KR102040362B1 - Markers for diagnosing ocular marginal zone lymphoma and uses thereof - Google Patents

Abstract

The present invention relates to a diagnostic marker for ocular marginal lymphoma and its use, and more particularly, to a diagnostic marker composition for ocular marginal lymphoma, including a specific gene mutation capable of causing ocular marginal lymphoma, comprising an agent for detecting the genetic mutation. It relates to a diagnostic composition for ocular marginal lymphoma, and a diagnostic method using the same.
In the present invention, gene mutations affecting the onset and progression of the disease are identified through sequencing of the entire genome and transcripts in ocular marginal lymphoma. The mutant gene and the protein encoded therein are diagnosed as ocular marginal lymphoma. It can be usefully used as a marker for this, and is expected to be used to diagnose the disease more quickly and accurately.

Description

Markers for diagnosing ocular marginal zone lymphoma and uses approximately}

The present invention relates to a diagnostic marker for ocular marginal lymphoma and its use, and more particularly, to a diagnostic marker composition for ocular marginal lymphoma, including a specific gene mutation capable of causing ocular marginal lymphoma, comprising an agent for detecting the genetic mutation. It relates to a diagnostic composition for ocular marginal lymphoma, and a diagnostic method using the same.

Extranodal marginal zone lymphoma (EMZL) is a heterogeneous disease belonging to low-grade mature B-cell lymphomas that exhibit different genetic changes depending on tumor origin. EMZL translocates t (11,18) (q21, q21), t (1,14) (p22, q32), t (3,14) (p14) of the three, 12, and 18 chromosomes It has been reported that various chromosomal abnormalities appear, such as .1; q32), and t (14; 18) (q32; q21). The frequency of chromosomal modifications depends on the site of the primary tumor, suggesting that genetic changes are associated with other causes. For example, t (11; 18) (q21; q21), including the API2 and MALT1 genes, occur most frequently in EMZL in the lungs (38-53%) and stomach (17-31%), and frequently in other sites. I never do that. T (14; 18) (q32; q21), including the IGH and MALT1 genes, are found in eye, saliva and cutaneous MZL, but not in the gastrointestinal tract, lungs and thyroid. T (3; 14) (p14.1; q32) containing FOXP1 has been reported to be found in ocular MZL (25%), thyroid MZL (50%), and cutaneous MZL (10%) but rarely in tumors elsewhere. Not found T (1; 14) (p22; q32) is also found in intestinal MZL (13%) and has not been reported elsewhere.

Ocular marginal zone lymphoma (Ocular MZL) is the second most common type of EMZL and occurs in the eyelids, conjunctiva, lacrimal glands, and orbits. Overall, 30% of ocular MZL cases are associated with specific chromosomal translocations associated with carcinogenic activity linked to the activation of antibody-receptor related NF-κB. In addition to chromosomal translocation, in 12-37% of ocular MZL cases, A20, a negative regulator of the NF-κB pathway, has been reported to be inactivated through somatic defects and / or mutations (Haematologica. 2012 Jun; 97 (6) : 926-30). However, these chromosomal translocations and somatic mutations have been identified in only a few ocular MZL cases, suggesting that additional genetic modifications are involved in the development and progression of ocular MZL.

Therefore, it is necessary to further identify markers for diagnosing ocular MZL, and for this purpose, studies on genes and modifications thereof related to the development of ocular MZL need to be made.

The present invention has been made to solve the above problems, the present inventors have performed whole genome sequencing, transcriptome sequence for the tumor sample to obtain comprehensive information about the gene expression pattern and genetic modification of ocular marginal lymphoma Analysis and target sequencing were conducted for the first time to search for changes in the whole genome and transcript in ocular marginal lymphoma. As a result, a marker for diagnosing the disease was identified, and thus the present invention was completed.

Thus, the present invention TNFAIP3 (Tumor necrosis factor, alpha- induced protein 3), TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1), and CREBBP (cAMP response element binding protein) one or more mutations selected from the group consisting of An object of the present invention is to provide a diagnostic marker composition for ocular marginal lymphoma, comprising mRNA of a gene or a protein encoded from the gene.

The present invention also provides a diagnostic composition for ocular marginal lymphoma, comprising an agent capable of detecting the expression of mRNA or at least one mutant gene selected from the group consisting of TNFAIP3 , TBL1XR1 , and CREBBP or a protein encoded by the gene. To provide for other purposes.

In addition, the present invention includes detecting an mRNA of at least one mutant gene selected from the group consisting of TNFAIP3, TBL1XR1 , and CREBBP or a protein encoded from the gene from a biological sample derived from an ocular marginal lymphoma patient. Another object is to provide a method for providing information for diagnosis of marginal lymphoma.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, the present invention, TNFAIP3 (Tumor necrosis factor, alpha-induced protein 3) , TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1), and Provided is a diagnostic marker composition for ocular marginal lymphoma, comprising mRNA of at least one mutant gene selected from the group consisting of CREBBP (cAMP response element binding protein) or a protein encoded from the gene.

Also, TNFAIP3(Tumor necrosis factor, alpha-induced protein 3), TBL1XR1(Transducin Beta Like 1 X-Linked Receptor 1), andCREBBPIt provides a diagnostic composition for ocular marginal lymphoma, comprising an agent capable of detecting the expression of the mRNA of at least one mutant gene selected from the group consisting of (cAMP response element binding protein) or a protein encoded from the gene.

The present invention also provides a kit for diagnosing ocular marginal lymphoma, comprising the composition.

In addition, the present invention provides a method for preparing a chromosomal lymphoma , comprising the steps of: a) measuring the expression of mRNA of at least one mutant gene selected from the group consisting of TNFAIP3 , TBL1XR1 , and CREBBP from a biological sample derived from an ocular marginal lymphoma patient or a protein encoded from the gene; And

b) diagnosing an ocular marginal lymphoma if the expression of mRNA of one or more mutant genes selected from the group consisting of TNFAIP3 , TBL1XR1 , and CREBBP or a protein encoded by said gene is detected in said sample, Provides a method of providing information for the diagnosis of ocular marginal lymphoma.

In one embodiment of the present invention, the TNFAIP3 The mutant gene is It may have one or more mutations selected from Table A below.

TABLE A

In another embodiment of the present invention, the TBL1XR1 The mutant gene is It may have one or more variations selected from Table B below.

TABLE B

In another embodiment of the present invention, the CREBBP The mutant gene is It may have one or more mutations selected from Table C below.

TABLE C

In another embodiment of the invention, the agent capable of detecting mRNA expression is TNFAIP3 , TBL1XR1 , or sense and antisense primers or probes that complementarily bind to mRNA of a CREBBP mutant gene.

In another embodiment of the invention, the agent capable of detecting the protein may be an antibody that specifically binds to the protein.

In another embodiment of the present invention, the expression of mRNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase) protection assay (RPA), microarray, and northern blotting.

In another embodiment of the present invention, the protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation) ), Flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip. It can be measured through.

In the present invention, gene mutations affecting the onset and progression of the disease are identified through sequencing of the entire genome and transcripts in ocular marginal lymphoma. The mutant gene and the protein encoded therein are diagnosed as ocular marginal lymphoma. It can be usefully used as a marker for this, and is expected to be used to diagnose the disease more quickly and accurately.

1 is a result of analyzing the copy number variation in the peripheral ocular lymphoma samples, Figure 1a will show that the 6q23.3 region including TNFAIP3 a significant deletion, Figure 1b is the effect on the degree of gene expression variability replication To show.
2 shows TNFAIP3 in ocular marginal lymphoma samples. This is the result of confirming that structural variation related to collapse, namely Insertion, Deletion, Inversion, Replication, and Translocation.
FIG. 3A shows five structural changes in the first intron of IL20RA in the 6q23.3 region in some of the ocular marginal lymphoma samples (3/10), and FIG. 3B shows the structural changes in the deletion, intrachromosome. And chromosomal translocations (Intra- and Inter-chromosomal translocation), and complex structural changes.
Figure 4 shows the results of identifying somatic single nucleotide variants (SNVs) in eye margin margin lymphoma samples.
5 is a heat map of gene mutations according to whole genome and target sequencing for ocular marginal lymphoma samples.
FIG. 6 shows the results of TBL1XR1 mutations in ocular marginal lymphoma samples. FIG. 6A shows the TBL1XR1 protein. Miss Sense The mutations are located in the WD40 domain, Figure 6b shows the structure of the TBL1XR1 protein in which the mutation occurred.
Figure 7 is a comparison of gene expression profiles of ocular marginal lymphoma and normal marginal B cell samples at the signaling pathway level.
8 is a result of analyzing the functional characteristics of the mutation in the WD40 domain of TBL1XR1, Figure 8a shows the position of the mutation in the protein of the TBL1XR1 mutant, Figure 8b through simultaneous immunoprecipitation and immunoblotting analysis As a result of confirming the increase in binding of the TBL1XR1 mutant and NCoR and HDAC3, Figure 8c is a result of confirming the decrease in the expression of NCoR protein in cells expressing the TBL1XR1 mutation, Figure 8d shows the intracellular location of the TBL1XR1 mutant through immunofluorescence analysis This is the confirmed result.
9 is a result of analyzing the effect of the TBL1XR1 mutation on gene expression, Figure 9a is a cell expressing wild type TBL1XR1 (WT) or mutations (MUT-2, MUT-3), and cells treated with TBL1XR1 siRNA (siRNA NF - κ B via microarray) And JUN Target 9B and 9C show upregulated NF-κB and JUN targets in patient samples with cell lines and somatic missense variants (n = 2) expressing TBL1XR1 mutations, respectively, via GSEA analysis. 9D is a result showing increased NF-κB activity in cells expressing the TBL1XR1 mutation through luciferase reporter analysis.
10 is a result of comparing the proliferation of cells expressing wild type TBL1XR1 (WT) or a mutation.

We performed whole genome sequencing, transcriptome sequencing, and target sequencing on the tumor samples to obtain comprehensive information about gene expression patterns and genetic modifications of ocular marginal lymphomas. For the first time, the change of the transcript was searched for, and as a result, a marker for diagnosing the disease was identified.

Thus, the present invention TNFAIP3 (Tumor necrosis factor, alpha- induced protein 3), TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1), and CREBBP (cAMP response element binding protein) one or more mutations selected from the group consisting of Provided is a diagnostic marker composition for ocular marginal lymphoma, comprising mRNA of a gene or a protein encoded from the gene.

The invention also TNFAIP3 (Tumor necrosis factor, alpha- induced protein 3), TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1), and CREBBP (cAMP response element binding protein) and a composition for diagnosing ocular marginal lymphoma comprising an mRNA capable of detecting the expression of at least one mutant gene selected from the group consisting of a gene or a protein encoded therefrom and an eye comprising the same Provided is a diagnostic kit for marginal lymphoma.

As used herein, the term "diagnosis" in the broad sense means to determine the actual condition of the patient in all aspects. The contents of the judgment include the name of the disease, the etiology, the type of disease, the severity, the details of the condition, and the presence or absence of complications. Diagnosis in the present invention is to determine the onset and progression level of ocular marginal lymphoma.

In the present invention, the TNFAIP3 The mutant gene is It may have one or more mutations selected from Table A below.

TABLE A

In the present invention, the TBL1XR1 The mutant gene may be one or more variants selected from Table B below.

TABLE B

In the present invention, the CREBBP The mutant gene is It may have one or more mutations selected from Table C below.

TABLE C

Agents capable of detecting the mRNA expression are TNFAIP3 , TBL1XR1 , or sense and antisense primers or probes that complementarily bind to mRNA of a CREBBP mutant gene, but are not limited thereto.

As used herein, the term "primer" refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, and the like, as a short gene sequence that is a starting point for DNA synthesis. The primers can be synthesized in a conventional length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, or the like by a known method.

As used herein, the term "probe" refers to a nucleic acid capable of specifically binding to mRNA of several bases to several hundred bases in length, which has been produced through enzymatic chemical separation or purification. The presence of mRNA can be confirmed by labeling radioisotopes or enzymes, and can be designed and modified by known methods.

The agent capable of detecting the protein may be an antibody that specifically binds to the protein, but is not limited thereto.

As used herein, the term “antibody” includes immunoglobulin molecules that are immunologically reactive with specific antigens, and include both monoclonal and polyclonal antibodies. In addition, such antibodies include forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

The diagnostic kit for ocular marginal lymphoma of the present invention consists of one or more other component compositions, solutions or devices suitable for analytical methods.

In one embodiment of the present invention, whole genome sequencing was performed on 10 pairs of ocular marginal lymphoma samples and normal samples to analyze somatic cell replication and structural changes, and 14 and 63 amplifications of the tumor samples, respectively. And the somatic cell number variation (CNV) in the deletion region, and the 6q23.3 region including TNFAIP3 , which is a negative regulator of the NF-κB signaling pathway, was significantly deleted (see Example 2). ).

In another embodiment of the present invention, it was confirmed that structural changes of Insertion, Deletion, Inversion, Replication, and Translocation occurred in the 10 tumor samples, respectively. In dog tumor samples (30%), deletion, intrachromosomal and chromosomal translocation, and complex structural changes were identified in the first intron of IL20RA in the 6q23.3 region (Example 3). Reference).

In another embodiment of the present invention, a single base mutation is analyzed in a total of 48 samples by performing target sequencing of 430 cancer-related genes on 38 tumor samples along with the results of whole genome sequencing on 10 tumor samples. It was. As a result, TNFAIP3 mutation, a negative regulator of the NF-κB pathway, was identified in 54% (n = 26) of 48 tumor samples, followed by chromatin modification and / or transcriptional regulation in ocular marginal lymphoma in 9 samples (19%). One of the genes involved in the TBL1XR1 mutation was confirmed to occur, most of the TBL1XR1 mutation was present in the WD40 domain known to be involved in NCoR and histone deacetylase binding. In addition, mutations in CREBBP were identified in 17% of tumor samples, as well as mutations in genes involved in B cell differentiation and tumor suppression (see Examples 4-6).

In another embodiment of the present invention, the gene expression profiles of ocular marginal lymphoma normal marginal B cell samples at the level of signaling pathways are compared, resulting in B cell proliferation and NF-κB pathways that induce abnormal activation of NF-κB, and It was confirmed that genes related to apoptosis-inhibition pathways were overexpressed (see Example 7).

In another embodiment of the present invention, TBL1XR1 As a result of various experiments to verify the functional characteristics of the mutation, TBL1XR1 The variant was found to increase the binding of NCoR and HDAC , thereby promoting the degradation of NCoR , and to change the intracellular location, thereby inducing the progression of inhibitory complex and anti-apoptosis, and NF-κB activity It was confirmed to increase and increase cell proliferation (see Example 8).

Therefore, through the embodiment of the present invention, TNFAIP3 , which frequently induces mutations in ocular marginal lymphoma, It can be seen that the detection of TBL1XR1 , and CREBBP mutations can be used as a genetic marker for diagnosing the disease.

Accordingly, as another aspect of the present invention, the present invention provides a) expression of mRNA of at least one mutant gene selected from the group consisting of TNFAIP3 , TBL1XR1 , and CREBBP from a biological sample derived from a patient with ocular marginal lymphoma or a protein encoded from the gene. Measuring; And

b) the eye, comprising the step of diagnosing in said sample to TNFAIP3, TBL1XR1, and if the first mutated gene or more kinds selected from the group consisting of CREBBP mRNA or the expression of the protein encoded by the gene detection, the eye margin lymphoma Provides a method of providing information for the diagnosis of marginal lymphoma.

Biological samples derived from the patient may include, but are not limited to, tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine.

The expression level of the mRNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase) by conventional methods known in the art protection assay (RPA), microarray, or northern blotting, or one or more methods selected from the group consisting of, but is not limited thereto.

The protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation) by conventional methods known in the art One or more methods selected from the group consisting of flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, or protein chip. It may be measured through, but is not limited thereto.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  1: Experiment Preparation and Experiment Method

1-1. Subject and Sample Preparation for Sequencing

Blood samples and 10 tumor samples matching them were obtained from ocular marginal lymphoma patients for whole-genome sequencing (WGS), and 38 MZL embedded in formalin fixation and paraffin for target sequencing. Tumor tissue samples were obtained and used. The information on the clinical patients is shown in detail in Table 1 below, and patient samples were collected with the consent of Samsung Seoul Hospital. The present invention was carried out after approval of the Institutional Review Board according to the Declaration of Helsinki. .

On the other hand, the potential mutation was selected by whole exome sequencing of the sample of the marginal lymphoma patient and verified by Sanger sequencing.

TABLE 1

1-2. Cell line and cell culture

BJAB (bucket lymphoma) and 293T cell lines were prepared at 37 ° C. and 5 in RPMI-1640 and DMEM medium containing 10% FBS, 100U / ml penicillin, 100 μg / ml streptomycin, and 250 ng / ml amphotericin B, respectively. Cultured in% CO ₂ conditions. In addition, the MycoAlert mycoplasma detection kit (Lonza) was used to regularly check for mycoplasma infection in the cell line.

1-3. TBL1XR1 Cloning

CDNA clones encoding full-length human TBL1XR1 via PCR amplification were isolated from human placental cDNA libraries. The DNA fragment obtained through the above method was cloned into the pcDNA3-GFP vector to express a protein linked with GFP at the N-terminus. The QuikChange kit (Stratagene) was also used to construct TBL1XR1 mutants, specifically Gly187Arg (GGA to GTA), Leu282Pro (CTA to CCA), His348Glu (CAT to CAA), and Ser459Asn (AGT to AAT). The mutant constructs were verified by sequencing.

1-4. Transfection ( Transfection )

To overexpress TBL1XR1 in BJAB cell lines, plasmids expressing wild-type TBL1XR1 and TBL1XR1 mutants (Gly187Arg, Leu282Pro, His348Glu and Ser459Asn) with GFP linked to N-terminus were subjected to Nucleofector I device (Amaxa) using Nucleofector Solution V and Program The cells were transfected with O-017.

1-5. Simultaneous Immunoprecipitation (Co- Immunoprecipitation ) And Immunoblotting (Immunoblotting assay)

Total cell lysates were clarified by centrifugation at 12,000 × g for 10 minutes, then mixed with Protein G magnetic beads (Thermo Scientific) and 2 μg of GFP antibody (Abcam) and incubated at 4 ° C. for 2 hours. Immune complexes were collected using magnets and washed twice with wash buffer, and beads associated with whole cell lysate and protein complexes were separated by SDS-PAGE and immunoblotted with the corresponding antibodies.

Specifically, TBL1XR1 (Novus), HDAC3, NCoR (Abcam), and α-tubulin (Santa Cruz) antibodies were used for protein blotting, and Horseradish peroxidase (HRP) -conjugated secondary antibody (Bio-Rad) was used. Primary antibody was detected.

1-6. Immunofluorescence  analysis( Immunofluorescence  analysis)

Lipofectamine 3000 (Life Technologies) was used to transfect 293T cells with GFP-linked TBL1XR1 expression plasmid. Next, the nuclei were stained with Nucblue (Hoechst dye, Thermo Scientific), and images were obtained using a confocal laser scanning microscope (CLSM-780, Zeiss).

1-7. Cell growth assays and NF - κB  Activity analysis

48 hours after transfection, BJAB cells were seeded in triplicate in 96-well plates at a density of 7.5 × 10 ³ cells / well with 100 μl of RPMI1640 medium containing 10% FBS and antibiotics. Cell growth was analyzed according to the manufacturer's instructions using Cell Counting Kit-8 (Dojindo).

Meanwhile, luciferase assay was performed for NF-κB activity assay. To this end, cells were transfected simultaneously with three NF-κB binding sites (3 × κBL) the luciferase vector and hRL (renilla luciferase). Next, 5 × 10 ⁵ cells were incubated for 24 hours in 24-well plates, and then the cells were lysed and fireflies and litters with Glomax 20/20 luminometer (Promega) using a double luciferase reporter assay system (Promega). Nila luciferase activity was measured. Firefly luciferase activity was corrected with Renilla luciferase.

1-8. Gene expression analysis

Cell lines expressing wild type TBL1XR1 or mutants (L282P and H348Q), and cell lines treated with TBL1XR1 siRNA, were subjected to gene expression analysis using the Agilent Gene Expression Hybridization Kit (GPL13497).

Example  2. Eyeball MZL  Somatic cells Clones  analysis

We performed whole genome sequencing on 10 pairs of tumor samples and normal samples in order to determine the number of somatic cloning and structural changes (SVs) of Ocular marginal zone lymphoma (Ocular MZL). Specifically, the tumors were sequenced with an average depth of 66x and the normal samples matched with 32x coverage.

As a result, somatic cell copy number variation (CNV) was identified in 14 and 63 amplification and deletion regions, respectively. Specifically, the increase region shows wide and low amplitude changes, while the deletion region shows narrow and high-amplitude changes. In addition, one region of high recurrence was found.

The inventors further confirmed that there is a common genetic variation as previously identified in ocular MZL. More specifically, as shown in Figure 1a, it was confirmed that the 6q23.3 region containing TNFAIP3 , a negative regulator of the NF-κB signaling pathway in the tumor sample was significantly deleted ( FDR = 1.02E -03 ). That is, 50% (5/10) of the tumor samples showed deletion of the 6q23.3 region, of which three had homozygous deletions and two had heterozygous deletions. In addition, as a result of RNA sequencing of the same sample, it was confirmed that the average gene expression was significantly lower in the 6q23.3 region of the tumor sample (n = 5) where the deletion occurred than the sample without the deletion. ( P = 2.70E -02 ). We also found trisomy of chromosome 3 and chromosome 18 in 50% (n = 5) and 10% (n = 1) of tumor samples, respectively. Trisomal chromosome 3 was found to increase the mRNA expression of the gene on the chromosome. Tumor samples with this amplification (n = 5) showed significantly higher gene expression compared to samples without amplification.

Example  3. TNFAIP3 of Identify recurrent structural variation associated with collapse

We found that TNFAIP3 in tumor samples As a result of analyzing the structural variation related to decay, as shown in Fig. 2, five types of structural changes in each of 10 tumor samples, namely, Insertion, Deletion, Inversion, and Duplication, are shown. , And translocation were found, confirming an average of 40 changes per sample. In addition, as shown in FIG. 3A, three tumor samples (30%) showed five structural change breakpoints in the first intron of IL20RA encoding a subunit of the Interleukin 20 receptor in the 6q23.3 region. Found. More specifically, as shown in detail in FIG. 3B, the structural changes included deletion, intrachromosomal and chromosomal translocations (Intra- and Inter-chromosomal translocation), and complex structural changes. For example, WG-06 samples were found to have multiple rearrangements involving chromosomal translocations with different chromosome and complex structural changes. Since IL20RA is not expressed in lymphoid organs, the first intron of IL20RA may be a hot spot where the structural genes of ocular MZL become unstable. In particular, all samples with structural changes (WG-04, WG-05, WG-06, IL20RA upstream of TNFAIP3 (specifically 1 mb) and can be seen in the whole-genome seq results of FIG. ) Has a homozygous deletion of TNFAIP3, so the structural change of the IL20RA first intron is due to TNFAIP3 It can be thought of as an important mechanism for complete inactivation. In the ten tumor samples of the present invention, t (11; 18) (q21; q21) API2 - MALT1 , t (14; 18) (q32; q21) IgH - MALT1 , t (1; 14) (p22; q32) No well-known chromosomal translocation was found in MALT lymphomas such as BCL10 - IgH , and t (3; 14) (p14.1; q32), and no corresponding fusion transcript was detected in RNA fusion data.

Example  4. Eyeball Margin  In lymphoma TNFAIP3 transition  Confirm

In addition to the results of Example 2, we analyzed the presence of somatic single base mutations (SNVs) through whole genome sequencing on 10 ocular marginal lymphoma samples. As a result, as shown in FIG. 4, an average of 22 non-silent substitutions per tumor sample were confirmed (range, 12-44). More specifically, 70% (n = 7) of the total samples showed disruptive deformations such as nonsense, frame_shift_indel, or deletion in TNFAIP3. It has been shown that it is associated with overexpression of the NF-κB pathway, which implies that the pathogenesis of ocular marginal lymphoma is associated with TNFAIP3 inactivation following continuous activation of the NF-κB pathway.

Further, to obtain information on more comprehensive genetic modifications for ocular marginal lymphomas, we performed target sequencing of 430 cancer related genes in 38 ocular marginal lymphoma samples (average sequencing depth 400x per sample). As a result, TNFAIP3 mutation was confirmed in 54% (n = 26) of a total of 48 tumor samples as shown in FIG. 5 and Tables 2 to 5 below. In addition, the clonaility analysis confirmed that all TNFAIP3 mutations of the sample sequenced for the entire genome were identical. In addition, the results confirmed that the modification of TNFAIP3 occurs early in the lymphoma formation process.

In addition, the sequencing confirmed the positive regulatory factors of NF-κB, CARD11 (4%), BCL10 (4%), CD79B (4%) and MYD88 (4%), as other recurrent mutations that activate the NF-κB pathway. It was. Most of the mutations that appear in the gene appears to be activated mutant containing the mutation with a cut obtained in the potential function BCL10. Modifications of BCL10 , CARD11 and MYD88 are known to be associated with the pathogenesis of ocular marginal lymphoma, and somatic mutations of CD79B have been reported in splenic marginal lymphoma. Although these mutations were mutually exclusive, most of the mutations were found to coincide in the sample with TNFAIP3 mutation, and 55% of the total samples confirmed that the genetic modification including activation of the NF-κB pathway occurred.

TABLE 2

TABLE 3

TABLE 4

TABLE 5

Example  5. TBL1XR1 of  Check for variation

In addition to the results of Example 4 above, the present inventors have determined TNFAIP3 as a result of total genome sequencing on 10 tumor samples and target sequencing on 38 tumor samples. Next, it was confirmed that many mutations occurred in TBL1XR1 . The TBL1XR1 gene is known to be essential for transcriptional inhibition mediated by unliganded nuclear receptors (NRs) and other regulated transcription factors. Specifically, as shown in FIG. 5, 9 (19%) of the 48 samples showed 13 TBL1XR1 mutations. The majority of the mutations (92.1%, n = 12) were missense variants and all variants were in the WD40 domain known to be involved in NCoR and histone deacetylase 3 (HDAC3) binding ( Figure 6a) All these somatic mutations were heterozygotes verified by Sanger sequencing. Furthermore, as can be seen in Figure 6b, many mutations are located near the top of the domain, some of which are directly related to DHSW tetrad, the core skeleton of the WD40 domain, which is expected to affect the binding of TBL1XR1 and partner proteins. Mutations in TBL1XR1 are markedly higher in conjunctival eye MZL (P = 4.63E-02 by χ2 test), suggesting a role in the development of the disease. Meanwhile, the clonal analysis of the present inventors confirmed that the TBL1XR1 mutations are identical.

Example  6. Identify Other Genetic Variations

The inventors of the present invention have shown that genome sequencing on 10 tumor samples and target sequencing on 38 tumor samples resulted in a second to genes involved in chromatin modification and / or transcriptional regulation in ocular marginal lymphoma, as shown in FIG. 5. It was confirmed that the mutation occurred frequently. More specifically, the cAMP response element binding protein (CREBBP) belonging to the KAT3 group of histone / protein lysine acetyltransferase confirmed that mutations occurred in 17% of samples ( n = 8). In addition, MLL2 encoding histone methyltransferase was mutated in 6% of samples (n = 3). The majority of such genetic mutations were truncated modifications involving disruptive structural changes in CREBBP. The results are consistent with previous findings that loss of function of these genes is associated with the development of B-cell lymphoma. ANKRD11 , BAP1 , WDR90 and CUX1 mutations were also observed in the group of genes involved in chromatin modification and / or transcriptional regulation. Overall, 44% of the patients in this gene group mutated, and chromatin modifications and / or modifications of transcriptional regulators mutated ocular marginal lymphomas. The results were the first to find that chromatin remodelers and / or transcriptional regulators were involved in ocular marginal lymphoma.

In addition to the above results, sequencing of 48 tumor samples confirmed that mutations occurred in genes related to B cell differentiation or tumor suppression as shown in FIG. 5. More specifically, 23% and 27% of the samples were mutated in the B cell differentiation and tumor suppression groups, respectively, and genes reported to be mutated in other types of lymphomas such as NOTCH1, POU2F2, CXCR4, and IGF1R. Was also confirmed.

Example  7. Eyeball At MZL  Gene expression signature  analysis

To further elucidate the molecular pathogenesis of ocular marginal lymphoma, we used a single sample Gene Set Enrichment analysis (ssGSEA), each of 10 ocular MZL samples and normal margin B, at the signaling pathway level. Gene expression profiles of cell (Normal MZB) samples were compared.

As a result, as shown in FIG. 7, 12 pathways differentially expressed in ocular marginal lymphoma samples were identified (p value <0.01), 11 pathways were up-regulated and the other down-regulated. Upregulated pathways included signaling pathways that play important roles in B cell proliferation (NF-κB, CD40, and IL10) and NF-κB linkage pathways (EGFR_SMRTE, TNFR1, and STRESS), which are associated with ocular marginal lymphoma. It is known to induce abnormal NF-κB abnormal activation. It was also confirmed that overexpression of apoptosis-inhibiting pathways (HIVNEF, HSP27, and VIP) conferring apoptosis resistance in lymphocytes. On the other hand, the role of other pathways differentially expressed in the development of ocular marginal lymphoma is still unknown.

Example  8. TBL1XR1 Functional Characterization of Variations

8-1. TBL1XR1 Wow  control Complement  Analysis of binding of complex protein

To verify the functional properties of the mutations, we sought to analyze the interaction of the TBL1XR1 mutants with the NCoR complex components. The mutation is involved in the interaction with NCoR and is associated with major inhibitory complement complexes including HDAC3 , G-protein pathway suppressor 2 ( GPS2 ), silent mediators of retinoic acid and thyroid hormone receptors ( SMRT ), and TBL1 . It is located in the WD40 domain, which is part of it. NCoR regulates the balance between transcriptional inhibition and activation through ubiquitin mediated degradation, and HDAC3 is known to be responsible for the deacetylation of histones that regulate transcriptional inhibition. TBL1XR1 is required for transcriptional activation and acts as an inhibitory complement / complement activator exchange factor that regulates the degradation of inhibitory complement components and recruitment of complement activator complexes.

By first using the TBL1XR1 so that each of the mutations four residues preserved mutant (G187R, L282P, H348Q, and S459N) in as shown by the illustration in Fig. 8a, co-immunoprecipitation described in the embodiment 1-5 And immunoblotting analysis to analyze whether binding of the inhibitory complement complex protein to TBL1XR1 . As a result, as shown in Figure 8b it was confirmed that the binding of the disease-specific TBL1XR1 mutation and NCoR and HDAC3 increased compared to wild type TBL1XR1. In addition, as shown in Figure 8c it was confirmed that the expression of NCoR protein in the total cell lysate of the cells transfected with the mutant TBL1XR1. The results show that the increase in binding between NCoR and TBL1XR1 promotes the degradation of NCoR . In contrast, there was no significant difference in HDAC3 expression levels in cells expressing mutant TBL1XR1 . The results indicate that these conservative residues are not directly involved in protein binding but serve to maintain local morphology. The fact that the mutation is not present in residues at the protein-protein interaction key of the WD40 domain also supports our interpretation that the mutant can induce structural changes that enhance rather than inhibit the protein-protein interaction. .

8-2. TBL1XR1 Intracellular Positional Change Analysis of Mutants

Next, we tried to analyze the intracellular location of wild type and mutant TBL1XR1 . In general, wild-type TBL1XR1 is located in both the nucleus and cytoplasm, and when TBL1XR1 is located in the nucleus, it is reported that cell growth decreases and cell death increases. To this end, immunofluorescence analysis was carried out according to the method described in Examples 1-6, and as shown in FIG. 8C, TBL1XR1. In 293T cells transfected with mutants, it was confirmed that the mutants were expressed at different positions from the wild-type protein. Specifically, whereas the position in the cytoplasm particular TBL1XR1 mutant (L282P, H348Q and S459N), other TBL1XR1 mutant (G187R) was confirmed to exhibit a tendency which is located in both the cytoplasm and nucleus. The results indicate that the change of the intracellular location of the mutant TBL1XR1 can induce the progression of inhibitory complex and anti-apoptosis.

8-3. TBL1XR1  Mutation NF - κ B And JUN on  Impact Analysis

Inhibition of complement complex NF - κ B And It exhibits a transcriptional inhibitory effect through interaction with transcription factors, including JUN . Thus we found that NF-κB and By confirming the expression pattern of JUN , TBL1XR1 The purpose of this study was to determine whether the complex affects inhibitory function. To this end, first, similarity of gene expression between samples was analyzed using microarray analysis data for cell lines expressing wild type TBL1XR1 and mutations (L282P and H348Q) and cell lines transfected with TBL1XR1 siRNA.

As a result, as shown in FIG. 9A, TBL1XR1 The cells expressing the mutations were more closely packed with the wild type than cells treated with TBL1XR1 siRNA, indicating that L282P and H348Q are mutations that increase function. In addition, we performed gene set enrichment analysis (GSEA) to show patients with cell lines and somatic missense variants (n = 2) expressing TBL1XR1 mutations, respectively, as shown in FIGS. 9B and 9C. in the sample compared to the wild type TBL1XR1 sample it was significantly detect the up-regulation of NF-κB target genes and JUN. In addition, wild-type TBL1XR1 in TBL1XR1 mutant in BJAB (human Burkitt's lymphoma) cells via NF-κB luciferase reporter assay as shown in FIG. 9D. It was confirmed that about 1.5-fold NF-κB activity was increased.

Overall, these results show that the TBL1XR1 mutation can activate the transcription of some transcription factors such as NF - κB and JUN by breaking down the inhibitory complement complex.

8-4. TBL1XR1 Analysis of Cell Proliferation Change by Mutation

Finally, the inventors of the present invention TBL1XR1 CCK-8 analysis was performed to analyze the effects of the mutations. As a result, as shown in FIG. 10, the BJAB cells expressing the TBL1XR1 mutant showed about 10-15% higher proliferation than the cells expressing the wild type TBL1XR1 .

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (17)

MRNA of one or more mutant genes selected from the group consisting of Tumor necrosis factor (alpha-induced protein 3) , TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1) , and cAMP response element binding protein ( CREBBP ) A diagnostic marker composition for ocular marginal lymphoma, comprising a protein encoded from a gene,
Wherein said TNFAIP3 mutant gene has one or more mutations selected from Table A below,
TABLE A

The TBL1XR1 mutant gene has one or more mutations selected from Table B below.
TABLE B

The CREBBP mutant gene is characterized in that it has one or more mutations selected from Table C, marker composition.
TABLE C

delete delete delete MRNA of one or more mutant genes selected from the group consisting of Tumor necrosis factor (alpha-induced protein 3) , TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1) , and cAMP response element binding protein ( CREBBP ) A diagnostic composition for ocular marginal lymphoma, comprising an agent capable of detecting the expression of a protein encoded from a gene,
Wherein said TNFAIP3 mutant gene has one or more mutations selected from Table A below,
TABLE A

The TBL1XR1 mutant gene has one or more mutations selected from Table B below.
TABLE B

The CREBBP mutant gene is characterized in that it has one or more mutations selected from Table C, diagnostic composition.
TABLE C

delete delete delete The method of claim 5,
Agents capable of detecting the mRNA expression are TNFAIP3 , TBL1XR1 , or a sense and antisense primer or probe that complementarily binds to the mRNA of the CREBBP mutant gene, diagnostic composition.
The method of claim 5,
The agent capable of detecting the protein is characterized in that the antibody that specifically binds to the protein, diagnostic composition.
A kit for diagnosing ocular marginal lymphoma, comprising the composition of claim 5.
a) From a biological sample from an ocular marginal lymphoma patient, from a group consisting of Tumor necrosis factor, alpha-induced protein 3 ( TNFAIP3 ) , TBL1XR1 (Transducin Beta Like 1 X-Linked Receptor 1), and CREBBP (cAMP response element binding protein) Measuring the expression of mRNA of at least one mutant gene selected or a protein encoded from said gene; And
b) diagnosing an ocular marginal lymphoma if the expression of mRNA of one or more mutant genes selected from the group consisting of TNFAIP3, TBL1XR1, and CREBBP or a protein encoded by said gene is detected in said sample, As a method of providing information for the diagnosis of ocular marginal lymphoma,
Wherein said TNFAIP3 mutant gene has one or more mutations selected from Table A below,
TABLE A

The TBL1XR1 mutant gene has one or more mutations selected from Table B below.
TABLE B

The CREBBP mutant gene is characterized in that it has one or more mutations selected from Table C below, information providing method.
TABLE C

delete delete delete The method of claim 12,
Expression of the mRNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase protection assay; RPA), microarray ( microarray), and northern blotting, and at least one method selected from the group consisting of: information providing method.
The method of claim 12,
The protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, flow cytometry, Characterized in that it is measured by one or more methods selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip, How to Provide Information.

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