WO2020138435A1 - Auxiliary diagnostic method for intraocular malignant lymphoma - Google Patents

Auxiliary diagnostic method for intraocular malignant lymphoma Download PDF

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WO2020138435A1
WO2020138435A1 PCT/JP2019/051482 JP2019051482W WO2020138435A1 WO 2020138435 A1 WO2020138435 A1 WO 2020138435A1 JP 2019051482 W JP2019051482 W JP 2019051482W WO 2020138435 A1 WO2020138435 A1 WO 2020138435A1
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mutation
cd79b
gene
myd88
btg2
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和城 田岡
綾子 唐川
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国立大学法人東京大学
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  • the present invention relates to an auxiliary diagnostic method for intraocular malignant lymphoma. More specifically, it relates to an auxiliary diagnostic method using a gene mutation specific to intraocular malignant lymphoma as an index.
  • Intraocular malignant lymphoma is a highly intractable disease with the worst prognosis among the eye diseases, with a high rate of 60-80% disseminated to the central nervous system and a 5-year survival rate of 30% to 40%. Reference 1). At present, there are 4.6 rare diseases in 1 million per year, and no standard treatment has been established worldwide or in Japan.
  • Diagnosis of intraocular malignant lymphoma is said to be difficult, and it has been reported that the average diagnosis period is 1 year or longer. Moreover, since the amount of the obtained sample is very small and the tissue diagnosis cannot be performed, it is difficult to perform a pathological diagnosis in normal lymphoma. Therefore, the diagnostic probability has been improved so far by using IgH clonality (immunoglobulin H chain) gene rearrangement), Il10/IL6, specificity and sensitivity of cytodiagnosis. Recently, it has been reported that gene mutations of MYD88 and CD79B are found in intraocular malignant lymphoma. However, a diagnostic method for efficiently diagnosing from a small amount of sample has not yet been established.
  • Non-patent documents 2 to Non-Patent Document 5 Apart from the above-mentioned diagnostic method, research is also being conducted on target gene analysis of intraocular malignant lymphoma. L265P mutation in MYD88 about 70% of the intraocular malignant lymphoma is detected, Y196 in CD79B about 35% (F, E, D , C and N) are observed mutations (non-patent documents 2 to Non-Patent Document 5 ). However, a gene diagnosis method capable of performing an auxiliary diagnosis of intraocular malignant lymphoma at a high rate has not yet been established.
  • an object of the present invention is to provide a method for assisting the diagnosis of intraocular malignant lymphoma, which comprises detecting a gene mutation specific to intraocular malignant lymphoma.
  • the present inventors have performed a search other than known intraocular malignant lymphoma-specific gene mutations and found that they are newly identified as intraocular malignant lymphoma-specific mutant genes. BTG2 and PIM1 were found. Further, in view of the extremely small amount of DNA that can be prepared for gene analysis of intraocular malignant lymphoma, the present inventors have made it possible to simultaneously detect multiple gene mutations in order to solve this point. In addition, before analysis by digital PCR (digital PCR; digital polymerase chain reaction), etc., pre-amplification of sample DNA from a small amount of sample by multiplex PCR method etc. enables multiple mutations in one analysis by digital PCR etc. It has been found that the genes can be detected simultaneously.
  • digital PCR digital PCR; digital polymerase chain reaction
  • the present invention is the following (1) to (9).
  • a method for assisting the diagnosis of intraocular malignant lymphoma which comprises detecting one or more of the following gene mutations (A) to (I) in a specimen derived from a subject.
  • the gene mutation is at least one of the above (E), (F) or (I).
  • a method for assisting diagnosis of intraocular malignant lymphoma which comprises detecting one or more of the following gene mutations (a) to (i) in a specimen derived from a subject (a) CD79B CD79B gene mutation with amino acid substitution of Y196N of protein, (B) CD79B mutations involving amino acid substitutions Y196H of CD79B proteins, (C) CD79B mutation involving an amino acid substitution Y196S of CD79B proteins, (D) CD79B mutations involving amino acid substitutions Y196C the CD79B proteins, (E) BTG2 protein involves amino acid substitution A45T the BTG2 gene mutation, (F) BTG2 gene mutation accompanied by E48K amino acid substitution of BTG2 protein, (G) MYD88 proteins involving amino acid substitutions L265P of MYD88 gene mutation, (H) MYD88 proteins involving amino acid substitutions S243N of MYD88 genetic mutations, and (i) PIM1 protein
  • the present invention it becomes possible to detect a mutant gene specific to an intraocular malignant lymphoma and a mutation thereof using a trace amount sample. Therefore, by carrying out the diagnostic assistance method according to the present invention, highly accurate diagnosis of intraocular malignant lymphoma having a poor prognosis becomes possible.
  • the method according to the present invention can detect 96% of cases by detecting mutation sites of 4 genes, and uses not only MYD88 and CD79B but also 4 disease-specific gene mutations of BTG2 and PIM1 mutant genes.
  • This is an auxiliary diagnostic method that can assist the diagnosis at a high rate. Therefore, by using the novel disease-specific gene disclosed for the first time in the present specification, including BTG2 and PIM1 mutant genes, in cases that could not be sufficiently diagnosed by histopathological analysis and pathological cytology. , Useful as an auxiliary diagnosis.
  • the first embodiment of the present invention is a diagnosis of an intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (A) to (I) in a specimen derived from a subject. It is a method to assist.
  • D CD79B c.587A>G mutation
  • E c.133G >A mutation of BTG2 (BTG Anti-proliferation Factor 2)
  • F c.142G >A mutation of BTG2
  • G MYD88 (MYD88 Innate Immune Signal Transduction Adaptor; Myeloid Differentiation Primary Response 88) of c.794T> C mutation
  • the first embodiment of the present invention includes detecting one or more of the gene mutations of (A) to (I) above, and when a gene mutation is detected, This is a method of determining that the subject is an intraocular malignant lymphoma and assisting in the diagnosis of the intraocular malignant lymphoma.
  • the second embodiment of the present invention is directed to the diagnosis of intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (a) to (i) in a specimen derived from a subject.
  • Assisting method (a) CD79B gene mutation involving amino acid substitution of Y196N of CD79B protein, (B) CD79B mutations involving amino acid substitutions Y196H of CD79B proteins, (C) CD79B mutation involving an amino acid substitution Y196S of CD79B proteins, (D) CD79B mutations involving amino acid substitutions Y196C the CD79B proteins, (E) BTG2 protein involves amino acid substitution A45T the BTG2 gene mutation, (F) BTG2 gene mutation accompanied by E48K amino acid substitution of BTG2 protein, (G) MYD88 proteins involving amino acid substitutions L265P of MYD88 gene mutation, (H) MYD88 gene mutation with S243N amino acid substitution of MYD88 protein, and (i) P
  • the second embodiment of the present invention comprises detecting one or more of the gene mutations of (a) to (i) above, and when a gene mutation is detected, This is a method of determining that the subject is an intraocular malignant lymphoma and assisting the diagnosis of the intraocular malignant lymphoma.
  • the gene name is underlined, and when the gene product is represented, the gene name is described without being underlined, or the gene name is followed by ""Protein" will be described.
  • the method for detecting a gene mutation may be any method and is not particularly limited, and examples thereof include a digital PCR method, an allele-specific PCR (ASPCR) method, and a metagenetic deep sequencing method.
  • ASPCR allele-specific PCR
  • a metagenetic deep sequencing method You can In particular, when the amount of sample is very small, a small amount of DNA extracted from the sample is amplified in advance by PCR (e.g., multiplex PCR method, multiplex PCR method using digital PCR method) before performing the above-described analysis ( Pre-amplification) and performing the above analysis using the obtained amplification product enables efficient detection and enables detection of a plurality of gene mutation sites at once. If a certain amount of sample can be secured, gene mutation can be detected by using Sanger sequencing or next generation sequencing (NGS).
  • NGS next generation sequencing
  • the subject-derived sample may be a sample containing chromosomal DNA derived from an intraocular malignant lymphoma, and is not particularly limited, and examples thereof include intraocular fluid (for example, vitreous humor and anterior chamber). Water solution), cerebrospinal fluid, peripheral blood and bone marrow.
  • intraocular fluid for example, vitreous humor and anterior chamber. Water solution
  • cerebrospinal fluid for example, peripheral blood and bone marrow.
  • the third embodiment of the present invention is a kit for carrying out the method according to the present embodiment.
  • the kit according to the third embodiment contains at least the elements necessary for detecting the gene mutation of any one of (A) to (I) or (a) to (i). included.
  • at least any one of the gene mutations of (E), (F) or (I) above or any of (e), (f) or (i) above It is preferable that an element necessary for detecting one gene mutation is included.
  • the above-mentioned elements are not particularly limited, but examples thereof include primers and probes for performing digital PCR and sequencing.
  • Method 1-1 Sample treatment of vitreous humor and DNA extraction Pass the cells through a 48 ⁇ m nylon mesh filter into the vitreous humor obtained by vitreous surgery of a patient diagnosed with intraocular malignant lymphoma, wash with MACS buffer, 1500 rpm, Centrifuge for 5 minutes at 4°C. The cells were washed twice more with MACS buffer, centrifuged at 2500 rpm and 4°C for 5 minutes, and the supernatant was discarded. The lower solution was subjected to DNA extraction using AllPrep DNA/RNA Micro Kit (QIAGEN, Valencia, CA, USA).
  • the above-mentioned intraocular malignant lymphoma was diagnosed using the following criteria; (1) Cytology class 4 or higher, or (2) Cytology class 3, (3) IL10/IL6>1, ( 4) IgH gene rearrangement (PCR method) positive (LSI mediaence), (5) kappa/lambda ratio >3 or ⁇ 05 by FACS (Davis et al, Am J Ophthalmol 2005, 140:822-29: Sagoo et al , Surv Ophthalmol 2014, 59:503-16) (2) to (5), 2 or more of the 4 items must be positive.
  • the cytodiagnosis class 3 is atypical and may be malignant, and the class 4 is likely to be malignant cells.
  • Tables 2 and 3 show the mutation regions of each gene and the set positions of primers and probes for Digital PCR analysis (note that the sequence regions shown in Tables 2 and 3 are CD79B (c.586T> A, c.586T>C, c.587A>C, c.587A>G: SEQ ID NO: 33), BTG2 (c.133G>A, c142G>A: SEQ ID NO: 34), MYD88 (c.794T>c: SEQ ID NO: 35, c.728G>A: SEQ ID NO: 36) and PIM1 (c.550C>T: SEQ ID NO: 37) in the sequence listing).
  • mutation sites are shown as [T/A] (mutation from T to A), [A/C] (mutation from A to C), and the like.
  • Detection of gene mutation When confirming the presence or absence of multiple gene mutations in a digital PCR analysis, it was necessary to perform a digital PCR analysis for each gene mutation until now. Was needed. However, the inventors have for the first time established a detection system for digital PCR analysis capable of simultaneously detecting a plurality of gene mutations from a small amount of sample that can obtain only a small amount of DNA. That is, a small amount of DNA extracted from a small amount of sample is pre-amplified by applying the multiplex PCR method in a digital PCR droplet to improve amplification efficiency, and 9 points of 4 genes selected as analysis points are analyzed once. It was successfully detected by digital PCR analysis. Specifically, the following method is used.
  • Pre-amplification When the total DNA amount was less than 10 ng in the digital PCR analysis, pre-amplification by the multiplex PCR method was performed with reference to the previous report. multiplex PCR is adjusted so that the final concentration of each primer is 5 ⁇ M, using 2xddPCR SuperMix For Probe (no dUTP; Bio-Rad Laboratories, Hercules, CA, USA) which is a master mix, template DNA, 5 ⁇ M primer, It was adjusted to 20 ⁇ l including nuclease free water.
  • 2xddPCR SuperMix For Probe no dUTP; Bio-Rad Laboratories, Hercules, CA, USA
  • Droplet Generator Oil For Probe (Bio-Rad Laboratories, Hercules, CA, USA) was added to 20 ⁇ l of this adjusted solution, and droplets were prepared using QX200 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA). Using a MiniAmp Thermal Cycler applied biosystems (Thermo Fisher Scientific, Waltham, MA, USA), this droplet was reacted at 95°C for 10 minutes, and then 94°C for 30 seconds and 60°C for 1 minute, 10 cycles were performed, and 98 The reaction was carried out at 0°C for 10 minutes, and preamplification by PCR was performed.
  • amplicon After pre-amplification was purified using DNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, CA, USA) and adjusted to a final volume of 20 ⁇ l.
  • 2xddPCR SuperMix For Probe (no dUTP; Bio-Rad Laboratories, Hercules, CA, USA), TaqMan Assay 40x (Thermo Fisher Scientific, Waltham, Massachusetts, USA; FAM or VIC) (8 ⁇ M probe, mix of 36 ⁇ M each primer) and nuclease free water were added to adjust the total volume to 20 ⁇ l.
  • 70 ⁇ l of Droplet Generator Oil For Probe Bio-Rad Laboratories, Hercules, CA, USA
  • droplets were created with the QX200 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA). ..
  • the present invention is expected to be used in the medical field.

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Abstract

The purpose of the present invention is to provide an auxiliary diagnostic method for intraocular malignant lymphomas that includes detecting an intraocular malignant lymphoma-specific gene mutation. Specifically, the present invention is an auxiliary diagnostic method for intraocular malignant lymphomas that includes, from a subject-derived sample, detecting one or more from among the gene mutations of (A) to (I) below: (A) c.586T>A mutation of <u>CD79B</u>; (B) c.586T>C mutation of <u>CD79B</u>; (C) c.587A>C mutation of <u>CD79B</u>; (D) c.587A>G mutation of <u>CD79B</u>; (E) c.133G>A mutation of <u>BTG2</u>; (F) c.142G>A mutation of <u>BTG2</u>; (G) c.794T>C mutation of <u>MYD88</u>; (H) c.728G>A mutation of <u>MYD88</u>; and (I) c.550C>T mutation of <u>PIM1</u>.

Description

眼内悪性リンパ腫の補助的診断法Auxiliary diagnostic method for intraocular malignant lymphoma
　本発明は、眼内悪性リンパ腫の補助的診断法に関する。より具体的には、眼内悪性リンパ腫に特異的な遺伝子変異を指標とする補助的診断方法に関する。 The present invention relates to an auxiliary diagnostic method for intraocular malignant lymphoma. More specifically, it relates to an auxiliary diagnostic method using a gene mutation specific to intraocular malignant lymphoma as an index.
　眼内悪性リンパ腫は、脳中枢神経系へ60-80%と高率に播種し、5年生存率は30%から40%と眼疾患の中でも最も生命予後の悪い難治性疾患である（非特許文献１）。現在のところ、年間100万人に4.6人の希少疾患であることもあり、標準治療は世界的および本邦においても確立されていない。 Intraocular malignant lymphoma is a highly intractable disease with the worst prognosis among the eye diseases, with a high rate of 60-80% disseminated to the central nervous system and a 5-year survival rate of 30% to 40%. Reference 1). At present, there are 4.6 rare diseases in 1 million per year, and no standard treatment has been established worldwide or in Japan.
　眼内悪性リンパ腫の診断は、診断自体が困難であるとされ、診断における平均期間は1年以上という報告がある。また、得られる検体が微量であり、組織診断が出来ないことから、通常のリンパ腫における病理診断が困難である。そのため、これまでに、IgHクローナリティ(免疫グロブリンH鎖)遺伝子再構成）、Il10/IL6、細胞診の特異度、感度を用いて診断確率を向上させてきた。近年では、MYD88やCD79Bの遺伝子変異が眼内悪性リンパ腫に認められることが報告されている。しかしながら、微量な検体から効率よく診断する診断方法は未だ確立されていない。 Diagnosis of intraocular malignant lymphoma is said to be difficult, and it has been reported that the average diagnosis period is 1 year or longer. Moreover, since the amount of the obtained sample is very small and the tissue diagnosis cannot be performed, it is difficult to perform a pathological diagnosis in normal lymphoma. Therefore, the diagnostic probability has been improved so far by using IgH clonality (immunoglobulin H chain) gene rearrangement), Il10/IL6, specificity and sensitivity of cytodiagnosis. Recently, it has been reported that gene mutations of MYD88 and CD79B are found in intraocular malignant lymphoma. However, a diagnostic method for efficiently diagnosing from a small amount of sample has not yet been established.
　上記診断方法とは別に、眼内悪性リンパ腫の標的遺伝子解析についても研究が進められている。眼内悪性リンパ腫の約7割でMYD88におけるL265P変異が検出され、約35％でCD79Bにおける Y196（F、E、D、CおよびN）変異をみとめている（非特許文献２～非特許文献５）。しかしながら、高率に眼内悪性リンパ腫と補助診断できる遺伝子診断方法は未だ確立されてない。 Apart from the above-mentioned diagnostic method, research is also being conducted on target gene analysis of intraocular malignant lymphoma. L265P mutation in MYD88 about 70% of the intraocular malignant lymphoma is detected, Y196 in CD79B about 35% (F, E, D , C and N) are observed mutations (non-patent documents 2 to Non-Patent Document 5 ). However, a gene diagnosis method capable of performing an auxiliary diagnosis of intraocular malignant lymphoma at a high rate has not yet been established.
　上記事情に鑑み、本発明は、眼内悪性リンパ腫に特異的な遺伝子変異を検出することを含む、眼内悪性リンパ種の診断を補助する方法の提供を目的とする。 In view of the above circumstances, an object of the present invention is to provide a method for assisting the diagnosis of intraocular malignant lymphoma, which comprises detecting a gene mutation specific to intraocular malignant lymphoma.
　本発明者らは、上記課題を解決するために、これまでに知られている眼内悪性リンパ腫特異的な遺伝子変異以外の検索を行ったところ、眼内悪性リンパ腫特異的な変異遺伝子として新規にBTG2およびPIM1を見出した。
　さらに、本発明者らは、眼内悪性リンパ腫の遺伝子解析のために調製できるDNA量が非常に微量であることに鑑み、この点を解決すべく、複数の遺伝子変異を同時に検出可能とするために、デジタルPCR（digital PCR；digital polymerase chain reaction）等による解析の前に、微量検体由来のサンプルDNAをmultiplex PCR法などにより前増幅することによって、1回のデジタルPCR等による解析で複数の変異遺伝子が同時に検出可能となることを見出した。 In order to solve the above-mentioned problems, the present inventors have performed a search other than known intraocular malignant lymphoma-specific gene mutations and found that they are newly identified as intraocular malignant lymphoma-specific mutant genes. BTG2 and PIM1 were found.
Further, in view of the extremely small amount of DNA that can be prepared for gene analysis of intraocular malignant lymphoma, the present inventors have made it possible to simultaneously detect multiple gene mutations in order to solve this point. In addition, before analysis by digital PCR (digital PCR; digital polymerase chain reaction), etc., pre-amplification of sample DNA from a small amount of sample by multiplex PCR method etc. enables multiple mutations in one analysis by digital PCR etc. It has been found that the genes can be detected simultaneously.
　すなわち、本発明は、以下の（１）～（９）である。
（１）被験者由来の検体において、以下の（Ａ）ないし（Ｉ）のいずれかの遺伝子変異の1または複数を検出することを含む、眼内悪性リンパ種の診断を補助する方法。
（Ａ）CD79Bのc.586T>A変異、
（Ｂ）CD79Bのc.586T>C変異、
（Ｃ）CD79Bのc.587A>C変異、
（Ｄ）CD79Bのc.587A>G変異、
（Ｅ）BTG2のc.133G>A変異、
（Ｆ）BTG2のc.142G>A変異、
（Ｇ）MYD88のc.794T>C変異、
（Ｈ）MYD88のc.728G>A変異、および
（Ｉ）PIM1のc.550C>T変異
（２）前記遺伝子変異が、少なくとも、前記（Ｅ）、（Ｆ）または（Ｉ）のいずれかであることを特徴とする上記（１）に記載の方法。
（３）被験者由来の検体において、以下の（ａ）ないし（ｉ）のいずれかの遺伝子変異の1または複数を検出することを含む、眼内悪性リンパ種の診断を補助する方法
（ａ）CD79Bタンパク質のY196Nのアミノ酸置換を伴うCD79B遺伝子変異、
（ｂ）CD79Bタンパク質のY196Hのアミノ酸置換を伴うCD79B遺伝子変異、
（ｃ）CD79Bタンパク質のY196Sのアミノ酸置換を伴うCD79B遺伝子変異、
（ｄ）CD79Bタンパク質のY196Cのアミノ酸置換を伴うCD79B遺伝子変異、
（ｅ）BTG2タンパク質のA45Tのアミノ酸置換を伴うBTG2遺伝子変異、
（ｆ）BTG2タンパク質のE48Kのアミノ酸置換を伴うBTG2遺伝子変異、
（ｇ）MYD88タンパク質のL265Pのアミノ酸置換を伴うMYD88遺伝子変異、
（ｈ）MYD88タンパク質のS243Nのアミノ酸置換を伴うMYD88遺伝子変異、および
（ｉ）PIM1タンパク質のL184Fのアミノ酸置換を伴うPIM1遺伝子変異
（４）前記遺伝子変異が、少なくとも、前記（ｅ）、（ｆ）または（ｉ）のいずれかであることを特徴とする上記（３）に記載の方法。
（５）前記遺伝子変異の検出を、デジタルPCR法により検出することを特徴とする上記（１）ないし（４）のいずれかに記載の方法。
（６）上記（５）の検出を行う前に、予め検体由来のDNAを増幅することを特徴とする（５）に記載の方法。
（７）前記検体が眼内液、髄液、末梢血または骨髄であることを特徴とする上記（１）ないし（６）のいずれかに記載の方法。
（８）眼内悪性リンパ種の診断を補助する方法を実施するためのキット。
（９）以下の（Ａ）ないし（Ｉ）のいずれかの遺伝子変異の1または複数を検出するためのプローブおよび／またはプライマーを含むことを特徴とする上記（８）に記載のキット。
（Ａ）CD79Bのc.586T>A変異、
（Ｂ）CD79Bのc.586T>C変異、
（Ｃ）CD79Bのc.587A>C変異、
（Ｄ）CD79Bのc.587A>G変異、
（Ｅ）BTG2のc.133G>A変異、
（Ｆ）BTG2のc.142G>A変異、
（Ｇ）MYD88のc.728G>A変異、
（Ｈ）MYD88のc.794T>C変異、および
（Ｉ）PIM1のc.550C>T変異 That is, the present invention is the following (1) to (9).
(1) A method for assisting the diagnosis of intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (A) to (I) in a specimen derived from a subject.
(A) CD79B c.586T>A mutation,
(B) CD79B c.586T>C mutation,
(C) CD79B of c.587A> C mutation,
(D) CD79B c.587A>G mutation,
(E) c.133G >A mutation of BTG2,
(F) c.142G >A mutation of BTG2,
(G) c.794T >C mutation of MYD88,
(H) c.728G >A mutation of MYD88, and (I) c.550C >T mutation of PIM1 (2) The gene mutation is at least one of the above (E), (F) or (I). The method according to (1) above, wherein
(3) A method for assisting diagnosis of intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (a) to (i) in a specimen derived from a subject (a) CD79B CD79B gene mutation with amino acid substitution of Y196N of protein,
(B) CD79B mutations involving amino acid substitutions Y196H of CD79B proteins,
(C) CD79B mutation involving an amino acid substitution Y196S of CD79B proteins,
(D) CD79B mutations involving amino acid substitutions Y196C the CD79B proteins,
(E) BTG2 protein involves amino acid substitution A45T the BTG2 gene mutation,
(F) BTG2 gene mutation accompanied by E48K amino acid substitution of BTG2 protein,
(G) MYD88 proteins involving amino acid substitutions L265P of MYD88 gene mutation,
(H) MYD88 proteins involving amino acid substitutions S243N of MYD88 genetic mutations, and (i) PIM1 protein PIM1 mutations involving amino acid substitutions L184F of (4) the gene mutation, at least, the (e), (f) Alternatively, the method according to (3) above, which is either (i).
(5) The method according to any one of (1) to (4) above, wherein the detection of the gene mutation is performed by a digital PCR method.
(6) The method according to (5), wherein the sample-derived DNA is amplified in advance before the detection in (5) above.
(7) The method according to any one of (1) to (6) above, wherein the sample is intraocular fluid, spinal fluid, peripheral blood, or bone marrow.
(8) A kit for carrying out a method for assisting the diagnosis of intraocular malignant lymphoma.
(9) The kit according to (8) above, which comprises a probe and/or a primer for detecting one or more of the following gene mutations (A) to (I).
(A) CD79B c.586T>A mutation,
(B) CD79B c.586T>C mutation,
(C) CD79B of c.587A> C mutation,
(D) CD79B c.587A>G mutation,
(E) c.133G >A mutation of BTG2,
(F) c.142G >A mutation of BTG2,
(G) c.728G >A mutation of MYD88,
(H) MYD88 of c.794T> C mutation, and (I) PIM1 of c.550C> T mutation
　本発明により、微量検体による眼内悪性リンパ種特異的な変異遺伝子およびその変異の検出が可能となる。従って、本発明による診断補助方法を実施することで、予後不良とされる眼内悪性リンパ種の確度の高い診断が可能となる。 According to the present invention, it becomes possible to detect a mutant gene specific to an intraocular malignant lymphoma and a mutation thereof using a trace amount sample. Therefore, by carrying out the diagnostic assistance method according to the present invention, highly accurate diagnosis of intraocular malignant lymphoma having a poor prognosis becomes possible.
　これまでのMYD88やCD79Bの遺伝子変異では、眼内悪性リンパ腫の70%程度しか補助診断の役割を果たしていなかった。本発明にかかる方法は、4遺伝子9箇所の変異箇所の検出により、症例の96%を網羅できており、MYD88やCD79B だけでなく、BTG2、PIM1変異遺伝子の4つの疾患特異的遺伝子変異を用いることで高率に診断を補助できる補助的診断方法である。従って、BTG2、PIM1変異遺伝子を含む、本明細書よって初めて開示された新規な疾患特異的遺伝子を用いることでこれまで病理組織学的解析、病理細胞診では十分に診断できなかった症例に対して、補助診断として有用である。 Up to now , only about 70% of intraocular malignant lymphomas have played a role as an auxiliary diagnosis in the gene mutations of MYD88 and CD79B . The method according to the present invention can detect 96% of cases by detecting mutation sites of 4 genes, and uses not only MYD88 and CD79B but also 4 disease-specific gene mutations of BTG2 and PIM1 mutant genes. This is an auxiliary diagnostic method that can assist the diagnosis at a high rate. Therefore, by using the novel disease-specific gene disclosed for the first time in the present specification, including BTG2 and PIM1 mutant genes, in cases that could not be sufficiently diagnosed by histopathological analysis and pathological cytology. , Useful as an auxiliary diagnosis.
　さらに、眼内悪性リンパ腫は、得られる検体量が微量であるため遺伝子解析が困難であったが、本発明において、multiplex PCR法を用いて、DNAの前増幅を行うことで、微量な検体量から変異遺伝子を検出する系を確立した。この増幅系を用いることで、微量な検体であってもデジタルPCR法等による測定可能となる。 Furthermore, in intraocular malignant lymphoma, it was difficult to carry out gene analysis because the amount of sample obtained was very small. A system for detecting mutant genes was established from. By using this amplification system, even a small amount of sample can be measured by the digital PCR method or the like.
眼内悪性リンパ種症例のデジタルPCR解析結果の代表例。プローブ6（BTG2 c.142G>A E48K）を用いて解析を行った。Representative example of digital PCR analysis results of cases of intraocular malignant lymphoma. Analysis was performed using probe 6 ( BTG2 c.142G>A E48K).
　本発明の第１の実施形態は、被験者由来の検体において、以下の（Ａ）ないし（Ｉ）のいずれかの遺伝子変異の1または複数を検出することを含む、眼内悪性リンパ種の診断を補助する方法である。
（Ａ）CD79B（CD79b Molecule）のc.586T>A変異、
（Ｂ）CD79Bのc.586T>C変異、
（Ｃ）CD79Bのc.587A>C変異、
（Ｄ）CD79Bのc.587A>G変異、
（Ｅ）BTG2（BTG Anti-proliferation Factor 2）のc.133G>A変異、
（Ｆ）BTG2のc.142G>A変異、
（Ｇ）MYD88（MYD88 Innate Immune Signal Transduction Adaptor；Myeloid Differentiation Primary Response 88）のc.794T>C変異
（Ｈ）MYD88のc.728G>A変異、および
（Ｉ）PIM1（Pim-1 Proto-oncogene, Serine/Threonine kinase）のc.550C>T変異 The first embodiment of the present invention is a diagnosis of an intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (A) to (I) in a specimen derived from a subject. It is a method to assist.
(A) CD79B (CD79b Molecule) c.586T>A mutation,
(B) CD79B c.586T>C mutation,
(C) CD79B of c.587A> C mutation,
(D) CD79B c.587A>G mutation,
(E) c.133G >A mutation of BTG2 (BTG Anti-proliferation Factor 2),
(F) c.142G >A mutation of BTG2,
(G) MYD88 (MYD88 Innate Immune Signal Transduction Adaptor; Myeloid Differentiation Primary Response 88) of c.794T> C mutation (H) MYD88 of c.728G> A mutation, and (I) PIM1 (Pim-1 Proto-oncogene, Serine/Threonine kinase) c.550C>T mutation
　より具体的には、本発明の第１の実施形態は、前記（Ａ）ないし（Ｉ）のいずれかの遺伝子変異の1または複数を検出することを含み、遺伝子変異が検出されたときに、当該被験者は眼内悪性リンパ種であると判定し、眼内悪性リンパ種の診断を補助する方法である。第１の実施形態における遺伝子検出工程において、少なくとも前記（Ｅ）、（Ｆ）または（Ｉ）のいずれか１つの遺伝子変異を検出することが好ましい。 More specifically, the first embodiment of the present invention includes detecting one or more of the gene mutations of (A) to (I) above, and when a gene mutation is detected, This is a method of determining that the subject is an intraocular malignant lymphoma and assisting in the diagnosis of the intraocular malignant lymphoma. In the gene detection step in the first embodiment, it is preferable to detect at least one of the gene mutations (E), (F) or (I).
　本発明の第２の実施形態は、被験者由来の検体において、以下の（ａ）ないし（ｉ）のいずれかの遺伝子変異の1または複数を検出することを含む、眼内悪性リンパ種の診断を補助する方法
（ａ）CD79Bタンパク質のY196Nのアミノ酸置換を伴うCD79B遺伝子変異、
（ｂ）CD79Bタンパク質のY196Hのアミノ酸置換を伴うCD79B遺伝子変異、
（ｃ）CD79Bタンパク質のY196Sのアミノ酸置換を伴うCD79B遺伝子変異、
（ｄ）CD79Bタンパク質のY196Cのアミノ酸置換を伴うCD79B遺伝子変異、
（ｅ）BTG2タンパク質のA45Tのアミノ酸置換を伴うBTG2遺伝子変異、
（ｆ）BTG2タンパク質のE48Kのアミノ酸置換を伴うBTG2遺伝子変異、
（ｇ）MYD88タンパク質のL265Pのアミノ酸置換を伴うMYD88遺伝子変異、
（ｈ）MYD88タンパク質のS243Nのアミノ酸置換を伴うMYD88遺伝子変異、および
（ｉ）PIM1タンパク質のL184Fのアミノ酸置換を伴うPIM1遺伝子変異 The second embodiment of the present invention is directed to the diagnosis of intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (a) to (i) in a specimen derived from a subject. Assisting method (a) CD79B gene mutation involving amino acid substitution of Y196N of CD79B protein,
(B) CD79B mutations involving amino acid substitutions Y196H of CD79B proteins,
(C) CD79B mutation involving an amino acid substitution Y196S of CD79B proteins,
(D) CD79B mutations involving amino acid substitutions Y196C the CD79B proteins,
(E) BTG2 protein involves amino acid substitution A45T the BTG2 gene mutation,
(F) BTG2 gene mutation accompanied by E48K amino acid substitution of BTG2 protein,
(G) MYD88 proteins involving amino acid substitutions L265P of MYD88 gene mutation,
(H) MYD88 gene mutation with S243N amino acid substitution of MYD88 protein, and (i) PIM1 gene mutation with L184F amino acid substitution of PIM1 protein
　　より具体的には、本発明の第２の実施形態は、前記（ａ）ないし（ｉ）のいずれかの遺伝子変異の1または複数を検出することを含み、遺伝子変異が検出されたときに、当該被験者は眼内悪性リンパ種であると判定し、眼内悪性リンパ種の診断を補助する方法である。第１の実施形態における遺伝子検出工程において、少なくとも前記（ｅ）、（ｆ）または（ｉ）のいずれか１つの遺伝子変異を検出することが好ましい。
　なお、本明細書においては遺伝子名を表す場合には、遺伝子名に下線を付し、その遺伝子産物を表す場合には、遺伝子名に下線を付さずに記載する、または遺伝子名の後に「タンパク質」を記載することとする。 More specifically, the second embodiment of the present invention comprises detecting one or more of the gene mutations of (a) to (i) above, and when a gene mutation is detected, This is a method of determining that the subject is an intraocular malignant lymphoma and assisting the diagnosis of the intraocular malignant lymphoma. In the gene detection step in the first embodiment, it is preferable to detect at least one gene mutation of (e), (f) or (i) above.
In the present specification, when a gene name is represented, the gene name is underlined, and when the gene product is represented, the gene name is described without being underlined, or the gene name is followed by ""Protein" will be described.
　本実施形態において、遺伝子変異を検出する方法は、いかなる方法であってもよく、特に限定はしないが、例えば、デジタルPCR法、allele-specific PCR（ASPCR）法、metagenomic deep sequencing法などを挙げることができる。特に、検体量が微量の場合には、上記例示した解析を行う前に、検体から抽出した微量DNAを、予めPCR法（例えばmultiplex PCR法、デジタルPCR法を用いたmultiplex PCR法）で増幅（前増幅）し、得られた増幅産物を使用して上記解析を行うことで、効率的な検出が可能となり、複数の遺伝子変異箇所を一度に検出することが可能となる。また、ある程度の検体量が確保できる場合には、サンガーシークエンスや次世代シークエンス（next generation sequencing：NGS）などを用いても遺伝子変異の検出が可能である。 In the present embodiment, the method for detecting a gene mutation may be any method and is not particularly limited, and examples thereof include a digital PCR method, an allele-specific PCR (ASPCR) method, and a metagenetic deep sequencing method. You can In particular, when the amount of sample is very small, a small amount of DNA extracted from the sample is amplified in advance by PCR (e.g., multiplex PCR method, multiplex PCR method using digital PCR method) before performing the above-described analysis ( Pre-amplification) and performing the above analysis using the obtained amplification product enables efficient detection and enables detection of a plurality of gene mutation sites at once. If a certain amount of sample can be secured, gene mutation can be detected by using Sanger sequencing or next generation sequencing (NGS).
　本実施形態において、被験者由来の検体は、眼内悪性リンパ種由来の染色体DNAが含まれている検体であればよく、特に限定はしないが、例えば、眼内液（例えば、硝子体液および前房水液など）、髄液、末梢血および骨髄などを挙げることができる。 In the present embodiment, the subject-derived sample may be a sample containing chromosomal DNA derived from an intraocular malignant lymphoma, and is not particularly limited, and examples thereof include intraocular fluid (for example, vitreous humor and anterior chamber). Water solution), cerebrospinal fluid, peripheral blood and bone marrow.
　本発明の第３の実施形態は、本実施形態にかかる方法を実施するためのキットである。第３の実施形態にかかるキットには、少なくとも、前記（Ａ）ないし（Ｉ）のいずれか、または、前記（ａ）ないし（ｉ）のいずれかの遺伝子変異を検出するために必要な要素が含まれる。特に、第２の実施形態のキットには、少なくとも、前記（Ｅ）、（Ｆ）または（Ｉ）のいずれか１つの遺伝子変異、あるいは、前記（ｅ）、（ｆ）または（ｉ）のいずれか１つの遺伝子変異を検出するために必要な要素が含まれることが好ましい。上記要素としては、特に限定はしないが、例えば、デジタルPCRやシークエンスを行うための、プライマーやプローブなどを挙げることができる。 The third embodiment of the present invention is a kit for carrying out the method according to the present embodiment. The kit according to the third embodiment contains at least the elements necessary for detecting the gene mutation of any one of (A) to (I) or (a) to (i). included. In particular, in the kit of the second embodiment, at least any one of the gene mutations of (E), (F) or (I) above or any of (e), (f) or (i) above It is preferable that an element necessary for detecting one gene mutation is included. The above-mentioned elements are not particularly limited, but examples thereof include primers and probes for performing digital PCR and sequencing.
　本明細書が英語に翻訳されて、単数形の「a」、「an」および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものも含むものとする。
　以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 When this specification is translated into English and includes the singular forms of the words "a", "an" and "the", the singular as well as the plural is used unless the context clearly indicates otherwise. It includes the thing of.
EXAMPLES The present invention will be further described below with reference to examples, but the examples are merely examples of the embodiments of the present invention and do not limit the scope of the present invention.
１．方法
１－１．硝子体液の検体処理とDNA抽出
　眼内悪性リンパ腫と診断された患者の硝子体手術により得た硝子体液中に細胞を48 μmのナイロンメッシュのフィルターに通過させ、MACS bufferで洗浄し、1500 rpm、4℃で5分間遠心分離した。MACS bufferで更に2回洗浄し、2500rpm、4℃で5分間遠心分離し上清を破棄した。下液をAllPrep DNA/RNA Micro Kit (QIAGEN, Valencia, CA, USA)を用いてDNA抽出した。
　上記眼内悪性リンパ種の診断は、以下の基準を用いて行った；（１）細胞診クラス４以上であること、または（２）細胞診クラス3、（３）IL10/IL6>1、（４）IgH遺伝子再構成（PCR法）陽性（LSIメディエンス）、（５）FACSでkappa/lambda ratio >3 or<05) (Davis et al, Am J Ophthalmol 2005, 140:822-29：Sagoo et al, Surv Ophthalmol 2014, 59:503-16)の（２）から（５）の4項目中2項目以上陽性であること。なお、細胞診クラス３とは異型性があり、悪性の疑いあり、クラス４とは悪性細胞の可能性が高いものをいう。 1. Method 1-1. Sample treatment of vitreous humor and DNA extraction Pass the cells through a 48 μm nylon mesh filter into the vitreous humor obtained by vitreous surgery of a patient diagnosed with intraocular malignant lymphoma, wash with MACS buffer, 1500 rpm, Centrifuge for 5 minutes at 4°C. The cells were washed twice more with MACS buffer, centrifuged at 2500 rpm and 4°C for 5 minutes, and the supernatant was discarded. The lower solution was subjected to DNA extraction using AllPrep DNA/RNA Micro Kit (QIAGEN, Valencia, CA, USA).
The above-mentioned intraocular malignant lymphoma was diagnosed using the following criteria; (1) Cytology class 4 or higher, or (2) Cytology class 3, (3) IL10/IL6>1, ( 4) IgH gene rearrangement (PCR method) positive (LSI mediaence), (5) kappa/lambda ratio >3 or<05 by FACS (Davis et al, Am J Ophthalmol 2005, 140:822-29: Sagoo et al , Surv Ophthalmol 2014, 59:503-16) (2) to (5), 2 or more of the 4 items must be positive. The cytodiagnosis class 3 is atypical and may be malignant, and the class 4 is likely to be malignant cells.
１－２．変異遺伝子および解析箇所の選定
　本実施例ではいくつかの眼内悪性リンパ腫と関連する候補遺伝子（CD79B、BTG2、MYD88および PIM1など）および変異箇所を選定し、表１に示す通り4遺伝子9箇所を解析箇所として選出した。4遺伝子9箇所はすべて一塩基置換である。
Figure JPOXMLDOC01-appb-T000001
 1-2. Mutant gene and selection of the analysis portion in this embodiment the candidate genes associated with several intraocular malignant lymphoma (CD79B, BTG2, such MYD88 and PIM1) and selects the mutation site, the street 4 gene nine shown in Table 1 Selected as an analysis point. All 9 positions of 4 genes have single nucleotide substitutions.
Figure JPOXMLDOC01-appb-T000001
１－３．プライマーおよびプローブの作成
　MYD88 L265P以外の4遺伝子8箇所のプローブおよびプライマー配列は、Custom TaqMan（登録商標）Assay Design Tool（Thermo Fisher Scientific, Waltham, MA, USA）を用いて合成した。MYD88 L265Pのプローブおよびプライマーは、中枢性悪性リンパ腫のMYD88 L265P検出を行った既報と同じ配列を設計した。
　プライマーおよびプローブ配列（FAMは変異体のプローブ、VICは野生型のプローブを表す）を以下に示す。また、表２および表３には、各遺伝子の変異領域とDigital PCR解析のためのプライマーおよびプローブの設定位置を示す（なお、表２および表３に示す配列領域は、CD79B（c.586T>A、c.586T>C、c.587A>C、c.587A>G：配列番号３３）、BTG2（c.133G>A、c142G>A：配列番号３４）、MYD88（c.794T>c：配列番号３５、c.728G>A：配列番号３６）およびPIM1（c.550C>T：配列番号３７）として配列表に示す）。表２および３において、変異箇所を[T/A]（TからAへの変異）、[A/C]（AからCへの変異）などのように示した。 1-3. Preparation of Primers and Probes Probes and primer sequences at 8 sites of 4 genes other than MYD88 L265P were synthesized using Custom TaqMan (registered trademark) Assay Design Tool (Thermo Fisher Scientific, Waltham, MA, USA). The MYD88 L265P probe and primer were designed with the same sequences as those previously reported for MYD88 L265P detection in central malignant lymphoma.
The primer and probe sequences (FAM stands for mutant probe, VIC stands for wild type probe) are shown below. Further, Tables 2 and 3 show the mutation regions of each gene and the set positions of primers and probes for Digital PCR analysis (note that the sequence regions shown in Tables 2 and 3 are CD79B (c.586T> A, c.586T>C, c.587A>C, c.587A>G: SEQ ID NO: 33), BTG2 (c.133G>A, c142G>A: SEQ ID NO: 34), MYD88 (c.794T>c: SEQ ID NO: 35, c.728G>A: SEQ ID NO: 36) and PIM1 (c.550C>T: SEQ ID NO: 37) in the sequence listing). In Tables 2 and 3, mutation sites are shown as [T/A] (mutation from T to A), [A/C] (mutation from A to C), and the like.
＜プライマー配列＞
CD79B c.586T>A
Forward；5’- GGGCCTGCCCCTCTC -3’（配列番号１）
Reverse；5’- AGCAAGGCTGGCATGGA -3’（配列番号２）
CD79B c.586T>C
Forward；5’- GGGCCTGCCCCTCTC -3’（配列番号１）
Reverse；5’- AGCAAGGCTGGCATGGA -3’（配列番号２）
CD79B c.587A>C
Forward；5’- CTGACCCCGAGGACTCAGA -3’（配列番号３）
Reverse；5’- GGCTGGCATGGAGGAAGA -3’（配列番号４）
CD79B c.587A>G
Forward；5’- CTGACCCCGAGGACTCAGA -3’（配列番号３）
Reverse；5’- GGCTGGCATGGAGGAAGA -3’（配列番号４）
BTG2 c.133G>A
Forward；5’- GAGCAGAGGCTTAAGGTCTTCA -3’（配列番号５）
Reverse；5’- GGCATGCGCTCACCTG -3’（配列番号６）
BTG2 c.142G>A
Forward；5’- GAGCAGAGGCTTAAGGTCTTCAG -3’（配列番号７）
Reverse；5’- AGGCCCCTCGGCATG -3’（配列番号８）
MYD88 c.794T>C
Forward；5’- CCTTGGCTTGCAGGT -3’（配列番号９）
Reverse；5’- TCTTTCTTCATTGCCTTGT -3’（配列番号１０）
MYD88 c.728G>A
Forward；5’- GGTGGTGGTTGTCTCTGATGATTAC -3’（配列番号１１）
Reverse；5’- TGAGTGCAAATTTGGTCTGGAAGT -3’（配列番号１２）
PIM1 c.550C>T
Forward；5’- GAAAACATCCTTATCGACCTCAATCG -3’（配列番号１３）
Reverse；5’- CGTGTAGACGGTGTCCTTGAG -3’（配列番号１４） <Primer sequence>
CD79B c.586T>A
Forward; 5'- GGGCCTGCCCCTCTC -3' (SEQ ID NO: 1)
Reverse; 5'- AGCAAGGCTGGCATGGA -3' (SEQ ID NO: 2)
CD79B c.586T>C
Forward; 5'- GGGCCTGCCCCTCTC -3' (SEQ ID NO: 1)
Reverse; 5'- AGCAAGGCTGGCATGGA -3' (SEQ ID NO: 2)
CD79B c.587A>C
Forward; 5'- CTGACCCCGAGGACTCAGA -3' (SEQ ID NO: 3)
Reverse; 5'- GGCTGGCATGGAGGAAGA -3' (SEQ ID NO: 4)
CD79B c.587A>G
Forward; 5'- CTGACCCCGAGGACTCAGA -3' (SEQ ID NO: 3)
Reverse; 5'- GGCTGGCATGGAGGAAGA -3' (SEQ ID NO: 4)
BTG2 c.133G>A
Forward; 5'- GAGCAGAGGCTTAAGGTCTTCA -3' (SEQ ID NO: 5)
Reverse; 5'- GGCATGCGCTCACCTG -3' (SEQ ID NO: 6)
BTG2 c.142G>A
Forward; 5'- GAGCAGAGGCTTAAGGTCTTCAG -3' (SEQ ID NO: 7)
Reverse; 5'- AGGCCCCTCGGCATG -3' (SEQ ID NO: 8)
MYD88 c.794T>C
Forward; 5'- CCTTGGCTTGCAGGT -3' (SEQ ID NO: 9)
Reverse; 5'- TCTTTCTTCATTGCCTTGT -3' (SEQ ID NO: 10)
MYD88 c.728G>A
Forward; 5'- GGTGGTGGTTGTCTCTGATGATTAC -3' (SEQ ID NO: 11)
Reverse; 5'- TGAGTGCAAATTTGGTCTGGAAGT -3' (SEQ ID NO: 12)
PIM1 c.550C>T
Forward; 5'- GAAAACATCCTTATCGACCTCAATCG -3' (SEQ ID NO: 13)
Reverse; 5'- CGTGTAGACGGTGTCCTTGAG -3' (SEQ ID NO: 14)
＜プローブ配列＞
CD79B c.586T>A
FAM；5’- AGATCACACCTTCGAGGTA -3’（配列番号１５）
VIC；5’- AAGATCACACCTACGAGGTA -3’（配列番号１６）
CD79B c.586T>C
FAM；5’- TCACACCTGCGAGGTA -3’（配列番号１７）
VIC；5’- AAGATCACACCTACGAGGTA -3’（配列番号１８）
CD79B c.587A>C
FAM；5’- CTTACCTCGTCGGTGTGA -3’（配列番号１９）
VIC；5’- TCCTTACCTCGTAGGTGTGA -3’（配列番号２０）
CD79B c.587A>G
FAM；5’- CTTACCTCGTGGGTGTG -3’（配列番号２１）
VIC；5’- CCTTACCTCGTAGGTGTG -3’（配列番号２２）
BTG2 c.133G>A
FAM；5’- CTCCAGGAGACACTCA -3’（配列番号２３）
VIC；5’- CTCCAGGAGGCACTCA -3’（配列番号２４）
BTG2 c.142G>A
FAM；5’- CACTCACAAGTGAGCG -3’（配列番号２５）
VIC；5’- CACTCACAGGTGAGCG -3’（配列番号２６）
MYD88 c.794T>C
FAM；5’- TGGGGATCGGTCGC -3’（配列番号２７）
VIC；5’- TGGGGATCAGTCGCTT -3’（配列番号２８）
MYD88 c.728G>A
FAM；5’- CACATTCCTTGTTCTGCA -3’（配列番号２９）
VIC；5’- CACATTCCTTGCTCTGCA -3’（配列番号３０）
PIM1 c.550C>T
FAM；5’- AAGTCGATGAACTTGAG -3’（配列番号３１）
VIC；5’- AAGTCGATGAGCTTGAG -3’（配列番号３２） <Probe sequence>
CD79B c.586T>A
FAM; 5'- AGATCACACCTTCGAGGTA -3' (SEQ ID NO: 15)
VIC; 5'-AAGATCACACCTACGAGGTA -3' (SEQ ID NO: 16)
CD79B c.586T>C
FAM; 5'- TCACACCTGCGAGGTA -3' (SEQ ID NO: 17)
VIC; 5'-AAGATCACACCTACGAGGTA -3' (SEQ ID NO: 18)
CD79B c.587A>C
FAM; 5'- CTTACCTCGTCGGTGTGA -3' (SEQ ID NO: 19)
VIC; 5'- TCCTTACCTCGTAGGTGTGA -3' (SEQ ID NO: 20)
CD79B c.587A>G
FAM; 5'- CTTACCTCGTGGGTGTG -3' (SEQ ID NO: 21)
VIC; 5'- CCTTACCTCGTAGGTGTG -3' (SEQ ID NO: 22)
BTG2 c.133G>A
FAM; 5'- CTCCAGGAGACACTCA -3' (SEQ ID NO: 23)
VIC; 5'- CTCCAGGAGGCACTCA -3' (SEQ ID NO: 24)
BTG2 c.142G>A
FAM; 5'- CACTCACAAGTGAGCG -3' (SEQ ID NO: 25)
VIC; 5'- CACTCACAGGTGAGCG -3' (SEQ ID NO: 26)
MYD88 c.794T>C
FAM; 5'-TGGGGATCGGTCGC -3' (SEQ ID NO: 27)
VIC; 5'-TGGGGATCAGTCGCTT -3' (SEQ ID NO: 28)
MYD88 c.728G>A
FAM; 5'- CACATTCCTTGTTCTGCA -3' (SEQ ID NO: 29)
VIC; 5'- CACATTCCTTGCTCTGCA -3' (SEQ ID NO: 30)
PIM1 c.550C>T
FAM; 5'- AAGTCGATGAACTTGAG -3' (SEQ ID NO: 31)
VIC; 5'- AAGTCGATGAGCTTGAG -3' (SEQ ID NO: 32)
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
１－４．遺伝子変異の検出
　デジタルPCR解析において、複数の遺伝子変異の有無を確認する場合、これまで各遺伝子変異について、各々デジタルPCR解析を行うことが必須であったが、これには解析に十分なDNA量が必要であった。しかし、発明者らは、微量なDNA量しか得ることができない微量検体から、複数の遺伝子変異を、同時に検出可能なデジタルPCR解析の検出系を初めて確立した。すなわち、微量検体より抽出した微量DNAを、デジタルPCRのドロップレット内で、multiplex PCR法を応用して前増幅することで、増幅効率を上げ、解析箇所として選定した4遺伝子9箇所を、一度のデジタルPCR解析で検出することに成功した。具体的には、以下の方法となる。 1-4. Detection of gene mutation When confirming the presence or absence of multiple gene mutations in a digital PCR analysis, it was necessary to perform a digital PCR analysis for each gene mutation until now. Was needed. However, the inventors have for the first time established a detection system for digital PCR analysis capable of simultaneously detecting a plurality of gene mutations from a small amount of sample that can obtain only a small amount of DNA. That is, a small amount of DNA extracted from a small amount of sample is pre-amplified by applying the multiplex PCR method in a digital PCR droplet to improve amplification efficiency, and 9 points of 4 genes selected as analysis points are analyzed once. It was successfully detected by digital PCR analysis. Specifically, the following method is used.
　１－４－１．前増幅
　デジタルPCR解析に当たり、DNA総量が10 ngに満たない場合、既報を参考にmultiplex PCR法による前増幅を行った。multiplex PCRは、各プライマーの終濃度が5μMになるよう調整し、Master mixである2xddPCR SuperMix For Probe (no dUTP; Bio-Rad Laboratories, Hercules, CA, USA)を用い、鋳型DNA、5μMのプライマー、nuclease free waterを含め20μlになるように調整した。この調整液20μlにDroplet Generator オイル For Probe (Bio-Rad Laboratories, Hercules, CA, USA)を70μl加え、QX200 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA)にてドロップレットを作成した。このドロップレットを、MiniAmp Thermal Cycler applied biosystems (Thermo Fisher Scientific, Waltham, MA, USA)を用いて、95℃で10分反応させた後、94℃30秒と60℃1分を10サイクル行い、98℃で10分反応させ、PCRによる前増幅を行った。 1-4-1. Pre-amplification When the total DNA amount was less than 10 ng in the digital PCR analysis, pre-amplification by the multiplex PCR method was performed with reference to the previous report. multiplex PCR is adjusted so that the final concentration of each primer is 5 μM, using 2xddPCR SuperMix For Probe (no dUTP; Bio-Rad Laboratories, Hercules, CA, USA) which is a master mix, template DNA, 5 μM primer, It was adjusted to 20 μl including nuclease free water. 70 μl of Droplet Generator Oil For Probe (Bio-Rad Laboratories, Hercules, CA, USA) was added to 20 μl of this adjusted solution, and droplets were prepared using QX200 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA). Using a MiniAmp Thermal Cycler applied biosystems (Thermo Fisher Scientific, Waltham, MA, USA), this droplet was reacted at 95°C for 10 minutes, and then 94°C for 30 seconds and 60°C for 1 minute, 10 cycles were performed, and 98 The reaction was carried out at 0°C for 10 minutes, and preamplification by PCR was performed.
１－４－２．アンプリコンの精製
　前増幅後のアンプリコンをDNA Clean & Concentrator -5 Kit（Zymo Research, Irvine, CA, USA）を用いて精製し、最終量が20 μlとなるように調整した。 1-4-2. Purification of amplicon The amplicon after pre-amplification was purified using DNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, CA, USA) and adjusted to a final volume of 20 μl.
１－４－３．デジタルPCR解析
　前増幅し生成したアンプリコンを鋳型として、以下のようにドロップレットを作製後、デジタルPCR解析を行い、蛍光強度を読み取り解析した。
　まず、プローブ7（MYD88 c.794T>C L265P）のアッセイについては、溶出した20 μlのアンプリコンの内5 μlを、それ以外の8アッセイについては溶出した20 μlのアンプリコンを10倍希釈した5 μlを鋳型DNAとして用い、この鋳型DNAに、2xddPCR SuperMix For Probe（no dUTP；Bio-Rad Laboratories, Hercules, CA, USA）、TaqMan Assay 40x（Thermo Fisher Scientific, Waltham, Massachusetts, USA；FAMまたはVICのプローブ8 μM、各プライマー36 μMのミックス）、nuclease free waterを加え、全量が20 μlになるように調整した。この調整液20 μlにDroplet Generator オイル For Probe（Bio-Rad Laboratories, Hercules, CA, USA）を70 μl加え、QX200 Droplet Generator（Bio-Rad Laboratories, Hercules, CA, USA）にてドロップレットを作成した。このドロップレットを、MiniAmp Thermal Cycler applied biosystems（Thermo Fisher Scientific, Waltham, MA, USA）を用いて、95℃で10分反応させた後、94℃30秒と60℃1分を40サイクル行い、98℃で10分反応させ、QX200 Droplet Reader（Bio-Rad Laboratories, Hercules, CA, USA）にて蛍光強度を読み取り、QuantaSoft Software version 1.7（Bio-Rad Laboratories, Hercules, CA, USA）にて解析した。 1-4-3. Digital PCR analysis Using the amplicon pre-amplified and generated as a template, droplets were prepared as follows, and then digital PCR analysis was performed to read and analyze the fluorescence intensity.
First, for the probe 7 ( MYD88 c.794T>C L265P) assay, 5 μl of the eluted 20 μl amplicon was diluted 10-fold with the eluted 20 μl amplicon for the other 8 assays. Using 5 μl as a template DNA, 2xddPCR SuperMix For Probe (no dUTP; Bio-Rad Laboratories, Hercules, CA, USA), TaqMan Assay 40x (Thermo Fisher Scientific, Waltham, Massachusetts, USA; FAM or VIC) (8 μM probe, mix of 36 μM each primer) and nuclease free water were added to adjust the total volume to 20 μl. 70 μl of Droplet Generator Oil For Probe (Bio-Rad Laboratories, Hercules, CA, USA) was added to 20 μl of this adjusted solution, and droplets were created with the QX200 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA). .. Using a MiniAmp Thermal Cycler applied biosystems (Thermo Fisher Scientific, Waltham, MA, USA), this droplet was reacted at 95°C for 10 minutes, and then 94°C for 30 seconds and 60°C for 1 minute for 40 cycles, and 98 The reaction was performed at 10° C. for 10 minutes, the fluorescence intensity was read by QX200 Droplet Reader (Bio-Rad Laboratories, Hercules, CA, USA), and analyzed by QuantaSoft Software version 1.7 (Bio-Rad Laboratories, Hercules, CA, USA).
２．結果
　デジタルPCR解析により、28例の眼の検体から変異遺伝子の検出を行った。プローブ6 （BTG2 c.142G>A E48K）を用いて解析した眼内悪性リンパ腫の代表的な解析結果を、図１に示す。本解析結果では、FAM陽性ドロップレットを多数みとめ、変異有りと判定できた。デジタルPCR解析結果から、変異遺伝子ごとに、変異をみとめた症例数と変異率を求めたところ、CD79Bは37%、BTG2は15%、MYD88は79%、PIM1は26%で変異をみとめ、眼内悪性リンパ腫と診断された症例の96%の症例で、4遺伝子9箇所のいずれかに少なくとも1つの変異が認められた。
　以上の結果はから、MYD88やCD79B だけでなく、BTG2、PIM1変異遺伝子を用いることで、高率に眼内悪性リンパ腫の診断を補助できる補助的診断方法と考えられた。 2. Results Mutant genes were detected in 28 eye samples by digital PCR analysis. A representative analysis result of intraocular malignant lymphoma analyzed using probe 6 (BTG2 c.142G>A E48K) is shown in FIG. In this analysis result, a large number of FAM-positive droplets were found, and it was possible to determine that there was a mutation. From the results of digital PCR analysis, the number of cases and mutation rate in which mutations were found for each mutant gene was calculated.37 % for CD79B , 15% for BTG2 , 79% for MYD88 , and 26% for PIM1. In 96% of cases diagnosed with internal malignant lymphoma, at least one mutation was found in any of the 9 positions of 4 genes.
These results suggest that not only MYD88 and CD79B but also BTG2 and PIM1 mutant genes may be used as a supplementary diagnostic method that can assist the diagnosis of intraocular malignant lymphoma at a high rate.
　本発明の方法を用いることで、眼内悪性リンパ種の診断確度が顕著に上昇すると考えられる。従って、本発明は、医療分野における利用が期待される。 It is considered that the accuracy of diagnosis of intraocular malignant lymphoma is significantly increased by using the method of the present invention. Therefore, the present invention is expected to be used in the medical field.

Claims (9)

  1. 　被験者由来の検体において、以下の（Ａ）ないし（Ｉ）のいずれかの遺伝子変異の1または複数を検出することを含む、眼内悪性リンパ種の診断を補助する方法。
    （Ａ）CD79Bのc.586T>A変異、
    （Ｂ）CD79Bのc.586T>C変異、
    （Ｃ）CD79Bのc.587A>C変異、
    （Ｄ）CD79Bのc.587A>G変異、
    （Ｅ）BTG2のc.133G>A変異、
    （Ｆ）BTG2のc.142G>A変異、
    （Ｇ）MYD88のc.794T>C変異、
    （Ｈ）MYD88のc.728G>A変異、および
    （Ｉ）PIM1のc.550C>T変異     A method for assisting the diagnosis of intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (A) to (I) in a specimen derived from a subject.
    (A) CD79B c.586T>A mutation,
    (B) CD79B c.586T>C mutation,
    (C) CD79B of c.587A> C mutation,
    (D) CD79B c.587A>G mutation,
    (E) c.133G >A mutation of BTG2,
    (F) c.142G >A mutation of BTG2,
    (G) c.794T >C mutation of MYD88,
    (H) MYD88 of c.728G> A mutation, and (I) PIM1 of c.550C> T mutation
  2. 　前記遺伝子変異が、少なくとも、前記（Ｅ）、（Ｆ）または（Ｉ）のいずれかであることを特徴とする請求項１に記載の方法。 The method according to claim 1, wherein the gene mutation is at least one of (E), (F) or (I).
  3. 　被験者由来の検体において、以下の（ａ）ないし（ｉ）のいずれかの遺伝子変異の1または複数を検出することを含む、眼内悪性リンパ種の診断を補助する方法
    （ａ）CD79Bタンパク質のY196Nのアミノ酸置換を伴うCD79B遺伝子変異、
    （ｂ）CD79Bタンパク質のY196Hのアミノ酸置換を伴うCD79B遺伝子変異、
    （ｃ）CD79Bタンパク質のY196Sのアミノ酸置換を伴うCD79B遺伝子変異、
    （ｄ）CD79Bタンパク質のY196Cのアミノ酸置換を伴うCD79B遺伝子変異、
    （ｅ）BTG2タンパク質のA45Tのアミノ酸置換を伴うBTG2遺伝子変異、
    （ｆ）BTG2タンパク質のE48Kのアミノ酸置換を伴うBTG2遺伝子変異、
    （ｇ）MYD88タンパク質のL265Pのアミノ酸置換を伴うMYD88遺伝子変異、
    （ｈ）MYD88タンパク質のS243Nのアミノ酸置換を伴うMYD88遺伝子変異、および
    （ｉ）PIM1タンパク質のL184Fのアミノ酸置換を伴うPIM1遺伝子変異     A method for assisting diagnosis of intraocular malignant lymphoma, which comprises detecting one or more of the following gene mutations (a) to (i) in a specimen derived from a subject (a) CD196B protein Y196N CD79B gene mutation with amino acid substitution of
    (B) CD79B mutations involving amino acid substitutions Y196H of CD79B proteins,
    (C) CD79B mutation involving an amino acid substitution Y196S of CD79B proteins,
    (D) CD79B mutations involving amino acid substitutions Y196C the CD79B proteins,
    (E) BTG2 protein involves amino acid substitution A45T the BTG2 gene mutation,
    (F) BTG2 gene mutation accompanied by E48K amino acid substitution of BTG2 protein,
    (G) MYD88 proteins involving amino acid substitutions L265P of MYD88 gene mutation,
    (H) MYD88 gene mutation with S243N amino acid substitution of MYD88 protein, and (i) PIM1 gene mutation with L184F amino acid substitution of PIM1 protein
  4. 　前記遺伝子変異が、少なくとも、前記（ｅ）、（ｆ）または（ｉ）のいずれかであることを特徴とする請求項３に記載の方法。 The method according to claim 3, wherein the gene mutation is at least any one of (e), (f) or (i).
  5. 　前記遺伝子変異の検出を、デジタルPCR法により検出することを特徴とする請求項１ないし４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the detection of the gene mutation is detected by a digital PCR method.
  6. 　請求項５の検出を行う前に、予め検体由来のDNAを増幅することを特徴とする請求項５に記載の方法。 The method according to claim 5, wherein the sample-derived DNA is amplified in advance before the detection according to claim 5.
  7. 　前記検体が眼内液、髄液、末梢血または骨髄であることを特徴とする請求項１ないし６のいずれかに記載の方法。 7. The method according to any one of claims 1 to 6, wherein the sample is intraocular fluid, spinal fluid, peripheral blood or bone marrow.
  8. 　眼内悪性リンパ種の診断を補助する方法を実施するためのキット。 A kit for carrying out a method for assisting the diagnosis of intraocular malignant lymphoma.
  9. 以下の（Ａ）ないし（Ｉ）のいずれかの遺伝子変異の1または複数を検出するためのプローブおよび／またはプライマーを含むことを特徴とする請求項８に記載のキット。
    （Ａ）CD79Bのc.586T>A変異、
    （Ｂ）CD79Bのc.586T>C変異、
    （Ｃ）CD79Bのc.587A>C変異、
    （Ｄ）CD79Bのc.587A>G変異、
    （Ｅ）BTG2のc.133G>A変異、
    （Ｆ）BTG2のc.142G>A変異、
    （Ｇ）MYD88のc.728G>A変異、
    （Ｈ）MYD88のc.794T>C変異、および
    （Ｉ）PIM1のc.550C>T変異     The kit according to claim 8, comprising a probe and/or a primer for detecting one or more of the following gene mutations (A) to (I).
    (A) CD79B c.586T>A mutation,
    (B) CD79B c.586T>C mutation,
    (C) CD79B of c.587A> C mutation,
    (D) CD79B c.587A>G mutation,
    (E) c.133G >A mutation of BTG2,
    (F) c.142G >A mutation of BTG2,
    (G) c.728G >A mutation of MYD88,
    (H) MYD88 of c.794T> C mutation, and (I) PIM1 of c.550C> T mutation
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JP7423090B2 (en) 2022-04-07 2024-01-29 学校法人藤田学園 B-cell lymphoma diagnostic aid kit and information provision method for patients with fever of unknown origin

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