KR102016806B1 - Method for predicting of resistance against therapeutic agent in diffuse large B-cell lymphoma patients - Google Patents

Abstract

The present invention relates to a composition for predicting drug resistance in patients with diffuse large B-cell lymphoma and a method for predicting resistance using the same. More specifically, the present invention relates to a diffuseness comprising an agent detecting a mutation of a specific gene and / or a copy number variation of a specific gene. The present invention relates to a composition for predicting drug resistance in patients with giant B-cell lymphoma and a prediction method using the same.
By predicting treatment resistance through mutation of a specific gene and / or increasing or decreasing the number of copies of a specific gene provided in the present invention, it is possible to quickly and accurately predict the drug reactivity of individual patients with diffuse large B-cell lymphoma, thereby preventing unnecessary drug treatment. It can alleviate the physical, mental and economic pain of the patient, which is very meaningful given the quality of life during the patient's treatment.

Description

Method for predicting of resistance against therapeutic agent in diffuse large B-cell lymphoma patients}

The present invention relates to a composition for predicting drug resistance in patients with diffuse large B-cell lymphoma and a method for predicting resistance using the same, and more specifically, to an agent for detecting mutation of a specific gene and / or copy number variation of a specific gene. The present invention relates to a composition for predicting drug resistance in patients with diffuse large B-cell lymphoma and a prediction method using the same.

Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma and includes pathological changes, cell of orising (COO), clinical course, and heredity. It is known as a heterogeneous disease in terms of enemy variation. Clinically, DLBCL is a treatable disease and 30-50% of all patients are rituximab (R) -cyclophosphamide, doxorubicin, vincristine, and prednisone ( CHOP) can be cured through treatment. However, 40% of patients may relapse or be treated with initial treatment and may have a lower survival rate.

Many clinical, immunohistochemical and molecular factors have been used to predict treatment response and prognosis following standard R-CHOP treatment, but reliable markers are not yet known. The International Prognostic Index (IPI) is regarded as a surrogate for the bioactivity and cancer of an individual, including 60 years of age or older, abnormal increases in LDH levels, Ann Arbor stage III or IV disease,> 1 extra-nodular site, and 2 or more European Cooperative It can be calculated as the performance status of Oncology Group patients. IPI is known as a good prognostic marker, but many patients do not predict prognosis and response.

 DLBCL is classified into germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL based on gene expression profile or immunohistochemistry. ABC-type DLBCL is characterized by continuous activation of the NF-κB signaling pathway and is known to have a poorer prognosis for standard R-CHOP chemotherapy when compared to GCB-type DLBCL patients. This difference is used in therapy, and signaling pathway target therapy has been developed to improve survival of patients with ABC-type DLBCL. In addition to these biological markers, expression and recombination of the BCL-2 gene, expression and mutation of the p53 gene, and the like have also been used to predict the prognosis of DLBCL patients.

Therefore, there is an increasing demand for researches to identify markers contributing to drug resistance and to determine drug resistance by using them, and research on this is being done (Domestic Publication No. 10-2016-0090345), It is still insignificant.

The present invention has been made to solve the above problems, the present inventors are mutated and through the whole whole-exome and transcriptome sequencing for two groups of DLBCL patients with different treatment response The molecular background of the refractory DLBCL was first identified by comparing the copy number variation, and thus the present invention was completed.

Thus, the present invention provides drug resistance in patients with diffuse large B-cell lymphoma, comprising a B2M mutant gene and / or an agent detecting one or more gene copy number mutations selected from the group consisting of POU2AF1 , REL , BCL11A , XPO1 and CACNA1D . It is an object to provide a composition for prediction.

The invention also includes detecting a B2M mutant gene and / or one or more gene copy number variations selected from the group consisting of POU2AF1 , REL , BCL11A , XPO1 and CACNA1D from biological samples from patients with diffuse large B-cell lymphoma. Another object is to provide a method for providing information for predicting drug resistance in patients with diffuse large B-cell lymphoma.

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

In order to achieve the above object, the present invention provides drug resistance in patients with diffuse large B-cell lymphoma, comprising an agent capable of detecting the expression of mRNA of a B2M (beta-2 microglobulin) mutant gene or a protein encoded therefrom. Provided is a composition for prediction.

In one embodiment of the present invention, the B2M mutant gene may have one or more mutations selected from Table A below.

TABLE A

In another embodiment of the present invention, the composition may further comprise a probe for detecting copy number variation of one or more genes present on chromosomal positions defined in Table B below.

TABLE B

In another embodiment of the invention, the gene may be selected from the group consisting of POU2AF1 , REL , BCL11A , XPO1 and CACNA1D .

In another embodiment of the invention, the agent capable of detecting mRNA expression may be sense and antisense primers, or probes that complementarily bind to the mRNA of the B2M 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.

The present invention also provides a kit for predicting drug resistance in patients with diffuse large B-cell lymphoma comprising the composition.

In addition, the present invention

1) Said B2M from biological samples from patients with diffuse large B-cell lymphoma Measuring the expression of mRNA of a mutant gene or a protein encoded from said gene; And

2) a diffuse large B-cell lymphoma comprising determining that the sample is unresponsive to diffuse large B-cell lymphoma agent when the sample of the mRNA of the B2M mutant gene or the protein encoded by the gene is detected in the sample It provides a method of providing information for predicting drug resistance of a patient.

In addition, the present invention

a) detecting copy number variation of one or more genes selected from the group consisting of genes present on the chromosomal positions defined in Table B from biological samples from patients with diffuse large B-cell lymphoma; And

TABLE B

b) in step a), if the number of copies increased compared to the control group, comprising determining to be non-responsive to diffuse large B-cell lymphoma agents for predicting drug resistance in patients with diffuse large B-cell lymphoma Provide a method of providing information.

In one embodiment of the invention, the gene may be selected from the group consisting of POU2AF1 , REL , BCL11A , XPO1 and CACNA1D .

By predicting treatment resistance through mutation of a specific gene and / or increasing copy number variation of a specific gene provided in the present invention, a rapid and accurate prediction of drug reactivity of individual patients with diffuse large B-cell lymphoma can be performed. Can alleviate the physical, mental and economic pain of the patient.

FIG. 1 shows the results of analysis of single nucleotide variants (SNVs) between seven patients (S group) and six drug-resistant patients (F group) responding to treatment through total exome sequencing (WES) and RNA sequencing.
2 shows the mutation distribution results between drug-resistant and reactive DLBCL patients.
3 isTP53(A) Mutant residue positions (b) Number of copies in S groups (S1 to S5) and F groups (F1 to F5) with respect to mutations in (C) Alleles at DNA and RNA levels of drug-resistant DLBCL patients Frequency is shown.
4 isMYD88 In terms of mutations,MYD88This is the result of confirming the variation in the conservative locus of.
Figure 5 shows the results of mutation distribution in BCR signaling, NF-κB, mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K) -protein kinase B (Akt), and JAK / STAT pathway.
6a and 6b respectivelyREL - BCL11A AndPIM1 - MAPK13 In order to predict the functional change caused by the fusion, the difference in expression at each partner gene breakpoint is confirmed through a read-depth pattern.
FIG. 7 shows the relation between SNVs and copy number variation in S groups S1 to S4 and F groups F1 to F4.
8 shows S groups S1 to S5 and F groups F1 to F5.POU2AF1 This is the result of checking the variation of the copy number.
9 shows the results of overexpression of the ATP binding cassette transporter gene in the S and F groups.

We compared two groups of DLBCL patients with different therapeutic responses through whole-exome and transcriptome sequencing, and we found that specific genes in drug-resistant (refractory) patients The molecular background of the refractory DLBCL was first identified by confirming that not only the mutation was frequently observed but also the copy number variation of the specific gene was increased, and thus the present invention was completed.

Accordingly, the present invention provides a composition for predicting drug resistance in patients with diffuse large B-cell lymphoma comprising an mRNA capable of detecting the mRNA of a B2M (beta-2 microglobulin) mutant gene or a protein encoded therefrom, and the same. Provided is a kit for predicting drug resistance of diffuse large B-cell lymphoma patients.

In the present invention, the B2M mutant gene may be one having one or more mutations selected from Table A below.

TABLE A

The composition according to the present invention may further include a probe for detecting copy number variation of one or more genes present on chromosomal positions defined in Table B below.

TABLE B

In one embodiment of the present invention, whole exome sequencing (WES) and RNA sequencing is performed on patients with reactive DLBCL who respond to treatment and patients with nonresponsive drug-resistant DLBCL, compared to patients with reactive DLBCL. Single base mutations (SNVs) and insertion / deletion (indels) occur frequently in patients, and B2M mutations in drug-resistant DLBCL are found to be more frequent.

In another embodiment of the present invention, copy number alterations (CNAs) are identified in drug-resistant DLBCL patients to increase BCL11A , REL , XPO1 , and FANCL on 2p16.3 -p15, 3p14.3-p21. CACNA1D at 1 An increase was confirmed.

Based on the above results, detection of copy number variation of one or more genes selected from the group consisting of POU2AF1 , REL , BCL11A , XPO1 and CACNA1D or expression of a B2M mutant gene or a protein encoded therefrom can be used in patients with diffuse large B-cell lymphoma. It can be seen that it can be used as a marker for predicting drug resistance.

In the present invention, the agent for measuring the mRNA level of the B2M mutant gene may be, but is not limited to, sense and antisense primers, or probes that bind to mRNA complementarily.

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 for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene, 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 kit for predicting drug resistance in patients with diffuse large B-cell lymphoma of the present invention may be composed of one or more other component compositions, solutions or devices suitable for the assay method.

As another aspect of the present invention,

1) Said B2M from biological samples from patients with diffuse large B-cell lymphoma Measuring the expression of mRNA of a mutant gene or a protein encoded from said gene; And

2) a diffuse large B-cell lymphoma comprising determining that the sample is unresponsive to diffuse large B-cell lymphoma agent when the sample of the mRNA of the B2M mutant gene or the protein encoded by the gene is detected in the sample It provides a method of providing information for predicting drug resistance of a patient.

Moreover, as another aspect of this invention, this invention is

a) detecting copy number variation of one or more genes selected from the group consisting of genes present on the chromosomal positions defined in Table B from biological samples from patients with diffuse large B-cell lymphoma; And

TABLE B

b) in step a), if the number of copies increased compared to the control group, comprising determining to be non-responsive to diffuse large B-cell lymphoma agents for predicting drug resistance in patients with diffuse large B-cell lymphoma Provide a method of providing information.

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. Next-generation sequencing; NGS And sample preparation for

To investigate the genetic changes associated with therapeutic response in patients with diffuse large B-cell lymphoma (DLBCL), Rituximab-Cyclophosphamide, Doxorubicin, Freshly frozen tumor tissues obtained from initial biopsies were collected from 13 patients who were treated with Vincristine and Prednisone (R-CHOP). Frozen tissues were obtained to evaluate the cytoplasm and cancer cells were used in excess of 50% of the sample tissue. In addition, patients who remained fully treated for one year and the remaining patients were classified into a responsive group (S group) and a drug resistance group (F group), respectively. Seven of the 13 tumor samples were from the S group (number S1-7) and the remaining six were from the F group (number F1-6). In addition, blood samples were obtained from four patients in each group (NS1-4 and NF1-4) and paired with respective tumor tissues S1-4 and F1-4. DNA was extracted from all 13 tumor tissues and 8 blood samples matching them, and RNA was extracted from 13 tumor tissues for deep sequencing analysis. COO subtypes were determined according to Han's classification by immunohistochemical staining (IHC) of CD10, Bcl-6, and MUM-1. All samples were negative for EBER in situ hybridization. The trial was approved by the Institutional Review Board and complied with the Declaration of Helsinki (Approval number: 2015-01-034-001).

1-2. Exome ( Exome ) And Transcript ( transcriptome ) Sequence analysis and alignment

Whole-exome sequencing (WES) was performed on 10 tumor samples (S1-5 and F1-5) and 8 normal serum samples (NS1-4 and NF1-4). In order to enrich the coding region, SureSelect Human All Exon 50M (Agilent Technologies) was used for tumor tissues, and SureSelect Human All Exon V4 was used for normal tissues. Sequence readings were performed using Illumina HiSeq2000 instruments at depths of 53 × (range 47-77 ×) and 102 × (range 96-106 ×) at the center for tumor and normal tissue, respectively. Standard genomes were aligned using BWA with default parameters and hg19.

In addition, RNA sequencing (RNA-seq) was performed on 13 tumor samples (S1-7 and F1-6). RNA concentration was determined using Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit), and Agilent 2100 bioanalyzer was used to confirm the purity / state of RNA samples. Sequencing libraries include previous studies (Ju YS, Kim JI, Kim S, Hong D, Park H, Shin JY, Lee S, Lee WC, Kim S, Yu SB, Park SS, Seo SH, Yun JY, Kim). HJ, Lee DS, Yavartanoo M, et al. Extensive genomic and transcriptional diversity identified through massively parallel DNA and RNA sequencing of eighteen Korean individuals.Natural genetics. 2011; 43: 745-752). Raw data obtained with Illumina HiSeq 2000 was aligned with human standard dielectric (hg19) using the STAR 2-pass method. Alignment results were evaluated using RNA-SeqQC (Supplementary Table S3), and two samples with low throughput (F3-4) (mapped read count <10 Mb) were excluded from downstream analysis.

1-3. Sequence Variation Analysis

PCR replicates were removed using Picard (http://broadinstitute.github.io/picard/). The Genome Analysis Tool Kit (GATK) was used for indel rearrangement and basic quality score readjustment. SNVs were identified using MuTect. Somatic cell insertion / deletion of eight tumor-normal pairs was confirmed using a GATK Somatic Indel Detector (https://www.broadinstitute.org/cancer/cga/indelocator) and an unpaired sample using GATK UnifiedGenotyper and HaplotypeCaller. The insertion / deletion of was predicted. Variant annotations show results for gene, variant type, and minor allele frequency (MAF) in Exome Aggregation Consortium Version 0.3 (ExAC03, http: //exac.broadinstitute.) And Combined Annotation Dependent Depletion (CADD) scores. Obtained using the software tool ANNOVAR integrated into the database tool.

After listing nonsynonymous SNVs (nsSNVs), splice site SNVs, and indels of coding regions from 8 pairs of samples, criteria and filters were applied to identify candidate mutations as follows:

1) tumor allele frequency of at least 10% (≧ 10%) and normal allele frequency of at most 1% (≦ 1%);

2) rare mutations in which MAF is less than 0.5% (<0.5%) in ExAC03; And

3) Noxious mutations with a CADD score of 20 or greater (≥20).

In addition, additional mutations of the somatic candidate genes in unpaired samples were also identified. In samples where both available DNA and RNA were present, mutations were considered when transcribed or edited in RNA.

1-4. Clones  Mutation analysis

Somatic cell copy number variations (CNAs) were identified in eight tumor-normal pairs using an Excavator in somatic mode. For two unpaired samples (S5 and F5), the Excavator was used in pooling mode to compare each tumor sample with the mean value of eight normal (NS1-4 and NF1-4) samples. The excavator generated a CNA call based on the ratio of tumor-normal readings (T / N ratio) and detected the part with similar copy number. The cases where the T / N ratios were> 1.25 and <0.75 were defined as increase and loss of replication, respectively. If the T / N ratio was> 1.5 or <0.5, the portion was considered high level increase and severe loss, respectively. Minimal common regions (MCRs) were identified using the Integrated Genome Browser (IGV). For MCR belonging to the F group, the CNAs of individual genes were examined. The B allele frequency was calculated as the alternative allele frequency of the MuTect results and was used to identify CN-LOH.

1-5. Fusion Gene Analysis

To identify fusion transcripts, deFuse, ChimeraScan, and pyPRADA were used. First, we excluded the paralog pair based on Ensembl version 75. Among the remaining results, the candidates supported by two or more (≧ 2) junction-spanning reads and predicted at least two of the three tools were selected as fusion candidates.

1-6. Calculate differential expression of genes in two groups

HTSeq was used to calculate sequence reads for the gene, and differential expression of the gene was calculated using DESeq2. DEG was defined as a gene with a q value of less than 0.05 (<0.05) and a log2 (fold change, FC) of at least 1 (≧ 1). To identify over-existing gene sets, MSigDB (http://software.broadinstitute.org/gsea/msigdb/index.jsp), which integrates gene oncology (GO) terms and KEGG pathways, was used. We also used previously proposed criteria to identify cancer outlier genes (COGs).

Example  2. Drug resistance and responsive DLBCL  Single nucleotide variants between patients; SNVs ) And insertion / deletion ( indels Number comparison

Total exome sequencing (WES) and RNA sequencing according to Examples 1-2 were performed on a total of 13 samples, 7 patients (Group S) and 6 drug-resistant patients (Group F) that responded to the treatment. Was carried out. Eight tumor-normal samples (S1-4, F1-4) were analyzed around the results, and as a result, it was confirmed that they have a highly reliable somatic mutation as shown in FIG. In addition, the number of pathological somatic single nucleotide mutations and insertions / deletions in the coding region was 38 in the S group (range 19-66) and 71 in the F group (range 28-120). The variation of the encryption domain tends to occur more frequently in group F, but there was no statistically significant difference ( P = 0.3429).

Example  3. Drug resistance and reactivity DLBCL  Identify Mutant Mutations in Patients

3-1. Drug resistance and reactivity DLBCL  Comparison of Mutations Between Patients

Gene mutations between drug-resistant and reactive DLBCL patients were compared. As a result, as shown in Figure 2, it was confirmed that a single base mutation of TP53 appears more frequently (50%) and exclusively in drug-resistant DLBCL patients. All single base mutations of TP53 occurred in the DNA-binding domain, and the frequency of mutant alleles of TP53 exceeded 60% at the DNA level and was higher when transcribed (<90%) (FIG. 3). ).

In addition, RAB11FIP5 , PRKCB , PRDM15 , FNBP4 , AHR , CEP128 , BRE , DHX16, MYO6 , and NMT1 Variations were identified in at least two samples (Tables 1-16).

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

TABLE 9

TABLE 10

TABLE 11

TABLE 12

TABLE 13

TABLE 14

TABLE 15

TABLE 16

Also, MYD88 , B2M , SORCS3 , and WDFY3 Mutations were found in two drug-resistant patient samples and one reactive patient sample (FIG. 2). Activation site variation of MYD88 (p.Leu265Pro) is known to be more common in non-GCB type DLBCLs. Four mutations were identified in the most conservative locus on the Toll-IL-1 receptor (TIR) domain of MYD88 (FIG. 4). Two of the four mutations in these patients occurred at hot spots and the rest included other loci.

B2M is a component of the class I major histocompatibility complex (MHC) and encodes β2 microglobulin, which is required for recognition by cytotoxic T cells. B2M mutations in drug-resistant DLBCL patients included missense mutations (n = 1) and frameshift deletions (n = 1).

In addition, major factors associated with the development of DLBCL, including BCR signaling, NF-kB, mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K) -protein kinase B (Akt), and JAK / STAT pathways The mutation distribution of the signaling pathway was evaluated. The signaling pathways are closely related to each other. For example, BCR activation is upstream of the PI3K / Akt and NF-kB pathways, and the mTOR pathway is activated by Akt, which can be activated by the NF-kB pathway. NF-kB and STAT3 cooperatively mediate tumorigenesis.

As a result, as shown in Figure 5, the F group showed more frequent mutations in the PI3K-Akt and mTOR pathway, PTEN belonging to the PI3K-Akt and mTOR pathway And TSC2 confirmed that a mutation occurred in F4. These are known as tumor-suppressor genes (TSGs). All patients in group F had one or more tumor suppressor gene mutations, whereas only half of patients in group S identified tumor suppressor gene mutations.

3-2. Drug resistance and reactivity DLBCL  Identify common mutations in patients

Of the 485 single base mutations and insertion / deletion candidates listed in Tables 1-16 above, four of the four genes known to be associated with the major genes of DLBCL ( TP53, MYD88 , PIM1, and B2M ) mutations were 3 of 13 patients. (23.1%). Most of the single base mutations (SNVs) and insertions / indels occurred at different locations except MYD88 encoding p.Leu265Pro and PIM1 encoding p.Glu226Lys .

In addition, PTPN6 , TRIP12 , SORCS3 , and WDFY3 are Mutation occurred in 3 of 13 patients (23.1%). PTPN6 and PTEN belongs to Class I classical Cys-based phosphatase family, PTPN6 encodes the SHP-1 phosphatase weaken the BCR signaling by dephosphorylation the ITAM motif of the BCR of CD79A and CD79B signaling subunit. All mutations identified in this experiment were missense mutations (p.Leu63Gln, p.Ser26Asn, p.Lys68Thr, and p.Pro105Leu) and PTPN6 Three quarters of the mutations were found in the SH2 domain. One patient (S1) had both missense mutations and copy-neutral loss of heterozygosity (CN-LOH), indicating loss of function of tumor suppressor genes. PTPN6 Loss-of-function mutations are known to promote downregulation of STAT3 via JAK3 kinase in DLBCL.

In addition, mutations in genes belonging to the Class I classical Cys-based phosphatase family were examined. As a result, eight mutations in various receptor type protein tyrosine phosphatase and one mutation in DUSP11 were identified . Additionally found. A total of 12 mutations were found in 7 of the 13 patients (53.8%). Five of the twelve mutations in the phosphatase group were found in the phosphatase domain and two variants were found in the fibronectin domain located in the extracellular-interacting part. Frequent mutations in the functional domain mean that phosphatase modifications play a very important role in the development of DLBCL.

SORCS3 encodes a type-I receptor transmembrane protein belonging to the vacuolar protein sorting 10 receptor family. Thyroid hormone receptor interactor 12 ( TRIP12 ), which encodes the HECT domain ubiquitin E3 ligase, is known to be critical for the homeostasis of ubiquitin-controlled events after DNA cleavage. WDFY encodes a phosphatidylinositol 3-phosphate-binding protein that functions as a major factor for aggregate clearance by autophagy.

Example  4. Drug resistance and reactivity DLBCL  Identifying Fusion Genes in Patients

First, to identify the fusion transcripts, three different fusion callers were used, and eight fusion genes with> 2 fusion callers in common were selected as candidate groups. Five of eight were identified in two S-group patients and three in two F-group patients (Table 17).

TABLE 17

Next, expression differences were identified at each partner gene breakpoint to predict functional changes caused by fusion. Most partner genes showed no difference in fusion status, but REL , BCL11A , and MAPK13 showed a clear read-depth pattern at the ends (FIGS. 6A and 6B). PIM1 - MAPK13 fusion transfer member is composed of the respective protein kinase domain (protein kinase domain) of PIM1 and MAPK13, fusion is under the assumption that leads to the acquisition function of the associated gene (gain of function) PIM1 of-two of MAPK13 Dog partner genes were upregulated compared to unfused samples. REL - BCL11A In two patients in group F with fusion, one had both fusion and copy number acquisition, PIM1 - MAPK13 The same result was confirmed in the case of fusion.

S-group is transposed of BCL6 and IGLL5 and PRDM1 Structural variants associated with the well-known DLBCL, such as inversion. Inactivation of PRDM1 is known to constitute predominantly truncating mutations in DLBCL patients. In this experiment PRDM1 - ATG5 It was confirmed that the fusion transcript was formed by inversion. Fusion transcripts are irregular, which is PRDM1 A new mechanism of inactivation is shown. BCL6 And IG are usually translocated in DLBCL, and this experiment also identified fusion transcripts of these genes in two samples of the S group.

Example  5. Drug resistance DLBCL  In the patient Clones  Copy number alterations (CNAs) analysis

Segmented copy number variation was defined based on a T / N ratio of> 1.25 or <0.75. The copy number variation was analyzed according to the method described in Examples 1-4 above, and as a result, as shown in FIG. CNA) was observed.

In addition, minimal common regions (MCRs) of segment copy number variation in drug-resistant DLBCL patients have been identified and specific information is presented in Table 18 below. The expression levels of the genes included in each MCR were confirmed, and the genes having the same variation between the copy number variation and expression are shown in the 'Genes validated by RNA-seq' column of Table 18.

TABLE 18

In addition, POU2AF1 in F1, F4, and F5 patients An increase in copy number variation was found (FIG. 8). POU2AF1 protein, also called Oct-binding factor 1 (OBF-1), Oct coactivator from B cells (OCA-B), or BOB.1, is a transcriptional coactivator involved in the transcription of immunoglobulin genes and is responsible for B cell development and immunity. It is known to play an important role in globulin expression.

In addition, an increase in BCL11A , REL , XPO1 , and FANCL on 2p16.3 -p15 was observed in 2 of 5 drug-resistant DLBCL patients. Also, CACNA1D was confirmed that in drug-resistant DLBCL patients as compared to the reactive DLBCL patients is overexpressed by increasing copy number, CACNA1D in the 3p14.3-p21.1 More specifically An increase was confirmed.

In addition, an increase in the modification / transcription domain related protein (TRRAP) and CUX1 (CUT-like homeobox 1) at 7q22.1-q21.3 was observed in three drug-resistant DLBCL patients. As a common component of many HAT complexes, TRRAP is known as an essential cofactor of two c-Myc and E1A / E2F carcinogenic transcription factor pathways. CUX1 is a homeobox gene that is involved in both tumor suppression and progression. Increased expression of CUX1 is associated with tumor progression.

RNA sequencing results showed that the mean gene expression of POU2AF1 , SLC1A4 , REL11, FANCL , CACNA1D , TRRAP , and CUX1 was significantly increased in the F group compared to the S group.

Example  6. Drug resistance DLBCL  Gene expression in patients signature  Confirm

Genes expressed differently between the F and S groups were identified. A total of 1531 differentially expressed genes (DEGs) were identified, including 743 up-regulated genes and 787 down-regulated genes using the criteria of log ₂ FC ≧ 1 and FDR <0.05 (Table 19). To Table 27)

TABLE 19

TABLE 20

TABLE 21

Table 22

TABLE 23

TABLE 24

TABLE 25

TABLE 26

TABLE 27

Upregulated DEG was present in many of the standard pathway gene sets involved in transcription / translation, replication / cell cycle / DNA repair, lipid synthesis, cell energy metabolism, B cell receptor signaling, and Notch signaling. The most significant abundance of gene sets existed in the 'transcription / translation' group, including genes that encode protein synthesis and degradation, S and L ribosomal proteins, RNA polymerase subunits, zinc finger proteins and exosome components. The 'replicate / cell cycle' group included E2F1 transcription activator, MCM7 with DNA helicase activity, telomerase reverse transcriptase (TERT), and centromere proteins. The second largest group includes NADH: ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase complex, and adenosine triphosphate (ATP) synthase components. It was 'cell energy metabolism'.

Downregulated DEG was present in many gene sets related to immune responses, particularly T cell receptor signaling, cytokine / chemokine signaling, and the complement cascade. A study using the whole genome microarray, conducted in 2005, involved three separate subsets of DLBCL: oxidative phosphorylation (OxPhos), B cell receptor / proliferation and host response (HR). Presented. Thus, a gene classifying three groups ('Consensus Clusters Markers') was extracted to determine the group to which drug-resistant DLBCL belongs. The log ₂ FC of these genes showed a significant difference between the three groups (P = 5.475e-09). The median log ₂ FC was highest in the OxPhos group and lowest in the HR group (Tables 28-30). Drug-resistant DLBCL group showed higher expression of OxPhos genes and lower HR gene expression than reactive group.

TABLE 28

TABLE 29

TABLE 30

In addition, cancer outlier genes (COGs) were identified and gene sets excessively expressed in the COG were identified. The top gene set overlaps with many up set genes. Further identified genes included the ATP-binding cassette transporter gene group (Tables 28-30). Three of the four drug resistant samples overexpressed at least one ATP binding cassette transporter, whereas one of six reactive samples overexpressed the transporter (FIG. 9). In addition, there were markedly many key signal transduction pathways such as mitogen-activated protein kinase (MAPK), insulin, adipocytokine, toll-like receptors, mTOR, and VEGF pathways (FDR <0.05). Tables 28-30. MAPK12 and MAPK13, which encode p38 gamma and delta, are members of the MAPK family, respectively, and the p38 signaling pathway is involved in resistance to cytotoxic drugs, and inhibition by rituximab makes cells susceptible to drug-induced apoptosis. Make it. Recent studies have reported increased p38 expression in CHOP resistant patients. In addition, the insulin, adipocytokine and toll-like receptor pathways sharing the PI3K-Akt pathway were overexpressed in the F group. Thus, the results of the above examples suggest that activation of the PI3K-Akt-mTOR pathway along with mutational results may be important for the therapeutic resistance of DLBCL.

Example 7 Identification of Genetic Variations Between Cell-of-Origin (COO) Groups

Known DLBCL related genetic modifications were identified and compared differences between COO groups. In a total of 13 samples, 8 samples were GCB and 6 were non-GCB types, regardless of the subtype of treatment response. Frequently mutated genes in the non-GCB type were CARD11, MYD88, CDKN2A, CD79A / B TNFAIP3, and PRDM1 , whereas the GCB type frequently showed mutations of GNA13, EZH2, BCL6 . TP53, B2M, MEF2B and CREBBP mutations were found in both types.

In addition, eight GCB patient MYD88 (2/8), cutting-type mutations in TNFAIP3 (1/8), miss sense mutation in the CD79B (1/8), B2M (3/8 ), and TP53 (2/8) The mutation of Whereas six non-GCB samples were MYD88 (1/6) mutant, TNFAIP3 (1/6) truncated mutation, missense mutation of CD79B (1/6), non-frameshift deletion of CARD11 (1/6), MEF2B (2/6), TP53 (1/6) mutations were shown. GNA13, EZH2 , and BCL6 mutations were not found in both cases.

In addition, increased replication of REL and BCL11A in the GCB subtype was found in two samples and in one case a copy neutral LOH of PTEN was found. In non-GCB subtypes, increased replication of BCL2 was found in four samples, and replication deletions in CDKN2A and CDKN2B were found in two samples, which is consistent with previous reports. In addition, up-regulated DEGs among 11 samples subjected to RNA sequencing were present in non-GCB type extracellular matrix protein gene sets and gene sets related to GCB type transcription / translation.

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 (10)

Rituximab-Cyclophosphamide in patients with diffuse large B-cell lymphoma, comprising a B2M (beta-2 microglobulin) mutant gene mRNA or an agent capable of detecting the expression of a protein encoded therefrom. ), As a composition for predicting resistance to doxorubicin, Vincristine, Prednisone (R-CHOP) drugs,
The B2M mutant gene is characterized in that it has one or more mutations selected from Table A below.
TABLE A

delete The method of claim 1,
The composition is characterized in that it further comprises a probe for detecting the copy number variation of one or more genes present on the chromosomal positions defined in Table B below.
TABLE B

The composition of claim 3, wherein the gene is selected from the group consisting of POU2AF1 , REL , BCL11A , XPO1 and CACNA1D . The composition of claim 1, wherein the agent capable of detecting mRNA expression is a sense and antisense primer or probe that complementarily binds to mRNA of a B2M mutant gene. The composition of claim 1, wherein the agent capable of detecting the protein is an antibody that specifically binds to the protein. Rituximab-Cyclophosphamide in patients with diffuse large B-cell lymphoma, comprising the composition of any one of claims 1, 3, 4, 5 or 6. Kit for predicting resistance to Doxorubicin, Vincristine, Prednisone (R-CHOP) drugs. Rituximab-Cyclophosphamide, Doxorubicin, Vincristine, Prednisone (R-CHOP) in patients with diffuse large B-cell lymphoma, comprising the following steps: Information about how to predict drug resistance:
1) measuring the expression of mRNA of the B2M mutant gene according to claim 1 or a protein encoded from said gene from a biological sample from a diffuse large B-cell lymphoma patient; And
2) determining that the sample is unresponsive to the R-CHOP drug of diffuse large B-cell lymphoma when the expression of the mRNA of the B2M mutant gene or the protein encoded by the gene is detected in the sample. The method of claim 8, wherein the information providing method is
a) detecting copy number variation of one or more genes selected from the group consisting of genes present on the chromosomal positions defined in Table B from biological samples from patients with diffuse large B-cell lymphoma; And
TABLE B

b) in step a), further comprising determining that if the copy number is increased compared to the control group, it is unresponsive to the R-CHOP drug of diffuse large B-cell lymphoma. The method of claim 9, wherein the gene is selected from the group consisting of POU2AF1, REL, BCL11A, XPO1, and CACNA1D .

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