilution series of chemically synthesized miRNA corresponding to the target sequence. This permits determination of the limit of detection and linear range of quantitation of each assay. Furthermore, when used as a standard curve, these data permit an estimate of the absolute abundance of miRNAs measured in the samples. Amplification curves may optionally be checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude. For each candidate miRNA assayed, a chemically synthesized version of the miRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, and the linear range of quantitation. Relative expression levels may be determined, for example, according to the 2(- ΔΔ C(T)) Method, as described by Livak et ah, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔ C(T)) Method. Methods (2001) Dec;25(4):402-8.

Rolling circle amplification is a DNA-polymerase driven reaction that can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (see, for example, Lizardi et al., Nat. Gen. (1998) 19(3):225-232; Gusev et al, Am. J. Pathol. (2001) 159(l):63-69; Nallur et al, Nucleic Acids Res. (2001) 29(23):E118). In the presence of two primers, one hybridizing to the (+) strand of DNA, and the other hybridizing to the (-) strand, a complex pattern of strand displacement results in the generation of over 109 copies of each DNA molecule in 90 minutes or less. Tandemly linked copies of a closed circle DNA molecule may be formed by using a single primer. The process can also be performed using a matrix- associated DNA. The template used for rolling circle amplification may be reverse transcribed. This method can be used as a highly sensitive indicator of miRNA sequence and expression level at very low miRNA concentrations (see, for example, Cheng et al., Angew Chem. Int. Ed. Engl. (2009) 48(18):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91).

miRNA quantification may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe. Typically, said method comprises a first step wherein the stem-loop primers are annealed to miRNA targets and extended in the presence of reverse transcriptase. Then miRNA- specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of miRNAs is estimated based on measured CT values. Many miRNA quantification assays are commercially available from Qiagen (S. A. Courtaboeuf, France), Exiqon (Vedbaek, Denmark) or Applied Biosystems (Foster City, USA).

Nucleic acids exhibiting sequence complementarity or homology to the miRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin). The probes and primers are "specific" to the miRNAs they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

miRNA may be detected using hybridization-based methods, including but not limited to hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization.

Microarrays can be used to measure the expression levels of large numbers of miRNAs simultaneously. Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre- made masks, photolithography using dynamic micromirror devices, inkjet printing, or electrochemistry on microelectrode arrays. Also useful are microfluidic TaqMan Low-Density Arrays, which are based on an array of microfluidic qRT-PCR reactions, as well as related microfluidic qRT-PCR based methods. In one example of microarray detection, various oligonucleotides (e.g., 200+ 5'- amino- modified-C6 oligos) corresponding to human sense miRNA sequences are spotted on three- dimensional CodeLink slides (GE Health/ Amersham Biosciences) at a final concentration of about 20 μΜ and processed according to manufacturer's recommendations. First strand cDNA synthesized from 20 μg TRIzol-purified total RNA is labeled with biotinylated ddUTP using the Enzo Bio Array end labeling kit (Enzo Life Sciences Inc.). Hybridization, staining, and washing can be performed according to a modified Affymetrix Antisense genome array protocol. Axon B-4000 scanner and Gene-Pix Pro 4.0 software or other suitable software can be used to scan images. Non-positive spots after background subtraction, and outliers detected by the ESD procedure, are removed. The resulting signal intensity values are normalized to per-chip median values and then used to obtain geometric means and standard errors for each miRNA. Each miRNA signal can be transformed to log base 2, and a one-sample t test can be conducted. Independent hybridizations for each sample can be performed on chips with each miRNA spotted multiple times to increase the robustness of the data.

Microarrays can be used for the expression profiling of miRNAs. For example, RNA can be extracted from the sample and, optionally, the miRNAs are size- selected from total RNA. Oligonucleotide linkers can be attached to the 5' and 3' ends of the miRNAs and the resulting ligation products are used as templates for an RT-PCR reaction. The sense strand PCR primer can have a fluorophore attached to its 5' end, thereby labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. A PCR product, referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA. Total RNA containing the miRNA extracted from the sample can also be used directly without size- selection of the miRNAs. For example, the RNA can be 3' end labeled using T4 RNA ligase and a fluorophore-labeled short RNA linker. Fluorophore-labeled miRNAs complementary to the corresponding miRNA capture probe sequences on the array hybridize, via base pairing, to the spot at which the capture probes are affixed. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA. Several types of microarrays can be employed including, but not limited to, spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays or spotted long oligonucleotide arrays.

Accordingly, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A "detectable label" is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook- A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1- sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 - (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), Dichlorotriazinylamino fluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'- difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitro tyro sine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912). In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281:2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif.).

RT-PCR is typically carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and thermal polymerase. The majority of the thermocyclers on the market now offer similar characteristics. Typically, thermocyclers involve a format of glass capillaries, plastics tubes, 96-well plates or 384-well plates. The thermocylcer also involves software analysis.

miPvNAs can also be detected without amplification using the nCounter Analysis System (NanoString Technologies, Seattle, WA). This technology employs two nucleic acid-based probes that hybridize in solution (e.g., a reporter probe and a capture probe). After hybridization, excess probes are removed, and probe/target complexes are analyzed in accordance with the manufacturer's protocol. nCounter miRNA assay kits are available from NanoString Technologies, which are capable of distinguishing between highly similar miRNAs with great specificity. The basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008. 26(3): 317- 325; the contents of which are each incorporated herein by reference in their entireties). The code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed. A pair of probes is designed for each oligonucleotide target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode. This system is also referred to, herein, as the nanoreporter code system. Specific reporter and capture probes are synthesized for each target. The reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over-lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence. Preferably, each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively. The capture probe can comprise a second target- specific sequence; and a first affinity tag. In some embodiments, the capture probe can also comprise one or more label attachment regions. Preferably, the first target- specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected. Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library". The relative abundance of each target is measured in a single multiplexed hybridization reaction. The method comprises contacting the sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex. The complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution. After hybridization, the tripartite hybridized complexes (probe pairs and target) are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes. This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target- specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample. All post hybridization steps are handled robotically on a custom liquid- handling robot (Prep Station, NanoString Technologies). Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe, electrophoresed to elongate the reporter probes, and immobilized. After processing, the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies). The expression level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface. Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance. Data is output in simple spreadsheet format listing the number of counts per target, per sample. This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No. WO 07/076129 and WO07/076132, and US Patent Publication No. 2010/0015607 and 2010/0261026, the contents of which are incorporated herein in their entireties. Further, the term nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No. 2010/0047924, incorporated herein by reference in its entirety.

Mass spectroscopy can be used to quantify miRNA using RNase mapping. Isolated

RNAs can be enzymatically digested with RNA endonucleases (RNases) having high specificity (e.g., RNase Tl, which cleaves at the 3'-side of all unmodified guanosine residues) prior to their analysis by MS or tandem MS (MS/MS) approaches. The first approach developed utilized the on-line chromatographic separation of endonuclease digests by reversed phase HPLC coupled directly to ESTMS. The presence of post-transcriptional modifications can be revealed by mass shifts from those expected based upon the RNA sequence. Ions of anomalous mass/charge values can then be isolated for tandem MS sequencing to locate the sequence placement of the post- transcriptionally modified nucleoside. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used as an analytical approach for obtaining information about post-transcriptionally modified nucleosides. MALDI- based approaches can be differentiated from EST-based approaches by the separation step. In MALDI-MS, the mass spectrometer is used to separate the miRNA. To analyze a limited quantity of intact miRNAs, a system of capillary LC coupled with nanoESI-MS can be employed, by using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) or a tandem-quadrupole time-of-flight mass spectrometer (QSTAR® XL, Applied Biosystems) equipped with a custom-made nanospray ion source, a Nanovolume Valve (Valco Instruments), and a splitless nano HPLC system (DiNa, KYA Technologies). Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated. Intact miRNAs are eluted from the trap column and directly injected into a CI 8 capillary column, and chromatographed by RP-HPLC using a gradient of solvents of increasing polarity. The chromatographic eluent is sprayed from a sprayer tip attached to the capillary column, using an ionization voltage that allows ions to be scanned in the negative polarity mode.

Additional methods for miRNA detection and measurement include, for example, strand invasion assay (Third Wave Technologies, Inc.), surface plasmon resonance (SPR), cDNA, MTDNA (metallic DNA; Advance Technologies, Saskatoon, SK), and single-molecule methods such as the one developed by US Genomics. Multiple miRNAs can be detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle- amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attamole levels. miRNAs can also be detected using branched DNA (bDNA) signal amplification (see, for example, Urdea, Nature Biotechnology (1994), 12:926-928). miRNA assays based on bDNA signal amplification are commercially available. One such assay is the QuantiGene® 2.0 miRNA Assay (Affymetrix, Santa Clara, CA). Northern Blot and in situ hybridization may also be used to detect miRNAs. Suitable methods for performing Northern Blot and in situ hybridization are known in the art. Advanced sequencing methods can likewise be used as available. For example, miRNAs can be detected using Illumina ® Next Generation Sequencing (e.g. Sequencing-By-Synthesis or TruSeq methods, using, for example, the HiSeq, HiScan, GenomeAnalyzer, or MiSeq systems (Illumina, Inc., San Diego, CA)). miRNAs can also be detected using Ion Torrent Sequencing (Ion Torrent Systems, Inc., Gulliford, CT), or other suitable methods of semiconductor sequencing.

The expression level of miR-125b may be expressed as absolute expression levels or normalized expression levels. Typically, expression levels are normalized by correcting the absolute expression level of miRNAs by comparing its expression to the expression of a mRNA that is not a relevant marker for determining whether a subject suffering from acute severe colitis (ASC) will be a responder or a non-responder to a corticosteroid, infliximab and cyclosporine, e.g., a housekeeping mRNA that is constitutively expressed. Suitable mRNAs for normalization include housekeeping mRNAs such as the U6, U24, U48 and S18 (RNU1A1 and SNORD44 pour nous ! !). This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, or between samples from different sources. In a particular embodiment, expression levels are normalized by correcting the absolute expression level of miRNAs by comparing its expression to the expression of a reference mRNA.

In some embodiments, the predetermined reference value is a threshold value or a cutoff value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of expression level of the gene in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured levels of the marker in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

The second object of the present invention relates to a method of treating NPM-ALK(+) anaplastic large cell lymphoma in a patient in need thereof comprising i) predicting whether the patient will achieve a response with chemotherapy by performing the method of the first object and ii) administering the patient with chemotherapy when it is predicted that the patient will achieve a response.

In some embodiments, when it is predicted that the patient will not achieve a response with chemotherapy, the patient is administered with at least one drug selected from the group consisting of aurora A kinase inhibitors (e.g. Alisertib (MLN8237), proteasome inhibitors (e.g. Bortezomib such as Velcade®), antibodies having specificity for CD30 (e.g. antibody-drug conjugates directed to the CD30 such as brentuximab vedotin), crizotinib, histone deacetylase inhibitors and DNA methyltransferase inhibitors. As used herein, the term "histone deacetylase inhibitor" or "HDAC inhibitor" in relation to the present invention is to be understood as meaning any molecule of natural, recombinant or synthetic origin capable of inhibiting the activity of at least one of the enzymes classified as histone deacetylases of class I, class II or class IV. HDAC inhibitors can be separated into several structurally distinct classes: short- chain fatty acids (i.e., valproic acid), hydroxamic acids (i.e., vorinostat, TSA, tubacin, and PCT 24781), benzamides (i.e., entinostat), cyclic tetrapeptides (i.e., romidepsin), and electrophilic ketones. For example, the HDAC inhibitors romidepsin, panobinostat, vorinostat (SAHA) and entinostat (MS-275) inhibit the class 1 HDACs 1 and 2. Entinostat and romidepsin are more selective than the hydroxamic acids panobinostat and SAHA, which inhibit the class 2 HDAC 6 in addition to inhibiting class 1 HDACs 1, 2 and 3, and, to a lesser degree, HDAC 8. Based on data from Bradner et al., Nat Chem Biol 6:238 (2010). Some of the histone deacetylase inhibitors currently at the clinical study stage are described, with other analogues thereof, in the following patents: WO 2004/092115, WO 2005/019174, WO 2003/076422, WO 1997/043251, WO 2006/010750, WO 2006/003068, WO 2002/030879, WO 2002/022577, WO 1993/007148, WO 2008/033747, WO 2004/069823, EP 0847992 and WO 2004/071400, the contents of which are incorporated herein by reference in their entirety. Diethyl- [6-(4-hydroxycarbamoyl- phenylcarbamoyloxymethyl)-naphthalen-2-yl methyl] -ammonium chloride, which is described in WO 97/43251 (anhydrous form) and in WO 2004/065355 (monohydrate crystal form), herein both incorporated by reference, is an HDAC inhibitor. As use herein, the term "DNA methyltransferase inhibitors" or "DNMTi" DNA methyltransferase inhibitor that can be sub-divided into nucleoside analogue (5-Azacytidine (azacytidine), 5-Aza-2'-deoxycytidine (decitabine, 5- Aza-Cd ), zebularine, 5-Fluoro-2'-deoxycytidine (5-F-CdR), 5 , 6-Dihydro-5- azacytidine (DHAC)) and non-nucleoside analogue families (Hydralazine, Procainamide, Procaine, EGCG ((-)-epigallocatechin-3-gallate), Psammaplin A, MG98, RG108) (8).

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

Typically, the agents of the present invention are administered to the patient in the form of pharmaceutical compositions. The pharmaceutical composition as provided herewith may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of provided herein, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The compounds described herein including pharmaceutically acceptable carriers can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. MiR125b down-expression as a biomarker for early prognosis of NPM- ALK-positive primary tumors. Quantitative real-time PCR analysis of (A) miR-125b expression relative to RNU1A and (B) BAK1 expression relative to GAPDH in reactive lymph nodes (RLN, n=14) and in ALCL NPM-ALK(+) primary biopsies from early relapsing (with relapse, n=15) and non-relapsing (no relapse, n=17) patients. RNU1A and GAPDH were used as an internal control and the relative ratio of miR125b or BAK1 expression were expressed as 2-AACt relative to reactive lymph nodes. (C and D) Quantitative real-time PCR analysis of the expression of pri-miR125bl (C) and mature miR-125b (D) in NPM-ALK(+) ALCL KARPAS- 299 cells treated or not (PBS) with doxorubicin Actin and SNORD44 mRNA was used as an internal control and the relative ratio pri-miR125bl and miR-125b expression respectively were expressed as 2~ΔΔα relative to untreated cells. Data represent mean + SEM (bars). **P < 0.001, ***P < 0.0001; unpaired 2-tailed Student's t test.

Figure 2: MiR-125b/BAKl signaling impairs the doxorubicin response of NPM- ALK635 positive ALCL cells. In NPM-ALK(+) ALCL KARPAS-299 and COST cells transfected with a mimic miR-125b (miR-125b) or negative control microRNA (miR-CTL). Measurement of caspase 3/7 activity in cells treated with doxorubicin and transfected with either miR-CTL or miR-125b mimics. Densitometric analysis was performed using GeneTools software from Syngene. *P<0.05 and **P<0.001; unpaired 2-tailed Student's t test.

Figure 3: BAK1 silencing mimics the overexpression of miR-125b and leads to doxorubicin resistance in NPM-ALK-positive ALCL cells. Assessment of caspase 3/7 activity in NPM-ALK(+) ALCL KARPAS-299 and COST cells transfected with si-CTL or si- BAK1 and treated for 48h with 200 or 400 nM doxorubicin. Data represent means + SEM (bars) from 3 independent experiments. *P<0.05 and **P<0.001; unpaired 2-tailed Student's t test.

Figure 4: DNA me thy transferase I and DNA topoisomerase II are mediators of miR125b silencing in NPM-ALK-positive ALCL cells. Quantitative real-time PCR (qRTPCR) analysis of miR-125b expression in NPM-ALK(+) ALCL KARPAS-299 cells treated for 48h or not (PBS) with 400 nM doxorubicin. EXAMPLE:

NPM/ALK acts as a driving force behind in the miR-125b downregulation.

Since recent researches suggested that miR-125b plays in hematological disorders (43- 45, 56, 57), we studied the miR-125b expression level in ALCL cells. The expressions level of miR-125b was analyzed by quantitative real-time PCR in human NPM/ALK+ ALCL cell lines KARPAS-299 and COST, as well as in as well as a NPM-ALK-negative cell line, FE-PD. MiR- 125b levels in these cells were compared with those in CD4 isolated lymphocytes stimulated (S, n=3) or not (NS, n=4) with phytohemagglutinin (PHA). MiR-125b expression was significantly decreased as compared with normal CD4 in ALCL cell lines. Using ALK-positive model systems (human ALCL cell lines and a CD4/NPM-ALK transgenic mouse) Merkel et al. identified miR-125b as being deregulated in ALK-positive cells (18). To corroborate the results of Merkel et al., using our previously published conditional NPM/ALK lymphoma transgenic mouse model (Tet-OFF-NPM/ALK murine model) (58), miR-125b expression was assessed by quantitative real-time PCR in the presence [NPM/ALK(-)] or in the absence [NPM/ALK(+)] of doxycycline or crizotinib. Upon the both treatment, in lymph nodes isolated from mice with NPM/ALK(+) lymphoma (NPM/ALK(+), n = 6 for each treatment), we found that miR-125b was significantly downregulated, when compared with lymph nodes isolated from either control normal age-matched WT littermate mice (n = 6); or healthy transgenic mice who had received doxycycline (n = 6) or crizotinib (n = 4) treatment [NPM-ALK(-)] . The miR-125b silencing observed in all of the NPM/ALK(+) models tested suggested that the NPM-ALK(+) protein account for a part in low miR-125b expression.

To test whether NPM-ALK is involved in miR-125b downregulation, NPM-ALK was silenced in human NPM-ALK+ ALCL cell line KARPAS-299 using either crizotinib (Figure ID) or siRNAs directed against ALK mRNA (Figure IE). The loss of NPM-ALK autophosphorylation on the tyrosine 1604 residue confirmed that the ALK kinase activity was properly inhibited upon crizotinib treatment (Figure IF). In addition, we showed by western- blotting that the knockdown of expression NPM-ALK had been performed efficiently (Figure 1G). Finally, we observed that the inhibition of NPM-ALK using either crizotinib or si-RNA directed against ALK mRNA (si- ALK) induced a clear increase of miR-125b in NPM-ALK(+) cells As ( (Figure ID and IE),. Together, these results suggest that ALK tyrosine kinase activity may partially contribute to the underexpression of miR-125b.

DNA methylation is partly responsible for miR-125b silencing in NPM/ALK- positive cells.

Recent studies showed that the methylation state of distal CpG-rich regions correlated with the expression pattern of miRNAs in cancer (59). Recently, it was reported that the hypermethylation of the MIR125B-1 promoter accounts for the reduction in miR-125b expression in solid cancers. To investigate whether methylation alteration of MIR125B-1 could be extrapolated to NPM/ALK(+) cells, we carried out quantitative methylation pyro sequencing to assess CpG methylation status within the -142 to -552 CpG island enriched site region upstream of MIR125B-1, known as methylated in solid cancers. We designed and validated bisulfate sequencing PCR for the promoter region of miR-125b including 17 CpGs. MiR-125b CpG methylation levels in the NPM/ALK(+) cell lines KARPAS-299 and COST were compared with those in CD4 isolated lymphocytes stimulated (S, n=3) or not (NS, n=4) with phytohemagglutinin (PHA). We identified that only the CpGl to the CpG7 sites were found to be heavily methylated. The methylation level varied from 79% to 98% in the NPM/ALK(+) cells with a mean of 93.55% and 89.25% in the KARPAS-299 and COST cell respectively. In contrast, the methylation level of miR-125b observed in the normal CD4 lymphocytes ranged from 6% to 28%, with a mean of 17.28% and 13.86% in the unstimulated (NS) and stimulated (S) normal CD4 lymphocytes respectively. Methylation levels >20.93% were considered as hypermethylation. CpGl to the CpG7 sites were found methylated in both NPM/ALK(+) cell lines. For the CpG12 to the CpG17, the methylation level of miR-125b varied from 40% to 83%, with a mean ratio of 64.09% to 65.04% in the normal CD4 lymphocytes, whereas the methylation level of miR-125b observed in the NPM/ALK(+) cells ranged from 29% to 93.86%, with a mean of 81.72 and 65% in the KARPAS-299 and COST cell respectively. Methylation levels >74.02% were considered as hypermethylation. In KARPAS-299 cell line, all CpGs sites were found to be methylated. To further determine whether DNA methylation contributes to the silencing of miR-125 in NPM/ALK(+) cells, we analyzed methylation status of CpGl to the CpG7 sites CpG in the KARPAS-299 and COST cells treated with decitabin (a DNA hypomethylating agent) to that in untreated (PBS) cells by quantitative methylation pyrosequencing. We observed that DNA methylation levels in CpGl to the CpG7 were significantly decreased in NPM/ALK(+) cells after treatment with DNA hypomethylating drug when compared to untreated cells (PBS). Collectively, these data suggest that miR-125b silencing is due at least in part to aberrant MIR 125b- 1 promoter methylation in NPM/ALK(+) cells.

BAKl is a direct target of miR-125 in NPM-ALK positive cells.

Because the pro-apoptotic protein BAKl, is a bona fide miR-125b target (42), we performed an in vitro pull-down assay using biotinylated miR-125b to evaluate if BAKl is a direct candidate gene, as miR-125b target. As a first step in the binding analysis, following transfection of biotin-labeled miR-125b into KARPAS-299 cells, cell lysates were exposed to avidin-coated beads. RNA was harvested from the pull-down material and amplified with BAKl primers by q-PCR. A biotin-labeled irrelevant miRNA, miR-39, served as a control in these experiments. The levels of BAKl mRNA was markedly elevated in the pull-down material isolated from KARPAS-299 cells following transfection with biotin-labeled miR-125b compared with control miRNA-treated cells as revealed by quantitative real-time PCR at 72 h after transfection.

In general, the expression of miRNA targets should decrease in cells after treatment with DNA hypomethylating drugs. To confirm this relationship, we monitored the expression levels of miR-125b and BAKl in extracts of decitabin-treated NPM/ALK(+) KARPAS-299 cells by Western-blotting analysis and by quantitative real-time PCR respectively. In the two NPM/ALK(+) cell lines, as compare to untreated cells (PBS) we found that miR-125b expression was restored after treatment with the decitabin. Concomitantly to miR-125b recovery we noted that BAKl protein was reduced (0.48 fold) in NPM/ALK(+) cells upon DNA hypomethylating agent treatment. Jointly, these results suggest that in NPM/ALK(+) cells BAKl may be a functional downstream target of miR-125b.

Interplay between miR-125b and BAKl in human NPM/ALK(+) ALCL biopsies. To assess whether link between miR125b methylation and BAKl mRNA expression was also present in NPM/ALK(+) ALCL in vivo, biologic samples taken from lymph nodes of 65 patients affected by NPM/ALK(+) were analyzing using quantitative real-time PCR. Despite the presence of a mixed population of neoplastic and normal cells in lymph nodes samples, as only cases with at least 50% of lymph node involvement by malignant cells were selected, evidence for inverse correlated between miR-125b and BAKl mRNA expression was detected in all cases as compare with reactive lymph node (RLN, n = 14). To further characterize miR- 125b methylation the same NPM/ALK(+) ALCL primary tissues (n=65) and reactive lymph node (RLN, n=14) samples we also analyzed by quantitative methylation pyro sequencing confirming the presence of methylation of CpGl to the CpG7 sites in NPM/ALK(+) ALCL. Methylation level varied from 10.20% to 34.5% with a mean of 21.95% in NPM-ALK(+) primary tissues and ranged from 9% to 25%, with a mean of 15.64% in the reactive lymph node. As we considered as hypermethylation the methylation levels >20.32% evidence for miR-125b methylation was detected in NPM/ALK(+) samples as compare reactive normal lymph nodes.

miR-125b/BAKl signaling impairs doxorubicin response of NPM-ALK(+) cells. As miR-125b promotes cell resistance to chemotherapeutic agent such as anthracyclin by inhibiting apoptosis through the suppression of pro-apoptotique BAKl expression (51) and anthracyclin doxorubicin induces apoptosis through a activation of BAKl (60) we examined whether overexpression of miR-125b could act as modulators of doxorubicin induced apoptosis. NPM/ALK(+) KARPAS-299 ALCL cells were transfected with mimic-miR-125b (miR-125b) or with mimic-miR-negative control (miR-CTL) and then treated with doxorubicin or with the drug vehicle alone (PBS). The activity of caspase 3, an important marker of apoptosis, was further detected using the Caspase-Glo® 3/7 assay system in miR-125b and miR-negative control (miR-CTL) -transfected KARPAS-299 cells under various concentrations of doxorubicin (0, 0.1, 0.2, 0.4, 0.6, 0.8 and ΙμΜ) for 24h. Upon anthracyclin doxorubicin caspase 3 activity was found significantly decreased without significantly change of cell proliferation in miR-125b-transfected cells compared with the control cells. Apoptosis modulation was accompanied by reduction of pro-apoptotic BAKl protein expression as assessed by Western-blotting. Moreover in NPM/ALK(+) cells we confirmed that BAKl protein expression was modulated by this miR-125b and that previously described by Panaretalis et al (60) we noted also that doxorubicin induces the modulation of BAKl expression (1.61 fold). Consequently, to mimic the overexpression of miR-125b, BAKl was silenced in NPM/ALK(+) KARPAS-299 cell line using siRNAs directed against BAKl mRNA. As expected, the knockdown of pro-apoptotic BAK-1, efficiently achieved as shown by Western-blotting, induced a clear protection against the doxorubicin-induced apoptosis as assessed by Caspase-Glo® 3/7 assay system. All together, these results indicated that miR- 125b overexpression by chemotherapy regulates pharmacoresistance in NPM/ALK(+) cancer cells by modulating tumor cell apoptosis through the suppression of BAKl expression.

Reversal of miR- 125b silencing by decitabin leads to dox cyclin resistance in NPM- ALK(+) cells.

Recent researches suggested that i) miR- 125b promotes resistance to chemotherapeutic agent such as anthracyclin by inhibiting apoptosis in hematological disorders (51)and ii) abrogation of BAKl function increases apoptotic resistance to chemotherapeutic drugs (61). In order to examine the possible role of miR- 125b in anthracyclin doxorubicin resistance, we induced an increase in miR- 125b expression with decitabin in human NPM/ALK(+) ALCL cell line KARPAS-299 upon doxorubicin treatment. We first validated that DNA demethylation was sufficient to induce transcription of pri-miR-125b in NPM/ALK(+). Using quantitative real-time PCR we examined KARPAS-299 treated with decitabin for expression of pri-miR- 125b. Decitabin-treated KARPAS-299 showed expression of endogenous pri-miR-125b at a much higher level (mean 22.51 +/- 8.48) than that seen in untreated cells (PBS) (data not shown). Secondly, we analyzed the effect on apoptosis of doxorubicin-treated NPM/ALK(+) cells upon decitabin using the Caspase-Glo® 3/7 assay system (data not shown). As expected, doxorubicin induces caspase 3 activation. By contrast after the addition of decitabin we noted a significant increase of apoptotic doxorubicin resistance in NPM/ALK(+) cells. To obtain insights into the relationship between decitabin, doxycyclin resistance, miR- 125b and BAKl statue we assessed the expression level of BAKl under the single or the co-treatment using KARPAS-299 cells by Western-blotting. As expected, in decitabin-treated cells the expression of pro-apoptotic BAKl protein was downregulated ( 0.51 fold). By contrast doxorubicin induces the positive modulation of BAKl expression (1.54 fold) as compared with untreated NPM/ALK(+) cells. Hence we observed a decrease of BAKl expression in doxorubicin-treated cells upon decitabin, co-treatment. Since the hypomethylating agent caused concomitantly the miR-125b up- and the BAK1 down-regulation expression in NPM-ALK(+) cells upon doxycyclin we considered that addition of miR-125b could lead to resistance to chemotherapeutic agent of NPM-ALK(-t-) cells.

miR125b down-expression as a biomarker for early prognosis of human NPM/ALK(+) ALCL biopsies.

Based on our results, we hypothesized that a perturbation of miR-125b expression during CHOP chemotherapy regimen which includes doxorubicin in patients with NPM/ALK(+) disease could regulate drug response. We used microarray microRNA- expression profiling of

ALCL samples obtained at the diagnosis (n=52) to identify micro RNA were differentially-expressed between NPM-ALK(+) patients who experienced an early relapse (n=28) and non-relapsing NPM-ALK(+) patients (n=24). After normalization with the expression level of miRNAs in reactive lymph nodes (RLN ; n=3), analysis revealed that the miR-125b expression levels were differentially regulated in who experienced an early relapse (n=28) and non-relapsing NPM-ALK(+) patients. To valid these results miR-125b expression was performed by quantitative real-time PCR in the same NPM-ALK(+) cohort. After normalization with the miR-125b expression level in reactive lymph nodes (RLN ; n=14), data generated by qRT-PCR, showed consistent down-regulation of miR-125b in early relapsing compared to non-relapsing patients (Figure 1A, p<0.001). Interestingly, we observed also a greater induction in BAK1 levels (Figure IB, p<0.001).

Collectively our findings suggest that miR-125b alteration could emerge as prediction of therapeutic outcomes. To support such a proposal using quantitative real-time PCR we evaluated pri-miR-125bl and mature miR-125b expression in NPM-ALK(+) ALCL cell line KARPAS-299 upon doxorubicin treatment. Doxorubicin-treated KARPAS-299 showed expression of both endogenous pri-miR-125bl and mature miR-125b at a much higher level (mean 4.86 + 2.28 and 13.95 + 8.56 respectively) than that seen in untreated cells (PBS) (Figure 1C and D). All together our data suggested that turn-on miR-125b expression might be linking to recurrence of NPM-ALK(-t-) ALCL after doxorubicin-containing polvchemotherapy. The reversal of miR-125b-silencing upon doxorubicin reduces BAK1 expression and induces doxorubicin resistance in NPM-ALK-positive ALCL cells.

We next investigated whether miR-125b overexpression and BAK1 repression affects the efficiency of doxorubicin in inducing apoptosis in ALCL cells. KARPAS-299 and COST cells were transfected with miR-125b or miR-CTL and then treated with either doxorubicin (concentrations of 0.1, 0.2 or 0.4 μΜ for 48 h) or vehicle (PBS). Caspase 3 activity was evaluated using the caspase-Glo® 3/7 assay system in both miR-125b and miR-CTL249 transfected KARPAS-299 and COST cells and was significantly reduced in cells transfected with miR-125b compared to miR-CTL- transfected cells (Figure 2). We noted that overexpression of miR-125b in the absence of doxorubicin did not affect proliferation of NPM- ALK(+) cells (data not shown). To quantify the percentage of apoptotic cells after drug treatment the NPM-ALK(+) cells were stained with Annexin V-pacific blue/IP. The rate of apoptotic cells (Annexin V-pacific blue positive cells) in miR-125b transfected COST cells upon doxorubicin was statistically reduced compared with that of miR-CTL-transfected cells (data not shown). Moreover, we observed that upon doxorubicin treatment cell viability was significantly enhanced in COST cells which ectopically expressed miR-125b (data not shown). By contrast, in miR-125b-transfected KARPAS-299 cells viability was not affected (data not shown). It is to note that the successful overexpression of miR-125b in the cells, confirmed by qPCR, revealed that both KARPAS-299 and COST cells contain the same amount of mimic miR-125b (data not shown). As we have previously shown, miR-125b is less abundant in COST cells than in KARPAS-299 cells (data not shown). We observed that doxorubicin treatment induces an increase of BAKl expression (to a relative level of 1.78 and 1.91 for KARPAS-299 and COST respectively) (data not shown). By contrast, the expression of BAKl was inhibited when KARPAS-299 and COST cells upon doxorubicin were transfected with the mimic miR- 125b (relative level 0.52 and 0.78 respectively, data not shown). Thus we considered whether an increase in miR-125b could induce NPM-ALK(+) cells to become resistant to chemotherapeutic agents through the downregulation of BAKl. Silencing of BAKl expression in KARPAS-299 and COST cells using siRNAs directed against BAKl mRNA (data not shown) clearly protected cells against doxorubicin-induced apoptosis, as assessed by a caspase- Glo® 3/7 assay (Figure 3). Interestingly, doxorubicin resistance was more pronounced in NPM-ALK(+) ALCL cells with low abundance of miR-125b (COST cells compared to KARPAS-299 cells). Taken together,these results suggest that miR-125b overexpression regulates pharmacoresistance in NPM-ALK(+) cancer cells by protecting against apoptosis through BAKl suppression.

DNA topoisomerase II and DNMT1 mediate miR125b silencing in NPM-ALK- positive ALCL cells

Next, since we observed that doxorubicin treatment induced a clear increase in miR- 125b expression in NPM-ALK(+) ALCL cells (Figure 4), and since its major target is DNA topoisomerase II (Topo II), a DNA-binding protein required for the maintenance of DNA methylation by DNMT1, we assessed the role of Topo Π in miR-125b regulation by treating cells with etoposide, a Topo II inhibitor that is part of the CHOEP (cyclophosphamide, doxorubicin, vincristine, etoposide and prednisone) chemotherapy regimen, at final concentrations ranging from 100 to 300 nM. Like doxorubicin, etoposide increased miR-125b expression (data not shown), showing that Topo II is involved in miR-125b regulation following doxorubicin treatment. By contrast, doxorubicin and etoposide didn't affected miR29a (data not shown), a miRNA down-regulated through DNA methylation in NPM289ALK(+) ALCL cell lines. We next investigated whether the DNMT1 protein directly binds to the CpG-rich region upstream of the MIR125B1 gene using chromatin immunoprecipitation (ChIP) experiments (data not shown). Inhibition of Topo II by doxorubicin markedly reduced DNMT1 binding to the MIR125B1 promoter (data not shown) (P<0.001). Total DMNT1 protein levels were markedly reduced in doxorubicin treated cells (data not shown). We also checked the cellular DNA methylation status of the CpGl to CpG7 region upstream of the MIR125B1 gene, which had been previously analyzed by ChIP (data not shown), and found that this was not significantly altered by doxorubicin treatment in KARPAS-299 and COST cells. This lack of detectable demethylation despite the inhibition of DNMT by doxorubicin can be explained by the fact that the passive loss of DNA methylation requires several cell divisions to dilute out the methylated parental DNA strands. Similar results were previously reported by Yokochi et al. in a human colon carcinoma cell line. In addition to its DNA methyltransferase activity, DNMT1 also represses transcription by cooperating with repressive histone modifiers. In KARPAS-299 cells, ChIP analysis showed that doxorubicin reduced the levels of histone H3 trimethylated on its Lys-27 site (H3K27me3) that bound to the MIR125B1 promoter (data not shown) (P<0.001), an epigenetic mark that is associated with transcriptional repression. H3K27me3 was present at the MIR125B1 promoter in NPM- ALK(+) cells not exposed to doxorubicin (data not shown), indicating the presence of non- permissive chromatin in this region. Since these data suggested that miR125b transcription was induced following doxorubicin treatment, we then evaluated pri-miR-125bl expression in KARPAS-299 cells upon doxorubicin treatment using qRT-PCR and found them to be significantly higher (mean 4.86+2.28, P<0.05) relative to levels in untreated cells (data not shown). These results suggest that the loss of DNMT1 modulated chromatin structure. We also considered the possibility that doxorubicin-induced upregulation of the MIR125B1 gene was due to its capacity to directly intercalate into DNA and compete for the putative DNA binding site of transcription factors, such as Specificity protein 1 (Spl). Spl binds to human DNMT1 and to sites that are often embedded within CpG islands, such as those that are overrepresented in the MIR125B1 promoter. However, we observed no change in miR125b expression in NPM- ALK(+) ALCL cells following Spl inhibition by mithramycin A or si-RNA directed against Spl mRNA (data not shown). Taken together, these results suggest that both Topo II and DNMT1 inhibition contribute to turn on miR- 125b expression in NPM-ALK(+) cells following doxorubicin exposure.

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Claims

CLAIMS:
1. A method for predicting whether a patient suffering from NPM-ALK(+) anaplastic large cell lymphoma (ALCL) will achieve a response with chemotherapy comprising i) determining the expression level of miR-125b in a tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value, and iii) concluding that the patient will achieve a response when the expression level of miR-125b determined at step i) is higher than the predetermined reference value or concluding that the patient will not achieve a response when the level of miR-125b is lower than the predetermined reference value.
2. The method of claim 1 wherein the chemotherapy is a polychemotherapy which includes doxorubicin and at least one additional chemotherapeutic agent.
3. The method of claim 2 wherein the polychemotherapy consists of a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone.
4. The method of claim 2 wherein the polychemotherapy consists of a combination of rituximab cyclophosphamide, doxorubicine, vincristine, and prednisone.
5. A method of treating NPM-ALK(+) anaplastic large cell lymphoma in a patient in need thereof comprising i) predicting whether the patient will achieve a response with chemotherapy by performing the method of the first object and ii) administering the patient with chemotherapy when it is predicted that the patient will achieve a response.
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