WO2006112483A1 - Diagnostic method and prognostic method for disease type of diffuse large b-cell lymphoma - Google Patents

Diagnostic method and prognostic method for disease type of diffuse large b-cell lymphoma Download PDF

Info

Publication number
WO2006112483A1
WO2006112483A1 PCT/JP2006/308235 JP2006308235W WO2006112483A1 WO 2006112483 A1 WO2006112483 A1 WO 2006112483A1 JP 2006308235 W JP2006308235 W JP 2006308235W WO 2006112483 A1 WO2006112483 A1 WO 2006112483A1
Authority
WO
WIPO (PCT)
Prior art keywords
region
type
nucleic acid
human chromosome
human
Prior art date
Application number
PCT/JP2006/308235
Other languages
French (fr)
Japanese (ja)
Inventor
Masao Seto
Hiroyuki Tagawa
Yasuko Yoshida
Shigeki Kira
Original Assignee
Aichi Prefecture
Ngk Insulators, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aichi Prefecture, Ngk Insulators, Ltd. filed Critical Aichi Prefecture
Priority to JP2007528177A priority Critical patent/JPWO2006112483A1/en
Publication of WO2006112483A1 publication Critical patent/WO2006112483A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to diagnosis of a disease type of diffuse large B-cell lymphoma (DLBCL) and prognosis of DLBCL.
  • DLBCL diffuse large B-cell lymphoma
  • Diffuse large B-cell lymphoma is the most common non-Hodgkin's lymphoma and is known to include pathophysiologically distinct groups (Harris NL, Jaffe Eb, btein H, et al. A revised European- American classification of lym phoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392, Offit K, Le Coco F, Louie DC, et N Engl J Med 1994; 331: 74—80, Kramer MHH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, B CL6, and MYC al.
  • BCL6 gene Rearrangement of BCL6 gene as a prognostic marker in diffuse large cell lymphoma. rearrangements in diffuse large B— cell lymphoma. Blood. 1998; 92: 3 152—3162, Gatter KC and Warnke RA. Diffuse large B— cell lymphoma, In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World health classification of tumors.Pathol ogy & Genetics of tumors of haematopoietic and lymphoid tissues.Washington: IAR C press, Lyon; 2001. 171 -174).
  • DLBCL is also known to have clinical heterogeneity because patients have significantly different clinical courses (Fisher RI, Gaynor ER, Dahlberg S, et al. Comparison of a standard regimen (CHOP) with Three intensive chemotherapy regimens for advanced non—Hodgkin s lymphoma. N Engl J Med. 1993; 328: 1002-10 06.). For this reason, there is interest in the importance of subgroup identification in heterogeneous DLBCL.
  • the ABC group expresses genes specific to activated B cells and plasma cells, while the GCB group maintains a normal germinal center B cell gene expression program (Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffiise lar ge B— cell lymphoma identified by gene expression profiling. Nature. 2000; 403: 503— 511, Wright G, Tan B, Rosenwald A, et al. A gene expression-based method to diag nose Clinically distinct subgroups of diffiise large B cell lymphoma.Proc Natl Acad S ci USA. 2003; 100: 9991-9996, Rosenwald A, Wright G, Chan WC, et al.
  • CD5 +, CD5-CD10 +, and CD5-CD10- have been identified (Harada S, Suzuki R, Uehira K, et al. Molecular and immunological dissection of large B cell lymphoma: CD5 + and CD5 with CD10 + groups may constitute clinically relevant subtypes. Leukemi a. 1999; 13: 1441-1447).
  • the CD5 + group has been shown to account for approximately 10% of all DLBCL cases. This has a CD5 + C10—CD19 + CD20—CD21—CD23—cyclin D1—phenotype and a prognosis over CD5 ”DLBCL.
  • CD5—CD10 + gnole has a lower expression frequency of BCL2 protein than other groups, and usually expresses BCL2. A clear association with normal germinal center cells lacking the CD5-CD10-group was observed most frequently, and the expression rate of BCL6 gene rearrangement was higher than the other two groups. Bur, the disclosure of the difference is not significant (Harada S et al) 0 invention
  • an object of the present invention is to provide a technique for easily diagnosing a subgroup of DLBCL, which greatly affects mortality and prognosis.
  • Another object of the present invention is to provide a method for diagnosing prognosis of DLBCL.
  • the present inventors used a comparative genomic hybridization (microarray CGH method) that uses an array to identify genomic imbalance features of different subgroups of DLBCL (Ota A, fagawa H, Karnan S, et ai. Iaentincation and cnaractenzation of a novel gene, shi 13orl25, as a target for 13q31— q32 amplification in malignant lymphoma. Cancer Res. 2004; 64: 3087-3095, Tagawa H, Tsuzuki S, Suzuki R, et al. Genome-wide array-based comparative genomic hybridization of diffuse large B— cell lymphoma: comparis on between CD5— Positive and CD5— negative cases. Cancer Res.
  • a diffuse large cell type comprising a detection step of detecting an amplified region and Z or a defective region of a human chromosome in a test sample containing a chromosome collected from a human.
  • Methods for diagnosing B cell lymphoma pathology are provided.
  • the amplified region force detected in the detection step is selected from 3, 8q21-q26, Ilq21-q25, 16pl22-q24, 18, 19ql3 and X of the human chromosome.
  • Z or the deletion region is in one or more regions selected from 2pl l, 6ql2-27, 8p22-p23, 9p21 and 17p of the human chromosome It is possible to provide a determination step that positively determines that the disease type of the diffuse large B-cell lymphoma is ABC type, using whether or not it is an index. Further, in the determination step, the amplification region is 3p23-q28, 18ql l of human chromosome. .2-q23 and 9ql3.41-ql3.43 force selected if one or more are selected and said defective region is selected from 6q22.31-q24.1 and 9p21.3 of human chromosome 1 It may be a process using as an index whether there are two or more species.
  • the determination step may be a step using as an index whether the amplification region is 3q23-q28 and Z of a human chromosome or the deletion region is 9p21.3 of a human chromosome. Furthermore, the determination step may be a step of positively determining that the disease type is CD5 + type when the disease type is positively determined to be ABC type.
  • the amplified region force detected in the detection step is 1q22-32, 2pl4-p24, 5pl2-p, 5ql5-q31, 6pl2-p25, 7, 8q22 of the human chromosome.
  • a determination step of positively determining that the disease type of diffuse large B-cell lymphoma is GCB type may be provided using whether or not it is an indicator.
  • the amplification region is selected from human chromosomes q2 1.1-q23.3, lq31.1-q42.13, 2pl5-pl6.1, 7q22.1-q36.2 and 12ql3.1-ql4.
  • the determination step which may be performed using one or two or more types as an index, may be a step using whether or not the amplified region is in 7q22-q36 of a human chromosome.
  • the determination step may include a step of positively determining that the disease type is CD5-DC10 + when the disease type is positively determined to be GCB type.
  • the amplified region detected in the detection step is 3, 6p22-p25, 7p22-q31, 8q24, llq22-q25, 12, 16pl3-q21 of the human chromosome, 18, 19 and X force selected from one or more selected and Z or from the lp36, 2pll, 6ql4-q27, 8p23, 9p21, 15ql3-ql4 and 17pll-pl3 of the human chromosome
  • a step of positively determining that the disease type is CD5 + can be provided by using whether the disease type is one or more selected as an index.
  • the determination step includes determining whether the amplification region is a force of 3 of human chromosome and Z or whether the deletion region is 9p2 of human chromosome. As a process that uses this as an index.
  • the amplification region detected in the detection step is human chromosome lq, 2pl3-p25, 6p21-p25, 7, 8q22-q24, 9q33-q34, 12, 13q31-q33, 15q, 16pl3, 19ql3.3-13.4 and whether it is in one or more selected from X and Z or the deleted region is 1 ⁇ 36, 1 ⁇ 22, 2pll, 3pl4, 4p, human chromosome A determination step of positively determining that the disease type is CD5-CD10 + using one or more selected from 6ql3-q27, 9p2 1 and 13ql4-q21 as an index may be provided .
  • the determination step includes determining whether the amplification region is one or more selected from 7q22-q36 and 12ql3-ql4 of human chromosome, and Z or 17 pl3 of the deletion region of human chromosome. It is good also as a process using as an index whether it is.
  • the detection step includes a step of performing any one of a PCR method, an RT-PCR method and a nucleic acid hybridization on the region in the test sample. And a step of hybridizing a probe containing a region on the human chromosome with a nucleic acid in the test sample.
  • the detection step may be a step of performing an array CGH method.
  • nucleic acid probe for diagnosing the disease type of diffuse large B-cell lymphoma, on the human chromosome in ABC-type diffuse large B-cell lymphoma.
  • nucleic acid probes capable of detecting the amplification region and the Z or deletion region.
  • This nucleic acid probe supplies a test sample collected from a human individual suffering from ABC type diffuse large B cell lymphoma to an array in which nucleic acid probes derived from a predetermined range of human chromosomes are immobilized. Based on the log ratio value of the fluorescence intensity obtained at the fixed site of the nucleic acid probe by performing nucleic acid hybridization.
  • Nucleic acid selected based on the magnitude of the log ratio with respect to a predetermined predetermined upper and lower threshold
  • a probe can be included.
  • nucleic acid probe for diagnosing a disease type of diffuse large B-cell lymphoma, on a human chromosome in a GCB-type diffuse large B-cell lymphoma.
  • nucleic acid probes capable of detecting the amplification region and the Z or deletion region.
  • This nucleic acid probe is a nucleic acid probe derived from a predetermined range of human chromosomes.
  • Immobilizing the nucleic acid probe by supplying a test sample collected from a diffuse large B-cell lymphoma affected by GCB to an array with fixed lobes and performing a nucleic acid hybridization Set based on the log ratio value of fluorescence intensity obtained at the site.
  • Nucleic acid selected based on the magnitude of the log ratio with respect to a predetermined predetermined upper and lower threshold
  • a probe can be included.
  • an array for diagnosing a disease type of diffuse large B-cell lymphoma to which the nucleic acid probe described above is immobilized.
  • This array is selected from human chromosomes 3, 8q21-q26, Ilq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3, X, 2pll, 6ql2-27, 8p22-p23, 9p21, 17p 1
  • a nucleic acid probe capable of detecting at least a part of a species or two or more regions may be immobilized.
  • This array also includes human chromosomes lq22-32, 2pl4-p24, 5pl2-pl5, 5ql5-q31, 6 pl2—p25, 7, 8q22—q26, 9q33—q34, llq, 12, 13q31—q33, 16pll — Pl3, 18q21—from q23, 1 9p, 19ql3, 21q, X, lp36, 2pll, 3pl4, 4pl2—pl3, 4q33—q34, 6ql4—ql6, 8p22—p23, 9p21, 13ql2-q22, 17pl2, and 18q22-q23
  • a nucleic acid probe capable of detecting at least a part of one or two or more selected regions may be immobilized.
  • a prognostic diagnosis method for diffuse large B-cell lymphoma wherein a test sample containing a chromosome collected from a human is subjected to 9p21 of human chromosome.
  • a method comprising a detection step of detecting a defect or mutation.
  • the detecting step may be a step of performing a hybridization on a probe including a region on the human chromosome and the test sample.
  • the detection step can be a step using array CGH.
  • the detection step can be a step of detecting a deletion or mutation of the pl6 INK4a gene.
  • the detection step may be a step of detecting the presence / absence, expression level or mutation of the protein encoded by the pl6 INK4a gene.
  • an array for prognosis of diffuse large B-cell lymphoma wherein an array of nucleic acid probes for detecting 9p21 of human chromosome is immobilized. Is provided.
  • At least a part of human chromosome 9p21 is detected.
  • a prognostic marker for diffuse large B-cell lymphoma is provided.
  • the polynucleotide can be a polynucleotide having a pl6 INK4a gene, a part thereof, or a base sequence complementary thereto.
  • a prognostic marker for diffuse large B-cell lymphoma which is a protein encoded by the pl6 INK4a gene, a part thereof, or an antibody thereto.
  • FIG. 1 Force plan Meyer analysis of different subgroups.
  • A shows force plan Meyer analysis of ABC (26 cases) and G CB (17 cases).
  • B shows force plan Meyer analysis of CD5 + (33 cases), CD5—CD10 + (19 cases) and CD5—CD10— (44 cases).
  • FIG. 2 Hierarchical cluster of DLBCL cases. 46 cases (22 cases of CD5 +, 7 cases of CD5_CD10 +, 17 cases of CD5—CD10—) force Clustered by Treeview software based on the expression of 67 out of 100 genes described by Rosenwald et al. It was done. The relative expression level of each specimen is shown in the lower part. Samples were divided into two subgroups: ABC (left side) and GC B (right side).
  • FIG. 3 Shows typical features of the genomic profile of DLBCL cases.
  • Two CD5-CD10 cases (A) shows ABC signature and (B) shows GCB signature.
  • Gene amplification 3p26.3— ql2.3, 3ql3.33— q29, 4q32.1-q35.1, 9p24.3-q22.33, 17pll.2-q21.1, 17q 21.32, 17q23.2 -q24.2, and 18q; gene deletion: 9p21.3 (arrow), 13ql4.3-q21.2, 15q21. 3, 17pll.2-pl3.3, and 17q21.33-q22 0 Occurs only in BAC RP11-149 12 (see arrow) containing pl6 INK1 ⁇ 2 .
  • FIG. 4 shows the characteristics of genomic imbalance of DLBCL ABC type and GCB type.
  • A shows a summary of chromosomal imbalances in 28 cases in the ABC group and (B) in 18 cases in the GCB group.
  • the left (red) line supports the deficit and the right (green) indicates amplification.
  • It left square red, homo-deficient (Lo g 2 draw - 1.0) indicates, right green squares, shows the high degree of amplification in the right (Log 2 ratio> +1.0).
  • FIG. 5 shows the genome-wide gene imbalance frequency of the distinguished DLBCL subgroups.
  • Horizontal line Clone 2213 BAC / PAC in order from chromosome 1 to chromosome 22 and chromosome X. Based on the information from the Institute Institute of Information (November 2004 version) within each chromosome [Konii ii, Ensembl Genome Data Resources of Sanger], it is shown in order from p telomer to q telomer.
  • Vertical line shows the frequency (%) of amplification and loss.
  • (A) shows ABC group (28 cases), GCB group (18 cases), and the total of ABC group and GCB group (46 cases).
  • FIG. B shows CD5 + group (36 cases), CD5-CD10 + group (19 cases) and CD5-CD10- group (44 cases).
  • the characteristics of genomic disequilibrium in the CD5 + and CD5-CD10 + groups were similar to those in the ABC and GCB groups, respectively.
  • the region of genomic imbalance in the CD5-CD10 group showed a pattern similar to that of the ABC group and GC B group.
  • FIG. 6 shows 9p21.3 gene deletion, overall survival rate, and pl6INK4a gene in ABC and GCB groups.
  • A shows the power plan Meyer survival rate in all cases, ABC cases, CD + cases and CD5-CD10-cases with or without 9p21.3 (pl6INK4a locus) deficiency.
  • B shows a representative individual genomic profile of 9p21 in 3 cases. The dots indicate the BAC / PAC clone log2 ratio for both p and q telomeres, and the bold line in each profile indicates the missing region.
  • MCR shows the smallest common region in the 9p region, the vertical line is log
  • the present invention relates to an amplification region of a human chromosome in a test sample containing a chromosome collected from a human and It is characterized by diagnosing a DLBCL disease type based on a defective region.
  • a DLBCL disease type can be easily diagnosed by examining human chromosome amplification regions and Z or deletion regions in test samples containing chromosomes collected from DLBCL-affected human individuals.
  • DLBCL disease types are already more malignant by expression analysis and have been classified into ABC type and GCB type. According to the present invention, these disease types can be easily diagnosed.
  • the diagnosis or determination of the disease type is to determine whether a DLBCL patient is ABC type or GCB type, or is DLBCL patient a CD5 + type force CD5-CD10 + type? Is to judge. Such a determination has a characteristic amplification region and Z or a defective region in one of the disease types to be determined, and whether or not it has high frequency in one disease type. Can be based on the number of detected regions.
  • the present invention can be implemented in the form of a diagnostic method and array.
  • the present invention is characterized in that the prognosis of DLBCL is diagnosed based on a 9p21 deletion or mutation of a human chromosome in a test sample containing a chromosome collected from a human.
  • DLBCL prognosis can be easily and accurately performed. Prognosis enables more appropriate selection of treatment.
  • the determination of whether or not the prognosis is made is to determine whether “good prognosis” or “poor prognosis”.
  • “Good prognosis” means that when the force-plan Meier curves are compared between DLBCL patients with two types of disease, the p-value of the log rank test is a significant difference of 0.05 or less and the survival rate indicates the patient group. Can be.
  • “poor prognosis” can indicate a patient group in which the p-value of the log-rank test comparing force-plan Meyer curves is a significant difference of 0.05 or less and the survival rate is poor. Since 9p21 of the human chromosome corresponds to the pl6 INK4a gene, the prognosis of DLBCL can also be diagnosed by detecting the presence of the gene, its expression level, and mutation.
  • the present invention can be implemented in the form of a diagnostic method, an array, and a diagnostic marker.
  • Chromosome amplification and deletion regions that serve as indicators for diagnosing DLBCL disease types are determined using chromosome-containing samples collected from human individuals with DLBCL whose disease type has been previously diagnosed by expression analysis, etc. be able to. Determine amplification and deletion regions as indicators To do so, it is preferable to use array CGH.
  • array CGH the words of high frequency amplification and high frequency loss are defined in detail in the examples described later.
  • Amplification regions useful for determining DLBCL ABC type include amplification regions characteristic of ABC type from the frequency graph of genomic imbalance shown in Table 3 and FIG. 5 of Examples described later. It is done. For example, 3, 8q21-q26, llq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3 of human chromosome and one or more regions selected from X force can be mentioned. These regions are also forces that are high frequency amplification regions in the ABC type. Among them, 3p23_q28, 18 qll.2-q23 and 9ql3.41-ql3.43 force One or more selected regions are more characteristic for the higher frequency or ABC type. Furthermore, 3q23_q28 is an amplification region characteristic of ABC type.
  • a defective region useful for determining that it is the ABC type of DLBCL a defective region characteristic to ABC from the frequency graph of genomic imbalance shown in Table 3 of the Example described later and FIG. Is mentioned. Examples thereof include one or more selected from 2pll, 6ql2-27, 8p22-p23, 9p21 and 17p of human chromosome. This is because these regions are frequently defective regions in the ABC type. Of these, 6q22.31-q24.1 and 9p21.3 are preferred. Furthermore, it is 9p21.3 of the human chromosome.
  • useful amplification regions for determining the DLBCL GCB type include human chromosomes lq22-32, 2pl4-p24, 5pl2-p, 5ql5-q31, 6pl2-p25, 7, 8q22- one or more selected from q26, 9q33-q34, 1 lq, 12, 13q31-q33, 16pll-pl3, 18q21-q23, 19p, 19ql3, 21q and X. This is because these regions are frequently amplified regions in the GCB type.
  • lq21.1-q23.3, lq31.1-q42.13, 2pl5-pl6.1, 7q22.1-q 36.2 and 12ql3.1-ql4 are selected, 7q22-q36.
  • deletion regions useful for determining DLBCL GCB type include human chromosomes lp36, 2pll, 3pl4, 4pl2-pl3, 4q33-q34, 6ql4-ql6, 8p22-p23, 9p21, Examples thereof include one or more selected from 13ql2-q22, 17pl2, and 18q22-q23. This is because these are frequently defective regions in the GCB type.
  • human amplification stains are useful for determining that DLBCL is CD5 + type. Examples thereof include one or more selected from 3, 6p22-p25, 7p22-q31, 8q24, llq22-q25, 12, 16pl3-q21, 18, 19 and X.
  • Human chromosome 3 is preferred.
  • useful deletion regions include lp36, 2pll, 6ql4-q27, 8p23, 9p21, 15q13-ql4 and 17pll-pl3 of the human chromosome. Preferably, it is 9p2 of the human chromosome.
  • useful amplification regions for determining DLBCL CD5 and DC10 + are lq, 2pl3-p25, 6p21-p25, 7, 8q22-q24, 9q33-q34, 12 on the human chromosome. , 13q31-q33, 15q, 16pl3, 19ql3.3-13.4 and X force One or more selected.
  • the human chromosomes 7q22-q36, 12ql3-ql4 and 17pl3 are also preferred.
  • useful deletion regions include 1 ⁇ 36, 1 ⁇ 22, 2pll, 3pl4, 4p, 6ql3-q27, 9p21 and 13ql4-q21 of human chromosomes.
  • the GBB type may be associated with the CD5 ⁇ DC10 + type due to the genomic imbalance in the DLBCL GCB type and the CD5 ⁇ DC10 + type (particularly, Asian, especially Japanese). Therefore, when the type is ABC type, it may be positively determined that the type is CD5-CD10 + type.
  • Various methods can be employed to detect the amplified region and the defective region on these chromosomes. For example, it can be detected by hybridization using a chromosome-containing test sample collected from a human individual suffering from DLBCL and a probe that hybridizes with various regions on these chromosomes. Nucleic acid samples from patients, etc., can also be obtained by standard DNA extraction methods, etc. for patient lymphoma specimens. On the other hand, any probe that can detect such amplification and deletion may be any probe that hybridizes with at least a part of the region on the chromosome.
  • a BAC clone and / or a PAC clone corresponding to a chromosomal region can be used as a probe by amplifying genomic DNA cloned by DOP-PCR or adapter PCR.
  • this is a BACZPAC clone containing the gene or P Genomic DNA cloned using the CR method can also be used.
  • the form of hybridization between the test sample and the probe is not particularly limited. It may be a liquid phase reaction or a method using a solid phase carrier such as a bead or a substrate.
  • a DNA probe immobilized on a solid phase carrier such as a chip or a bead can be used.
  • Amplification of a specific region on a chromosome and detection of a defect can be performed by preparing a DNA array according to the array CGH method in the Examples.
  • such an amplified region and a defective region on a chromosome are detected by performing PCR using a predetermined primer on a predetermined region on the chromosome of a human individual suffering from DLB CL to obtain a PCR product. It is also possible to detect by the presence or absence and its identification.
  • Primers can be designed based on sequences such as the pi 6 gene.
  • the base length of the primer is preferably 15 to 40 bases, desirably 15 to 30 bases. However, when LA (longaccurate) PCR is performed, it is preferable to use at least 30 bases. It is preferable to select a base sequence so that the sense strand and the antisense strand do not anneal with each other and can avoid the formation of a hairpin-like structure.
  • the detection of the amplified region and the defective region on the chromosome can also be based on the expression level of the genes contained in these chromosomal regions. That is, it is also possible to determine the disease type based on whether the expression level of the gene in each chromosome region is significantly higher or lower than that of a healthy subject.
  • the expression level is significantly higher than 10%, preferably 30% or more, more preferably 50% or more, and even more preferably 70% compared to the expression level of the same gene in healthy individuals. Above, more preferably 100% or more.
  • the expression level is significantly less than 60%, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less compared to the expression level of the same gene in healthy individuals.
  • Each target gene can be easily obtained by a known method.
  • a cDNA library can be formed and each cDNA can be isolated using a DNA probe prepared based on a known nucleotide sequence.
  • a primer based on the cDNA base sequence may be used to obtain the required amount of cDNA by RT-PCR using mRNA as a saddle.
  • the analysis based on the expression level of the gene can be performed by a known method on a sample collected from a human. For example, in situ hybridization, Northern blotting, dot blot, RT-PCR, real-time PCR, DNA array methods, etc. can be used.
  • an antibody specific to such a protein can be used.
  • a polyclonal antibody or a monoclonal antibody can be used. It may be an antibody molecule or a part thereof. In the case of a polyclonal antibody, for example, such an antibody can be obtained with serum after immunizing an animal using a protein or a partial fragment thereof as an immunogen.
  • eukaryotic cell expression vector can be prepared by introducing the above eukaryotic cell expression vector into an animal muscle or skin by injection or gene gun, and then collecting serum.
  • animals include mice, rats, rabbits, goats, and birds.
  • Monoclonal antibodies are known monoclonal antibody production methods ("monoclonal antibodies”, Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; "Monoclonal Antibody James W. Coding, third edition, Academic Press, 1996) [This can be made.
  • the antibody may be appropriately labeled with a labeling substance!
  • a labeling substance an enzyme, a radioisotope or a fluorescent dye can be used.
  • the enzyme include, but are not limited to, enzymes used in normal EIA, such as peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose 6-phosphate dehydrogenase, Malate dehydrogenase or the like can also be used.
  • an enzyme inhibitor, a coenzyme, etc. can also be used. Binding of these enzymes and antibodies can be performed by a known method using a cross-linking agent such as maleimide compound.
  • the substrate a known substance can be used depending on the type of enzyme used.
  • the fluorescent dye those used in the usual fluorescent antibody method such as fluorescens isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) can be used.
  • fluorescent antibody method such as fluorescens isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) can be used.
  • FITC fluorescens isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • immunostaining such as tissue or cell staining, competitive or non-competitive radioimmunoassay (RIA), fluorescent immunoassay (FIA ), Luminescent immunoassay (LIA), enzyme immunoassay (EIA, ELISA), and other detection methods can be used.
  • the antigen-antibody reaction in such a measurement method may be performed in a liquid
  • the antigen-antibody reaction product is preferably separated.
  • a solid support such as chromatography or beads or plates may be used.
  • Western blotting may be used.
  • ELISA method etc. can be used.
  • an array in which antibodies are immobilized on a solid support such as a substrate can be used.
  • a marker for diagnosis of a DLBCL disease type comprising a protein or a part thereof or an antibody against them.
  • antibodies are preferred as detection reagents for detecting such proteins.
  • a kit for diagnosing a DLBCL disease type containing such an antibody is provided.
  • the diagnostic kit of the present invention may contain an antibody or a labeled antibody in the liquid phase, or may be one in which the antibody or labeled antibody is bound to a solid phase carrier. It may also contain a fixed antigen or a part thereof. When the antibody is labeled with an enzyme, the diagnostic kit may contain the substrate. Furthermore, in the case of a substance containing a solid phase carrier, a washing solution for washing away non-bound molecules from the solid phase may be contained.
  • elements that can generally be included in diagnostic kits containing antibodies can be included.
  • the nucleic acid probe of the present invention includes a nucleic acid probe capable of detecting a chromosomal amplified region and a defective region, which serve as an index for diagnosing DLBCL disease type. According to such a nucleic acid probe, amplification or deletion of these regions can be easily detected using a hybridization. For example, such a nucleic acid probe should be able to detect a region characteristic of a disease type on the genome-wide chromosome imbalance graph shown in Table 3 and FIG. In other words, BAC / PAC clones containing these regions or DNA obtained from these can be used as probes.
  • the amplification region on the human chromosome and the ABC type DLBCL described above and And a defective region force A probe capable of detecting one or more selected regions, more specifically, a BACZPAC clone containing these regions or a genomic DNA obtained from these forces.
  • a probe suitable for determining ABC type is similar to that already described in the diagnosis method. Which region contains DNA to be used as a probe can be determined, for example, by using an ABC type DLBCL-affected human against an array in which nucleic acid probes derived from human chromosomes in a predetermined range (preferably genome-wide range) are immobilized.
  • a predetermined upper and lower threshold value is obtained.
  • a nucleic acid probe selected based on the magnitude of the log ratio value relative to the threshold value
  • the threshold of the log ratio of fluorescence intensity can also be included.
  • the threshold of the log ratio of fluorescence intensity can also be included.
  • level amplification high level amplification, low level defects and high level defects.
  • the amplification region and deletion region force on the human chromosome in the already described GCB type DLBCL are probes capable of detecting one or more selected regions, and more specifically, these probes. BACZPAC clones containing these regions or genomic DNA obtained from these. Suitable probes for determining the GCB type are similar to those already described in the diagnostic method.
  • a test sample collected from a human individual suffering from DLBCL of the above-mentioned GCB type is supplied to an array in which nucleic acid probes derived from human chromosomes in a predetermined range (preferably genome-wide range) are immobilized. Based on the log ratio value of the fluorescence intensity obtained at the fixed site of the nucleic acid probe by carrying out dialysis
  • Selected nucleic acid probes can also be included.
  • the amplification region and the lacking region force useful for determining CD5 + and CD5 + CD10- described above are nucleic acid probes capable of detecting one or more selected regions.
  • the nucleic acid probe of the present invention is preferably a nucleic acid probe set that can determine two types of DLBCL, ABC type and GCB type.
  • ABC type determination for example, One or more selected from 3, 8q21-q26, llq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3, X, 2pll, 6ql2-27, 8p22-p23, 9p21, 17p
  • a nucleic acid probe capable of detecting at least a part of the region can be used.
  • a nucleic acid probe capable of detecting at least a part of one or two or more regions selected from can be used.
  • the nucleic acid probe of the present invention also includes an expression analysis probe.
  • a probe need not be completely complementary to a gene as long as it is specifically hybridized to a nucleic acid sample derived from a given gene.
  • Such slightly mutated polynucleotides are ⁇
  • the probe hybridizes with the nucleic acid sample derived from the gene under stringent conditions.
  • the stringent condition is a condition that enables selective and detectable specific binding between the probe and the nucleic acid sample.
  • Stringent conditions are defined by salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, stringency is increased by decreasing the salt concentration, increasing the organic solvent concentration, or increasing the hybridization temperature.
  • stringent salt concentrations are typically about 750 mM or less NaCl and about 75 mM or less trisodium citrate, more preferably about 500 mM or less NaCl and about 50 mM or less trisodium citrate, most preferably about 250 mM or less NaCl and citrate. Trisodium is about 25 mM or less.
  • the stringent organic solvent concentration is about 35% or more of formamide, most preferably about 50% or more.
  • Stringent temperature conditions are about 30 ° C or higher, more preferably about 37 ° C or higher, and most preferably about 42 ° C or higher.
  • Other conditions include hybridization time, washing time.
  • Various stringency can be set by combining these conditions, such as the concentration of detergent (eg, SDS) and the presence or absence of carrier DNA.
  • the hybridization conditions described above are merely examples, and those skilled in the art will consider the conditions such as the nucleotide sequence, concentration and length of the probe, reaction time, reaction temperature, reagent concentration, etc. Appropriate hybridization conditions can be set.
  • the probe may be appropriately labeled as necessary.
  • labeling it is preferable to use the force non-RI method which can be performed by radioisotope (RI) method or non-RI method.
  • RI radioisotope
  • non-RI method include a fluorescent labeling method, a piotine labeling method, a chemiluminescence method, and the like.
  • an immobilization medium in which various nucleic acid probes are immobilized on a solid phase carrier is also provided.
  • the solid phase carrier typically includes a flat substrate such as a slide glass and a granular material such as beads.
  • the immobilized form of the probe is not particularly limited, and includes immobilized forms by various bonds of covalent bond and non-covalent bond such as z or electrostatic or hydrophobic interaction.
  • the solid phase carrier on which such a nucleic acid probe is immobilized is typically a DNA array called a CGH array.
  • the nucleic acid probe applied to such a fixed body includes all embodiments of the nucleic acid probe used in the diagnostic method of the present invention.
  • a nucleic acid probe having a complementary base sequence can be preferably used as a marker for diagnosis of disease type.
  • primers (sets) that can amplify the region and the corresponding gene can be used as markers for diagnosis of disease type.
  • an immobilized body (array or the like) in which such a nucleic acid probe is immobilized on a solid phase carrier can be preferably used for the prognosis of DLBCL.
  • a marker for diagnosing a disease type is a protein encoded by a corresponding gene of an amplified region or a defective region on a human chromosome, or a part thereof, or an antibody against them, and a fixed enzyme in which the antibody is immobilized on a solid phase carrier. It may be a body.
  • a disease type diagnosis pro A kit for diagnosing a disease type including at least one of a probe, a primer (set), an antibody, and an array is also provided.
  • the prognosis diagnosis method of the present invention comprises a detection step of detecting a deletion or mutation of human chromosome 9p21 in a test sample collected from a human. This is because if 9p21 is deficient, D LBCL has a high grade of malignancy and a poor prognosis.
  • a probe such as a BACZPAC clone corresponding to this chromosomal region or genomic DNA obtained from this clone can be used.
  • a DNA array such as array CGH can be used. It can also be detected by site-specific amplification by PCR
  • the deletion or mutation of the pi 6 INK4a gene which is the 9p21 gene, can be directly detected by a noble hybridization method, or PCR, RT-PCR, Northern blotting, ReaKTime PCR It can also be detected by expression analysis using a method or the like.
  • expression analysis when the expression level of the gene in the test sample is significantly smaller than the expression level of healthy subjects, it can be diagnosed that the prognosis is poor in DLBCL.
  • the expression level is significantly less than 60% or less, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less compared to the expression level of the same gene in healthy individuals.
  • the presence / absence, expression level, and mutation of the protein encoded by the pl6 INK4a gene may be detected.
  • an antibody that specifically binds to such a protein can be used.
  • the same mode as described above can be applied.
  • a nucleic acid probe having at least a part of this region or a nucleotide sequence complementary thereto can be detected so that 9p21 of human chromosome or a part thereof can be detected. It can be preferably used as a marker.
  • the probe may have a pl6 INK 4a gene, a part thereof, or a base sequence complementary thereto.
  • primers (sets) that can amplify the region or responsible gene can also be used as prognostic markers.
  • an immobilized carrier in which such a nucleic acid probe is immobilized on a solid phase carrier can be preferably used for the prognosis of DLBCL.
  • the prognostic marker may be a protein encoded by the pl6 INK4a gene, a part thereof, an antibody thereto, or an immobilized antibody in which the antibody is immobilized on a solid phase carrier.
  • a DBCL prognosis kit including at least one of such a DBCL prognosis probe, a primer (set), an antibody, an array and the like is also provided.
  • Lymph node samples and clinical data were obtained from 99 patients (CD5 + 36 cases, CD-CD10 + 19 cases, CD5-CD10-44 cases) according to the protocol approved by the institutional review board. Three CD5 + cases had CD10 +. Notably, a total of 36 cases of CD5 + DLBCL, a higher proportion than in the general population, were collected for assessment of the genetic status of the disease. The 99 cases of DL BCL were analyzed for genomic imbalance using the array CGH method. Of these, 46 cases (CD 5+ 22 cases, CD5—CD10 + 7 cases, CD5—CD10—17 cases) were followed by gene expression profiling. In addition, these patients had no history of malignant lymphoma.
  • All DLBCL patients will receive therapeutic doses such as cyclophosphamide, adriamycin, vintalistin, and predonin (CHOP) after diagnosis, and all force DNA and RNA samples will be administered before treatment at diagnosis. From the tumor. Table 1 shows data on age, stage, performance status, lactate dehydrogenase (LDH), number of extranodal lesions, and International Prognostic Index (IPI) at diagnosis for all patients.
  • therapeutic doses such as cyclophosphamide, adriamycin, vintalistin, and predonin (CHOP) after diagnosis, and all force DNA and RNA samples will be administered before treatment at diagnosis. From the tumor. Table 1 shows data on age, stage, performance status, lactate dehydrogenase (LDH), number of extranodal lesions, and International Prognostic Index (IPI) at diagnosis for all patients.
  • LDH lactate dehydrogenase
  • IPI International Prognostic Index
  • Cy5- or Cy3-labeled complementary RNA was synthesized from total RNA using the Low RNA Input Amplification Kit (Agilent Technologies, Palo Alto, Calif.).
  • the probe was a mix of experimental Cy5-labeled cRNA and control Cy3-labeled cRNA.
  • the latter is a form of lymph node hyperplasia 1
  • the pool power of the total RNA extracted as well as 0 sample power was prepared.
  • the glass slide microarray used was an Agilent oligonucleotide array (Agilent Technologies) spotted with a total of 21619 genes custom-made for the Cancer Research Institute of the Foundation for Cancer Research.
  • the probe was hybridized on a glass slide using the manufacturer's protocol using In Situ Hybridization Kit Plus (Agilent Technologies). Fluorescence images of hybridized microarrays were acquired with an Agilent scanner G2565AA (Agilent Technologies), analyzed using Feature Extraction software (Agilent Technologies), and the fluorescence ratio of experimental Cy5-labeled samples to Cy3-labeled controls. was calculated. All the flags are raised! /,, (Non-flagged) The fluorescence ratio was logarithmically converted (bottom 2), and the center value of each gene calculated by the cluster analysis was subtracted.
  • DLBCL gene expression profile data obtained from Lymp hochip microarrays: http: ⁇ l It was obtained from the supplementary information contained in reference material 8 of the lmpp.nih.gov/DLBCL site. We have confirmed that the 274 DLBCLs in the Lymphochip microarray dataset can be divided into ABC and GCB and type 3. The distribution of the above 67 genes was almost the same as the 100 genes reported by Rosenwald et af.
  • Array CGH analysis was performed on DLBCL cases using a previously reported method using ACC array slide version 4.0 glass slides.
  • This array consists of 2304 BAC (bacterial artificial chromosomes) and PAC (P-1 derived artificial chromosomes) clones (BAC / PAC clones) that cover the entire human genome with a resolution of about 1.3 Mb (megabase). It is. BAC clones were obtained for RP11 and RP13 libraries, and PAC clones were also obtained for RP1, RP3, RP4, and RP5 libraries.
  • PCR oligonucleotide primed
  • 10 ng of BAC / PAC DNA as a template and degenerate oligonucleotides (5,-CCGACTCGAGNNNNNNATGTGG-3, N is one of A, T, C and G) as primers PCR
  • Amplification was obtained from TaKaRa PCR thermal Cycler MP (Takara, Tokyo, Japan) and ExTaq polymerase (TaKaRa).
  • the PCR product is concentrated by ethanol precipitation, dissolved in distilled water, followed by addition of an equal volume of DNA spotting solution DSP0050 (MATSUNAMI, Osaka, Japan) (; ⁇ 1 g / 1), inkjet technology (NGK, (Nagoya, Japan) was spotted in duplicate on a CodeLink TM active slide (Amersham Biosciences, Piscataway, NJ) by a robot.
  • DSP0050 MATSUNAMI, Osaka, Japan
  • NNK inkjet technology
  • DNA preparation, labeling, array generation and hybridization were performed according to previous reports (Ota A, I'agawa H, Karnan 3 ⁇ 4, et al. Identincation and characterization or a nove 1 gene, C13orf25 , as a target for 13q31— q32 amplification in malignant lymphoma, Cancer Res. 2004; 64: 3087-3095, Tagawa H, Tsuzuki S, Suzuki R, et al. Genome— wide array-based comparative genomic hybridization of diffuse large B— cell lymphoma: Comparison between CD5— Positive and CD5— negative cases. Cancer Res.
  • test DNA and control DNA (1 ⁇ g each) were digested with Dpnll, and Bio prime DNA Laoeling system (Invitrogen Life (Technology, Inc, Tokyo, Japan) [From this, Cy3-dUTP and Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ) were used for labeling.
  • the array was subjected to simultaneous normalization of healthy men 10 times with respect to healthy men and revealed normal fluctuations in the log ratio. Fluorescence intensity is not 10% of the average of all clones
  • the definition of the region of amplification or deletion is: (a) 3 consecutive clones exhibit amplification or deletion, or (b) one clone repeats high copy number amplification (log ratio> + 1.0) or homology Joining
  • Loss of body 11 was to show (log ratio ⁇ 1.0) '12. High level gain Z amplification area, and
  • the genomic change was amplified by copy number amplification with log ratio + 0.2 or more, and log ratio
  • the data set was constructed with 2 2 defined as a copy number loss of -0.2 or less.
  • Amplification log ratio
  • ⁇ +0.2 is ⁇ 1 '' for clones that show no amplification (+0.2 compared to log)
  • the Mann-Whit-1 U test was used to detect significant differences in pl6 INK4a expression levels between the ABC and GCB groups.
  • Statistical analysis is all STATA ver.8 statistical analysis package (StataCorp, College
  • DLBCL types can be characterized and classified by gene expression profiling and cell surface phenotyping.
  • Clinically ABC DLBC L behaves more malignantly than GCB DLBCL.
  • CD5 + DLBCL cases have a short survival time.
  • CD10 + DLBCL is relatively slow ( Figure 1).
  • Figure 2 To determine whether D LBCL with CD5 and / or CD10 markers is associated with ABC and GCB subgroups, a total of 46 DLB CLs (22 CD5 +, 7 CD5—CD10 +, 17 CD5—CD10—17) Gene expression profiling was performed. The results show that these 46 cases are clearly divided into either ABC or GCB groups ( Figure 2), and most importantly, the CD5 + and CD5—CD10 + phenotypes are ABC and GCB, respectively.
  • CD5 CD10 "9 8 0.5335
  • a P value is based on Fisher's exact test.
  • CD5 + DLBCL cases include 3 cases of CD10 +.
  • DLBCL of ABC and GCB are molecularly different because it is subgroup has been demonstrated previously 6 - 8, we first compared the genomic profiles of these subgroups.
  • Figure 3 shows two typical genomic profiles of one CD5—CD10— case with ABC signature and one case with GCB signature.
  • ABC group is more frequently amplified 3p23-q28, 18qll.2-q23, 9ql3.41-ql3.43, and 6q22.
  • the GCB group has the genomic features of higher frequency lq2 1.1-q23.3, lq31.1-q42.13, 2pl5-pl6.1, 7q22.1-q36.2 and 12ql3.1-ql4 (Fischer's exact method, P ⁇ 0.05).
  • the ABC and GCB D LBCL genomic imbalance ideograms are shown in Figure 4A-B and the ABC and GCB DLBCL groups of genomic imbalances are shown in Figure 5A.
  • CD5 expression did not affect the genomic imbalance observed in the ABC and GCB groups.
  • the genomic imbalance detected in the ABC group in this study reflects the prevalence of cases classified by CD5. That is, 67% (19/28) of the ABC group were CD5 + type.
  • the frequency and region of genomic imbalance between CD5 + and CD5 ⁇ in the ABC group was similar. There was no significant difference in the area or frequency of genomic imbalance between CD5 + and CD5- in the ABC group (data not shown).
  • CD5—CD10—DLBCL genomic imbalance was very similar to that of the “ABC plus GCB” group, as shown in FIG. There was no significant difference in the region or frequency of genomic imbalance between the CD5-CD10 + and “ABC plus GCB” groups (data not shown). These findings correlate well with the results of gene expression profiling that showed that CD5-CD10-DLCL was evenly distributed in ABC and GCB groups.
  • the CD5—CD10 + case also showed no signs suggesting a homozygous defect at 9p21.3.
  • Thirteen of the 37 patients with 9p21 deletion showed a defect in the restriction site of the genome surrounding one RP11-14912 containing the BAC, pl6 INK4a tumor suppressor gene.
  • DLBCL and GCB DLBCL transformed from FL can be used to program genomic abnormalities during lymphoma development. There may be a common step.
  • CD5 + and CD10 + DLBCL constitute clinically relevant subtypes.
  • CD5 + DLBCL is characterized by ABC expression and genomic patterns.
  • Katzenberger et al. (2003) conducted a cytogenetic and LOH study on a new CD5 + DLBCL, which showed a high frequency of loss of the D1 3S25 locus and pl6 INK4a tumor suppressor. Therefore, we suppose that CD5 + DLB CL may be derived from the same progenitor cells as B-chronic lymphocytic leukemia (CLL) (7) (Katzenberger er, Lohr A, 3 ⁇ 4cnwarz 3 ⁇ 4, et al.
  • CLL B-chronic lymphocytic leukemia
  • CD5 + DLBCL differs from CLL and mantle cell lymphoma (MC), which also express CD5, in both origin and route. It is thought that. Both CLL and MCL are characterized by loss of 1 ⁇ 22, 6q, 9p21, l lq22- q23, 13ql4-q21 (recognized in 30-50% of cases) (24. Bentz M, Plesch A, Bullinger L, et al.
  • Genomic DNA chip hybridization in t (ll; 14) — positive mantle cell lymphomas sho ws a high frequency of aberrations and allows a refined characterization of consensus regions.
  • Oncogene. 2005; 24: 1348- 1358) less than 10% of CD5 + DLBCL cases we examined showed Ip22, llq22-q23, and 13ql4-q21 deficiencies. Only the lack of pl6 INK4a appears to be a common feature.
  • CD10 + DLBCL is characterized by GCB expression and genomic patterns.
  • Huang et al. (2002) showed that it is characterized by CD10 + DLBCL force S GCB signature using gene expression profiling, as is evident from our results (Huang JZ , Sanger WG, Greiner TC, et al. The t (14; 18) defines a unique subset of diffiise large B- cell lymphoma with a germinal center B-cell gene expression profile.Blood. 2002; 99: 2285-2290, Iqbal
  • CD5—CD10—DLBCL subtypes ie, CD5 + and CD—CD10 + subtypes
  • CD5—CD10—DLBCL is equal to either ABC or GCB group. It is closely related to the results of gene expression profiling that has been observed, suggesting that CD5—CD 10 ”DLBCL is a genetically heterogeneous disease.
  • 9p21 (pl6 INK4a ) deficiency may be the most aggressive disease feature. Of particular interest in our case is that the cases of ABC and CD5 + DLBCL lacking 9p21 had a worse outcome than those not lacking. 9p21 deficiency may therefore be a factor that may represent the most aggressive form of DLBCL. 9p21.3 (pl6 INK4a locus) deficiency is more common in aggressive lymphomas and acute lymphoblastic leukemia (Serrano M, Hannon GJ, B each D. A new regulatory motif in cell-cycle control causing specific inhibition of cy clin D / CDK4. Nature. 1993; 366: 704—707, Nobori T, Miura K, Wu DJ, et al.
  • Pl6 (INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas .Blood. 1998; 91: 2977-2984)
  • 0 CD5 + DLBCL is also much aggressive Pl6 INK4a inactivity and 9p21 deficiency are likely to be characteristic of CD5 + DLB CL, but in fact, are very frequent in CD5 + DLBCL.
  • 9 p21 deficiency has been reported by us and other groups (12. Tagawa H, Tsuzuk l S, buz uki R, et al.
  • the present invention is useful for diagnosis of DLBCL disease type and prognosis.

Abstract

An object of the invention is to easily diagnose a subgroup of DLBCL. For this object, the invention provides a method for diagnosing a disease type of diffuse large B-cell lymphoma comprising a detection step of detecting an amplified region and/or a deleted region of a human chromosome in a test specimen containing a chromosome collected from a human.

Description

明 細 書  Specification
びまん性大細胞型 B細胞リンパ腫の病型の診断方法及び予後診断の方 法  Methods for diagnosing and prognosing the type of diffuse large cell B-cell lymphoma
技術分野  Technical field
[0001] 本発明は、びまん性大細胞型 B細胞リンパ腫 (DLBCL)の病型の診断及び DLBCL の予後診断に関する。 背景技術  [0001] The present invention relates to diagnosis of a disease type of diffuse large B-cell lymphoma (DLBCL) and prognosis of DLBCL. Background art
[0002] びまん性大細胞型 B細胞リンパ腫 (DLBCL)は最も一般的な非ホジキンリンパ腫で あり、これには病態生理学的に異なるグループが含まれていることが知られている（ H arris NL, Jaffe Eb, btein H, et al. A revised European- American classification of lym phoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994; 84: 1361-1392, Offit K, Le Coco F, Louie DC, et al. Rearrangement of BCL6 gene as a prognostic marker in diffuse large cell lymphoma. N Engl J Med 1994; 331 ： 74—80, Kramer MHH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, B CL6, and MYC rearrangements in diffuse large B— cell lymphoma. Blood. 1998; 92: 3 152—3162, Gatter KC and Warnke RA. Diffuse large B— cell lymphoma, In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World health classification of tumors. Pathol ogy & Genetics of tumors of haematopoietic and lymphoid tissues. Washington: IAR C press, Lyon; 2001. 171-174)。また、 DLBCLは、患者が著しく異なる臨床経過を迪 ることから臨床的異質性があることも知られている (Fisher RI, Gaynor ER, Dahlberg S, et al. Comparison of a standard regimen (CHOP)with three intensive chemotherapy regimens for advanced non— Hodgkin s lymphoma. N Engl J Med. 1993; 328: 1002—10 06.)。このため、異質性の DLBCLにおけるサブグループ同定の重要性に関心が集ま つている。  [0002] Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma and is known to include pathophysiologically distinct groups (Harris NL, Jaffe Eb, btein H, et al. A revised European- American classification of lym phoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392, Offit K, Le Coco F, Louie DC, et N Engl J Med 1994; 331: 74—80, Kramer MHH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, B CL6, and MYC al. Rearrangement of BCL6 gene as a prognostic marker in diffuse large cell lymphoma. rearrangements in diffuse large B— cell lymphoma. Blood. 1998; 92: 3 152—3162, Gatter KC and Warnke RA. Diffuse large B— cell lymphoma, In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World health classification of tumors.Pathol ogy & Genetics of tumors of haematopoietic and lymphoid tissues.Washington: IAR C press, Lyon; 2001. 171 -174). DLBCL is also known to have clinical heterogeneity because patients have significantly different clinical courses (Fisher RI, Gaynor ER, Dahlberg S, et al. Comparison of a standard regimen (CHOP) with Three intensive chemotherapy regimens for advanced non—Hodgkin s lymphoma. N Engl J Med. 1993; 328: 1002-10 06.). For this reason, there is interest in the importance of subgroup identification in heterogeneous DLBCL.
[0003] Alizadeh et al (2000)が実施した遺伝子発現プロフアイリングから、 B細胞分化の異 なるステージを示す遺伝子発現パターンを伴う 2つの分子的に区別される DLBCLの 型、すなわち活性化 B細胞様 (ABC)型、及び胚中心 B細胞様 (GCB)型が明らかにさ れ 7こ (Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B— cell 1 ymphoma identified by gene expression profiling. Nature. 2000; 403: 503—511, Wrig ht G, Tan B, Rosenwald A, et al. A gene expression-based method to diagnose clini cally distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad bci USA. 2 003; 100: 9991-9996)。 ABCグループは活性化 B細胞及びプラズマ細胞に特有な遺 伝子を発現し、一方、 GCBグループは正常な胚中心 B細胞の遺伝子発現プログラム を維持している (Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffiise lar ge B— cell lymphoma identified by gene expression profiling. Nature. 2000; 403: 503— 511, Wright G, Tan B, Rosenwald A, et al. A gene expression-based method to diag nose clinically distinct subgroups of diffiise large B cell lymphoma. Proc Natl Acad S ci USA. 2003; 100: 9991-9996, Rosenwald A, Wright G, Chan WC, et al. The use o f molecular profiling to predict survival after chemotherapy for diffuse large— B— cell ly mphoma. N Engl J Med. 2002; 346: 1937-1947)。この著者らは ABCグループの全生 存は GCBグループと比較して有意に劣って 、たことも報告して 、る。 [0003] From the gene expression profiling performed by Alizadeh et al (2000), two molecularly distinct DLBCL with gene expression patterns showing different stages of B cell differentiation 7 types (Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B—), namely activated B cell-like (ABC) type and germinal center B cell-like (GCB) type. cell 1 ymphoma identified by gene expression profiling.Nature. 2000; 403: 503—511, Wrig ht G, Tan B, Rosenwald A, et al. A gene expression-based method to diagnose clini cally distinct subgroups of diffuse large B cell lymphoma Proc Natl Acad bci USA. 2 003; 100: 9991-9996). The ABC group expresses genes specific to activated B cells and plasma cells, while the GCB group maintains a normal germinal center B cell gene expression program (Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffiise lar ge B— cell lymphoma identified by gene expression profiling. Nature. 2000; 403: 503— 511, Wright G, Tan B, Rosenwald A, et al. A gene expression-based method to diag nose Clinically distinct subgroups of diffiise large B cell lymphoma.Proc Natl Acad S ci USA. 2003; 100: 9991-9996, Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large — B— cell ly mphoma. N Engl J Med. 2002; 346: 1937-1947). The authors also report that the overall survival of the ABC group was significantly inferior to that of the GCB group.
さらに、本発明者らは、表現型の異なる 3つのサブグループ、 CD5+、 CD5— CD10+、 及び CD5— CD10—の DLBCLを同定したことを報告した（Harada S, Suzuki R, Uehira K, et al. Molecular and immunological dissection of diffiise large B cell lymphoma: CD5 + and CD5 with CD10+ groups may constitute clinically relevant subtypes. Leukemi a. 1999; 13: 1441-1447)。 CD5+グループは全 DLBCL症例の約 10%を占めることが 明らかにされている力 これは CD5+C10—CD19+CD20— CD21—CD23—サイクリン D1—表 現型を有し、 CD5" DLBCLよりも予後が不良で、節外病変が多く生じ、パフオーマン スステータスが不良で、また乳酸脱水素酵素濃度が高 ヽと ヽぅ特徴を持つ (Yamaguc hi M, beto M, Okamoto M, et al. De novo CD5+ diffuse large B— cell lympnoma: a cli nicopathologic study of 109 patients. Blood. 2002; 99: 815—821.)。 CD5— CD10+グノレ ープはその他のグループより BCL2タンパク質の発現頻度が低ぐ通常は BCL2発現 を欠く正常胚中心細胞との明確な関連性が示唆されている。最後に、 CD5— CD10—グ ループは最も多く認められ、 BCL6遺伝子再配列の発現率が他の 2グループよりも高 いが、その差は有意ではない（Harada S et al ) 0 発明の開示 In addition, the inventors have reported that DLBCL of three subgroups with different phenotypes, CD5 +, CD5-CD10 +, and CD5-CD10- have been identified (Harada S, Suzuki R, Uehira K, et al. Molecular and immunological dissection of large B cell lymphoma: CD5 + and CD5 with CD10 + groups may constitute clinically relevant subtypes. Leukemi a. 1999; 13: 1441-1447). The CD5 + group has been shown to account for approximately 10% of all DLBCL cases. This has a CD5 + C10—CD19 + CD20—CD21—CD23—cyclin D1—phenotype and a prognosis over CD5 ”DLBCL. , Poor extra-nodal lesions, poor performance status, and high lactate dehydrogenase concentration (Yamaguc hi M, beto M, Okamoto M, et al. De novo CD5 + Blood; 2002; 99: 815—821.) CD5—CD10 + gnole has a lower expression frequency of BCL2 protein than other groups, and usually expresses BCL2. A clear association with normal germinal center cells lacking the CD5-CD10-group was observed most frequently, and the expression rate of BCL6 gene rearrangement was higher than the other two groups. Bur, the disclosure of the difference is not significant (Harada S et al) 0 invention
[0005] DLBCLの各サブグループは分子的または表現型的に異なるにもかかわらず、遺伝 的特徴及びそれらの関連性については十分な検討が行われていない。そこで、本発 明は、死亡率や予後に大きく影響する DLBCLのサブグループを容易に診断する技 術を提供することを一つの目的とする。また、本発明は、 DLBCLの予後の診断方法 を提供することを他の一つの目的とする。  [0005] Although each DLBCL subgroup is molecularly or phenotypically different, the genetic features and their associations have not been fully studied. Therefore, an object of the present invention is to provide a technique for easily diagnosing a subgroup of DLBCL, which greatly affects mortality and prognosis. Another object of the present invention is to provide a method for diagnosing prognosis of DLBCL.
[0006] 本発明者らは、 DLBCLの異なるサブグループのゲノム不均衡の特徴の同定にァレ ィを用いる比較ゲノムハイブリダィゼーシヨン（マイクロアレイ CGH法）を使用した（Ota A, fagawa H, Karnan S, et ai. Iaentincation and cnaractenzation of a novel gene, し 13orl25, as a target for 13q31— q32 amplification in malignant lymphoma. Cancer Res . 2004; 64: 3087—3095、 Tagawa H, Tsuzuki S, Suzuki R, et al. Genome-wide array- based comparative genomic hybridization of diffuse large B— cell lymphoma: comparis on between CD5— Positive and CD5— negative cases. Cancer Res. 2004; 64: 5948—59 55)。また、遺伝子発現プロフアイリングも使用し、 ABCと GCB、及び CD5+と CD5— CD1 0+サブグループ間の関連性を解明した。さらに、本発明者らは、 DLBCLが特定の染 色体領域の欠損との関連性を見出した。これらの知見によれば、以下の手段が提供 される。  [0006] The present inventors used a comparative genomic hybridization (microarray CGH method) that uses an array to identify genomic imbalance features of different subgroups of DLBCL (Ota A, fagawa H, Karnan S, et ai. Iaentincation and cnaractenzation of a novel gene, shi 13orl25, as a target for 13q31— q32 amplification in malignant lymphoma. Cancer Res. 2004; 64: 3087-3095, Tagawa H, Tsuzuki S, Suzuki R, et al. Genome-wide array-based comparative genomic hybridization of diffuse large B— cell lymphoma: comparis on between CD5— Positive and CD5— negative cases. Cancer Res. 2004; 64: 5948-59 55). Gene expression profiling was also used to elucidate the association between ABC and GCB, and CD5 + and CD5—CD10 + subgroups. Furthermore, the present inventors have found that DLBCL is associated with a defect in a specific chromosomal region. These findings provide the following means.
[0007] 本発明の一つの形態によれば、ヒトから採取した染色体を含む被験試料中のヒト染 色体の増幅領域及び Z又は欠損領域を検出する検出工程を備える、びまん性大細 胞型 B細胞リンパ腫の病型の診断方法が提供される。この診断方法においては、前 記検出工程で検出された前記増幅領域力 ヒト染色体の 3、 8q21-q26、 I lq21-q25、 1 6pl l-pl3、 16q22-q24、 18、 19ql3及び Xから選択される 1種又は 2種以上にあるかど うか及び Z又は前記欠損領域がヒト染色体の 2pl l、 6ql2-27、 8p22-p23、 9p21及び 1 7pから選択される 1種又は 2種以上の領域にあるかどうかを指標としてびまん性大細 胞型 B細胞リンパ腫の病型が ABC型であると肯定的に判定する判定工程を備えるこ とができる。さらに、前記判定工程は、前記増幅領域がヒト染色体の 3p23-q28、 18ql l .2-q23及び 9ql3.41-ql3.43力 選択される 1種又は 2種以上にあるかどうか及び前記 欠損領域がヒト染色体の 6q22.31-q24.1及び 9p21.3から選択される 1種又は 2種以上 にあるかどうかを指標とする工程としてもよい。また、前記判定工程は、前記増幅領域 がヒト染色体の 3q23-q28及び Z又は前記欠損領域がヒト染色体の 9p21.3であるかど うかを指標とする工程としてもよい。さら〖こ、前記判定工程は、前記病型が ABC型で あると肯定的に判定されるとき、前記病型が CD5+型であると肯定的に判定する工程 としてちよい。 [0007] According to one aspect of the present invention, a diffuse large cell type comprising a detection step of detecting an amplified region and Z or a defective region of a human chromosome in a test sample containing a chromosome collected from a human. Methods for diagnosing B cell lymphoma pathology are provided. In this diagnosis method, the amplified region force detected in the detection step is selected from 3, 8q21-q26, Ilq21-q25, 16pl22-q24, 18, 19ql3 and X of the human chromosome. Z or the deletion region is in one or more regions selected from 2pl l, 6ql2-27, 8p22-p23, 9p21 and 17p of the human chromosome It is possible to provide a determination step that positively determines that the disease type of the diffuse large B-cell lymphoma is ABC type, using whether or not it is an index. Further, in the determination step, the amplification region is 3p23-q28, 18ql l of human chromosome. .2-q23 and 9ql3.41-ql3.43 force selected if one or more are selected and said defective region is selected from 6q22.31-q24.1 and 9p21.3 of human chromosome 1 It may be a process using as an index whether there are two or more species. The determination step may be a step using as an index whether the amplification region is 3q23-q28 and Z of a human chromosome or the deletion region is 9p21.3 of a human chromosome. Furthermore, the determination step may be a step of positively determining that the disease type is CD5 + type when the disease type is positively determined to be ABC type.
[0008] また、本発明の診断方法においては、前記検出工程で検出された前記増幅領域 力 ヒト染色体の lq22- 32、 2pl4- p24、 5pl2- p 、 5ql5- q31、 6pl2- p25、 7、 8q22- q26、 9q33— q34、 llq、 12、 13q31— q33、 16pll— pl3、 18q21— q23、 19p、 19ql3、 21q及び Xか ら選択される 1種又は 2種以上にあるかどうか及び Z又は前記欠損領域がヒト染色体 の lp36、 2pll、 3pl4、 4pl2- pl3、 4q33- q34、 6ql4- ql6、 8p22- p23、 9p21、 13ql2-q22 、 17pl2及び 18q22-q23から選択される 1種又は 2種以上にあるかどうかを指標として びまん性大細胞型 B細胞リンパ腫の病型が GCB型であると肯定的に判定する判定 工程を備えることとしてもよい。また、前記判定工程は、前記増幅領域がヒト染色体 q2 1.1- q23.3、 lq31.1- q42.13、 2pl5- pl6.1、 7q22.1- q36.2及び 12ql3.1- ql4から選択さ れる 1種又は 2種以上にあるかどうかを指標とする工程としてもよぐ前記判定工程は 、前記増幅領域がヒト染色体の 7q22-q36にあるかどうかを指標とする工程としてもよ い。さらにまた、前記判定工程は、前記病型が GCB型であることが肯定的に判定さ れるとき、前記病型が CD5—DC10+であると肯定的に判定する工程を備えることもで きる。  [0008] Further, in the diagnostic method of the present invention, the amplified region force detected in the detection step is 1q22-32, 2pl4-p24, 5pl2-p, 5ql5-q31, 6pl2-p25, 7, 8q22 of the human chromosome. -q26, 9q33—q34, llq, 12, 13q31—q33, 16pll—pl3, 18q21—q23, 19p, 19ql3, 21q The region is one or more selected from lp36, 2pll, 3pl4, 4pl2-pl3, 4q33-q34, 6ql4-ql6, 8p22-p23, 9p21, 13ql2-q22, 17pl2 and 18q22-q23 of human chromosome A determination step of positively determining that the disease type of diffuse large B-cell lymphoma is GCB type may be provided using whether or not it is an indicator. In the determining step, the amplification region is selected from human chromosomes q2 1.1-q23.3, lq31.1-q42.13, 2pl5-pl6.1, 7q22.1-q36.2 and 12ql3.1-ql4. The determination step, which may be performed using one or two or more types as an index, may be a step using whether or not the amplified region is in 7q22-q36 of a human chromosome. Furthermore, the determination step may include a step of positively determining that the disease type is CD5-DC10 + when the disease type is positively determined to be GCB type.
[0009] また、本発明の診断方法においては、前記検出工程で検出された前記増幅領域 が、ヒト染色体の 3、 6p22- p25、 7p22- q31、 8q24、 llq22- q25、 12、 16pl3- q21、 18、 19 及び X力 選択される 1種又は 2種以上にあるかどうか及び Z又は前記欠損領域がヒ ト染色体の lp36、 2pll、 6ql4- q27、 8p23、 9p21、 15ql3- ql4及び 17pll- pl3から選択 される 1種又は 2種以上にあるかどうかを指標として前記病型が CD5+であることを肯 定的に判定する工程を備えることができる。この場合、前記判定工程は、前記増幅領 域がヒト染色体の 3である力及び Z又は前記欠損領域がヒト染色体の 9p2であるかどう かを指標とする工程としてもょ 、。 [0009] Further, in the diagnostic method of the present invention, the amplified region detected in the detection step is 3, 6p22-p25, 7p22-q31, 8q24, llq22-q25, 12, 16pl3-q21 of the human chromosome, 18, 19 and X force selected from one or more selected and Z or from the lp36, 2pll, 6ql4-q27, 8p23, 9p21, 15ql3-ql4 and 17pll-pl3 of the human chromosome A step of positively determining that the disease type is CD5 + can be provided by using whether the disease type is one or more selected as an index. In this case, the determination step includes determining whether the amplification region is a force of 3 of human chromosome and Z or whether the deletion region is 9p2 of human chromosome. As a process that uses this as an index.
[0010] また、本発明の診断方法においては、前記検出工程で検出された前記増幅領域 が、ヒト染色体の lq、 2pl3- p25、 6p21- p25、 7、 8q22- q24、 9q33- q34、 12、 13q31- q33 、 15q、 16pl3、 19ql3.3-13.4及び Xから選択される 1種又は 2種以上にあるかどうか及 び Z又は前記欠損領域がヒト染色体の 1ρ36、 1ρ22、 2pll、 3pl4、 4p、 6ql3-q27、 9p2 1及び 13ql4-q21から選択される 1種又は 2種以上にあるかどうかを指標として前記病 型が CD5—CD10+であると肯定的に判定する判定工程を備えていてもよい。この場 合、前記判定工程は、前記増幅領域がヒト染色体の 7q22-q36及び 12ql3-ql4から選 択される 1種又は 2種以上にあるかどうか及び Z又は前記欠損領域がヒト染色体の 17 pl3であるかどうかを指標とする工程としてもよい。  [0010] Further, in the diagnostic method of the present invention, the amplification region detected in the detection step is human chromosome lq, 2pl3-p25, 6p21-p25, 7, 8q22-q24, 9q33-q34, 12, 13q31-q33, 15q, 16pl3, 19ql3.3-13.4 and whether it is in one or more selected from X and Z or the deleted region is 1ρ36, 1ρ22, 2pll, 3pl4, 4p, human chromosome A determination step of positively determining that the disease type is CD5-CD10 + using one or more selected from 6ql3-q27, 9p2 1 and 13ql4-q21 as an index may be provided . In this case, the determination step includes determining whether the amplification region is one or more selected from 7q22-q36 and 12ql3-ql4 of human chromosome, and Z or 17 pl3 of the deletion region of human chromosome. It is good also as a process using as an index whether it is.
[0011] 本発明の診断方法においては、前記検出工程は、被験試料中の前記領域につい て、 PCR法、 RT— PCR法及び核酸ハイブリダィゼーシヨンのいずれかを実施するェ 程を含むことができ、さらに、前記ヒト染色体上の領域を含むプローブと前記被験試 料中の核酸とをハイブリダィゼーションする工程を含むことができる。前記検出工程 は、アレイ CGH法を実施する工程とすることもできる。  [0011] In the diagnostic method of the present invention, the detection step includes a step of performing any one of a PCR method, an RT-PCR method and a nucleic acid hybridization on the region in the test sample. And a step of hybridizing a probe containing a region on the human chromosome with a nucleic acid in the test sample. The detection step may be a step of performing an array CGH method.
[0012] 本発明の他の一つの形態によれば、びまん性大細胞型 B細胞リンパ腫の病型診断 用の核酸プローブであって、 ABC型のびまん性大細胞型 B細胞リンパ腫におけるヒト 染色体上の増幅領域及び Z又は欠損領域を検出可能な 1種又は 2種以上の核酸プ ローブが提供される。この核酸プローブは、所定範囲のヒト染色体に由来する核酸プ ローブを固定ィ匕したアレイに対して前記 ABC型のびまん性大細胞型 B細胞リンパ腫 罹患ヒト個体カゝら採取した被験試料を供給し核酸ハイブリダィゼーシヨンを実施して 前記核酸プローブの固定部位において得られる蛍光強度の log比値に基づいて設  [0012] According to another aspect of the present invention, there is provided a nucleic acid probe for diagnosing the disease type of diffuse large B-cell lymphoma, on the human chromosome in ABC-type diffuse large B-cell lymphoma. There are provided one or more nucleic acid probes capable of detecting the amplification region and the Z or deletion region. This nucleic acid probe supplies a test sample collected from a human individual suffering from ABC type diffuse large B cell lymphoma to an array in which nucleic acid probes derived from a predetermined range of human chromosomes are immobilized. Based on the log ratio value of the fluorescence intensity obtained at the fixed site of the nucleic acid probe by performing nucleic acid hybridization.
2  2
定した所定の上下の閾値に対する前記 log比値の大小に基づいて選択される核酸  Nucleic acid selected based on the magnitude of the log ratio with respect to a predetermined predetermined upper and lower threshold
2  2
プローブを含むことができる。  A probe can be included.
[0013] 本発明の他の一つの形態によれば、びまん性大細胞型 B細胞リンパ腫の病型診断 用の核酸プローブであって、 GCB型のびまん性大細胞型 B細胞リンパ腫におけるヒト 染色体上の増幅領域及び Z又は欠損領域を検出可能な 1種又は 2種以上の核酸プ ローブが提供される。この核酸プローブは、所定範囲のヒト染色体に由来する核酸プ ローブを固定ィ匕したアレイに対して前記 GCB型のびまん性大細胞型 B細胞リンパ腫 罹患ヒト個体カゝら採取した被験試料を供給し核酸ハイブリダィゼーシヨンを実施して 前記核酸プローブの固定部位において得られる蛍光強度の log比値に基づいて設 [0013] According to another aspect of the present invention, there is provided a nucleic acid probe for diagnosing a disease type of diffuse large B-cell lymphoma, on a human chromosome in a GCB-type diffuse large B-cell lymphoma. There are provided one or more nucleic acid probes capable of detecting the amplification region and the Z or deletion region. This nucleic acid probe is a nucleic acid probe derived from a predetermined range of human chromosomes. Immobilizing the nucleic acid probe by supplying a test sample collected from a diffuse large B-cell lymphoma affected by GCB to an array with fixed lobes and performing a nucleic acid hybridization Set based on the log ratio value of fluorescence intensity obtained at the site.
2  2
定した所定の上下の閾値に対する前記 log比値の大小に基づいて選択される核酸  Nucleic acid selected based on the magnitude of the log ratio with respect to a predetermined predetermined upper and lower threshold
2  2
プローブを含むことができる。  A probe can be included.
[0014] 本発明の他の一つの形態によれば、びまん性大細胞型 B細胞リンパ腫の病型診断 用アレイであって、上記いずれかに記載の核酸プローブが固定ィ匕されたアレイが提 供される。このアレイには、ヒト染色体の 3、 8q21-q26、 Ilq21-q25、 16pll-pl3、 16q22 - q24、 18、 19ql3、 X、 2pll、 6ql2- 27、 8p22- p23、 9p21、 17pから選択される 1種又は 2 種以上の領域の少なくとも一部を検出可能な核酸プローブが固定化されていてもよ い。また、このアレイには、ヒト染色体の lq22-32、 2pl4-p24、 5pl2-pl5、 5ql5-q31、 6 pl2— p25、 7、 8q22— q26、 9q33— q34、 llq、 12、 13q31— q33、 16pll— pl3、 18q21— q23、 1 9p、 19ql3、 21q、 X、 lp36、 2pll、 3pl4、 4pl2— pl3、 4q33— q34、 6ql4— ql6、 8p22— p23、 9p21、 13ql2-q22、 17pl2及び 18q22-q23から選択される 1種又は 2種以上の領域の少 なくとも一部を検出可能な核酸プローブが固定ィ匕されていてもよい。  [0014] According to another embodiment of the present invention, there is provided an array for diagnosing a disease type of diffuse large B-cell lymphoma, to which the nucleic acid probe described above is immobilized. Provided. This array is selected from human chromosomes 3, 8q21-q26, Ilq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3, X, 2pll, 6ql2-27, 8p22-p23, 9p21, 17p 1 A nucleic acid probe capable of detecting at least a part of a species or two or more regions may be immobilized. This array also includes human chromosomes lq22-32, 2pl4-p24, 5pl2-pl5, 5ql5-q31, 6 pl2—p25, 7, 8q22—q26, 9q33—q34, llq, 12, 13q31—q33, 16pll — Pl3, 18q21—from q23, 1 9p, 19ql3, 21q, X, lp36, 2pll, 3pl4, 4pl2—pl3, 4q33—q34, 6ql4—ql6, 8p22—p23, 9p21, 13ql2-q22, 17pl2, and 18q22-q23 A nucleic acid probe capable of detecting at least a part of one or two or more selected regions may be immobilized.
[0015] 本発明の他の一つの形態によれば、びまん性大細胞型 B細胞リンパ腫の予後の診 断方法であって、ヒトから採取した染色体を含む被験試料について、ヒト染色体の 9p2 1の欠損又は変異を検出する検出工程を備える方法が提供される。この予後診断方 法において、前記検出工程は、前記ヒト染色体上の領域を含むプローブと前記被験 試料とをノヽイブリダィゼーシヨンする工程とすることができる。また、前記検出工程は、 アレイ CGHを用いる工程とすることができる。さらに、前記検出工程は、 pl6INK4a遺 伝子の欠損又は変異を検出する工程とすることができる。前記検出工程は、 pl6INK4a 遺伝子によってコードされるタンパク質の発現の有無、発現量又は変異を検出する 工程としてちよい。 [0015] According to another aspect of the present invention, there is provided a prognostic diagnosis method for diffuse large B-cell lymphoma, wherein a test sample containing a chromosome collected from a human is subjected to 9p21 of human chromosome. A method is provided comprising a detection step of detecting a defect or mutation. In this prognostic method, the detecting step may be a step of performing a hybridization on a probe including a region on the human chromosome and the test sample. The detection step can be a step using array CGH. Furthermore, the detection step can be a step of detecting a deletion or mutation of the pl6 INK4a gene. The detection step may be a step of detecting the presence / absence, expression level or mutation of the protein encoded by the pl6 INK4a gene.
[0016] 本発明の他の一つの形態によれば、びまん性大細胞型 B細胞リンパ腫の予後診断 用アレイであって、ヒト染色体の 9p21を検出するための核酸プローブを固定ィ匕したァ レイが提供される。  [0016] According to another aspect of the present invention, there is provided an array for prognosis of diffuse large B-cell lymphoma, wherein an array of nucleic acid probes for detecting 9p21 of human chromosome is immobilized. Is provided.
[0017] 本発明の他の一つの形態によれば、ヒト染色体 9p21の少なくとも一部を検出するた めのポリヌクレオチドである、びまん性大細胞型 B細胞リンパ腫の予後診断用マーカ 一が提供される。前記ポリヌクレオチドは、 pl6INK4a遺伝子若しくはその一部又はこれ らに相補的な塩基配列を有するポリヌクレオチドとすることができる。 [0017] According to another embodiment of the present invention, at least a part of human chromosome 9p21 is detected. A prognostic marker for diffuse large B-cell lymphoma is provided. The polynucleotide can be a polynucleotide having a pl6 INK4a gene, a part thereof, or a base sequence complementary thereto.
[0018] 本発明の他の一つの形態によれば、 pl6INK4a遺伝子によってコードされるタンパク 質若しくはその一部又はこれらに対する抗体である、びまん性大細胞型 B細胞リンパ 腫の予後診断用マーカーが提供される。 図面の簡単な説明 [0018] According to another aspect of the present invention, there is provided a prognostic marker for diffuse large B-cell lymphoma, which is a protein encoded by the pl6 INK4a gene, a part thereof, or an antibody thereto. Provided. Brief Description of Drawings
[0019] [図 1]異なるサブグループの力プランマイヤー解析である。 (A)は ABC (26症例）と G CB ( 17症例）の力プランマイヤー解析を示す。（B)は CD5+ (33症例）、 CD5—CD10 + (19症例）および CD5—CD10— (44症例）の力プランマイヤー解析を示す。 p値は、 ログランクテストによって得られた。 96症例の全体の生存率が解析され、 CD5+の生 存率が CD5_CdlO+及び CD5—CD10—よりも悪いことが示された（ログランクテスト、 p =0. 0238)。一方、 CD5—CD10+と CD5—CD10—との間には有意な生存率の差力 S なかった（ログランクテスト、 p = 0. 5045)。 46症例について発現プロフアイリングが 実施され、 43症例について生存率が解析され、 ABC群の生存率が GCB群よりも悪い ことを示した（p = 0. 0031)。  [0019] [Fig. 1] Force plan Meyer analysis of different subgroups. (A) shows force plan Meyer analysis of ABC (26 cases) and G CB (17 cases). (B) shows force plan Meyer analysis of CD5 + (33 cases), CD5—CD10 + (19 cases) and CD5—CD10— (44 cases). The p-value was obtained by a log rank test. Overall survival of 96 cases was analyzed and showed that the survival rate of CD5 + was worse than CD5_CdlO + and CD5—CD10— (log rank test, p = 0.0238). On the other hand, there was no significant survival difference S between CD5-CD10 + and CD5-CD10- (log rank test, p = 0.5045). Expression profiling was performed on 46 cases, and survival rates were analyzed on 43 cases, indicating that the survival rate in the ABC group was worse than that in the GCB group (p = 0.0031).
[図 2]DLBCL症例の階層的クラスターである。 46症例（22症例の CD5+、 7症例の CD 5_CD10+、 17症例の CD5—CD10—)力 ローゼンワルドらによって記載あ sれた 100 遺伝子のうちの 67遺伝子の発現に基づいてツリービユウソフトウェアによりクラスター 化された。各検体の相対的な発現程度を下段に示す。検体は、 ABC (左側)及び GC B (右側）の二つのサブグループに区別された。  [Figure 2] Hierarchical cluster of DLBCL cases. 46 cases (22 cases of CD5 +, 7 cases of CD5_CD10 +, 17 cases of CD5—CD10—) force Clustered by Treeview software based on the expression of 67 out of 100 genes described by Rosenwald et al. It was done. The relative expression level of each specimen is shown in the lower part. Samples were divided into two subgroups: ABC (left side) and GC B (right side).
[図 3]DLBCL症例のゲノムプロファイルの典型的な特徴を示す。二つの CD5—CD10 —症例、（A)は、 ABC signatureを示し、（B)は、 GCB signatureを示す。注：遺伝子増 幅： 3p26.3— ql2.3, 3ql3.33— q29、 4q32.1-q35.1, 9p24.3-q22.33, 17pll.2-q21.1, 17q 21.32, 17q23.2-q24.2, and 18q;遺伝子欠損： 9p21.3 (矢印), 13ql4.3-q21.2, 15q21. 3, 17pll.2-pl3.3, and 17q21.33-q220 9p21の欠損は、 pl6INK½を含む BAC RP11- 149 12 (矢印参照)においてのみ生じた。 (B)遺伝子増幅： Iq21.3-q44, 2pl3.2-p25.1, 7, Ilql3.5-q25, 15q24.3-q26.3,16,及び 21q;遺伝子欠損： 1ρ36.22- p36.33, 1ρ13.1- p 31.2,及び13913.1-914.3。 [Figure 3] Shows typical features of the genomic profile of DLBCL cases. Two CD5-CD10 cases, (A) shows ABC signature and (B) shows GCB signature. Note: Gene amplification: 3p26.3— ql2.3, 3ql3.33— q29, 4q32.1-q35.1, 9p24.3-q22.33, 17pll.2-q21.1, 17q 21.32, 17q23.2 -q24.2, and 18q; gene deletion: 9p21.3 (arrow), 13ql4.3-q21.2, 15q21. 3, 17pll.2-pl3.3, and 17q21.33-q22 0 Occurs only in BAC RP11-149 12 (see arrow) containing pl6 INK½ . (B) Gene amplification: Iq21.3-q44, 2pl3.2-p25.1, 7, Ilql3.5-q25, 15q24.3-q26.3, 16, and 21q; gene deletion: 1ρ36.22-p36.33, 1ρ13.1-p 31.2, and 13913.1-914.3.
[図 4]DLBCLの ABC型及び GCB型のゲノム不均衡の特徴を示す。（A)は、 ABC群の 28症例の、（B)は、 GCB群の 18症例との染色体不均衡の要約を示す。左側（赤）の ラインは欠損を支援し、右側（緑）は、増幅を示す。左側の赤の四角は、ホモ欠損 (Lo g2比く- 1.0)を示し、右側の緑四角は、右側における高度の増幅 (Log2比〉 +1.0)を示 す。 FIG. 4 shows the characteristics of genomic imbalance of DLBCL ABC type and GCB type. (A) shows a summary of chromosomal imbalances in 28 cases in the ABC group and (B) in 18 cases in the GCB group. The left (red) line supports the deficit and the right (green) indicates amplification. It left square red, homo-deficient (Lo g 2 draw - 1.0) indicates, right green squares, shows the high degree of amplification in the right (Log 2 ratio> +1.0).
[図 5]区別される DLBCLサブグループのゲノムワイドな遺伝子不均衡頻度を示す。水 平のライン： 1番染色体から 22番染色体及び X染色体の順で 2213 BAC/PACクロー ンを す。各染色体内【こお ヽ飞 ii、 Ensembl Genome Data Resources of Sangerし en ter Instituteの情報（November 2004 version)に基づいて pテロメァから qテロメァの順 で示す。垂直のライン:増幅と欠損との頻度（％)を示す。 （A)は ABC群（28症例)、 GCB群（18症例)及び ABC群と GCB群との合計 (46症例）を示す。 （B)は CD5+群 (36症例)， CD5—CD10+群（19症例)及び CD5—CD10—群（44症例)を示す。 CD5+ 群及び CD5—CD10+群のゲノム不均衡の特徴は、それぞれ ABC群及び GCB群のそ れに類似していた。また、 CD5—CD10—群のゲノム不均衡の領域は、 ABC群及び GC B群のそれに類似したパターンを示した。  FIG. 5 shows the genome-wide gene imbalance frequency of the distinguished DLBCL subgroups. Horizontal line: Clone 2213 BAC / PAC in order from chromosome 1 to chromosome 22 and chromosome X. Based on the information from the Institute Institute of Information (November 2004 version) within each chromosome [Konii ii, Ensembl Genome Data Resources of Sanger], it is shown in order from p telomer to q telomer. Vertical line: shows the frequency (%) of amplification and loss. (A) shows ABC group (28 cases), GCB group (18 cases), and the total of ABC group and GCB group (46 cases). (B) shows CD5 + group (36 cases), CD5-CD10 + group (19 cases) and CD5-CD10- group (44 cases). The characteristics of genomic disequilibrium in the CD5 + and CD5-CD10 + groups were similar to those in the ABC and GCB groups, respectively. The region of genomic imbalance in the CD5-CD10 group showed a pattern similar to that of the ABC group and GC B group.
[図 6]9p21.3の遺伝子欠損、全生存率及び ABC群と GCB群とにおける pl6INK4a遺伝 子を示す。 (A)は、全症例、 ABC症例、 CD+症例及び CD5—CD10—症例の 9p21.3 ( pl6INK4a座)の欠損の有無での力プラン マイヤー生存率を示す。（B)は、 3症例 における 9p21の代表的な個別のゲノムプロファイルを示す。ドットは、 BAC/PACクロ ーンの log2比を pテロメァカも qテロメァにわたつて示し、各プロファイルにおける太線 は、欠損領域を示す。 MCRは 9p領域における最小共通領域を示し、垂直線は、 log  FIG. 6 shows 9p21.3 gene deletion, overall survival rate, and pl6INK4a gene in ABC and GCB groups. (A) shows the power plan Meyer survival rate in all cases, ABC cases, CD + cases and CD5-CD10-cases with or without 9p21.3 (pl6INK4a locus) deficiency. (B) shows a representative individual genomic profile of 9p21 in 3 cases. The dots indicate the BAC / PAC clone log2 ratio for both p and q telomeres, and the bold line in each profile indicates the missing region. MCR shows the smallest common region in the 9p region, the vertical line is log
2 比を示す。水平線は、メガベース単位である。 log比く— 0. 2から log比がく +0. 2  2 Indicates the ratio. The horizontal line is in megabase units. log ratio — from 0.2 to log ratio +0.2
2 2  twenty two
の範囲を示す。（C)ABC群及び GCB群における pl6INK4a遺伝子の発現の比較を示 す。統計的有意差は、マン ウィット-一 Uテストにより算出した。 Indicates the range. (C) Comparison of pl6INK4a gene expression in ABC and GCB groups. Statistical significance was calculated using the Mann Wit-1 U test.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明は、ヒトから採取した染色体を含む被験試料中のヒト染色体の増幅領域及び /又は欠損領域に基づ 、て DLBCLの病型を診断することを特徴として 、る。本発明 によれば、 DLBCL罹患ヒト個体から採取した染色体を含む被験試料中のヒト染色体 の増幅領域及び Z又は欠損領域を検査することによって DLBCLの病型を簡易に診 断できる。 DLBCLの病型としては発現解析によって既により悪性度の高 、ABC型と G CB型とに分類されている力 本発明によれば、これらの病型を容易に診断できる。な お、本発明において、病型の診断ないし判定とは、 DLBCL患者が ABC型であるか G CB型であるかを判断すること又は DLBCL患者が CD5+型である力 CD5—CD10+型 であるかを判断するものである。こうした判断は、判断対象となる病型のうち一方の病 型にぉ 、て特徴的な増幅領域及び Z又は欠損領域を有して 、るか否かある 、は一 方の病型に高頻度な領域の検出数に基づくことができる。本発明は、診断方法、ァレ ィの形態で実施することができる。 The present invention relates to an amplification region of a human chromosome in a test sample containing a chromosome collected from a human and It is characterized by diagnosing a DLBCL disease type based on a defective region. According to the present invention, a DLBCL disease type can be easily diagnosed by examining human chromosome amplification regions and Z or deletion regions in test samples containing chromosomes collected from DLBCL-affected human individuals. DLBCL disease types are already more malignant by expression analysis and have been classified into ABC type and GCB type. According to the present invention, these disease types can be easily diagnosed. In the present invention, the diagnosis or determination of the disease type is to determine whether a DLBCL patient is ABC type or GCB type, or is DLBCL patient a CD5 + type force CD5-CD10 + type? Is to judge. Such a determination has a characteristic amplification region and Z or a defective region in one of the disease types to be determined, and whether or not it has high frequency in one disease type. Can be based on the number of detected regions. The present invention can be implemented in the form of a diagnostic method and array.
[0021] また、本発明は、ヒトから採取した染色体を含む被験試料中のヒト染色体の 9p21の 欠損又は変異に基づ 、て DLBCLの予後を診断することを特徴として 、る。本発明に よれば、 DLBCLの予後診断を簡易に、また精度よく実施できる。予後診断により、治 療法等についてより適切な選択が可能となる。なお、本発明において予後の診断な いし判定とは、「予後良好」か「予後不良」かのいずれかを判断するものである。「予後 良好」とは、 2つの病型の DLBCL患者群間で力プランマイヤー曲線を比較したとき、 ログランクテストの p値が 0. 05以下の有意差をもって生存率がょ 、患者群を示すもの とすることができる。また、「予後不良」とは、力プランマイヤー曲線を比較したログラン クテストの p値が 0. 05以下の有意差で生存率が悪 、患者群を示すものとすることが できる。ヒト染色体の 9p21は、 pl6INK4a遺伝子に対応しているため、当該遺伝子の発 現の有無や、発現量や変異を検出することにより DLBCLの予後を診断することもでき る。本発明は、診断方法、アレイ、診断用マーカーの形態で実施することができる。 [0021] Further, the present invention is characterized in that the prognosis of DLBCL is diagnosed based on a 9p21 deletion or mutation of a human chromosome in a test sample containing a chromosome collected from a human. According to the present invention, DLBCL prognosis can be easily and accurately performed. Prognosis enables more appropriate selection of treatment. In the present invention, the determination of whether or not the prognosis is made is to determine whether “good prognosis” or “poor prognosis”. “Good prognosis” means that when the force-plan Meier curves are compared between DLBCL patients with two types of disease, the p-value of the log rank test is a significant difference of 0.05 or less and the survival rate indicates the patient group. Can be. In addition, “poor prognosis” can indicate a patient group in which the p-value of the log-rank test comparing force-plan Meyer curves is a significant difference of 0.05 or less and the survival rate is poor. Since 9p21 of the human chromosome corresponds to the pl6 INK4a gene, the prognosis of DLBCL can also be diagnosed by detecting the presence of the gene, its expression level, and mutation. The present invention can be implemented in the form of a diagnostic method, an array, and a diagnostic marker.
[0022] 以下、本発明の実施の形態について詳細に説明する。  Hereinafter, embodiments of the present invention will be described in detail.
[0023] (DLBCLの病型の診断方法）  [0023] (Diagnosis method of DLBCL disease type)
DLBCLの病型を診断するための指標となる染色体の増幅領域及び欠損領域とは、 予め発現解析等により病型が診断された DLBCL罹患ヒト個体から採取した染色体含 有試料を利用して決定することができる。指標となる増幅領域及び欠損領域を決定 するには、アレイ CGHを利用することが好ましい。なお、以下の説明において、高頻 度の増幅及び高頻度の欠損の語については、後述する実施例に詳細な定義がなさ れている。 Chromosome amplification and deletion regions that serve as indicators for diagnosing DLBCL disease types are determined using chromosome-containing samples collected from human individuals with DLBCL whose disease type has been previously diagnosed by expression analysis, etc. be able to. Determine amplification and deletion regions as indicators To do so, it is preferable to use array CGH. In the following description, the words of high frequency amplification and high frequency loss are defined in detail in the examples described later.
[0024] DLBCLの ABC型であると判定するのに有用な増幅領域としては、後述する実施例 の表 3及び図 5に示すゲノム不均衡の頻度グラフから ABC型に特徴的な増幅領域が 挙げられる。例えば、ヒト染色体の 3、 8q21- q26、 llq21- q25、 16pll- pl3、 16q22-q24 、 18、 19ql3及び X力 選択される 1種又は 2種以上の領域が挙げられる。これらの領 域は、 ABC型において高頻度の増幅領域である力もである。なかでも、 3p23_q28、 18 qll.2-q23及び 9ql3.41-ql3.43力 選択される 1種又は 2種以上の領域はさらに高頻 度若しくは ABC型に特徴的である。さらに、 3q23_q28は ABC型に特徴的な増幅領域 である。  [0024] Amplification regions useful for determining DLBCL ABC type include amplification regions characteristic of ABC type from the frequency graph of genomic imbalance shown in Table 3 and FIG. 5 of Examples described later. It is done. For example, 3, 8q21-q26, llq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3 of human chromosome and one or more regions selected from X force can be mentioned. These regions are also forces that are high frequency amplification regions in the ABC type. Among them, 3p23_q28, 18 qll.2-q23 and 9ql3.41-ql3.43 force One or more selected regions are more characteristic for the higher frequency or ABC type. Furthermore, 3q23_q28 is an amplification region characteristic of ABC type.
[0025] また、 DLBCLの ABC型であると判定するのに有用な欠損領域としては、後述する実 施例の表 3及び図 5に示すゲノム不均衡の頻度グラフから ABCに特徴的な欠損領域 が挙げられる。例えば、ヒト染色体の 2pll、 6ql2-27、 8p22-p23、 9p21及び 17pから選 択される 1種又は 2種以上が挙げられる。これらの領域は、 ABC型において高頻度の 欠損領域であるからである。なかでも、 6q22.31-q24.1及び 9p21.3から選択されること が好ましい。さらには、ヒト染色体の 9p21.3である。  [0025] Further, as a defective region useful for determining that it is the ABC type of DLBCL, a defective region characteristic to ABC from the frequency graph of genomic imbalance shown in Table 3 of the Example described later and FIG. Is mentioned. Examples thereof include one or more selected from 2pll, 6ql2-27, 8p22-p23, 9p21 and 17p of human chromosome. This is because these regions are frequently defective regions in the ABC type. Of these, 6q22.31-q24.1 and 9p21.3 are preferred. Furthermore, it is 9p21.3 of the human chromosome.
[0026] 一方、 DLBCLの GCB型であると判定するのに有用な増幅領域としては、ヒト染色体 の lq22- 32、 2pl4- p24、 5pl2- p 、 5ql5- q31、 6pl2- p25、 7、 8q22- q26、 9q33- q34、 1 lq、 12、 13q31- q33、 16pll- pl3、 18q21- q23、 19p、 19ql3、 21q及び Xから選択される 1種又は 2種以上が挙げられる。これらの領域は、 GCB型において高頻度の増幅領 域であるからである。なかでも、 lq21.1- q23.3、 lq31.1- q42.13、 2pl5- pl6.1、 7q22.1-q 36.2及び 12ql3.1-ql4が選択されることが好ましぐより好ましくは、 7q22-q36である。  [0026] On the other hand, useful amplification regions for determining the DLBCL GCB type include human chromosomes lq22-32, 2pl4-p24, 5pl2-p, 5ql5-q31, 6pl2-p25, 7, 8q22- one or more selected from q26, 9q33-q34, 1 lq, 12, 13q31-q33, 16pll-pl3, 18q21-q23, 19p, 19ql3, 21q and X. This is because these regions are frequently amplified regions in the GCB type. Among them, it is preferable that lq21.1-q23.3, lq31.1-q42.13, 2pl5-pl6.1, 7q22.1-q 36.2 and 12ql3.1-ql4 are selected, 7q22-q36.
[0027] また、 DLBCLの GCB型であると判定するのに有用な欠損領域としては、ヒト染色体 の lp36、 2pll、 3pl4、 4pl2- pl3、 4q33- q34、 6ql4- ql6、 8p22- p23、 9p21、 13ql2-q22 、 17pl2及び 18q22-q23から選択される 1種又は 2種以上が挙げられる。これらは GCB 型にぉ 、て高頻度の欠損領域であるからである。  [0027] In addition, deletion regions useful for determining DLBCL GCB type include human chromosomes lp36, 2pll, 3pl4, 4pl2-pl3, 4q33-q34, 6ql4-ql6, 8p22-p23, 9p21, Examples thereof include one or more selected from 13ql2-q22, 17pl2, and 18q22-q23. This is because these are frequently defective regions in the GCB type.
[0028] さらに、 DLBCLの CD5 +型であると判定するのに有用な増幅領域としては、ヒト染 色体の 3、 6p22- p25、 7p22- q31、 8q24、 llq22- q25、 12、 16pl3- q21、 18、 19及び Xか ら選択される 1種又は 2種以上が挙げられる。好ましくはヒト染色体 3である。また、同 様に有用な欠損領域としては、ヒト染色体の lp36、 2pll、 6ql4-q27、 8p23、 9p21、 15q 13- ql4及び 17pll- pl3が挙げられる。好ましくは、ヒト染色体の 9p2である。本発明者 らによれば、 DLBCLの ABC型と CD5+型におけるゲノム不均衡から、 ABC型であると き、 CD5+型との関連付けが可能である場合があるといえる（特に、アジア人、なかで も日本人について）。したがたつて、 ABC型であるときには、 CD5+型であると肯定的 に判定するようにしてもよい。 [0028] Furthermore, human amplification stains are useful for determining that DLBCL is CD5 + type. Examples thereof include one or more selected from 3, 6p22-p25, 7p22-q31, 8q24, llq22-q25, 12, 16pl3-q21, 18, 19 and X. Human chromosome 3 is preferred. Similarly useful deletion regions include lp36, 2pll, 6ql4-q27, 8p23, 9p21, 15q13-ql4 and 17pll-pl3 of the human chromosome. Preferably, it is 9p2 of the human chromosome. According to the present inventors, it can be said that there is a case where association with CD5 + type is possible when it is ABC type due to genomic imbalance between ABC type and CD5 + type of DLBCL (especially Asians, among others). Also about the Japanese). Therefore, when it is the ABC type, it may be positively determined that it is the CD5 + type.
[0029] また、 DLBCLの CD5一 DC10+型であると判定するのに有用な増幅領域としては、ヒト 染色体上の lq、 2pl3- p25、 6p21- p25、 7、 8q22- q24、 9q33- q34、 12、 13q31- q33、 15q 、 16pl3、 19ql3.3-13.4及び X力 選択される 1種又は 2種以上が挙げられる。また、ヒ ト染色体の 7q22-q36、 12ql3-ql4及び 17pl3も好ましい。また、同様に有用な欠損領 域としては、ヒト染色体の 1ρ36、 1ρ22、 2pll、 3pl4、 4p、 6ql3-q27、 9p21及び 13ql4-q 21が挙げられる。また、本発明者らによれば、 DLBCLの GCB型と CD5—DC10+型に おけるゲノム不均衡から、 GBC型であるとき、 CD5—DC10+型との関連付けが可能で ある場合があるといえる（特に、アジア人、なかでも日本人について）。したがって、 AB C型であるときには、 CD5—CD10+型であると肯定的に判定するようにしてもよい。  [0029] In addition, useful amplification regions for determining DLBCL CD5 and DC10 + are lq, 2pl3-p25, 6p21-p25, 7, 8q22-q24, 9q33-q34, 12 on the human chromosome. , 13q31-q33, 15q, 16pl3, 19ql3.3-13.4 and X force One or more selected. The human chromosomes 7q22-q36, 12ql3-ql4 and 17pl3 are also preferred. Similarly, useful deletion regions include 1ρ36, 1ρ22, 2pll, 3pl4, 4p, 6ql3-q27, 9p21 and 13ql4-q21 of human chromosomes. Further, according to the present inventors, it can be said that the GBB type may be associated with the CD5−DC10 + type due to the genomic imbalance in the DLBCL GCB type and the CD5−DC10 + type (particularly, Asian, especially Japanese). Therefore, when the type is ABC type, it may be positively determined that the type is CD5-CD10 + type.
[0030] これらの染色体上における増幅領域及び欠損領域を検出するには、各種方法を採 用することができる。たとえば、 DLBCLに罹患したヒト個体から採取される染色体含有 被験試料とこれらの染色体上の各種領域とハイブリダィズするプローブを用いたハイ ブリダィゼーシヨンにより検出することができる。症例等からの核酸試料としては、患 者のリンパ腫標本など力も標準的な DNA抽出法等により取得することができる。一 方、これらの増幅や欠損を検出することができるプローブとしては、こうした染色体上 の領域の少なくとも一部とハイブリダィズするものであればよい。たとえば、プローブと しては、染色体領域に対応した BACクローン及び/又は PACクローンある ヽはこれ を铸型として DOP— PCRやアダプター PCR法によりクローン化したゲノム DNAを増 幅して用いることができる。また、増幅領域または欠損領域に対応する遺伝子が特定 される場合〖こは、当該遺伝子を含む BACZPACクローンまたはこれを铸型として P CR法を利用してクローンィ匕したゲノム DNAを用いることもできる。 [0030] Various methods can be employed to detect the amplified region and the defective region on these chromosomes. For example, it can be detected by hybridization using a chromosome-containing test sample collected from a human individual suffering from DLBCL and a probe that hybridizes with various regions on these chromosomes. Nucleic acid samples from patients, etc., can also be obtained by standard DNA extraction methods, etc. for patient lymphoma specimens. On the other hand, any probe that can detect such amplification and deletion may be any probe that hybridizes with at least a part of the region on the chromosome. For example, as a probe, a BAC clone and / or a PAC clone corresponding to a chromosomal region can be used as a probe by amplifying genomic DNA cloned by DOP-PCR or adapter PCR. . In addition, when a gene corresponding to an amplified region or a defective region is identified, this is a BACZPAC clone containing the gene or P Genomic DNA cloned using the CR method can also be used.
[0031] 被験試料とプローブとのハイブリダィゼーシヨンの形態は、特に限定されない。液相 反応であっても、ビーズや基板などの固相担体を用いた方法であってもよい。好まし くは、 DNAプローブをチップやビーズなどの固相担体に固定したものを用いることが できる。染色体上の特定領域の増幅や欠損の検出は、実施例におけるアレイ CGH 法に準じて DNAアレイを作成し、実施することができる。 [0031] The form of hybridization between the test sample and the probe is not particularly limited. It may be a liquid phase reaction or a method using a solid phase carrier such as a bead or a substrate. Preferably, a DNA probe immobilized on a solid phase carrier such as a chip or a bead can be used. Amplification of a specific region on a chromosome and detection of a defect can be performed by preparing a DNA array according to the array CGH method in the Examples.
[0032] 本診断方法において、こうした染色体上の増幅領域及び欠損領域の検出は、 DLB CL罹患ヒト個体の染色体上の所定領域について所定のプライマーを利用した PCR 法を実施し、 PCR産物の取得の有無やその同定によって検出することも可能である 。プライマーは、 pi 6遺伝子等の配列に基づいて設計することができる。プライマー の塩基長は、好ましくは、 15〜40塩基、望ましくは 15〜30塩基である。ただし、 LA( longaccurate) PCRを行う場合には、少なくとも 30塩基とすることが好ましい。なそ、 センス鎖とアンチセンス鎖が互いにァニールしないように、また、ヘアピン状構造の形 成を回避できるような塩基配列を選択することが好ましい。 [0032] In the present diagnostic method, such an amplified region and a defective region on a chromosome are detected by performing PCR using a predetermined primer on a predetermined region on the chromosome of a human individual suffering from DLB CL to obtain a PCR product. It is also possible to detect by the presence or absence and its identification. Primers can be designed based on sequences such as the pi 6 gene. The base length of the primer is preferably 15 to 40 bases, desirably 15 to 30 bases. However, when LA (longaccurate) PCR is performed, it is preferable to use at least 30 bases. It is preferable to select a base sequence so that the sense strand and the antisense strand do not anneal with each other and can avoid the formation of a hairpin-like structure.
[0033] さらに、本診断方法において、染色体上の増幅領域および欠損領域の検出は、これ らの染色体領域に含まれる遺伝子の発現量に基づくこともできる。すなわち、各染色 体領域の遺伝子の発現量が健常者の生体試料として比較して有意に多いか又は少 ないかで病型を判断することも可能である。なお、この場合、発現量が有意に多いと は、健常者の同一遺伝子の発現量と比較して 10%以上、好ましくは、 30%以上、よ り好ましくは 50%以上、さらに好ましくは 70%以上、一層好ましくは 100%以上であ る。また、発現量が有意に少ないとは、健常者の同一遺伝子の発現量と比較して 60 %以下、好ましくは 50%以下、さらに好ましくは 20%以下、一層好ましくは 10%以下 である。 [0033] Further, in the present diagnostic method, the detection of the amplified region and the defective region on the chromosome can also be based on the expression level of the genes contained in these chromosomal regions. That is, it is also possible to determine the disease type based on whether the expression level of the gene in each chromosome region is significantly higher or lower than that of a healthy subject. In this case, the expression level is significantly higher than 10%, preferably 30% or more, more preferably 50% or more, and even more preferably 70% compared to the expression level of the same gene in healthy individuals. Above, more preferably 100% or more. The expression level is significantly less than 60%, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less compared to the expression level of the same gene in healthy individuals.
[0034] 対象となる各遺伝子は公知の方法によって容易に取得することができる。たとえば 、 cDNAライブラリを形成して、公知の塩基配列に基づいて作成した DNAプローブ を用いて各 cDNAを単離することができる。また、 cDNAの塩基配列に基づいたプラ イマ一を用いて mRNAを铸型として RT—PCR法によって必要量の cDNAを取得し てもよい。 [0035] 遺伝子の発現量に基づく解析は、ヒトから採取した試料につき公知の方法で実施 することができる。たとえば、 in situハイブリダィゼーシヨン、ノザンブロッテイング、ドッ トブロット、 RT—PCR、 real-Time PCR、 DNAアレイによる方法などを用いることがで きる。 [0034] Each target gene can be easily obtained by a known method. For example, a cDNA library can be formed and each cDNA can be isolated using a DNA probe prepared based on a known nucleotide sequence. Alternatively, a primer based on the cDNA base sequence may be used to obtain the required amount of cDNA by RT-PCR using mRNA as a saddle. [0035] The analysis based on the expression level of the gene can be performed by a known method on a sample collected from a human. For example, in situ hybridization, Northern blotting, dot blot, RT-PCR, real-time PCR, DNA array methods, etc. can be used.
[0036] 本診断方法において、染色体上の増幅領域および欠損領域を検出するには、これ らの領域に対応する遺伝子によってコードされるタンパク質の発現の有無、発現量又 は変異を検出することによつてもよい。このためには、例えば、こうしたタンパク質に対 して特異的な抗体を用いることができる。抗体としては、ポリクローナル抗体やモノク ローナル抗体を用いることができる。抗体分子又はその一部であってもよい。このよう な抗体は、例えばポリクローナル抗体の場合には、タンパク質やその一部断片を免 疫原として動物を免役した後、血清力 得ることができる。あるいは、上記の真核細胞 用発現ベクターを注射や遺伝子銃によって、動物の筋肉や皮膚に導入した後、血清 を採取することによって作製することができる。動物としては、マウス、ラット、ゥサギ、 ャギ、 -ヮトリなどが用いられる。また、モノクローナル抗体は、公知のモノクローナル 抗体作製法（「単クローン抗体」、長宗香明、寺田弘共著、廣川書店、 1990年; "Mono clonal Antibody James W. Coding, third edition, Academic Press, 1996)【こ従 ヽ作 製することができる。  [0036] In this diagnostic method, in order to detect an amplified region and a defective region on a chromosome, the presence or absence of expression of a protein encoded by a gene corresponding to these regions, the expression level, or a mutation is detected. You may do it. For this purpose, for example, an antibody specific to such a protein can be used. As the antibody, a polyclonal antibody or a monoclonal antibody can be used. It may be an antibody molecule or a part thereof. In the case of a polyclonal antibody, for example, such an antibody can be obtained with serum after immunizing an animal using a protein or a partial fragment thereof as an immunogen. Alternatively, it can be prepared by introducing the above eukaryotic cell expression vector into an animal muscle or skin by injection or gene gun, and then collecting serum. Examples of animals include mice, rats, rabbits, goats, and birds. Monoclonal antibodies are known monoclonal antibody production methods ("monoclonal antibodies", Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; "Monoclonal Antibody James W. Coding, third edition, Academic Press, 1996) [This can be made.
[0037] 抗体は、適宜標識物質によって標識化されて!/ヽてもよ ヽ。標識物質は、酵素、放射 性同位体または蛍光色素を使用することができる。酵素としては、例えば、特に限定 しないで、通常の EIAに用いられる酵素、例えば、ペルォキシダーゼ、 β ガラクトシ ダーゼ、アルカリフォスファターゼ、グルコースォキシダーゼ、アセチルコリンエステラ ーゼ、グルコース 6—リン酸ィ匕脱水素酵素、リンゴ酸脱水素酵素等を用いることもで きる。また、酵素阻害物質や補酵素等を用いることもできる。これら酵素と抗体との結 合は、マレイミドィ匕合物等の架橋剤を用いる公知の方法によって行うことができる。基 質としては、使用する酵素の種類に応じて公知の物質を使用することができる。蛍光 色素としては、フルォレツセンスイソチオシァネート（FITC)ゃテトラメチルローダミン イソチオシァネート (TRITC)等の通常の蛍光抗体法に用いられるものを使用するこ とがでさる。 [0038] こうした抗体を用いてタンパク質の発現量等を検出するには、組織あるいは細胞染 色などの免疫染色、競合型又は非競合型による放射免疫測定法 (RIA)、蛍光免疫 測定法 (FIA)、ルミネッセント免疫測定法 (LIA)、酵素免疫測定法 (EIA、 ELISA) 等の各種検出方法による測定方法を用いることができる。なお、こうした測定方法に おける抗原抗体反応は、液相で行っても固相で行ってもよい。検出には、抗原抗体 反応産物が分離されていることが好ましい。このためには、例えば、クロマトグラフィー やビーズやプレートなどの固相担体を用いてもよい。また、ウェスタンブロッテイング の手法を用いてもよい。さらに、 ELISA法などを用いることができる。さらには、抗体 を基板などの固相担体に固定ィ匕したアレイを用いることでもできる。 [0037] The antibody may be appropriately labeled with a labeling substance! As the labeling substance, an enzyme, a radioisotope or a fluorescent dye can be used. Examples of the enzyme include, but are not limited to, enzymes used in normal EIA, such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose 6-phosphate dehydrogenase, Malate dehydrogenase or the like can also be used. Moreover, an enzyme inhibitor, a coenzyme, etc. can also be used. Binding of these enzymes and antibodies can be performed by a known method using a cross-linking agent such as maleimide compound. As the substrate, a known substance can be used depending on the type of enzyme used. As the fluorescent dye, those used in the usual fluorescent antibody method such as fluorescens isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) can be used. [0038] To detect the amount of protein expression using these antibodies, immunostaining such as tissue or cell staining, competitive or non-competitive radioimmunoassay (RIA), fluorescent immunoassay (FIA ), Luminescent immunoassay (LIA), enzyme immunoassay (EIA, ELISA), and other detection methods can be used. The antigen-antibody reaction in such a measurement method may be performed in a liquid phase or a solid phase. For detection, the antigen-antibody reaction product is preferably separated. For this purpose, for example, a solid support such as chromatography or beads or plates may be used. Further, Western blotting may be used. Furthermore, ELISA method etc. can be used. Furthermore, an array in which antibodies are immobilized on a solid support such as a substrate can be used.
[0039] なお、この態様によれば、タンパク質若しくはその一部又はこれらに対する抗体を 含む DLBCLの病型の診断用のマーカーが提供される。特に、抗体は、こうしたタンパ ク質を検出するための検出試薬として好ましい。また、この態様によれば、こうした抗 体を含む DLBCLの病型診断用キットが提供される。本発明の診断キットは、抗体や 標識化抗体を液相中に含むものであってもよく、あるいは抗体や標識化抗体を固相 担体に結合したものであってもよい。また、固定ィ匕された抗原又はその一部を含んで いてもよい。診断用キットは、抗体が酵素で標識されている場合には、その基質を含 んでいてもよい。さらに、固相担体を含むものの場合には、固相への非結合分子を洗 浄除去するための洗浄液を含んでいてもよい。このほか、一般に抗体を含む診断用 キットに含まれることのできる要素を含むことができる。  [0039] It should be noted that according to this aspect, a marker for diagnosis of a DLBCL disease type comprising a protein or a part thereof or an antibody against them is provided. In particular, antibodies are preferred as detection reagents for detecting such proteins. In addition, according to this embodiment, a kit for diagnosing a DLBCL disease type containing such an antibody is provided. The diagnostic kit of the present invention may contain an antibody or a labeled antibody in the liquid phase, or may be one in which the antibody or labeled antibody is bound to a solid phase carrier. It may also contain a fixed antigen or a part thereof. When the antibody is labeled with an enzyme, the diagnostic kit may contain the substrate. Furthermore, in the case of a substance containing a solid phase carrier, a washing solution for washing away non-bound molecules from the solid phase may be contained. In addition, elements that can generally be included in diagnostic kits containing antibodies can be included.
[0040] (DLBCLの病型の診断用の核酸プローブ）  [0040] (Nucleic acid probe for diagnosis of DLBCL disease type)
本発明の核酸プローブは、 DLBCLの病型を診断するための指標となる染色体の増 幅領域及び欠損領域を検出可能な核酸プローブを含んで 、る。こうした核酸プロ一 ブによれば、これらの領域の増幅や欠損をハイブリダィゼーシヨンを用いて容易に検 出することができる。こうした核酸プローブは、例えば、表 3や図 5に示すゲノムワイド な染色体不均衡グラフにぉ 、て病型に特徴的な領域を検出可能であればよ 、。す なわち、これらの領域を含む BAC/PACクローンあるいはこれらから得られた DNA をプローブとして利用できる。  The nucleic acid probe of the present invention includes a nucleic acid probe capable of detecting a chromosomal amplified region and a defective region, which serve as an index for diagnosing DLBCL disease type. According to such a nucleic acid probe, amplification or deletion of these regions can be easily detected using a hybridization. For example, such a nucleic acid probe should be able to detect a region characteristic of a disease type on the genome-wide chromosome imbalance graph shown in Table 3 and FIG. In other words, BAC / PAC clones containing these regions or DNA obtained from these can be used as probes.
[0041] 具体的には、既に説明した ABC型の DLBCLにおけるヒト染色体上の増幅領域及 び欠損領域力 選択される 1種又は 2種以上の領域を検出可能なプローブであり、よ り具体的には、これらの領域を含有する BACZPACクローンまたはこれら力 得られ るゲノム DNAである。 ABC型を判定するのに適したプローブは、すでに診断方法に おいて説明したのと同様である。どの領域を含む DNAをプローブとして用いるかは、 たとえば、所定範囲 (好ましくはゲノムワイドな範囲）のヒト染色体に由来する核酸プロ ーブを固定ィ匕したアレイに対して前記 ABC型の DLBCL罹患ヒト個体力も採取した染 色体を含む被験試料を供給し核酸ハイブリダィゼーシヨンを実施して前記核酸プロ ーブの固定部位において得られる蛍光強度の log比値に基づいて所定の上下の閾 [0041] Specifically, the amplification region on the human chromosome and the ABC type DLBCL described above and And a defective region force A probe capable of detecting one or more selected regions, more specifically, a BACZPAC clone containing these regions or a genomic DNA obtained from these forces. A probe suitable for determining ABC type is similar to that already described in the diagnosis method. Which region contains DNA to be used as a probe can be determined, for example, by using an ABC type DLBCL-affected human against an array in which nucleic acid probes derived from human chromosomes in a predetermined range (preferably genome-wide range) are immobilized. Based on the log ratio value of the fluorescence intensity obtained at the fixed site of the nucleic acid probe by supplying a test sample containing a chromosome that has also been collected and performing nucleic acid hybridization, a predetermined upper and lower threshold value is obtained.
2  2
値を設定し、該閾値に対する前記 log比値の大小に基づいて選択した核酸プローブ  A nucleic acid probe selected based on the magnitude of the log ratio value relative to the threshold value
2  2
を含むこともできる。例えば、後述する実施例に示すように、蛍光強度の log比の閾  Can also be included. For example, as shown in the examples described later, the threshold of the log ratio of fluorescence intensity
2 値を +0. 2から— 0. 2に設定し、この閾値を基準として蛍光強度 log比の大小で低  2 Set the value from +0.2 to —0.2 and use this threshold as a reference for the fluorescence intensity log ratio.
2  2
レベルの増幅、高レベルの増幅、低レベルの欠損及び高レベルの欠損を定義するこ とがでさる。  It is possible to define level amplification, high level amplification, low level defects and high level defects.
[0042] また、既に説明した GCB型の DLBCLにおけるヒト染色体上の増幅領域及び欠損領 域力 選択される 1種又は 2種以上の領域を検出可能なプローブであり、より具体的 には、これらの領域を含有する BACZPACクローンまたはこれら力 得られるゲノム DNAである。 GCB型を判定するのに適したプローブは、すでに診断方法において 説明したのと同様である。一方、所定範囲 (好ましくはゲノムワイドな範囲)のヒト染色 体に由来する核酸プローブを固定ィ匕したアレイに対して前記 GCB型の DLBCL罹患 ヒト個体カゝら採取した被験試料を供給し核酸ハイブリダィゼーシヨンを実施して前記 核酸プローブの固定部位において得られる蛍光強度の log比値に基づいて所定の  [0042] Further, the amplification region and deletion region force on the human chromosome in the already described GCB type DLBCL are probes capable of detecting one or more selected regions, and more specifically, these probes. BACZPAC clones containing these regions or genomic DNA obtained from these. Suitable probes for determining the GCB type are similar to those already described in the diagnostic method. On the other hand, a test sample collected from a human individual suffering from DLBCL of the above-mentioned GCB type is supplied to an array in which nucleic acid probes derived from human chromosomes in a predetermined range (preferably genome-wide range) are immobilized. Based on the log ratio value of the fluorescence intensity obtained at the fixed site of the nucleic acid probe by carrying out dialysis
2  2
上下の閾値を設定し、該設定した閾値に対する前記 log比値の大小に基づいて選  Set upper and lower thresholds, and select based on the log ratio value relative to the set threshold.
2  2
択した核酸プローブを含むこともできる。  Selected nucleic acid probes can also be included.
[0043] さらに、既に説明した CD5+や CD5+CD10—を判定するのに有用な増幅領域及び欠 損領域力 選択される 1種又は 2種以上の領域を検出可能な核酸プローブであって ちょい。 [0043] Further, the amplification region and the lacking region force useful for determining CD5 + and CD5 + CD10- described above are nucleic acid probes capable of detecting one or more selected regions.
[0044] 本発明の核酸プローブとしては、 DLBCLの 2つの病型である ABC型と GCB型とを判 定できる核酸プローブセットであることが好ましい。 ABC型判定用としては、例えば、ヒ ト染色体の 3、 8q21- q26、 llq21- q25、 16pll- pl3、 16q22- q24、 18、 19ql3、 X、 2pll、 6ql2-27、 8p22-p23、 9p21、 17pから選択される 1種又は 2種以上の領域の少なくとも 一部を検出可能な核酸プローブを用いることができる。また、 GCB型判定用としては 、ヒト染色体の lq22- 32、 2pl4- p24、 5pl2- p 、 5ql5- q31、 6pl2- p25、 7、 8q22- q26、 9 q33— q34、 llq、 12、 13q31— q33、 16pll— pl3、 18q21— q23、 19p、 19ql3、 21q、 X、 lp36、 2pll、 3pl4、 4pl2- pl3、 4q33- q34、 6ql4- ql6、 8p22- p23、 9p21、 13ql2- q22、 17pl2 及び 18q22-q23から選択される 1種又は 2種以上の領域の少なくとも一部を検出可能 な核酸プローブを用いることができる。 [0044] The nucleic acid probe of the present invention is preferably a nucleic acid probe set that can determine two types of DLBCL, ABC type and GCB type. For ABC type determination, for example, One or more selected from 3, 8q21-q26, llq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3, X, 2pll, 6ql2-27, 8p22-p23, 9p21, 17p A nucleic acid probe capable of detecting at least a part of the region can be used. For GCB type determination, human chromosomes lq22-32, 2pl4-p24, 5pl2-p, 5ql5-q31, 6pl2-p25, 7, 8q22-q26, 9 q33—q34, llq, 12, 13q31—q33 , 16pll—pl3, 18q21—q23, 19p, 19ql3, 21q, X, lp36, 2pll, 3pl4, 4pl2-pl3, 4q33-q34, 6ql4-ql6, 8p22-p23, 9p21, 13ql2-q22, 17pl2 and 18q22-q23 A nucleic acid probe capable of detecting at least a part of one or two or more regions selected from can be used.
また、本発明の核酸プローブとしては、発現解析用プローブも含まれる。こうしたプ ローブとしては、所定の遺伝子由来の核酸試料に対して特異的にノ、イブリダィズす ればよぐ遺伝子に対して完全に相補的である必要はない。このような多少変異した ポリヌクレオチドは、咅 |5位特異的変異 (Current protocols in Molecular Biology.Ausub elら編,（1987)John
Figure imgf000018_0001
、？じ1^ ^ protocols in Molecular Biology,Ausubelら編ズ 1987)John
Figure imgf000018_0002
、通常のハイブリダィゼー シヨン (Current protocols in Molecular Biology'Ausubelら編, (1987)John Wiley&Sons, 第 6.3-6.4章）等により得ることができる。プローブはストリンジェント（stringent)な条件 下で遺伝子由来の核酸試料とハイブリダィゼーシヨンする。ここで、ストリンジェントな 条件とは、プローブと前記核酸試料との選択的かつ検出可能な特異的結合を可能と する条件である。ストリンジェント条件は、塩濃度、有機溶媒 (例えば、ホルムアミド）、 温度、およびその他公知の条件によって定義される。すなわち、塩濃度を減じるか、 有機溶媒濃度を増加させる力、またはハイブリダィゼーシヨン温度を上昇させるかに よってストリンジエンシー（stringency)は増加する。例えば、ストリンジヱントな塩濃度 は、通常、 NaCl約 750mM以下およびクェン酸三ナトリウム約 75mM以下、より好ま しくは NaCl約 500mM以下およびクェン酸三ナトリウム約 50mM以下、最も好ましく は NaCl約 250mM以下およびクェン酸三ナトリウム約 25mM以下である。ストリンジ ェントな有機溶媒濃度は、ホルムアミド約 35%以上、最も好ましくは約 50%以上であ る。ストリンジェントな温度条件は、約 30°C以上、より好ましくは約 37°C以上、最も好 ましくは約 42°C以上である。その他の条件としては、ハイブリダィゼーシヨン時間、洗 浄剤（例えば、 SDS)の濃度、およびキャリアー DNAの存否等であり、これらの条件 を組み合わせることによって、様々なストリンジエンシーを設定することができる。なお 、上記のハイブリダィゼーシヨン条件は単なる例示であり、当業者であれば、プローブ のヌクレオチド配列、濃度、及び長さ、反応時間、反応温度、試薬濃度等の条件を考 慮して、適当なハイブリダィゼーシヨン条件を設定することができる。 The nucleic acid probe of the present invention also includes an expression analysis probe. Such a probe need not be completely complementary to a gene as long as it is specifically hybridized to a nucleic acid sample derived from a given gene. Such slightly mutated polynucleotides are 咅 | 5-position specific mutation (Current protocols in Molecular Biology. Edited by Ausubel et al., (1987) John
Figure imgf000018_0001
,? 1 ^^ protocols in Molecular Biology, edited by Ausubel et al. 1987) John
Figure imgf000018_0002
Ordinary hybridization (Edited by Current protocols in Molecular Biology 'Ausubel et al., (1987) John Wiley & Sons, Chapter 6.3-6.4). The probe hybridizes with the nucleic acid sample derived from the gene under stringent conditions. Here, the stringent condition is a condition that enables selective and detectable specific binding between the probe and the nucleic acid sample. Stringent conditions are defined by salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, stringency is increased by decreasing the salt concentration, increasing the organic solvent concentration, or increasing the hybridization temperature. For example, stringent salt concentrations are typically about 750 mM or less NaCl and about 75 mM or less trisodium citrate, more preferably about 500 mM or less NaCl and about 50 mM or less trisodium citrate, most preferably about 250 mM or less NaCl and citrate. Trisodium is about 25 mM or less. The stringent organic solvent concentration is about 35% or more of formamide, most preferably about 50% or more. Stringent temperature conditions are about 30 ° C or higher, more preferably about 37 ° C or higher, and most preferably about 42 ° C or higher. Other conditions include hybridization time, washing time. Various stringency can be set by combining these conditions, such as the concentration of detergent (eg, SDS) and the presence or absence of carrier DNA. The hybridization conditions described above are merely examples, and those skilled in the art will consider the conditions such as the nucleotide sequence, concentration and length of the probe, reaction time, reaction temperature, reagent concentration, etc. Appropriate hybridization conditions can be set.
[0046] なお、プローブは、必要に応じて適宜標識されて 、てもよ 、。標識は、ラジオァイソ トープ (RI)法または非 RI法によって行うことができる力 非 RI法を用いることが好まし い。非 RI法としては、蛍光標識法、ピオチン標識法、化学発光法等が挙げられるが、 蛍光標識法が挙げられる。  [0046] The probe may be appropriately labeled as necessary. For labeling, it is preferable to use the force non-RI method which can be performed by radioisotope (RI) method or non-RI method. Examples of the non-RI method include a fluorescent labeling method, a piotine labeling method, a chemiluminescence method, and the like.
[0047] (DLBCLの病型の診断用の核酸プローブが固相担体に固定ィ匕した固定ィ匕体） 本発明によれば、各種の核酸プローブを固相担体に固定ィ匕した固定ィ匕体も提供さ れる。固相担体は、典型的には、スライドガラスなど平坦な基板状体やビーズなどの 粒状体が挙げられる。プローブの固定形態は特に限定しないで、共有結合性及び z 又は静電的、疎水性相互作用等の非共有結合性の各種の結合による固定形態が包 含される。なお、こうした核酸プローブを固定ィ匕した固相担体は、典型的には CGHァ レイと称される DNAアレイである。こうした固定ィ匕体に適用される核酸プローブは、本 発明の診断方法にぉ 、て用いられる核酸プローブのすべての態様を含んで 、る。  (Immobilized body in which a nucleic acid probe for diagnosis of DLBCL disease type is immobilized on a solid phase carrier) According to the present invention, an immobilization medium in which various nucleic acid probes are immobilized on a solid phase carrier. A body is also provided. The solid phase carrier typically includes a flat substrate such as a slide glass and a granular material such as beads. The immobilized form of the probe is not particularly limited, and includes immobilized forms by various bonds of covalent bond and non-covalent bond such as z or electrostatic or hydrophobic interaction. The solid phase carrier on which such a nucleic acid probe is immobilized is typically a DNA array called a CGH array. The nucleic acid probe applied to such a fixed body includes all embodiments of the nucleic acid probe used in the diagnostic method of the present invention.
[0048] (DLBCLの病型診断用キット等） [0048] (DLBCL disease type diagnosis kit, etc.)
以上のことから、 DLBCLの病型診断には、 ABC型および GCB型に関連付けられる ヒト染色体上の増幅領域もしくは対応遺伝子および欠損領域もしくはその対応遺伝 子を検出可能に、これら領域の少なくとも一部あるいはこれと相補的な塩基配列を有 する核酸プローブを病型診断用マーカーとして好ましく用いることができる。また、当 該領域や対応遺伝子を増幅可能なプライマー (セット)等も病型診断用マーカーとし て用いることができる。さらに、こうした核酸プローブを固相担体に固定ィ匕した固定ィ匕 体 (アレイなど）を DLBCLの予後診断に好ましく用いることができる。さらに、病型診断 用マーカーは、ヒト染色体上の増幅領域や欠損領域の対応遺伝子によってコードさ れるタンパク質若しくはその一部又はこれらに対する抗体、該抗体を固相担体に固 定ィ匕した固定ィ匕体であってもよい。また、本発明によれば、こうした病型診断用プロ ーブ、プライマー（セット）、抗体、アレイなどの少なくとも 1つを含む病型診断用キット も提供される。 Based on the above, for DLBCL disease type diagnosis, it is possible to detect the amplified region or the corresponding gene and the defective region or the corresponding gene on the human chromosome associated with ABC type and GCB type. A nucleic acid probe having a complementary base sequence can be preferably used as a marker for diagnosis of disease type. In addition, primers (sets) that can amplify the region and the corresponding gene can be used as markers for diagnosis of disease type. Furthermore, an immobilized body (array or the like) in which such a nucleic acid probe is immobilized on a solid phase carrier can be preferably used for the prognosis of DLBCL. Furthermore, a marker for diagnosing a disease type is a protein encoded by a corresponding gene of an amplified region or a defective region on a human chromosome, or a part thereof, or an antibody against them, and a fixed enzyme in which the antibody is immobilized on a solid phase carrier. It may be a body. In addition, according to the present invention, such a disease type diagnosis pro A kit for diagnosing a disease type including at least one of a probe, a primer (set), an antibody, and an array is also provided.
[0049] (DLBCLの予後の診断方法）  [0049] (DLBCL prognosis diagnosis method)
本発明の予後の診断方法は、ヒトから採取した被験試料について、ヒト染色体 9p21 の欠損又は変異を検出する検出工程を備えている。 9p21に欠損がある場合には、 D LBCLの悪性度が高ぐ予後が不良であるからである。ヒト染色体 9p21の欠損を検出 するには、すでに説明したように、この染色体領域に対応する BACZPACクローン またはこのクローンから取得したゲノム DNA等などのプローブとのハイブリダィゼー シヨンを用いることができる。この場合、アレイ CGHなどの DNAアレイを用いることが できる。また、 PCR法による部位特異的増幅によって検出することができる  The prognosis diagnosis method of the present invention comprises a detection step of detecting a deletion or mutation of human chromosome 9p21 in a test sample collected from a human. This is because if 9p21 is deficient, D LBCL has a high grade of malignancy and a poor prognosis. In order to detect the deletion of human chromosome 9p21, as described above, hybridization with a probe such as a BACZPAC clone corresponding to this chromosomal region or genomic DNA obtained from this clone can be used. In this case, a DNA array such as array CGH can be used. It can also be detected by site-specific amplification by PCR
[0050] また、 9p21の遺伝子である pi 6INK4a遺伝子についてその欠損や変異をノヽイブリダィ ゼーシヨン等によって直接検出することもできるし、あるいは、 PCR法、 RT-PCR法、ノ ザンブロッテイング、 ReaKTime PCR法などを利用した発現解析によって検出すること もできる。発現解析による場合、被験試料中の当該遺伝子の発現量が健常者の発現 量よりも有意に少ない場合、 DLBCLにおいて予後不良であると診断することができる 。発現量が有意に少ないとは、健常者の同一遺伝子の発現量と比較して 60%以下、 好ましくは 50%以下、さらに好ましくは 20%以下、一層好ましくは 10%以下である。 [0050] In addition, the deletion or mutation of the pi 6 INK4a gene, which is the 9p21 gene, can be directly detected by a noble hybridization method, or PCR, RT-PCR, Northern blotting, ReaKTime PCR It can also be detected by expression analysis using a method or the like. In the expression analysis, when the expression level of the gene in the test sample is significantly smaller than the expression level of healthy subjects, it can be diagnosed that the prognosis is poor in DLBCL. The expression level is significantly less than 60% or less, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less compared to the expression level of the same gene in healthy individuals.
[0051] さらに、 pl6INK4a遺伝子によってコードするタンパク質の発現の有無、発現量、変異 を検出することによつてもよい。たとえば、こうしたタンパク質と特異的に結合する抗体 を用いることができる。抗体の取得等についてはすでに説明したのと同様の態様を適 用することができる。 [0051] Furthermore, the presence / absence, expression level, and mutation of the protein encoded by the pl6 INK4a gene may be detected. For example, an antibody that specifically binds to such a protein can be used. For obtaining antibodies, etc., the same mode as described above can be applied.
[0052] 以上のことから、 DLBCLの予後診断には、ヒト染色体の 9p21又はその一部を検出 可能に、この領域の少なくとも一部あるいはこれと相補的な塩基配列を有する核酸プ ローブを予後診断用マーカーとして好ましく用いることができる。プローブは、 pl6INK 4a遺伝子若しくはその一部又はこれらに相補的な塩基配列を有するものであってもよ い。また、当該領域や責任遺伝子を増幅可能なプライマー (セット)等も予後診断用 マーカーとして用いることができる。さらに、こうした核酸プローブを固相担体に固定 化した固定ィ匕体 (アレイなど）を DLBCLの予後診断に好ましく用いることができる。さ らに、予後診断用マーカーは、 pl6INK4a遺伝子によってコードされるタンパク質若しく はその一部又はこれらに対する抗体または該抗体を固相担体に固定ィ匕した固定ィ匕 体であってもよい。また、本発明によれば、こうした DBCL予後診断用プローブ、ブラ イマ一（セット）、抗体、アレイなどの少なくとも 1つを含む DBCL予後診断用キットも提 供される。 [0052] Based on the above, for the prognosis of DLBCL, a nucleic acid probe having at least a part of this region or a nucleotide sequence complementary thereto can be detected so that 9p21 of human chromosome or a part thereof can be detected. It can be preferably used as a marker. The probe may have a pl6 INK 4a gene, a part thereof, or a base sequence complementary thereto. In addition, primers (sets) that can amplify the region or responsible gene can also be used as prognostic markers. Furthermore, an immobilized carrier (array or the like) in which such a nucleic acid probe is immobilized on a solid phase carrier can be preferably used for the prognosis of DLBCL. The Further, the prognostic marker may be a protein encoded by the pl6 INK4a gene, a part thereof, an antibody thereto, or an immobilized antibody in which the antibody is immobilized on a solid phase carrier. Further, according to the present invention, a DBCL prognosis kit including at least one of such a DBCL prognosis probe, a primer (set), an antibody, an array and the like is also provided.
実施例  Example
[0053] 以下、開示された方法、及び請求の範囲に記載された方法の実施可能性を説明 するために実施例を示すが、本発明はこれらの実施例により限定されるものではなく 、本発明の範囲は、請求の範囲の記載された内容に基づき、その均等物も含むより 包括的なものとして判断される。  [0053] Examples are provided below to illustrate the feasibility of the disclosed methods and the methods described in the claims, but the present invention is not limited by these examples. The scope of the invention is determined to be more comprehensive, including equivalents thereof, based on the contents described in the claims.
[0054] (実験方法）  [0054] (Experimental method)
(患者及び試料）  (Patient and sample)
リンパ節の試料及び臨床データを施設内審査委員会の承認を得たプロトコルにより 患者 99名（CD5+36例、 CD— CD10+19例、 CD5— CD 10— 44例）から得た。 CD5+3例は CD1 0+を有していた。特記すべきことは、一般集団におけるよりも高い比率となった合計 36 症例の CD5+DLBCLを本疾病の遺伝的状態の評価用に収集したことである。この DL BCL 99例にアレイ CGH法を用いてゲノム不均衡の解析を行った。この中の 46例（CD 5+ 22例、 CD5— CD10+ 7例、 CD5— CD10—の 17例）に引き続き遺伝子発現プロフアイリン グを行った。また、これらの患者は悪性リンパ腫の既往歴を有していな力つた。全て の DLBCL患者には診断後適切な用量のシクロホスフアミド、アドリアマイシン、ビンタリ スチン、及びプレドニン（CHOP)などの治療薬が投与され力 DNA及び RNA試料は 全て、診断時に治療薬が投与される前に腫瘍から採取した。表 1に全患者の年齢、 病期、パフォーマンスステータス、乳酸脱水素酵素 (LDH)、節外病変数、及び診断 時の国際予後指標（International Prognostic Index (IPI))のデータを示してある。  Lymph node samples and clinical data were obtained from 99 patients (CD5 + 36 cases, CD-CD10 + 19 cases, CD5-CD10-44 cases) according to the protocol approved by the institutional review board. Three CD5 + cases had CD10 +. Notably, a total of 36 cases of CD5 + DLBCL, a higher proportion than in the general population, were collected for assessment of the genetic status of the disease. The 99 cases of DL BCL were analyzed for genomic imbalance using the array CGH method. Of these, 46 cases (CD 5+ 22 cases, CD5—CD10 + 7 cases, CD5—CD10—17 cases) were followed by gene expression profiling. In addition, these patients had no history of malignant lymphoma. All DLBCL patients will receive therapeutic doses such as cyclophosphamide, adriamycin, vintalistin, and predonin (CHOP) after diagnosis, and all force DNA and RNA samples will be administered before treatment at diagnosis. From the tumor. Table 1 shows data on age, stage, performance status, lactate dehydrogenase (LDH), number of extranodal lesions, and International Prognostic Index (IPI) at diagnosis for all patients.
[0055] 表 1 DLBCLサブグループの臨床的特徴 CD5+ CD5"CD10+ CD5"CD10" Total ABC GCB Total[0055] Table 1 Clinical characteristics of DLBCL subgroup CD5 + CD5 "CD10 + CD5" CD10 "Total ABC GCB Total
Characteristic no. (%) no. (%) no. (%) no. (%) no. (%) Characteristic no. (%) No. (%) No. (%) No. (%) No. (%)
Age (yr)  Age (yr)
Median (yr) 62 65 57 60 67 63 64  Median (yr) 62 65 57 60 67 63 64
Range 36-82 48-89 26-91 26-91 36-91 38-89 36-91 Range 36-82 48-89 26-91 26-91 36-91 38-89 36-91
No. of patients a n=36 n=19 n=44 n=9 n=28 n=18 n=46b No. of patients a n = 36 n = 19 n = 44 n = 9 n = 28 n = 18 n = 46 b
Ann Arbor stage Ann Arbor stage
I-II 5(15) 8(42) 17(49) 30 5(20) 7(41) 12 I-II 5 (15) 8 (42) 17 (49) 30 5 (20) 7 (41) 12
III-IV 29 (85) 11 (58) 18(51) 58 20 (SO) 10(59) 30III-IV 29 (85) 11 (58) 18 (51) 58 20 (SO) 10 (59) 30
No. of patients a 34 19 35 88 25 17 42No. of patients a 34 19 35 88 25 17 42
Performance status Performance status
0-1 24 (71) 16(84) 30 (97) 70 16(67) 16(100) 32 0-1 24 (71) 16 (84) 30 (97) 70 16 (67) 16 (100) 32
>2 10(29) 3(16) 1 (3) 14 8(33) 0(0) 8> 2 10 (29) 3 (16) 1 (3) 14 8 (33) 0 (0) 8
No. of patients 3 34 19 31 84 24 16 40No. of patients 3 34 19 31 84 24 16 40
LDHC LDH C
Normal 8(24) 12 (63) 12(38) 32 6(25) 3(19) 9 Normal 8 (24) 12 (63) 12 (38) 32 6 (25) 3 (19) 9
High 25 (76) 7(37) 20 (62) 52 18(75) 13 (81) 31High 25 (76) 7 (37) 20 (62) 52 18 (75) 13 (81) 31
No. of patients a 33 19 32 84 24 16 40No. of patients a 33 19 32 84 24 16 40
No. of extranodal sites No. of extranodal sites
<1 24 (73) 13(41) 25 (78) 62 17(71) 13 (81) 30 <1 24 (73) 13 (41) 25 (78) 62 17 (71) 13 (81) 30
>1 9(27) 6(59) 7(22) 22 7(29) 3(19) 10> 1 9 (27) 6 (59) 7 (22) 22 7 (29) 3 (19) 10
No. of patients a 33 19 32 84 24 16 40No. of patients a 33 19 32 84 24 16 40
IPI index IPI index
L/LI 13 (54) 9(47) 20 (65) 42 5(19) 7(41) 12 謹 I 21 (46) !0(53) 11(35) 42 21 (81) 10(59) 31 L / LI 13 (54) 9 (47) 20 (65) 42 5 (19) 7 (41) 12 謹 I 21 (46)! 0 (53) 11 (35) 42 21 (81) 10 (59) 31
No. of patients 1 34 19 31 84 26 17 43 a臨床データが入手できた患者数。 No. of patients 1 34 19 31 84 26 17 43 aNumber of patients for whom clinical data were available.
b 99症例にっ 、てアレイ CGH解析を行 1、（左 、'ネル）、その内 46症例につ V、て遺伝子発現 プロフアイリングを行った (右ノ ネル)。  b Array CGH analysis was performed in 99 cases, 1 (left, 'nel), and 46 of them were subjected to gene expression profiling (right).
c LDH:乳酸脱水素酵素。  c LDH: lactate dehydrogenase.
(遺伝子発現プロフアイリング） (Gene expression profiling)
塩ィ匕セシウム遠心法を用いて各試料力も全 RNAを抽出した。 Cy5-又は Cy3-を標識 した相補的 RNA(cRNA)を全 RNAから Low RNA Input Amplification Kit (Agilent Tech nologies, Palo Alto, CA)を用いて合成した。プローブは実験用の Cy5-標識 cRNA及 びコントロールの Cy3-標識 cRNAのミクスチヤ一であった。後者はリンパ節過形成の 1 0試料力も抽出した全 RNAのプール力も調製した。ガラススライドマイクロアレイは、財 団法人癌研究会の癌研究所のために注文作成された合計 21619の遺伝子がスポット されている Agilentオリゴヌクレオチドアレイ（Agilent Technologies)を使用した。プロ一 ブを In Situ Hybridization Kit Plus (Agilent Technologies)を用いて製造者のプロトコ ノレ〖こ従 、ガラススライド上でー晚ハイブリダィズした。ハイブリダィズしたマイクロアレイ の蛍光イメージを Agilent社スキャナ G2565AA (Agilent Technologies)により取得し、さ らに Feature Extractionソフトウェア（Agilent Technologies)を用いて解析し、 Cy3-標 識コントロールに対する実験用 Cy5_標識試料の蛍光比を算出した。全てのフラグが 立てられて！/、な 、 (non-flagged)蛍光比は対数変換 (底は 2)し、クラスタ解析カも算出 した各遺伝子の中央値を引くことにより中央ィ匕した。階層的クラスタアルゴリズムを Clu ster ana !YeeViewソフトゥエフ (http://rana.lbl. gov/Eisen¾oftware.ntm)を禾 lj用して、 これらの遺伝子の発現レベルに従い DLBCLの症例に用いた（Eisen MB, Spellman P Ί , Brown ΡΟ, et al. し luster analysis and display of genome-wide expression pattern s. Proc Natl Acad Sci U S A. 1998; 95: 14863— 1486813。 Rosenwald et al (2002 ; http:/ /llmpp.nih.gov/DLBCL)によって ABC及び GCBグループのクラスタリング用に特定さ れた 100の遺伝子の中で、 67種がクラスタ解析に利用可能であった（Rosenwald A, W right G, し han Wし, et al. The use of molecular proniing to predict survival after che motherapy for diffiise large— B— cell lymphoma. N Engl J Med.2002; 346: 1937— 1947) 8 。これらの 67の遺伝子とは、 BARD1、 PIK3CG、 LRMP、 Hs. l098、 BCL6、 HDAC1、 M YBL2、 MME (CD10)、 STAG3、 LM02、 APS、 Hs. l51051、 ADPRT、 ITPKB、 REL、 FLJ 20094、 Hs.211563、 MEF2B、 CD44、 Hs.75765、 IL6、 PTPN2、 PTPN12、 BMI1、 Hs.12 8003、 BACH2、 HIVEP1、 CFLAR、 APAF1、 RYKゝ EDG1、 KIAA0874, Hs.153649、 M ADH4、 PTPN1、 Hs.93213、 DCTDゝ Hs.193857、 IL16、 SP140、 SH3BP5、 IRF4 (MUM 1)、 TLK1、 KCNA3、 TCL1A、 PAK1、 Hs. l88、 CXCR4、 SLAゝ CCND2、 TGFBR2、 ET V6、 SPAP1、 PM5、 PDIR、 IGHM、 CD22、 Hs.296938、 Hs. l565、 Hs.83126、 MAPKAP K3、 RUNX1、 Hs.55947, S100A4、 TFAP4、 IRF2、及び OPA1である。この 67の遺伝子 を用いて報告されて ヽるマイクロアレイデータにクラスタリング解析を実施した。 Lymp hochipマイクロアレイから得られた DLBCL遺伝子発現プロファイルのデータを http:〃l lmpp.nih.gov/DLBCLのサイトの参照資料 8に含まれる補足情報から取得した。我々 は Lymphochipマイクロアレイのデータセットの 274の DLBCLが ABC及び GCBそして、 タイプ 3に分けることが可能であることを確認した。上記の 67の遺伝子を持つ腫瘍の 分布は Rosenwald et afによって報告されている 100の遺伝子とほぼ同じであった。 Total RNA was also extracted for each sample force using a salted cesium centrifuge. Cy5- or Cy3-labeled complementary RNA (cRNA) was synthesized from total RNA using the Low RNA Input Amplification Kit (Agilent Technologies, Palo Alto, Calif.). The probe was a mix of experimental Cy5-labeled cRNA and control Cy3-labeled cRNA. The latter is a form of lymph node hyperplasia 1 The pool power of the total RNA extracted as well as 0 sample power was prepared. The glass slide microarray used was an Agilent oligonucleotide array (Agilent Technologies) spotted with a total of 21619 genes custom-made for the Cancer Research Institute of the Foundation for Cancer Research. The probe was hybridized on a glass slide using the manufacturer's protocol using In Situ Hybridization Kit Plus (Agilent Technologies). Fluorescence images of hybridized microarrays were acquired with an Agilent scanner G2565AA (Agilent Technologies), analyzed using Feature Extraction software (Agilent Technologies), and the fluorescence ratio of experimental Cy5-labeled samples to Cy3-labeled controls. Was calculated. All the flags are raised! /,, (Non-flagged) The fluorescence ratio was logarithmically converted (bottom 2), and the center value of each gene calculated by the cluster analysis was subtracted. The cluster cluster algorithm Cluster ana! YeeView software (http://rana.lbl.gov/Eisen¾oftware.ntm) was used for DLBCL cases according to the expression level of these genes (Eisen MB, Spellman PΊ, BrownΡΟ, et al. And luster analysis and display of genome-wide expression pattern s. Proc Natl Acad Sci US A. 1998; 95: 14863— 14868 13. Rosenwald et al (2002; http: / / llmpp Of the 100 genes identified for clustering ABC and GCB groups by .nih.gov / DLBCL), 67 were available for cluster analysis (Rosenwald A, W right G, and han W N Engl J Med. 2002; 346: 1937— 1947) 8. These 67 genes are BARD1, PIK3CG, and the use of molecular proniing to predict survival after chemotherapy for diffiise large— B— cell lymphoma. , LRMP, Hs. L098, BCL6, HDAC1, M YBL2, MME (CD10), STAG3, LM02, APS, Hs. L51051, ADPRT, ITPKB, REL, FLJ 20094, Hs.211563, MEF2B, CD44, Hs.75765, IL6, PTPN2, PTPN12, BMI1, Hs.12 8003, BACH2, HIVEP1, CFLAR, APAF1, RYK ゝ EDG1, KIAA0874, Hs.153649, MADH4, PTPN1, Hs.93213, DCTD ゝ Hs.193857, IL16, SP140, SH3BP5, IRF4 (MUM 1), TLK1, KCNA3, TCL1A, PAK1, Hs.l88, CXCR4, SLA ゝ CCND2, TGFBR2, ET V6, SPAP1, PM5, PDIR IGHM, CD22, Hs.296938, Hs.l565, Hs.83126, MAPKAP K3, RUNX1, Hs.55947, S100A4, TFAP4, IRF2, and OPA1. Clustering analysis was performed on the microarray data reported using these 67 genes. DLBCL gene expression profile data obtained from Lymp hochip microarrays: http: 〃l It was obtained from the supplementary information contained in reference material 8 of the lmpp.nih.gov/DLBCL site. We have confirmed that the 274 DLBCLs in the Lymphochip microarray dataset can be divided into ABC and GCB and type 3. The distribution of the above 67 genes was almost the same as the 100 genes reported by Rosenwald et af.
[0057] (アレイ CGH法）  [0057] (Array CGH method)
ACCアレイスライドバージョン 4.0のスライドガラスを用いた以前に報告された方法に より、 DLBCL症例についてアレイ CGH解析を実施した。このアレイは 2304種の BAC ( 細菌人工染色体）と PAC (P-1由来人工染色体）のクローン（BAC/PACクローン）から 成り、全ヒトゲノムを約 1.3Mb (メガベース）の解像度でカバーしているものである。 BA Cクローンは RP11及び RP13ライブラリ力 入手し、 PACクローンは RP1、 RP3、 RP4、及 び RP5ライブラリ力も入手した。 10ngの BAC/PAC DNAをテンプレートに用いて縮重 オリゴヌクレオチド（5，- CCGACTCGAGNNNNNNATGTGG- 3，、 Nは、 A,T,Cおよび G のいずれかである。）をプライマーとして用いた PCR (oligonucleotide primed PCR) (H akan, T. et al" Genomics, 13:718-725, 1992)を行った。増幅は、 TaKaRa PCR ther mal Cycler MP(Takara、 Tokyo, Japan)と ExTaqポリメラーゼ (TaKaRa)得られた PCR産 物をエタノール沈殿により濃縮し、蒸留水で溶解し、続いて等量の DNAスポッティン グ溶液 DSP0050 (MATSUNAMI, Osaka, Japan)を添加し (；〜 1 g/ 1)、インクジェット 技術（NGK, Nagoya, Japan)によりロボットで CodeLink™活性スライド（Amersham Bios ciences, Piscataway, NJ)上にデュプリケートでスポットした。なお、使用した BAC/PA しクロ■ ~"ンは NIBC (http://www.ncbi.mm.nih.gov/)及び Ensembl uenome Data Resou rces (http://www.ensembl.org/)からの情報に基づ 、て選択した。これらのクローンは ,J、児病! ¾ (Children s Hospital) (Oakland Research Institute, Oa land, し A: http://b acpa chori.org/)の BACPACリリースセンターから入手した。  Array CGH analysis was performed on DLBCL cases using a previously reported method using ACC array slide version 4.0 glass slides. This array consists of 2304 BAC (bacterial artificial chromosomes) and PAC (P-1 derived artificial chromosomes) clones (BAC / PAC clones) that cover the entire human genome with a resolution of about 1.3 Mb (megabase). It is. BAC clones were obtained for RP11 and RP13 libraries, and PAC clones were also obtained for RP1, RP3, RP4, and RP5 libraries. PCR (oligonucleotide primed) using 10 ng of BAC / PAC DNA as a template and degenerate oligonucleotides (5,-CCGACTCGAGNNNNNNATGTGG-3, N is one of A, T, C and G) as primers PCR) (Hakan, T. et al "Genomics, 13: 718-725, 1992). Amplification was obtained from TaKaRa PCR thermal Cycler MP (Takara, Tokyo, Japan) and ExTaq polymerase (TaKaRa). The PCR product is concentrated by ethanol precipitation, dissolved in distilled water, followed by addition of an equal volume of DNA spotting solution DSP0050 (MATSUNAMI, Osaka, Japan) (; ~ 1 g / 1), inkjet technology (NGK, (Nagoya, Japan) was spotted in duplicate on a CodeLink ™ active slide (Amersham Biosciences, Piscataway, NJ) by a robot.The BAC / PA Shimuro used was used by NIBC (http: //www.ncbi .mm.nih.gov /) and Ensembluenome Data Resources (http://www.ensembl.org/) Selected. These clones were obtained from the BACPAC Release Center of J, Children's Disease! ¾ (Childrens Hospital) (Oakland Research Institute, Oa land, A: http: // b acpa chori.org/).
[0058] DNA調製、標識、アレイ作製及びハイブリダィゼーシヨンは以前の報告に従って実施 し 7こ (Ota A, I'agawa H, Karnan ¾, et al. Identincation and characterization or a nove 1 gene, C13orf25, as a target for 13q31— q32 amplification in malignant lymphoma, C ancer Res. 2004; 64: 3087—3095、 Tagawa H, Tsuzuki S, Suzuki R, et al. Genome— w ide array-based comparative genomic hybridization of diffuse large B— cell lymphoma: comparison between CD5— Positive and CD5— negative cases. Cancer Res. 2004; 64: 5948-5955) oすなわち、試験 DNAおよび対照 DNA (各 1 μ g)を Dpnllで消化し、 Bio prime DNA Laoeling system (Invitrogen Life technologies, Inc, Tokyo, Japan)【こより 、 Cy3— dUTP及び Cy5— dUTP(Amersham Pharmacia Biotech, Piscataway, NJ)を用い て標識した。標識化された 1 μ gの試験 DNAおよび対照 DNAを、 50〜： LOO /z g (どち らでも同じ結果がでるので 50〜100 /z gとしました:瀬戸）の Cot-1 DNA(Life Technol ogies, Inc., Gaithersburg, MD)と混合し沈殿させて、 45 1のハイブリダィゼーシヨン 溶液（50vol%ホルムアミド、 10%硫酸デキストラン、 2x SCC、 4%SDS及び 10 μ &/ μ 1 tRNA)に再懸濁させた。このハイブリダィゼーシヨン容器を、 73°Cで 5分間加熱して DNAを変性させ、続!、て 37°Cで 45分間インキュベートして反復配列のブロッキング がなされるようにした。 DNAをスポットしたスライドは、 70%ホルムアミド Z2xSSCを含 む溶液中で 73°Cで 4分間にわたり変性させ、続いて 5分づつ、 70%、 85%、 100% 冷エタノール中で脱水して風乾させた。緩やかに振動するテーブル上で、湿度調節 のために 200 μ 1の 50%ホルムアミドと 2xSSCを含む容器中において 48〜66時間の ハイブリダィゼーシヨン後、スライドを洗浄（50%ホルムアミド Z2xSSC中、 50°Cで 15分 間、 2xSSCZ0.1%SDS中、 50°Cで 30分間、 0.1M Na HPO 、 pH8.0の 0.1M Na HPO [0058] DNA preparation, labeling, array generation and hybridization were performed according to previous reports (Ota A, I'agawa H, Karnan ¾, et al. Identincation and characterization or a nove 1 gene, C13orf25 , as a target for 13q31— q32 amplification in malignant lymphoma, Cancer Res. 2004; 64: 3087-3095, Tagawa H, Tsuzuki S, Suzuki R, et al. Genome— wide array-based comparative genomic hybridization of diffuse large B— cell lymphoma: Comparison between CD5— Positive and CD5— negative cases. Cancer Res. 2004; 64: 5948-5955) o That is, test DNA and control DNA (1 μg each) were digested with Dpnll, and Bio prime DNA Laoeling system (Invitrogen Life (Technology, Inc, Tokyo, Japan) [From this, Cy3-dUTP and Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ) were used for labeling. Label 1 μg of test DNA and control DNA with 50–: LOO / zg (50-100 / zg because both gave the same result: Seto) Cot-1 DNA (Life Technologies ogies , Inc., Gaithersburg, MD) and precipitated into 45 1 hybridization solution (50 vol% formamide, 10% dextran sulfate, 2x SCC, 4% SDS and 10 μ & / μ 1 tRNA). Resuspended. The hybridization vessel was heated at 73 ° C for 5 minutes to denature the DNA, followed by incubation at 37 ° C for 45 minutes to allow repetitive sequence blocking. Slides spotted with DNA were denatured in a solution containing 70% formamide Z2xSSC for 4 minutes at 73 ° C, followed by dehydration in 70%, 85%, 100% cold ethanol and air-dried every 5 minutes. It was. Wash the slide after hybridization for 48-66 hours in a container containing 200 μl 50% formamide and 2xSSC for humidity control on a gently vibrating table (50% in formamide Z2xSSC, 50 15 min at ° C, in 2xSSCZ 0.1% SDS, 30 min at 50 ° C, 0.1M Na HPO, pH 8.0, 0.1M Na HPO
2 4 2 4 2 4 2 4
、 0.1%NP-40により構成される NP緩衝液中、室温で 15分間）を行い、 2xSSC中室温 ですすぎを行い、最後に室温にてそれぞれ 2分づっ 70%、 85%及び 100%エタノー ル中で脱水して風乾した。スキャニング分析は、アジレントマイクロアレイスキャナー（ Agilent Technologies, Palo Alto, CA)によりスキャンし、得られたアレイイメージを Gen epix Pro 4.1 (Axon Instruments, Inc., Foster City, CA)により解析した。すなわち、 D ポットを自動的に分割し、局所的なバックグラウンドを減算して、シグナル強度 を決定した。引き続き、 2種の色素（Cy3強度 ZCy5強度）のシグナル強度の比を各 スポットにつ 、て計算し、エタセルシート上にぉ 、て染色体における位置の順で log Rinsing in NP buffer composed of 0.1% NP-40 for 15 minutes at room temperature), rinsing in 2xSSC at room temperature, and finally 70%, 85% and 100% ethanol for 2 minutes each at room temperature It was dehydrated and air dried. Scanning analysis was scanned with an Agilent microarray scanner (Agilent Technologies, Palo Alto, Calif.), And the resulting array images were analyzed with Gen epix Pro 4.1 (Axon Instruments, Inc., Foster City, Calif.). That is, the signal intensity was determined by automatically dividing the D pot and subtracting the local background. Subsequently, the ratio of the signal intensity of the two dyes (Cy3 intensity ZCy5 intensity) is calculated for each spot, and the log is displayed in the order of the positions on the chromosomes on the etacel sheet.
2 比に変換した。  Converted to 2 ratio.
このアレイには、健常男性に対する健常男性の同時ノヽイブリダィゼーシヨンを 10回実 施し、 log比の正常な変動を明らかにした。蛍光強度が全クローンの平均値の 10%未 The array was subjected to simultaneous normalization of healthy men 10 times with respect to healthy men and revealed normal fluctuations in the log ratio. Fluorescence intensity is not 10% of the average of all clones
2  2
満で、参照群に対する試験群の比の最も極端な 1.0からの平均偏差を有し、この健常 対照者のデータセットにおいて最大の標準偏差を有する合計で 91クローンをさらなる 解析から除外した。従って、合計 2213クローン（1.3Mbの解像度で 2988Mbをカバー） に次の解析を行った。この 2213の中の 2158クローン（2834Mbをカバー）は染色体 lp のテロメァから 22qのテロメァ由来であった。残る 55クローンは X染色体由来であった。 With a mean deviation from the most extreme 1.0 of the ratio of the test group to the reference group, this healthy A total of 91 clones with the largest standard deviation in the control data set were excluded from further analysis. Therefore, the following analysis was performed on a total of 2213 clones (covering 2988 Mb at 1.3 Mb resolution). Of these 2213 clones, 2158 clones (covering 2834Mb) were derived from telomeres of chromosome lp to 22q. The remaining 55 clones were derived from the X chromosome.
[0060] 各スポット（2 X 2191クローン）に測定した蛍光強度の log比の値の 96%超が + 0.2か [0060] More than 96% of the log ratio of fluorescence intensity measured for each spot (2 X 2191 clones) is +0.2
2  2
らー 0.2までの範囲であったことから、この増幅と欠損の log比の閾値をそれぞれ +0.  Since it was in the range up to 0.2, the log ratio threshold of this amplification and deletion was +0.
2  2
2と 0.2に設定した。低レベルのゲイン Z増幅の領域を +0.2から + 1.0、ヘテロ接合 体のロス Z欠損を含む領域を 1.0から 0.2、高レベルのゲイン Z増幅を示す領域 を〉 + 1.0、ホモ接合体のロス Z欠損を示唆する領域をく— 1.0の log比と定めた。我  Set to 2 and 0.2. Low-level gain Z amplification region +0.2 to +1.0, heterozygous loss Z-defect region 1.0 to 0.2, high-level gain Z amplification region> +1.0, homozygous loss Z The region suggesting a defect was defined as a log ratio of 1.0. I
2  2
々は増幅又は欠損の領域の定義を、（a)連続する 3クローンが増幅又は欠損を示す、 又は (b) 1つのクローンが反復して高コピー数の増幅 (log比 > + 1.0)又はホモ接合  The definition of the region of amplification or deletion is: (a) 3 consecutive clones exhibit amplification or deletion, or (b) one clone repeats high copy number amplification (log ratio> + 1.0) or homology Joining
2  2
体の欠損 (log比 < 1.0)を示すこととした11'12。高レベルのゲイン Z増幅の領域、及 Loss of body 11 was to show (log ratio <1.0) '12. High level gain Z amplification area, and
2  2
びホモ接合性のロス Z欠損の領域も容易に検出され、また低レベルのゲイン Z増幅 及びへテロ接合性のロス z欠損の領域も同様であった。  Homozygous loss Z-deficient areas were easily detected, as were low-level gain Z amplification and heterozygous loss z-deficient areas.
[0061] (統計解析）  [0061] (Statistical analysis)
DLBCLの各サブグループの全生存率の統計解析をログランク検定により実施した 。有意性を示すため P< 0.05とした。  Statistical analysis of the overall survival of each subgroup of DLBCL was performed by log rank test. P <0.05 to show significance.
[0062] (アレイ CGH解析の統計解析） [0062] (Statistical analysis of array CGH analysis)
2つの患者群 (すなわち、 ABC群と GCB群）間のゲノム領域に統計的有意差がある かを解析するため、ゲノムの変化を log比が + 0.2以上のコピー数の増幅、及び log比  To analyze whether there is a statistically significant difference in the genomic region between the two patient groups (ie ABC group and GCB group), the genomic change was amplified by copy number amplification with log ratio + 0.2 or more, and log ratio
2 2 がー 0.2以下のコピー数の欠損として規定してデータセットを構築した。増幅 (log比  The data set was constructed with 2 2 defined as a copy number loss of -0.2 or less. Amplification (log ratio
2 2
≥ +0.2)を示すクローンに「1」、これに対して増幅を示さないクローン (log比く +0.2 ≥ +0.2) is `` 1 '' for clones that show no amplification (+0.2 compared to log)
2  2
)に「0」を各症例の Excelテンプレートに入力した。同様に、欠損を示したクローン (log 比≤ -0.2)に「1」、欠損を示さな力つたクローン (log比 >—0.2)に「0」を各症例毎の ) Was entered in the Excel template for each case. Similarly, "1" is assigned to clones showing a defect (log ratio ≤ -0.2) and "0" is assigned to clones showing no defect (log ratio> -0.2) for each case.
2 2 twenty two
もう 1つの Excelテンプレートに入力した。その後、データ解析を次の目的、すなわち i) クローン毎の増幅又は欠損の頻度の 2群 (すなわち ABC群と GCB群）間の比較、 ii) 1 つのクローンの増幅又は欠損を示す症例と増幅又は欠損を示さない症例間の全生 存率の比較をするために実施した。前者の比較にはフィッシャーの直接確率検定を 用い、後者の 2群間の生存曲線の比較にはログランク検定を用いた。 Entered in another Excel template. Data analysis is then performed for the following purposes: i) comparison of the frequency of amplification or deletion per clone between two groups (ie ABC group and GCB group), ii) case of amplification or deletion of one clone This was done to compare the overall survival rate among cases that did not show defects. Fisher's exact test is used for the former comparison. A log rank test was used to compare the survival curves between the latter two groups.
[0063] (遺伝子発現の統計解析） [0063] (Statistical analysis of gene expression)
ABC群と GCB群間の pl6INK4a発現レベルの有意差の検出にマンホイット-一の U検 定を用いた。統計解析は全て STATA ver.8統計解析パッケージ（StataCorp, CollegeThe Mann-Whit-1 U test was used to detect significant differences in pl6 INK4a expression levels between the ABC and GCB groups. Statistical analysis is all STATA ver.8 statistical analysis package (StataCorp, College
Station, TX)により実施した。 Station, TX).
[0064] (結果） [0064] (Result)
1. CD5+、 CD5— CD10+、及び CD5— CD10—の DLBCLの遺伝子発現プロフアイリング： AB C DLBCLと GCB DLBCLの関連性  1. CDB +, CD5—CD10 +, and CD5—CD10— DLBCL gene expression profiling: the relationship between AB C DLBCL and GCB DLBCL
DLBCLの種類は遺伝子発現プロフアイリング及び細胞表面フエノタイピング（cell su rface phenotyping)によって特徴付け及び分類が可能である。臨床的に、 ABC DLBC Lは GCB DLBCLよりも悪性の挙動を示す。 CD5+ DLBCL症例の生存期間は短ぐ C D10+ DLBCLは比較的緩慢である（図 1)。 CD5及び/又は CD10マーカーを有する D LBCLが ABC及び GCBサブグループと関連があるかどうかを決定するため、合計 DLB CL46例（CD5+ 22例、 CD5— CD10+ 7例、 CD5— CD10— 17例）に遺伝子発現プロファイリ ングを行った。この結果から、これら 46例は ABC群又は GCB群のどちらかに明確に分 けられることが示され（図 2)、また特に重要なことに、 CD5+及び CD5— CD10+表現型が それぞれ ABC及び GCBサブグループと密接に関連することが示された (表 2)。 CD5+ DLBCL 22例中、 19例が ABC signatureを示し、 3例のみ（CD5+CD10+が 2例、 CD5+CD 10一が 1例）が GCB signatureによって特徴付けられた（P= 0.0009)。これとは著しく対 照的に、 CD5— CD10+ DLBCLの 7例全て力 GCB signatureを示した（P=0.0031)。 CD5 "CD 10" DLBCLの症例では、 ABC (9例）又は GCB (8例）の signatureが示されると!ヽぅ 混合した結果が得られ (P= 0.5335)、これが異質性の疾患であることが示唆された。  DLBCL types can be characterized and classified by gene expression profiling and cell surface phenotyping. Clinically, ABC DLBC L behaves more malignantly than GCB DLBCL. CD5 + DLBCL cases have a short survival time. CD10 + DLBCL is relatively slow (Figure 1). To determine whether D LBCL with CD5 and / or CD10 markers is associated with ABC and GCB subgroups, a total of 46 DLB CLs (22 CD5 +, 7 CD5—CD10 +, 17 CD5—CD10—17) Gene expression profiling was performed. The results show that these 46 cases are clearly divided into either ABC or GCB groups (Figure 2), and most importantly, the CD5 + and CD5—CD10 + phenotypes are ABC and GCB, respectively. It was shown to be closely related to subgroups (Table 2). Of the 22 CD5 + DLBCL cases, 19 showed ABC signatures, and only 3 cases (2 CD5 + CD10 + cases and 1 CD5 + CD10 case) were characterized by GCB signature (P = 0.0009). In marked contrast, all seven cases of CD5—CD10 + DLBCL showed a strong GCB signature (P = 0.0031). In the case of CD5 "CD 10" DLBCL, a signature of ABC (9 cases) or GCB (8 cases) is shown! ヽ ぅ Mixed results are obtained (P = 0.5335), which is a heterogeneous disease Was suggested.
[0065] 表 2 遺伝子プロファイルによるサブグループ間の関連性 DLBCL subtype ABC GCB P a value [0065] Table 2 Association between subgroups by gene profile DLBCL subtype ABC GCB P a value
CD5+ b 19 3 0.0009 CD5 + b 19 3 0.0009
CD5 CD10+ 0 7 0.0031  CD5 CD10 + 0 7 0.0031
CD5 CD10" 9 8 0.5335  CD5 CD10 "9 8 0.5335
a P値はフィッシャーの直接確率検定による。  a P value is based on Fisher's exact test.
b CD5+ DLBCL症例は、 CD10+の 3症例を含む。  b CD5 + DLBCL cases include 3 cases of CD10 +.
[0066] 2. ABC DLBCL及び GCB DLBCLのゲノム不均衡 [0066] 2. Genomic imbalance of ABC DLBCL and GCB DLBCL
ABC及び GCBの DLBCLは分子的に異なるサブグループであることが以前に証明さ れているため6—8、我々は最初にこれらのサブグループのゲノムプロファイルを比較し た。 CD5— CD10—の 2症例の ABC signatureを有する 1例と GCB signatureを有する 1例の 典型的な 2つのゲノムプロファイルを図 3に示す。 DLBCL of ABC and GCB are molecularly different because it is subgroup has been demonstrated previously 6 - 8, we first compared the genomic profiles of these subgroups. Figure 3 shows two typical genomic profiles of one CD5—CD10— case with ABC signature and one case with GCB signature.
[0067] ABC群（≥ 6例）における高頻度のゲノム不均衡 (コピー数の変化）は、染色体 3、 8q 21- q26、 llq21- q25、 16pll- pl3、 16q22- q24、 18、 19ql3、 Xの増幅、及び染色体 2pl 1 (Igk遺伝子座）、 6ql2- 27、 8p22- p23、 9p21、 17pの欠損であった。 GCB群（≥4例） における高頻度のゲノム不均衡は、 lq22_32、 2pl4_p24、 5pl2_p 、 5ql5_q31、 6pl2- p25、 7、 8q22— q26、 9q33— q34、 llq、 12、 13q31— q33、 16pll— pl3、 18q21— q23、 19p、 1 9ql3、 21q、 Xの増幅、及び 1ρ36、 2pll、 3pl4、 4pl2- pl3、 4q33- q34、 6ql4- ql6、 8p2 2-p23、 9p21、 13ql2-q22、 17pl2、 18q22-q23の欠損であった。ゲノムの高頻度の増 幅及び欠損とは各群の 20%以上と定義した。  [0067] Frequent genomic imbalances (copy number changes) in the ABC group (≥6 cases) are chromosome 3, 8q 21-q26, llq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3, X And the loss of chromosome 2pl 1 (Igk locus), 6ql2-27, 8p22-p23, 9p21, 17p. Frequent genomic imbalances in the GCB group (≥4 cases) are lq22_32, 2pl4_p24, 5pl2_p, 5ql5_q31, 6pl2-p25, 7, 8q22—q26, 9q33—q34, llq, 12, 13q31—q33, 16pll—pl3, 18q21— q23, 19p, 1 9ql3, 21q, X amplification, and 1ρ36, 2pll, 3pl4, 4pl2-pl3, 4q33-q34, 6ql4-ql6, 8p2 2-p23, 9p21, 13ql2-q22, 17pl2, 18q22-q23 Deficiency. High-frequency genomic amplification and deletion were defined as 20% or more of each group.
[0068] ABC群はより高頻度の 3p23- q28、 18qll.2- q23、 9ql3.41- ql3.43の増幅、及び 6q22.  [0068] ABC group is more frequently amplified 3p23-q28, 18qll.2-q23, 9ql3.41-ql3.43, and 6q22.
31- q24.1と 9p21.3の欠損というゲノム的特徴を有していた。 GCB群はより高頻度の lq2 1.1- q23.3、 lq31.1- q42.13、 2pl5- pl6.1、 7q22.1- q36.2及び 12ql3.1- ql4の増幅という ゲノム的特長を有していた（フィッシャーの直接確率法、 P< 0.05)。 ABC及び GCB D LBCLのゲノム不均衡のイデォグラムを図 4A-Bに、 ABC及び GCB DLBCLの各群の ゲノム不均衡のゲノムワイドな頻度を図 5Aに示してある。  31- q24.1 and 9p21.3 were deficient in genomic character. The GCB group has the genomic features of higher frequency lq2 1.1-q23.3, lq31.1-q42.13, 2pl5-pl6.1, 7q22.1-q36.2 and 12ql3.1-ql4 (Fischer's exact method, P <0.05). The ABC and GCB D LBCL genomic imbalance ideograms are shown in Figure 4A-B and the ABC and GCB DLBCL groups of genomic imbalances are shown in Figure 5A.
我々は ABC及び GCB DLBCLのゲノム不均衡のパターンが明確に異なることを見出 した。例えば、染色体 3q23-q28の増幅は ABC群の 25〜36%に認められ、 GCB群で は認められなかったが（0%)、 7q22-q36の増幅は GCB群の 50〜61%に認められ、 AB C群では遥かに少な力つた（< 5%)。 We found that the pattern of genomic imbalance between ABC and GCB DLBCL is distinctly different. For example, chromosome 3q23-q28 amplification is observed in 25-36% of the ABC group, and in the GCB group. Was not observed (0%), but 7q22-q36 amplification was observed in 50-61% of the GCB group, with much less power in the ABC group (<5%).
[0069] CD5の発現は ABC及び GCB群に認められたゲノム不均衡に影響を及ぼさなかったこ とは注目すべきである。本研究で ABC群に検出されたゲノム不均衡は CD5で分類さ れる症例数が優勢であることを反映している。すなわち、 ABC群の 67% (28例中の 19 例）は CD5+型であった力 ABC群内の CD5+及び CD5—間のゲノム不均衡の頻度と領 域は同様であった。 ABC群内の CD5+及び CD5-間のゲノム不均衡の領域又は頻度 のどちらにも有意差は認められな力つた (データは示さず)。 1つの典型例は 9p21の 欠損である。この欠損は ABC群のみに見出され、この ABC群内で CD5+DLBCL 19例 中の 13例（68%)、及び CD5— DLBCL 9例中の 6例 (66%)にこの欠損が認められた（P = 0.999)。 [0069] It should be noted that CD5 expression did not affect the genomic imbalance observed in the ABC and GCB groups. The genomic imbalance detected in the ABC group in this study reflects the prevalence of cases classified by CD5. That is, 67% (19/28) of the ABC group were CD5 + type. The frequency and region of genomic imbalance between CD5 + and CD5− in the ABC group was similar. There was no significant difference in the area or frequency of genomic imbalance between CD5 + and CD5- in the ABC group (data not shown). One typical example is a 9p21 deficiency. This defect was found only in the ABC group, and in this ABC group, 13 (68%) of 19 CD5 + DLBCL cases and 6 (66%) of 9 CD5—DLBCL cases were found to have this defect. (P = 0.999).
[0070] 3. CD5+、 CD5— CD10+、及び CD5— CD10— DLBCLのゲノム不均衡  [0070] 3. Genomic imbalance of CD5 +, CD5—CD10 +, and CD5—CD10—DLBCL
我々は CD5+ (36例）、 CD5— CD10+(19例）、及び CD5— CD10— (44例）の群に増幅及び 欠損領域の頻度を検討した。 CD5+群における高頻度のゲノム不均衡（≥8例）は染色 体 3、 6p22- p25、 7p22- q31、 8q24、 llq22- q25、 12、 16pl3- q21、 18、 19、 Xの増幅、及 び 1ρ36、 2pll、 6ql4- q27、 8p23、 9p21、 15ql3- ql4、 17pll-pl3の欠損であった。 lq2 l-q32、 7p22_q36、及び 12の増幅は GCB群の特徴であつたが、これらの増幅は CD5+ 群の≤ 20%にも認められた。  We examined the frequency of amplified and defective regions in the CD5 + (36 cases), CD5-CD10 + (19 cases), and CD5-CD10- (44 cases) groups. High frequency genomic imbalances (≥8 cases) in the CD5 + group are chromosomal 3, 6p22-p25, 7p22-q31, 8q24, llq22-q25, 12, 16pl3-q21, 18, 19, X amplification, and 1ρ36 2pll, 6ql4-q27, 8p23, 9p21, 15ql3-ql4 and 17pll-pl3. Amplifications of lq2 l-q32, 7p22_q36, and 12 were characteristic of the GCB group, but these amplifications were also found in ≤ 20% of the CD5 + group.
[0071] CD5—CD10+群における高頻度のゲノム不均衡（≥4例）は、 lq、 2pl3-p25、 6p21-p2 5、 7、 8q22- q24、 9q33- q34、 12、 13q31- q33、 15q、 16pl3、 19qll3.3- 13.4、 Xの増幅、 及び 1ρ36、 1ρ22、 2pll、 3pl4、 4p、 6ql3- q27、 9p21、 13ql4-q21の欠損であった。 CD5+及び CD5— CD10+サブグループ間の比較から、 CD5+ DLBCLでは染色体 3の増 幅及び染色体 9p21の欠損が CD5— CD10+DLBCLよりも高頻度に認められた力 CD5" CD10+DLBCLでは 7q22- q36、 12ql3- ql4及び 17pl3の増幅力 CD5+DLBCLよりも高頻 度に認められた（表 3)。 CD5+及び CD5— CD10+ DLBCLの各群のゲノム不均衡のゲノ ムワイドな頻度を図 5Bに示してある。特に重要なことに、これらの CD5+及び CD5— CD1 0+DLBCLのゲノムプロファイルの特徴は、それぞれ ABC及び GCB DLBCLのそれと 極めて類似している（図 5A-B)。 CD5+群と ABC群間、又は CD5— CD10+群と GCB群間 のどちらにも、ゲノム不均衡の領域又は頻度に有意差は認められな力つた (表 3)。 表 3 特徴的なゲノム不均衡と頻度の比較 [0071] Frequent genomic imbalances in the CD5—CD10 + group (≥4 cases) are lq, 2pl3-p25, 6p21-p2 5, 7, 8q22-q24, 9q33-q34, 12, 13q31-q33, 15q, 16pl3, 19qll3.3-13.4, X amplification, and 1ρ36, 1ρ22, 2pll, 3pl4, 4p, 6ql3-q27, 9p21, 13ql4-q21. Comparison between CD5 + and CD5—CD10 + subgroups showed that CD5 + DLBCL showed chromosome 3 amplification and chromosome 9p21 loss more frequently than CD5—CD10 + DLBCL CD5 ”CD10 + DLBCL 7q22-q36, Amplification power of 12ql3-ql4 and 17pl3 was observed more frequently than CD5 + DLBCL (Table 3) Genome-wide frequency of genomic imbalance in each group of CD5 + and CD5—CD10 + DLBCL is shown in Figure 5B Of particular importance, the genomic profile characteristics of these CD5 + and CD5—CD10 + DLBCL are very similar to those of ABC and GCB DLBCL, respectively (Figure 5A-B). Or CD5— Between CD10 + group and GCB group In both cases, no significant difference was observed in the region or frequency of genomic imbalance (Table 3). Table 3 Comparison of characteristic genomic imbalance and frequency
Minimum ABC (n-28) GCB (n=18) CD5+ (n-36)Minimum ABC (n-28) GCB (n = 18) CD5 + (n-36)
Chr. Gain/loss Common region No. of cases % No. of cases % No. of cases % Chr. Gain / loss Common region No. of cases% No. of cases% No. of cases%
Chr. l gain lq2Ll-q23.3 0 0% 8 44% 6-7 17-19% gain Iq31.l-q42.13 2 0-7% 8-9 44-50% 5-6 14-17%Chr. L gain lq2Ll-q23.3 0 0% 8 44% 6-7 17-19% gain Iq31.l-q42.13 2 0-7% 8-9 44-50% 5-6 14-17%
Chr.2 gain 2pl5-pl6.1 2 7% 6 33% 4-8 11-22%Chr.2 gain 2pl5-pl6.1 2 7% 6 33% 4-8 11-22%
Chr.3 gain 3q23-q28 7-10 25-36% 0 0% 11 31%Chr.3 gain 3q23-q28 7-10 25-36% 0 0% 11 31%
Chr.6 loss 6q22 1-q24.1 11-13 39-46% 2 11% 16 44%Chr.6 loss 6q22 1-q24.1 11-13 39-46% 2 11% 16 44%
Chr.7 gain 7q22.1-q36.2 1 4% 9-11 50-61% 4-6 11-22%Chr.7 gain 7q22.1-q36.2 1 4% 9-11 50-61% 4-6 11-22%
Chr.9 loss 9p21.3 19 68% 5 28% 18 50%Chr.9 loss 9p21.3 19 68% 5 28% 18 50%
Chr.12 gain 12ql3.13- ql4.1 4 11% 8 44% 5-6 14-17%Chr.12 gain 12ql3.13- ql4.1 4 11% 8 44% 5-6 14-17%
Chr. I7 gain 17pl3.1 5 18% 3 17% 2 6%Chr. I7 gain 17pl3.1 5 18% 3 17% 2 6%
Chr.18 gain 18ql l.2-q23 13-15 46-54% 3-4 17-22% 14-15 38-42%Chr.18 gain 18ql l.2-q23 13-15 46-54% 3-4 17-22% 14-15 38-42%
Chr.19 gain 19ql3.32-ql3.33 13 46% 3 17% 12 33% gain 19ql3.41-ql3.42 15 54% 3 17% 17 47%Chr.19 gain 19ql3.32-ql3.33 13 46% 3 17% 12 33% gain 19ql3.41-ql3.42 15 54% 3 17% 17 47%
NS： not significant NS: not significant
b増幅/欠損の最小共通領域 表 3つづき bMinimum common area for amplification / deletion Table 3 continued
CD5 CD10+{n-19) Fisher's P CD5 CD10 + (n-19) Fisher's P
No. of cases % ABC vs. GCB CD5 " vs. CD10+ No. of cases% ABC vs. GCB CD5 "vs. CD10 +
5- 7 26-37% 0.01 NS5- 7 26-37% 0.01 NS
6- 7 32-37% <0.01 NS 5-8 21-32% 0.04 NS 0-1 0-5% 0.03 0.01-0.04 12 63% 0.02-0.04 NS6- 7 32-37% <0.01 NS 5-8 21-32% 0.04 NS 0-1 0-5% 0.03 0.01-0.04 12 63% 0.02-0.04 NS
9-11 42 58% <0.01 <0.019-11 42 58% <0.01 <0.01
4 21% 0.01 0.04 9-13 47-68% 0.04 0.02 37% NS <0.01 26% 0.03 NS4 21% 0.01 0.04 9-13 47-68% 0.04 0.02 37% NS <0.01 26% 0.03 NS
12 63% NS NS 12 63% 0.01 NS 増幅 Z欠損領域と BACクローン等との関係 12 63% NS NS 12 63% 0.01 NS Amplified Relationship between Z-deficient region and BAC clone
cytogeneviccytogenevic
ID BAG MB old MB new GENE position marker
Figure imgf000032_0001
ID BAG MB old MB new GENE position marker
Figure imgf000032_0001
つづさ Spelling
cytogeneyiccytogeneyic
ID BAG MB old MB new GENE position marker
Figure imgf000033_0001
ID BAG MB old MB new GENE position marker
Figure imgf000033_0001
づさ Tsusa
cytogeneyiccytogeneyic
ID BAG MB old MB GENE osition
Figure imgf000034_0001
Figure imgf000035_0001
CD5—CD10—群（≥9例）における高頻度なゲノム不均衡は、 Iq21-q42 3q21-q29pl3- pl5 6p21- p25 7 llq22 12ql3- ql4 18 19ql3.1- ql3.4 Xの増幅、及び 2p1 3pl4.2 6ql2- q27 9p21.3 15ql4- ql5 17pll-pl3の欠損であった。 CD5— CDIO DLBCLのゲノム不均衡は、これらの増幅及び欠損が CD5+及び CD5— CD10+群にお いて認められたため、混合型であった。さらに、 CD5—CD10— DLBCLのゲノム不均衡 の頻度と領域は、図 5に示されている通り「ABCプラス GCB」群のそれと極めて類似し ていた。 CD5—CD10+と「ABCプラス GCB」群間にゲノム不均衡の領域又は頻度に有 意差は認められなかった（データは示していない）。これらの知見は、 CD5—CD10— D LBCLが ABC及び GCB群に均等に分布していたことを示した遺伝子発現プロファイリ ングの結果と良好な相関を示す。 ID BAG MB old MB GENE osition
Figure imgf000034_0001
Figure imgf000035_0001
Frequent genomic imbalances in the CD5—CD10—group (≥9 cases) are: Iq21-q42 3q21-q29pl3- pl5 6p21- p25 7 llq22 12ql3- ql4 18 19ql3.1- ql3.4 X amplification and 2p1 3pl4 .2 6ql2- q27 9p21.3 15ql4- ql5 17pll-pl3 deficiency. CD5—CDIO The DLBCL genomic imbalance was mixed because these amplifications and deletions were observed in the CD5 + and CD5-CD10 + groups. In addition, the frequency and region of CD5—CD10—DLBCL genomic imbalance was very similar to that of the “ABC plus GCB” group, as shown in FIG. There was no significant difference in the region or frequency of genomic imbalance between the CD5-CD10 + and “ABC plus GCB” groups (data not shown). These findings correlate well with the results of gene expression profiling that showed that CD5-CD10-DLCL was evenly distributed in ABC and GCB groups.
[0075] 4.強力な予後マーカーとしての 9ρ21 (ρ16ΙΝΚ½遺伝子座)の欠損の同定 [0075] 4. Identification of 9ρ21 (ρ16 ΙΝΚ½ locus) deficiency as a strong prognostic marker
最後に、我々はアレイ CGH法によって検出された予後変数の同定を試み、 9p21の欠 損が患者の生存に有害な影響を与えたことを見出した。 9p21の欠損を有する 37例は この欠損を有さない 59例よりも生存が有意に低力つた (ログランク検定、 P=0.0003) ( 図 6A)。  Finally, we attempted to identify prognostic variables detected by the array CGH method and found that 9p21 deficiency had a detrimental effect on patient survival. The 37 cases with 9p21 deficiency had significantly lower survival than the 59 cases without this deficit (log rank test, P = 0.0003) (Figure 6A).
[0076] 9p21の欠損は ABC群（19例）で GCB群 (5例）よりも有意に多く検出された (フイツシャ 一の直接確率検定、 P=0.0147) o 9p21が欠損している ABC症例の生存はこのような 欠損をもたない ABC症例よりも有意に劣っていたが（ログランク検定、 P=0.0138)、 G CB群の同じ領域の欠損は生存に影響を及ぼさな力つた。同様に、 9p21を欠損してい る CD5+症例の生存は、この欠損を持たない症例よりも有意に劣っていた（ログランク 検定、 P=0.0048)。従って、我々は 9p21 (pl6INK4a遺伝子座）の欠損が DLBCLの最も 悪性の型を示すことを立証することができた。 CD5—CD10— DLBCL症例の中で、 9p21 の欠損は生存にマイナスの影響を及ぼす傾向があつたが、これは統計的有意性に 達しなかった（ログランク検定、 P= 0.0684)。 [0076] 9p21 deficiency was detected significantly more in the ABC group (19 cases) than in the GCB group (5 cases) (Fitzia direct probability test, P = 0.0147) o In the ABC cases lacking 9p21 Although survival was significantly inferior to ABC cases without such deficits (log rank test, P = 0.0138), deficiencies in the same region of the GCB group had no effect on survival. Similarly, the survival of CD5 + cases lacking 9p21 was significantly inferior to those without this deficiency (Logrank test, P = 0.0048). Thus, we were able to demonstrate that the 9p21 (pl6 INK4a locus) deficiency represents the most malignant form of DLBCL. Among CD5—CD10—DLBCL cases, 9p21 deficiency tended to have a negative impact on survival, but this did not reach statistical significance (log rank test, P = 0.0684).
[0077] 図 6Bに示されている通り、 9pの欠損が最小である共通の領域は 9p21.3の 2.2Mb以 内に位置していた。 9p21のホモ接合性の欠損を示唆する証拠力 ¾例に認められたが (log比く—1.0として定義、 CD5+及び CD5— CD10—の各群に 3例）、一方でどの GCB又 [0077] As shown in FIG. 6B, the common region where the 9p loss was minimal was located within 2.2 Mb of 9p21.3. Evidence suggesting a homozygous deficiency of 9p21 was found in ¾ cases (compared to log—defined as 1.0, 3 cases in each of the CD5 + and CD5—CD10— groups), while any GCB or
2 2
は CD5— CD10+の症例にも 9p21.3にホモ接合性の欠損を示唆する徴候は示されなか つた。 9p21の欠損を有する 37例中の 13例が一つの BAC、 pl6INK4a腫瘍抑制遺伝子を 含む RP11-14912を取り囲むゲノムの制限部位に欠損を示した。 ABC群における pl6INK 4aの発現レベルは GCB群よりも有意に低力つた（図 6C) (マンホイット-一の U検定、 P = 0.001)。これらの結果は ABC DLBCL症例において 9p21.3の欠損の頻度が高かつ たという知見とよく一致する。 The CD5—CD10 + case also showed no signs suggesting a homozygous defect at 9p21.3. Thirteen of the 37 patients with 9p21 deletion showed a defect in the restriction site of the genome surrounding one RP11-14912 containing the BAC, pl6 INK4a tumor suppressor gene. The expression level of pl6 INK 4a in the ABC group was significantly lower than that in the GCB group (Figure 6C) (Mann Whit-One U test, P = 0.001). These results are in good agreement with the finding that the frequency of 9p21.3 deficiency was high in ABC DLBCL cases.
[0078] (考察） [0078] (Discussion)
一部の研究者らは、従来の CGH法又はアレイ CGH法を用いて検出した DLBCLに おけるゲノムの変化を報告している（Monni 0, Joensuu H, Franssila K, Knuutila S. D NA copy number changes in diffuse large B— cell lymphoma— comparative genomic hyb ridization study. Blood. 1996; 87:5269—5278、 Rao PH, Houldsworth J, Dyomina K, et al. Chromosomal and gene amplification in diffuse large B— cell lymphoma. Blood. 1998; 92: 234—240、 Berglund M, Enblad G, Flordal E, et al. Chromosomal imbalan ces in diffuse large B— cell lymphoma detected by comparative genomic hybridization. Some researchers have reported genomic changes in DLBCL detected using conventional CGH or array CGH methods (Monni 0, Joensuu H, Franssila K, Knuutila S. D NA copy number changes in diffuse large B— cell lymphoma— comparative genomic hybridization study. Blood. 1996; 87: 5269—5278, Rao PH, Houldsworth J, Dyomina K, et al. Chromosomal and gene amplification in diffuse large B— cell lymphoma. Blood. 1998; 92: 234-240, Berglund M, Enblad G, Flordal E, et al. Chromosomal imbalan ces in diffuse large B— cell lymphoma detected by comparative genomic hybridization.
Mod Pathol, 2002; 15: 807-816、 . Bea S, Colomo L, Lopez- Guillermo A, et al. Clin icopathologic significance and prognostic value of chromosomal imbalances in diffuse large B- cell lymphomas. J Clin Oncol. 2004; 22: 3498-3560、 Martinez- Climent JA,Mod Pathol, 2002; 15: 807-816, .Bea S, Colomo L, Lopez- Guillermo A, et al. Clin icopathologic significance and prognostic value of chromosomal imbalances in diffuse large B-cell lymphomas. J Clin Oncol. 2004; : 3498-3560, Martinez- Climent JA,
Alizadeh AA, Segraves R., et al. Transformation of follicular lymphoma to diffuse lar ge cell lymphoma is associated with a heterogeneous set of DNA copy number and g ene expression alterations. Blood. 2003; 101: 3109—3117)。し力し、 DLBCLのサブタ イブのゲノム比較解析を実施した研究は極めて少ない（Tagawa H, Tsuzuki S, SuzukiAlizadeh AA, Segraves R., et al. Transformation of follicular lymphoma to diffuse large cell lymphoma is associated with a heterogeneous set of DNA copy number and gene expression alterations. Blood. 2003; 101: 3109-3117). However, very few studies have conducted comparative genome analysis of DLBCL subtypes (Tagawa H, Tsuzuki S, Suzuki)
R, et al. enome-wide array— based comparative genomic hybridization or diffuse lar ge B— cell lymphoma: comparison between CD5— Positive and CD5— negative cases. C ancer Res. 2004; 64: 5948—5955、 Zang X, Karnan S, Tagawa H, et al. Comparison o f genetic aberrations in CDlO+diffuse large B— cell lymphoma and follicular lymphoma by comparative genomic hybridization and tissue-fluorescence in situ hybridization.R, et al. Enome-wide array— based comparative genomic hybridization or diffuse lar ge B— cell lymphoma: comparison between CD5— Positive and CD5— negative cases. C ancer Res. 2004; 64: 5948-5955, Zang X, Karnan S, Tagawa H, et al. Comparison of genetic aberrations in CDlO + diffuse large B— cell lymphoma and follicular lymphoma by comparative genomic hybridization and tissue-fluorescence in situ hybridization.
Cancer Sci. 2004; 95:809-814)。 Cancer Sci. 2004; 95: 809-814).
[0079] 本研究では、我々のアレイ CGH法により、 ABC及び GCBサブグループ間にゲノム 不均衡のパターンの明確な差を検出することが可能であった。 ABC DLBCLは 3q、 18 q、 19qの増幅、及び 6qと 9p21の欠損というゲノム的特徴を有し、 GCB DLBCLは lq、 2 p、 7q、 12qの増幅というゲノム的特徴を有する。従って、これらの結果から、 ABC及び GCB群は遺伝的に異なっているという証拠が得られ、これは ABC及び GCB DLBCL では腫瘍が異なるゲノム経路によって発生することを示唆している。 [0079] In this study, our array CGH method was able to detect distinct differences in the pattern of genomic imbalance between ABC and GCB subgroups. ABC DLBCL has the genomic features of 3q, 18q, 19q amplification and 6q and 9p21 deletion, and GCB DLBCL has the genomic features of lq, 2p, 7q, 12q amplification. Therefore, these results provide evidence that the ABC and GCB groups are genetically different, which are ABC and GCB DLBCL Suggests that tumors arise by different genomic pathways.
[0080] 4グループの研究者らが濾胞性リンパ腫 (FL)力 転ィ匕した DLBCLのゲノム不均衡 について研究報告を発表している（Martinez- Climent JA, Alizadeh AA, Segraves R.， et al. Transformation of follicular lymphoma to diffuse large cell lymphoma is associa ted with a heterogeneous set of DNA copy number and gene expression alterations. Blood. 2003; 101: 3109—3117、 Goff LK, Neat MJ, Crawley CR, et al. The use of real -time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma. Br J Haematol. 200 0; 111: 618—625、 Nagy M, Balazs M, Adam Z, et al., Genetic instability is associate d with follicle center lymphoma. Leukemia. 2000; 14: 2142—2148、 Hough RE, Goep el JR, Alcock HE, Hancock BW, Lorigan PC, Hammond DW. Copy number gain at 1 2ql2— 14 may be important in the transformation from follicular lymphoma to diffiise large B cell lymphoma. Br J Cancer. 2001; 84:499-503)。これらの報告によれば、 FL 力 転化した DLBCLは 2p、 7p、 12p、 12qの増幅、及び 4qと 13qの欠損からなるゲノム 不均衡を有して 、る。これらの FLから転ィ匕した DLBCLに特徴的なゲノム不均衡は GC B群のそれと類似しているため、 FLから転化した DLBCLと GCB DLBCLには、リンパ 腫発生の過程におけるゲノム異常のプログラムにある共通のステップが存在する可 能性がある。 [0080] Four groups of researchers have published research reports on genomic imbalances in DLBCL with follicular lymphoma (FL) force (Martinez-Climent JA, Alizadeh AA, Segraves R., et al. Transformation of follicular lymphoma to diffuse large cell lymphoma is associa ted with a heterogeneous set of DNA copy number and gene expression alterations.Blood. 2003; 101: 3109-3117, Goff LK, Neat MJ, Crawley CR, et al. The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma. Br J Haematol. 200 0; 111: 618—625, Nagy M, Balazs M, Adam Z, et al., Genetic instability is associate d with follicle center lymphoma. Leukemia. 2000; 14: 2142-2148, Hough RE, Goep el JR, Alcock HE, Hancock BW, Lorigan PC, Hammond DW. Copy number gain at 1 2ql2— 14 may be important in the transformation from follicular lymphoma to diffiise large B cell lymphoma. Br J Cancer. 2001; 84: 499-503). According to these reports, FLB-converted DLBCL has a 2p, 7p, 12p, 12q amplification and a genomic imbalance consisting of 4q and 13q deletions. The genomic disequilibrium characteristic of DLBCL transformed from these FLs is similar to that of GC B group, so DLBCL and GCB DLBCL transformed from FL can be used to program genomic abnormalities during lymphoma development. There may be a common step.
[0081] 我々は以前に CD5+及び CD10+ DLBCLが臨床的関連性のあるサブタイプを構成す ることを報告している。本稿で、我々は CD5+DLBCLが ABCの発現とゲノムパターンに よって特徴付けられることを初めて報告する。最近、 Katzenberger et al. (2003)は新 規の CD5+DLBCLを対象とする細胞遺伝及び LOH研究を実施し、 CD5+ DLBCLが D1 3S25遺伝子座及び pl6INK4a癌抑制因子の高頻度の欠損を示したことから、 CD5+DLB CLは B-慢性リンパ性白血病 (CLL)と同じ前駆細胞に由来する可能性があると推測 し 7こ (Katzenberger Ί , Lohr A, ¾cnwarz ¾, et al. Genetic analysis or de novoし D5+ d iffuse large B— cell lymphomas suggests an origin from a somatically mutated CD5+ p rogenitor B cell. Blood. 2003; 101: 699—702)。し力し、 CD5+ DLBCLはその起源と経 路の両方において、 CD5も発現する CLL及びマントル細胞リンパ腫 (MC)とは異なつ ていると考えられる。 CLL及び MCLは共に、 1ρ22、 6q、 9p21、 l lq22- q23、 13ql4-q21 の欠損という特徴を有するが（症例の 30〜50%に認められる） (24. Bentz M, Plesch A, Bullinger L, et al. 丄丄； 14)— positive mantle cell lymphomas exnibit complex karyo types and share similarities with B— cell chronic lymphocytic leukemia. Genes Chrom osomes Cancer. 2000; 27: 285—294、 Schwaenen C, Nessling M, Wessendorf S, et al . Automated array-based genomic profiling in chronic lymphocytic leukemia: develop ment of a clinical tool and discovery of recurrent genomic alterations. Proc Natl Aca d Sci U S A. 2004; 101:1039-1044、 Kohlhammer H, Schwaenen C, Wessendorf S, et al. Genomic DNA— chip hybridization in t(l l; 14)— positive mantle cell lymphomas sho ws a high frequency of aberrations and allows a refined characterization of consensus regions. Blood. 2004;104: 795—801、 Tagawa H, Karnan S, Suzuki R, et al. Genome -wide array-based CGH for mantle cell lymphoma: identification of homozygous dele tions of the proapoptotic gene BIM. Oncogene. 2005; 24: 1348- 1358)、我々が検討 した CD5+ DLBCL症例の 10%未満が Ip22、 l lq22- q23、及び 13ql4- q21の欠損を示 し、 pl6INK4aの欠損のみが共通の特徴であるとみられる。 [0081] We have previously reported that CD5 + and CD10 + DLBCL constitute clinically relevant subtypes. In this article, we report for the first time that CD5 + DLBCL is characterized by ABC expression and genomic patterns. Recently, Katzenberger et al. (2003) conducted a cytogenetic and LOH study on a new CD5 + DLBCL, which showed a high frequency of loss of the D1 3S25 locus and pl6 INK4a tumor suppressor. Therefore, we suppose that CD5 + DLB CL may be derived from the same progenitor cells as B-chronic lymphocytic leukemia (CLL) (7) (Katzenberger er, Lohr A, ¾cnwarz ¾, et al. Genetic analysis or de D5 + d iffuse large B—cell lymphomas suggests an origin from a somatically mutated CD5 + progenitor B cell. Blood. 2003; 101: 699—702). However, CD5 + DLBCL differs from CLL and mantle cell lymphoma (MC), which also express CD5, in both origin and route. It is thought that. Both CLL and MCL are characterized by loss of 1ρ22, 6q, 9p21, l lq22- q23, 13ql4-q21 (recognized in 30-50% of cases) (24. Bentz M, Plesch A, Bullinger L, et al. 丄 丄; 14) — positive mantle cell lymphomas exnibit complex karyo types and share similarities with B— cell chronic lymphocytic leukemia. Genes Chrom osomes Cancer. 2000; 27: 285—294, Schwaenen C, Nessling M, Wessendorf S, et al. Automated array-based genomic profiling in chronic lymphocytic leukemia: develop ment of a clinical tool and discovery of recurrent genomic alterations.Proc Natl Aca d Sci US A. 2004; 101: 1039-1044, Kohlhammer H, Schwaenen C, Wessendorf S, et al. Genomic DNA— chip hybridization in t (ll; 14) — positive mantle cell lymphomas sho ws a high frequency of aberrations and allows a refined characterization of consensus regions. Blood. 2004; 104: 795—801, Tagawa H , Karnan S, Suzuki R, et al. Genome -wide array-based CGH for mantle cell lymphoma: identif ication of homozygous dele- tions of the proapoptotic gene BIM. Oncogene. 2005; 24: 1348- 1358), less than 10% of CD5 + DLBCL cases we examined showed Ip22, llq22-q23, and 13ql4-q21 deficiencies. Only the lack of pl6 INK4a appears to be a common feature.
我々はまた、 CD10+ DLBCLが GCBの発現とゲノムパターンによって特徴付けられる こと示すことができた。我々は以前に、正常な濾胞中心を持つ細胞は CD5—及び CD1 0+の免疫表現型を有し、 BCL2をほとんど発現しないことから、 CD10+DLBCL力胚中 心前駆細胞に由来する可能性を報告した。最近、 Huang et al. (2002)は、我々の結 果からも明らかであるように、遺伝子発現プロフアイリングを用いて、 CD10+DLBCL力 S GCB signatureによって特徴付けられることを示した（Huang JZ, Sanger WG, Greiner TC, et al. The t(14;18) defines a unique subset of diffiise large B- cell lymphoma with a germinal center B - cell gene expression profile. Blood. 2002; 99: 2285 - 2290、 Iqbal We could also show that CD10 + DLBCL is characterized by GCB expression and genomic patterns. We have previously shown that cells with normal follicular centers have CD5- and CD10 + immunophenotypes and rarely express BCL2, suggesting that they originate from CD10 + DLBCL embryonic central progenitor cells. reported. Recently, Huang et al. (2002) showed that it is characterized by CD10 + DLBCL force S GCB signature using gene expression profiling, as is evident from our results (Huang JZ , Sanger WG, Greiner TC, et al. The t (14; 18) defines a unique subset of diffiise large B- cell lymphoma with a germinal center B-cell gene expression profile.Blood. 2002; 99: 2285-2290, Iqbal
J, Sanger WG, Horsman DE, et al. BCL2 translocation defines a unique rumor subset within the germinal center B— celHike diffuse large B— ell lymphoma. A Jour Pathol. 2004; 165: 159-166)。我々は CD5— CD10— DLBCLカ^つのサブタイプ（すな わち CD5+及び CD— CD10+サブタイプ）のゲノム不均衡の混合パターンを示すことを認 めた。この結果は CD5— CD10— DLBCLが ABC又は GCBグループのどちらにも均等に 認められたことを示した遺伝子発現プロフアイリングの結果とよく関連しており、 CD5— CD 10" DLBCLが遺伝的に異質性の疾患であることを示唆している。 J, Sanger WG, Horsman DE, et al. BCL2 translocation defines a unique rumor subset within the germinal center B—celHike diffuse large B—ell lymphoma. A Jour Pathol. 2004; 165: 159-166). We have found that CD5—CD10—DLBCL subtypes (ie, CD5 + and CD—CD10 + subtypes) exhibit a mixed pattern of genomic imbalances. This result shows that CD5—CD10—DLBCL is equal to either ABC or GCB group. It is closely related to the results of gene expression profiling that has been observed, suggesting that CD5—CD 10 ”DLBCL is a genetically heterogeneous disease.
9p21 (pl6INK4a)の欠損は最も侵攻性の疾患を示す特徴であるだろう。我々の症例に おいて特に興味深いのは、 9p21を欠損している ABC及び CD5+ DLBCLの症例はこれ を欠損していない症例よりも転帰が不良であったことである。 9p21の欠損は、従って、 DLBCLの最も侵攻性の型を示す可能性のある因子であろう。 9p21.3 (pl6INK4a遺伝子 座)の欠損は侵攻性のリンパ腫及び急性リンパ芽球性白血病にお ヽて多く認められ る力 低悪性度のリンパ腫においてはそれほど多くはない（Serrano M, Hannon GJ, B each D. A new regulatory motif in cell-cycle control causing specific inhibition of cy clin D/CDK4. Nature. 1993; 366: 704—707、 Nobori T, Miura K, Wu DJ, et al. Deleti ons of the cyclin— dependent kinase— 4 inhibitor gene in multiple human cancers. Nat ure. 1994; 368: 753—756、 Koduru PR, Zariwala M, Soni M, et al. Deletion of cyclin— dependent Kinase 4 inhibitor genes pl5 and plo in non-Hodg in s lymphoma. Blood.9p21 (pl6 INK4a ) deficiency may be the most aggressive disease feature. Of particular interest in our case is that the cases of ABC and CD5 + DLBCL lacking 9p21 had a worse outcome than those not lacking. 9p21 deficiency may therefore be a factor that may represent the most aggressive form of DLBCL. 9p21.3 (pl6 INK4a locus) deficiency is more common in aggressive lymphomas and acute lymphoblastic leukemia (Serrano M, Hannon GJ, B each D. A new regulatory motif in cell-cycle control causing specific inhibition of cy clin D / CDK4. Nature. 1993; 366: 704—707, Nobori T, Miura K, Wu DJ, et al. Deleti ons of the cyclin — Dependent kinase— 4 inhibitor gene in multiple human cancers. Nat ure. 1994; 368: 753—756, Koduru PR, Zariwala M, Soni M, et al. Deletion of cyclin— dependent Kinase 4 inhibitor genes pl5 and plo in non- Hodg in s lymphoma. Blood.
1995; 86:2900—2905、 Stranks G, HeightSE, Mitchell P, et al. Deletions and rearra ngement of CDKN2 in lymphoid malignancy. Blood.1995; 85: 893—901、 Ogawa S, H angaishi A, Miyawaki S, et al. Loss of the cyclin- dependent kinase 4- inhibitor (pl6; MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies. Blood. 1995; 86: 1548—1556、 Pinyol M, Cobo F, Bea S, et al. pl6(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas . Blood. 1998; 91: 2977-2984) 0また CD5+ DLBCLも多くの侵攻性の臨床的特徴又 はパラメーターと密接に関連しており、 pl6INK4aの不活性ィ匕と 9p21の欠損は CD5+ DLB CLの特徴である可能性が高いが、実際に、 CD5+ DLBCLにおける極めて高頻度の 9 p21の欠損が我々及び他のグループによって報告されている（12. Tagawa H, Tsuzuk l S, buzuki R, et al. Genome-wide array-based comparative genomic nybnaization of diffuse large B— cell lymphoma: comparison between CD5— Positive and CD5— negativ e cases. Cancer Res. 2004; 64: 5948—5955. 23. Katzenberger T, Lohr A, Schwarz S, et al. enetic analysis of de novo CD5+ diffuse large B— cell lymphomas suggests an origin from a somatically mutated CD5+ progenitor B cell. Blood. 2003; 101: 699—7 02)。 1995; 86: 2900-2905, Stranks G, HeightSE, Mitchell P, et al. Deletions and rearrangement of CDKN2 in lymphoid malignancy. Blood. 1995; 85: 893-901, Ogawa S, Hangaishi A, Miyawaki S, et al. Loss of the cyclin-dependent kinase 4- inhibitor (pl6; MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies.Blood. 1995; 86: 1548—1556, Pinyol M, Cobo F, Bea S, et al. Pl6 (INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas .Blood. 1998; 91: 2977-2984) 0 CD5 + DLBCL is also much aggressive Pl6 INK4a inactivity and 9p21 deficiency are likely to be characteristic of CD5 + DLB CL, but in fact, are very frequent in CD5 + DLBCL. 9 p21 deficiency has been reported by us and other groups (12. Tagawa H, Tsuzuk l S, buz uki R, et al. Genome-wide array-based comparative genomic nybnaization of diffuse large B— cell lymphoma: comparison between CD5— Positive and CD5— negativ e cases. Cancer Res. 2004; 64: 5948—5955. 23. Katzenberger T , Lohr A, Schwarz S, et al. Enetic analysis of de novo CD5 + diffuse large B— cell lymphomas suggests an Origin from a somatically mutated CD5 + progenitor B cell. Blood; 2003; 101: 699-7 02).
[0084] 要約すると、我々は ABC及び GCB DLBCLは遺伝子発現のみが異なって!/、るので はなぐゲノム不均衡もまた異なっていること、並びに CD5+及び CD5— CD10+DLBCL症 例の殆どが ABC群及び GCB群にそれぞれ含まれることを示すことができた。さらに、 患者の予後に影響するゲノム不均衡を模索する際に、我々は 9p21 (pl6INK4a遺伝 子座)の欠損が DLBCLの最も侵攻性の型を示すことを見出した。遺伝子発現プロフ アイリグとアレイ CGH法を併用することで、 DLBCLだけではなぐ一般の異質性腫瘍 に関する知識をさらに深めることが可能である。  [0084] In summary, we found that ABC and GCB DLBCL differ only in gene expression! / And thus the genomic imbalances are also different, and that most cases of CD5 + and CD5-CD10 + DLBCL cases are ABC It was possible to show that they were included in the group and GCB group, respectively. Furthermore, in exploring genomic imbalances that affect patient prognosis, we found that 9p21 (pl6INK4a locus) deficiency represents the most aggressive form of DLBCL. By combining the gene expression profile and array CGH method, it is possible to further deepen the knowledge of general heterogeneous tumors that are not possible with DLBCL alone.
[0085] 本明細書に記載の参考文献に記載の全内容は、引用により本明細書の一部に組 み込まれる。 産業上の利用の可能性  [0085] The entire contents of the references described herein are hereby incorporated by reference. Industrial applicability
[0086] 本発明は、 DLBCLの病型や予後の診断に有用である。 [0086] The present invention is useful for diagnosis of DLBCL disease type and prognosis.

Claims

請求の範囲 The scope of the claims
[1] ヒトから採取した染色体を含む被験試料中のヒト染色体の増幅領域及び Z又は欠 損領域を検出する検出工程を備える、びまん性大細胞型 B細胞リンパ腫の病型の診 断方法。  [1] A method for diagnosing a disease type of diffuse large B-cell lymphoma, comprising a detection step of detecting an amplified region and Z or a defective region of a human chromosome in a test sample containing a chromosome collected from a human.
[2] 前記検出工程で検出された前記増幅領域が、ヒト染色体の 3、 8q21-q26、 Ilq21-q2 5、 16pll-pl3、 16q22-q24、 18、 19ql3及び Xから選択される 1種又は 2種以上にある かどうか及び Z又は前記欠損領域がヒト染色体の 2pll、 6ql2-27、 8p22-p23、 9p21及 び 17pから選択される 1種又は 2種以上にあるかどうかを指標としてびまん性大細胞型 B細胞リンパ腫の病型が ABC型であると肯定的に判定する判定工程を備える、請求 項 1に記載の診断方法。  [2] The amplification region detected in the detection step is one or two selected from 3, 8q21-q26, Ilq21-q2 5, 16pll-pl3, 16q22-q24, 18, 19ql3 and X of the human chromosome As an indicator, whether or not there is more than one species and whether Z or the deletion region is one or more species selected from 2pll, 6ql2-27, 8p22-p23, 9p21 and 17p of human chromosomes The diagnostic method according to claim 1, further comprising a determination step of positively determining that the disease type of the cell type B-cell lymphoma is ABC type.
[3] 前記判定工程は、前記増幅領域がヒト染色体の 3p23-q28、 18qll.2-q23及び 9ql3.  [3] In the determination step, the amplification region is 3p23-q28, 18qll.2-q23 and 9ql3.
41-ql3.43から選択される 1種又は 2種以上にあるかどうか及び前記欠損領域がヒト染 色体の 6q22.31-q24.1及び 9p21.3から選択される 1種又は 2種以上にあるかどうかを 指標とする工程である、請求項 2に記載の診断方法。  Whether one or more selected from 41-ql3.43 and one or more selected from 6q22.31-q24.1 and 9p21.3 of the human chromosome The diagnostic method according to claim 2, wherein the method is a step using as an index whether or not it is present.
[4] 前記判定工程は、前記増幅領域がヒト染色体の 3q23-q28及び Z又は前記欠損領 域がヒト染色体の 9p21.3であるかどうかを指標とする工程である、請求項 3に記載の 診断方法。  [4] The determination step according to claim 3, wherein the determination step is a step using as an index whether the amplified region is 3q23-q28 and Z of a human chromosome or the deletion region is 9p21.3 of a human chromosome. Diagnosis method.
[5] 前記判定工程は、前記病型が ABC型であると肯定的に判定されるとき、前記病型が CD5+型であると肯定的に判定する工程である、請求項 1〜4のいずれかに記載の 診断方法。  [5] The determination step according to any one of claims 1 to 4, wherein when the disease type is positively determined to be ABC type, the disease type is positively determined to be CD5 + type The diagnostic method according to the above.
[6] 前記検出工程で検出された前記増幅領域が、ヒト染色体の lq22-32、 2pl4-p24、 5p 12— p 、 5ql5— q31、 6pl2— p25、 7、 8q22— q26、 9q33— q34、 llq、 12、 13q31— q33、 16pll— pl3、 18q21-q23、 19p、 19ql3、 21q及び Xから選択される 1種又は 2種以上にあるかど うか及び Z又は前記欠損領域がヒト染色体の lp36、 2pll、 3pl4、 4pl2-pl3、 4q33-q3 4、 6ql4- ql6、 8p22- p23、 9p21、 13ql2- q22、 17pl2及び 18q22- q23から選択される 1種 又は 2種以上にあるかどうかを指標としてびまん性大細胞型 B細胞リンパ腫の病型が GCB型であると肯定的に判定する判定工程を備える、請求項 1に記載の診断方法。  [6] The amplified regions detected in the detection step are human chromosomes lq22-32, 2pl4-p24, 5p 12—p, 5ql5—q31, 6pl2—p25, 7, 8q22—q26, 9q33—q34, llq 12, 13q31—q33, 16pll—pl3, 18q21-q23, 19p, 19ql3, 21q and whether there is one or more selected from X and Z or the deletion region is lp36, 2pll of the human chromosome, 3pl4, 4pl2-pl3, 4q33-q3 4, 6ql4-ql6, 8p22-p23, 9p21, 13ql2-q22, 17pl2, and 18q22-q23 The diagnostic method according to claim 1, further comprising a determination step of positively determining that the disease type of the cell type B cell lymphoma is GCB type.
[7] 前記判定工程は、前記増幅領域がヒト染色体 q21.1-q23.3、 Iq31.1-q42.13、 2pl5- pl6.1、 7q22.1_q36.2及び 12ql3.1_ql4から選択される 1種又は 2種以上にあるかどう かを指標とする工程である、請求項 6に記載の診断方法。 [7] In the determination step, the amplification region is human chromosome q21.1-q23.3, Iq31.1-q42.13, 2pl5- 7. The diagnostic method according to claim 6, which is a step using as an index whether or not one or more selected from pl6.1, 7q22.1_q36.2 and 12ql3.1_ql4.
[8] 前記判定工程は、前記増幅領域がヒト染色体の 7q22-q36にあるかどうかを指標とす る工程である、請求項 7に記載の診断方法。 [8] The diagnostic method according to claim 7, wherein the determination step is a step of using as an index whether or not the amplified region is in 7q22-q36 of a human chromosome.
[9] 前記判定工程は、前記病型が GCB型であることが肯定的に判定されるとき、前記病 型が CD5—CD10+であると肯定的に判定する工程を備える、請求項 1、 6〜8のいず れかに記載の診断方法。 [9] The determination step includes a step of positively determining that the disease type is CD5-CD10 + when the disease type is positively determined to be GCB type. The diagnostic method according to any one of -8.
[10] 前記検出工程で検出された前記増幅領域が、ヒト染色体の 3、 6p22_p25、 7p22_q31、 [10] The amplification region detected in the detection step is a human chromosome 3, 6p22_p25, 7p22_q31,
8q24、 Ilq22-q25、 12、 16pl3-q21、 18、 19及び Xから選択される 1種又は 2種以上に あるかどうか及び Z又は前記欠損領域がヒト染色体の lp36、 2pll、 6ql4-q27、 8p23、 8q24, Ilq22-q25, 12, 16pl3-q21, 18, 19 and whether it is in one or more selected from X and Z or the deletion region is lp36, 2pll, 6ql4-q27, 8p23 of human chromosome ,
9p21、 15ql3_ql4及び 17pll_pl3から選択される 1種又は 2種以上にあるかどうかを指 標として前記病型が CD5+であることを肯定的に判定する工程を備える、請求項 1に 記載の診断方法。 The diagnostic method according to claim 1, comprising a step of affirmatively determining that the disease type is CD5 + using as an index whether or not one or more selected from 9p21, 15ql3_ql4, and 17pll_pl3.
[11] 前記判定工程は、前記増幅領域がヒト染色体の 3であるか及び Z又は前記欠損領域 がヒト染色体の 9p2であるかどうかを指標として前記病型が CD5+であると肯定的に判 定する工程を備える、請求項 1に記載の診断方法。  [11] In the determination step, it is positively determined that the disease type is CD5 + using as an index whether the amplified region is 3 of human chromosome and Z or the deletion region is 9p2 of human chromosome. The diagnostic method according to claim 1, further comprising:
[12] 前記検出工程で検出された前記増幅領域が、ヒト染色体の lq、 2pl3_p25、 6p21-p25 、 7、 8q22- q24、 9q33- q34、 12、 13q31- q33、 15q、 16pl3、 19ql3.3- 13.4及び Xから選 択される 1種又は 2種以上にあるかどうか及び Z又は前記欠損領域がヒト染色体の lp 36、 1ρ22、 2pll、 3pl4、 4p、 6ql3- q27、 9p21及び 13ql4- q21から選択される 1種又は 2 種以上にあるかどうかを指標として前記病型が CD5—CD10+であると肯定的に判定 する判定工程を備える、請求項 1に記載の診断方法。  [12] The amplified region detected in the detection step is lq, 2pl3_p25, 6p21-p25, 7, 8q22-q24, 9q33-q34, 12, 13q31-q33, 15q, 16pl3, 19ql3.3- Selected from 13.4 and X or not, and Z or the deletion region is selected from lp 36, 1ρ22, 2pll, 3pl4, 4p, 6ql3-q27, 9p21 and 13ql4-q21 of human chromosome The diagnostic method according to claim 1, further comprising a determination step of positively determining that the disease type is CD5-CD10 + using as an index whether or not there is one or more types.
[13] 前記判定工程は、前記増幅領域がヒト染色体の 7q22-q36、 12ql3-ql4及び 17pl3 力も選択される 1種又は 2種以上にあることを指標として前記病型が CD5—CD10+で あると肯定的に判定する判定工程を備える、請求項 1に記載の診断方法。  [13] In the determination step, the disease type is CD5-CD10 +, using as an index that the amplification region is present in one or more selected 7q22-q36, 12ql3-ql4 and 17pl3 forces of human chromosomes. The diagnostic method according to claim 1, further comprising a determination step of positive determination.
[14] 前記検出工程は、被験試料中の前記領域にっ 、て、 PCR法、 RT— PCR法及び 核酸ノヽイブリダィゼーシヨンのいずれかを実施する工程を含んでいる、請求項 1〜13 の!、ずれかに記載の診断方法。 [14] The detection step includes a step of performing any one of a PCR method, an RT-PCR method, and a nucleic acid hybridization according to the region in the test sample. 13! The diagnostic method described in any of the above.
[15] 前記検出工程は、前記ヒト染色体上の領域を含むプローブと前記被験試料とをハ イブリダィゼーションする工程である、請求項 1〜 14に記載の方法。 [15] The method according to any one of [1] to [14], wherein the detection step is a step of hybridizing a probe containing a region on the human chromosome and the test sample.
[16] 前記検出工程は、アレイ CGH法を実施する工程である、請求項 1〜15のいずれか に記載の診断方法。  [16] The diagnostic method according to any one of claims 1 to 15, wherein the detection step is a step of performing an array CGH method.
[17] びまん性大細胞型 B細胞リンパ腫の病型診断用の核酸プローブセットであって、  [17] A nucleic acid probe set for diagnosing the type of diffuse large B-cell lymphoma,
ABC型のびまん性大細胞型 B細胞リンパ腫におけるヒト染色体上の増幅領域及び Z又は欠損領域を検出可能な 1種又は 2種以上の核酸プローブ。  One or more nucleic acid probes capable of detecting amplified regions and Z or defective regions on human chromosomes in ABC-type diffuse large B-cell lymphoma.
[18] 所定範囲のヒト染色体に由来する核酸プローブを固定ィ匕したアレイに対して前記 A BC型のびまん性大細胞型 B細胞リンパ腫罹患ヒト個体から採取した被験試料を供給 し核酸ハイブリダィゼーシヨンを実施して前記核酸プローブの固定部位にぉ 、て得ら れる蛍光強度の log比値に基づ 、て設定した所定の上下の閾値に対する前記 log  [18] A test sample collected from a human individual suffering from the above-mentioned ABC type diffuse large B-cell lymphoma is supplied to an array in which nucleic acid probes derived from a predetermined range of human chromosomes are immobilized. Based on the log ratio value of the fluorescence intensity obtained by performing the determination and fixing the nucleic acid probe, the log with respect to the predetermined upper and lower thresholds set
2 2 比値の大小に基づいて選択される核酸プローブを含む、請求項 17に記載の核酸プ ローブ 0 Comprising a nucleic acid probe which is selected based on the magnitude of 2 2 ratio value, the nucleic acid probe 0 of Claim 17
[19] びまん性大細胞型 B細胞リンパ腫の病型診断用の核酸プローブセットであって、  [19] A nucleic acid probe set for diagnosing the type of diffuse large B-cell lymphoma,
GCB型のびまん性大細胞型 B細胞リンパ腫におけるヒト染色体上の増幅領域及び Z又は欠損領域を検出可能な 1種又は 2種以上の核酸プローブ。  One or more nucleic acid probes capable of detecting the amplified region and the Z or defective region on the human chromosome in GCB-type diffuse large B-cell lymphoma.
[20] 所定範囲のヒト染色体に由来する核酸プローブを固定ィ匕したアレイに対して前記 G CB型のびまん性大細胞型 B細胞リンパ腫罹患ヒト個体から採取した被験試料を供給 し核酸ハイブリダィゼーシヨンを実施して前記核酸プローブの固定部位にぉ 、て得ら れる蛍光強度の log比値に基づ 、て設定した所定の上下の閾値に対する前記 log  [20] A test sample collected from a human individual suffering from a diffuse large B-cell lymphoma of the above-mentioned GCB type is supplied to an array in which nucleic acid probes derived from a predetermined range of human chromosomes are immobilized. Based on the log ratio value of the fluorescence intensity obtained by performing the determination and fixing the nucleic acid probe, the log with respect to the predetermined upper and lower thresholds set
2 2 比値の大小に基づいて選択される核酸プローブを含む、請求項 19に記載の核酸プ ローブ 0 The nucleic acid probe 0 according to claim 19, comprising a nucleic acid probe selected based on the magnitude of the 2 2 ratio value.
[21] びまん性大細胞型 B細胞リンパ腫の病型診断用アレイであって、  [21] An array for diagnosing the type of diffuse large B-cell lymphoma,
請求項 17〜20のいずれかに記載の核酸プローブが固定ィ匕されたアレイ。  An array on which the nucleic acid probe according to any one of claims 17 to 20 is immobilized.
[22] ヒト染色体の 3、 8q21- q26、 llq21- q25、 16pll- pl3、 16q22- q24、 18、 19ql3、 X、 2pl 1、 6ql2-27、 8p22-p23、 9p21、 17pから選択される 1種又は 2種以上の領域の少なくと も一部を検出可能な核酸プローブが固定ィ匕された、請求項 21に記載のアレイ。  [22] Human chromosome 3, 8q21-q26, llq21-q25, 16pll-pl3, 16q22-q24, 18, 19ql3, X, 2pl 1, 6ql2-27, 8p22-p23, 9p21, 17p The array according to claim 21, wherein a nucleic acid probe capable of detecting at least a part of two or more kinds of regions is immobilized.
[23] ヒト染色体の lq22- 32、 2pl4- p24、 5pl2- p 、 5ql5- q31、 6pl2- p25、 7、 8q22- q26、 9 q33— q34、 llq、 12、 13q31— q33、 16pll— pl3、 18q21— q23、 19p、 19ql3、 21q、 X、 lp36、[23] lq22-32, 2pl4-p24, 5pl2-p, 5ql5-q31, 6pl2-p25, 7, 8q22-q26, 9 of human chromosome q33—q34, llq, 12, 13q31—q33, 16pll—pl3, 18q21—q23, 19p, 19ql3, 21q, X, lp36,
2pll、 3pl4、 4pl2- pl3、 4q33- q34、 6ql4- ql6、 8p22- p23、 9p21、 13ql2- q22、 17pl2 及び 18q22-q23から選択される 1種又は 2種以上の領域の少なくとも一部を検出可能 な核酸プローブが固定ィ匕された、請求項 21又は 22に記載のアレイ。 Can detect at least part of one or more regions selected from 2pll, 3pl4, 4pl2-pl3, 4q33-q34, 6ql4-ql6, 8p22-p23, 9p21, 13ql2-q22, 17pl2 and 18q22-q23 23. The array according to claim 21 or 22, wherein a specific nucleic acid probe is immobilized.
[24] びまん性大細胞型 B細胞リンパ腫の予後の診断方法であって、 [24] A prognostic method for the diagnosis of diffuse large B-cell lymphoma,
ヒトから採取した染色体を含む被験試料について、ヒト染色体の 9p21の欠損又は変 異を検出する検出工程を備える、方法。  A method comprising a detection step of detecting a 9p21 deletion or mutation in a human chromosome for a test sample containing a chromosome collected from a human.
[25] 前記検出工程は、前記ヒト染色体上の領域を含むプローブと前記被験試料とをハ イブリダィゼーションする工程である、請求項 24に記載の方法。 [25] The method according to claim 24, wherein the detecting step is a step of hybridizing a probe including a region on the human chromosome and the test sample.
[26] 前記検出工程は、アレイ CGHを用いる工程である、請求項 25に記載の方法。 26. The method according to claim 25, wherein the detection step is a step using array CGH.
[27] 前記検出工程は、 pl6INK4a遺伝子の欠損又は変異を検出する工程である、請求項[27] The detection step is a step of detecting a deletion or mutation of the pl6 INK4a gene.
24〜26の!、ずれかに記載の方法。 24 to 26!
[28] 前記検出工程は、 pl6INK4a遺伝子によってコードされるタンパク質の発現の有無、 発現量又は変異を検出する工程である、請求項 27に記載の方法。 [28] The method according to claim 27, wherein the detection step is a step of detecting the presence / absence, expression level or mutation of the protein encoded by the pl6 INK4a gene.
[29] びまん性大細胞型 B細胞リンパ腫の予後診断用アレイであって、 [29] An array for prognosis of diffuse large B-cell lymphoma,
ヒト染色体の 9p21を検出するための核酸プローブを固定ィ匕したアレイ。  An array with immobilized nucleic acid probes to detect 9p21 on human chromosomes.
[30] ヒト染色体 9p21を検出するためのポリヌクレオチドである、びまん性大細胞型 B細胞 リンパ腫の予後診断用マーカー。 [30] A prognostic marker for diffuse large B-cell lymphoma, a polynucleotide for detecting human chromosome 9p21.
[31] 前記ポリヌクレオチドは、 P16INK4a遺伝子若しくはその一部又はこれらに相補的な 塩基配列を有するポリヌクレオチドである、請求項 30に記載のマーカー。 [31] the polynucleotide is a polynucleotide having a P 16 INK4a gene or a portion thereof, or a nucleotide sequence complementary to, markers of claim 30.
[32] pl6INK4a遺伝子によってコードされるタンパク質若しくはその一部又はこれらに対す る抗体である、びまん性大細胞型 B細胞リンパ腫の予後診断用マーカー。 [32] A prognostic marker for diffuse large B-cell lymphoma, which is a protein encoded by the pl6 INK4a gene, a part thereof, or an antibody against them.
PCT/JP2006/308235 2005-04-19 2006-04-19 Diagnostic method and prognostic method for disease type of diffuse large b-cell lymphoma WO2006112483A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2007528177A JPWO2006112483A1 (en) 2005-04-19 2006-04-19 Method for diagnosing disease type and prognosis of diffuse large B-cell lymphoma

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US67251605P 2005-04-19 2005-04-19
US60/672,516 2005-04-19

Publications (1)

Publication Number Publication Date
WO2006112483A1 true WO2006112483A1 (en) 2006-10-26

Family

ID=37115195

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/308235 WO2006112483A1 (en) 2005-04-19 2006-04-19 Diagnostic method and prognostic method for disease type of diffuse large b-cell lymphoma

Country Status (2)

Country Link
JP (1) JPWO2006112483A1 (en)
WO (1) WO2006112483A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101968491A (en) * 2010-09-29 2011-02-09 上海生物芯片有限公司 Molecular pathological classification method of diffuse large B-cell lymphomata, kit and application thereof
US20150004158A1 (en) * 2012-02-29 2015-01-01 Dana-Farber Cancer Institute, Inc. Compositions, Kits, and Methods for the Identification, Assessment, Prevention, and Therapy of Cancer
CN111655868A (en) * 2018-03-14 2020-09-11 深圳华大生命科学研究院 Malignant lymphoma marker and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BEA S. ET AL.: "Diffuse large B-cell lymphoma subgroups have distinct genetic profiles that influence tumor biology and improve gene-expression-based survival prediction", BLOOD, vol. 106, no. 9, November 2005 (2005-11-01), pages 3183 - 3190, XP003004678 *
CHEN W. ET AL.: "Array comparative genomic hybridization reveals genomic copy number changes associated with outcome in diffuse large B-cell lymphomas", BLOOD, vol. 107, no. 6, March 2006 (2006-03-01), pages 2477 - 2485, XP003004679 *
TAGAWA H. ET AL.: "Comparison of genome profiles for identification of distinct subroups of diffuse large B-cell lymphoma", BLOOD, vol. 106, no. 5, September 2005 (2005-09-01), pages 1770 - 1777, XP003004677 *
TAGAWA H. ET AL.: "Genome Wide na Array CGH ni yoru diffuse large B-cell lymphoma (DLBCL) no Genome Ijo. (Genome wide array based CGH for diffuse large B-cell lymphoma)", HEMATOLOGY & ONCOLOGY, vol. 50, May 2005 (2005-05-01), pages 293 - 301, XP003004674 *
ZETTL A. ET AL.: "Chromosomal imbalance in germinal center B-cell like and activate B-cell-like diffuse large B-cell lymphoma", BLOOD, vol. 104, no. 11, 2004, pages 122A (ABSTRACT 415), XP003004675 *
ZETTL A. ET AL.: "Different subtypes of diffuse large B-cell lymphoma defined by gene expression profiling are genetically distinct", BLOOD, vol. 102, no. 11, 2003, pages 178A (ABSTRACT 619), XP003004676 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101968491A (en) * 2010-09-29 2011-02-09 上海生物芯片有限公司 Molecular pathological classification method of diffuse large B-cell lymphomata, kit and application thereof
US20150004158A1 (en) * 2012-02-29 2015-01-01 Dana-Farber Cancer Institute, Inc. Compositions, Kits, and Methods for the Identification, Assessment, Prevention, and Therapy of Cancer
US9890429B2 (en) * 2012-02-29 2018-02-13 Dana-Farber Cancer Institute, Inc. Compositions, kits, and methods for the identification, assessment, prevention, and therapy of cancer
CN111655868A (en) * 2018-03-14 2020-09-11 深圳华大生命科学研究院 Malignant lymphoma marker and application thereof

Also Published As

Publication number Publication date
JPWO2006112483A1 (en) 2008-12-11

Similar Documents

Publication Publication Date Title
US20210108266A1 (en) Method for discovering pharmacogenomic biomarkers
Wang et al. Molecular inversion probes: a novel microarray technology and its application in cancer research
JP4938672B2 (en) Methods, systems, and arrays for classifying cancer, predicting prognosis, and diagnosing based on association between p53 status and gene expression profile
US8722331B2 (en) Method for selecting a treatment for non-small cell lung cancer using gene expression profiles
Cheung et al. High resolution analysis of follicular lymphoma genomes reveals somatic recurrent sites of copy‐neutral loss of heterozygosity and copy number alterations that target single genes
Chang et al. Comparison of genomic signatures of non-small cell lung cancer recurrence between two microarray platforms
JP2016047044A (en) Methods and compositions for identifying, classifying and monitoring subject having bcl-2 family inhibitor-resistant tumors and cancers
JP2009506777A (en) Chemotherapy sensitivity assay using tumor cells with resistant phenotypic characteristics
CA2679208A1 (en) Method for predicting the occurrence of metastasis in breast cancer patients
JP2007527238A (en) Classification, diagnosis, and prognosis of acute myeloid leukemia by gene expression profiling
KR20140105836A (en) Identification of multigene biomarkers
US20090098538A1 (en) Prognostic and diagnostic method for disease therapy
US20120141603A1 (en) Methods and compositions for lung cancer prognosis
CA2660857A1 (en) Prognostic and diagnostic method for disease therapy
KR101501826B1 (en) Method for preparing prognosis prediction model of gastric cancer
WO2009150256A1 (en) Markers for predicting response and survival in anti-egfr treated patients
JP2006014739A (en) Method for assessing and treating cancer
JP2005333987A (en) Prognosis of hematologic malignancies
CA2939539A1 (en) Prostate cancer survival and recurrence
WO2006066240A2 (en) Methods for assessing patients with acute myeloid leukemia
WO2009156858A1 (en) Molecular signature of liver tumor grade and use to evaluate prognosis and therapeutic regimen
WO2016057852A1 (en) Markers for hematological cancers
US20220136065A1 (en) High-grade serous ovarian carcinoma (hgsoc)
WO2006112483A1 (en) Diagnostic method and prognostic method for disease type of diffuse large b-cell lymphoma
JP2011500017A (en) Differentiation of BRCA1-related and sporadic tumors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2007528177

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06745467

Country of ref document: EP

Kind code of ref document: A1