WO2023236189A1 - 利用非完整重组的t细胞受体核苷酸序列诊断t细胞淋巴瘤的方法及试剂盒 - Google Patents

利用非完整重组的t细胞受体核苷酸序列诊断t细胞淋巴瘤的方法及试剂盒 Download PDF

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WO2023236189A1
WO2023236189A1 PCT/CN2022/098132 CN2022098132W WO2023236189A1 WO 2023236189 A1 WO2023236189 A1 WO 2023236189A1 CN 2022098132 W CN2022098132 W CN 2022098132W WO 2023236189 A1 WO2023236189 A1 WO 2023236189A1
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sequence
gene
cell
cell receptor
recombinant
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刘宗霖
何中良
陈怡伶
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刘宗霖
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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    • 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

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  • the present disclosure relates to a method and kit for diagnosing T-cell lymphoma. More specifically, the present disclosure uses digital polymerase chain reaction (digital PCR) or real-time PCR (real-time PCR) technology to quantify non-complete recombinant T cell receptor nucleotide sequences as biomarkers for the diagnosis of T cell lymphoma methods and kits.
  • digital PCR digital polymerase chain reaction
  • real-time PCR real-time PCR
  • T cells have the ability to recognize multiple antigens and therefore play an important role in adaptive immunity.
  • the ability of T cells to recognize antigens comes from the intergenic recombination of the V gene, J gene or D gene during the cell development and maturation process to form genes encoding various T cell receptors (TCR) ( Schatz D.G.et al.,Recombination centers and the orchestration of V(D)J recombination.Nat.Rev.Immunol.2011;11(4):251-63), as shown in Figure 1A to Figure 1C.
  • TCR T cell receptors
  • the TCR ⁇ gene has 67 V, 2 D, and 13 J coding segments, which are modified by nucleotide deletions and nucleosides at the V-D and D-J junctions during V(D)J recombination in each T cell.
  • the addition of acid produces variations in the complementary determining region (CDR), thereby enhancing the diversity of antigen recognition.
  • CDR complementary determining region
  • the immune repertoire of a study individual is often characterized by recombinant VJ and complementarity determining region 3 (CDR3) sequences, distinguishing multiple T cell clones.
  • T cell lymphoma When a certain T cell becomes malignant and expands abnormally (ie, T cell lymphoma), its unique T cell receptor gene frequency will also increase abnormally, so it can be used as a biomarker for T cell lymphoma (Gazzola A. et al.,The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies.Ther.Adv.Hematol.2014;5(2):35-47;Scheijen B.et al.,Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.Leukemia 2019;33(9):2227-2240).
  • Abnormal amplification represents a high frequency of the corresponding clone, that is, clonality.
  • the diagnosis of T-cell lymphoma is performed by looking for high-frequency T cell receptor recombinant genes, which is called clonality assessment.
  • the standard method for clonality assessment is to use multiple pairs of primers proposed by the EuroClonality Joint Conference (ie, BIOMED-2 primers) to detect T cell receptor recombination.
  • Multiplex T cell receptor analysis will use multiple pairs of primers (i.e., multiplex polymerase chain reaction).
  • the present disclosure provides a method of diagnosing T cell lymphoma in an individual, comprising: providing a biological sample taken from the individual; detecting non-intact recombinant T cell receptor nucleotides in the biological sample The expression level of the first target sequence, the second target sequence, and the reference sequence in the sequence; compare the ratio of the expression level of the first target sequence to the expression level of the reference sequence, or the expression level of the second target sequence The ratio of the expression level to the reference sequence is used to diagnose the individual with T-cell lymphoma; wherein the incomplete recombinant T-cell receptor nucleotide sequence is located between the exon upstream segments of the T-cell receptor gene The non-coding region; and the non-complete recombinant T cell receptor nucleotide sequence includes at least one of the following groups: J gene intron sequence, pseudogene (pseudogene), D gene upstream intron sequence and the intergenic segment sequence preceding the D1 gene.
  • the individual's J gene intron sequence, pseudo
  • the non-complete recombinant T cell receptor nucleotide sequence further includes at least one of the group consisting of: J gene segment sequence, pseudogene, D gene segment sequence and C Gene fragment sequence.
  • the incomplete recombinant T cell receptor nucleotide sequence includes multiple J gene segment sequences, pseudogenes, J gene intron sequences and C gene segment sequences. In other specific embodiments, the incomplete recombinant T cell receptor nucleotide sequence includes a D gene upstream intron sequence, a D gene fragment sequence, a J gene fragment sequence, a pseudogene, and a C gene fragment. sequence. In other specific embodiments, the incomplete recombinant T cell receptor nucleotide sequence includes an intergenic fragment sequence before the D1 gene and a C gene fragment sequence.
  • the non-complete recombinant T cell receptor nucleotide sequence includes at least one of the nucleotide sequences shown in SEQ ID NO: 4 to 6, and SEQ ID NO: 8 to 11 At least one of the nucleotide sequences. In other embodiments, the non-complete recombinant T cell receptor nucleotide sequence is represented by any one of SEQ ID NOs: 1 to 3.
  • the first target sequence is the J2-2P gene sequence (SEQ ID NO: 8), and the reference sequence is the J2-3 gene sequence (SEQ ID NO: 9).
  • the second target sequence is the intergenic fragment sequence before the D1 gene (SEQ ID NO: 6), and the reference sequence is the J2-3 gene sequence (SEQ ID NO: 9).
  • the ratio of the expression level of the first target sequence to the expression level of the reference sequence is greater than 15%, or the ratio of the expression level of the second target sequence to the expression level of the reference sequence is greater than 50%. , indicating that the individual has T-cell lymphoma.
  • the ratio of the expression level of the J2-2P gene to the expression level of the J2-3 gene is greater than 15%, or the expression level of the intergenic fragment before the D1 gene is less than the expression level of the J2-3 gene.
  • the expression ratio is greater than 50%, it indicates that the individual has T-cell lymphoma.
  • the present disclosure also provides a kit for diagnosing T-cell lymphoma in an individual, including a first primer pair and a probe for detecting a first target sequence, a second target sequence for detecting a second primer pair and probe and a reference primer pair and probe for detecting a reference sequence, wherein the first target sequence and the second target sequence are located in the non-completely recombinant T cell receptor nucleotides of the individual sequence, and the first target sequence is different from the second target sequence.
  • the first target sequence and the second target sequence are respectively selected from the group consisting of: J gene intron sequence, pseudogene (pseudogene), D gene upstream intron sequence, D1 gene The former intergenic segment sequence, J gene segment sequence, D gene segment sequence and C gene segment sequence.
  • the first target sequence is a J2-2P gene sequence
  • the second target sequence is an intergenic segment sequence before the D1 gene
  • the reference sequence is a J2-3 gene sequence.
  • the present disclosure provides biomarkers for the detection of T cell lymphoma in non-intact recombinant T cell receptor nucleotide sequences by measuring the amount of each type of non-intact recombinant T cell receptor nucleotide sequence present (e.g. The expression ratio of the J2-2P gene/J2-3 gene or the intergenic fragment before the D1 gene/the expression ratio of the J2-3 gene) can be used for diagnostic evaluation of T-cell lymphoma, and can be compared with known T-cell lymphomas.
  • the expression ratio of the J2-2P gene/J2-3 gene or the intergenic fragment before the D1 gene/the expression ratio of the J2-3 gene can be used for diagnostic evaluation of T-cell lymphoma, and can be compared with known T-cell lymphomas.
  • lymphoma Additional procedures for lymphoma (such as cell marker resolution by flow cytometry, pathological sections, immunochemical staining, or BIOMED-2 multiplex PCR testing) are combined to provide a complete detection platform and assist in the diagnosis of T-cell lymphoma and Follow-up treatment.
  • Figure 1A is a schematic diagram of the interaction between antigen (Ag)-T cells-histocompatibility antigen (MHC) (cited from Woodsworth et al. Genome Medicine 2013, 5:98);
  • Figure 1B is a schematic diagram of T cells presenting foreign antigens , where the V region contains V ⁇ and V ⁇ ; the J region contains J ⁇ and J ⁇ ; the D region contains C ⁇ and C ⁇ and the CDR3 ⁇ region;
  • Figure 1C shows the V(D)J recombination process in T cells, TCR- ⁇ VDJ Gene recombination produces TCR diversity.
  • Figure 2 is a schematic diagram of the experimental flow chart using the RACE method (rapid amplification of cDNA ends) combined with second-generation sequencing technology.
  • Figure 3 shows the RACE of TCR ⁇ in peripheral blood cells of 21 healthy individuals and 2 lymphoma patients (I11a and I13) and bone marrow cells (I11b) of one lymphoma patient with non-completely recombinant T cell receptor nucleotide sequences. Sequencing has non-completely recombined TCR sequences (non-completely recombined sequences), and the J2-2P genotype accounts for the majority of non-completely recombined sequences. The proportion of J2-2P genotype in complete sequences and incomplete recombinant sequences was highest in blood and bone marrow cells of I11 lymphoma patients. Results also from a lymphoma patient (I11) showed the similarity in TCR genotype composition between bone marrow cells and blood cells.
  • Figure 4 shows that the lymphoma patient (I11) had negative results for BIOMED-2 clonality assessment twice.
  • Figure 7 shows the relative expression of J2-2P genotype (left) and intD1 genotype (right) in 6 lymphoma patients (left), including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma. , mature T-cell lymphoma and extranodal NK/T-cell lymphoma and other types of T-cell lymphoma) to the tissue of a person without lymphoma (right).
  • Figure 8 shows the detection results of the J2-2P to J2-3 segment sequences in peripheral blood cells of healthy individuals using Sanger sequencing.
  • Figure 9 shows that the T cell line was verified by Sanger sequencing to have J2-2P ⁇ J2-3 sequences, including intron sequences in the middle.
  • sample refers to cells or tissues that are removed from an individual and tested in vitro, that is, in vitro (in vitro) or ex vivo (ex vivo). ) cells or tissues.
  • the individual is an animal, such as a mammal. In some embodiments of the present disclosure, the individual is a human.
  • target sequence refers to a sequence that is amplified, detected, or amplified and detected, which is either complementary to a sequence provided herein or has at least one intron in its natural state, i.e. As genomic DNA or extrachromosomal DNA.
  • multiple target sequences may be included, and each of the multiple target sequences may have a number (eg, first target sequence, second target sequence). The numbering of target sequences is only used to indicate different target sequences and has no sequential relationship.
  • the first target sequence and the second target sequence may be amplified, detected, or amplified and detected before, after, or simultaneously with the reference sequence.
  • intersegment non-coding region refers to the intersegment non-coding region between the various V, D, and J coding segments of a T cell receptor gene (e.g., TCR ⁇ gene). Sequence segments that are not used to encode amino acids. Under normal circumstances, the nucleotide sequence located in the "non-coding region between exon upstream segments" will be clipped during the V(D)J recombination process and will not Used to encode the amino acids that form complementarity determining regions.
  • the "inter-segment non-coding region upstream of the exon” is the sequence other than the normal exon used for V(D)J recombination, including: introns, pseudogenes (pseudogenes), and intergenic regions before genes. segments etc.
  • upstream when the term “upstream” is placed before or after a reference position, it refers to the sequence segment from the reference position toward the 5' end of the nucleic acid (the forward strand, if it is a double-stranded nucleic acid).
  • the upstream of the D gene refers to the sequence segment in the nucleic acid from the D gene to the 5' end of the forward strand.
  • This disclosure uses the rapid amplification of cDNA ends (RACE) method to avoid the bias of the PCR method, and uses second-generation sequencing and analysis for detection.
  • the RACE method can avoid primer bias.
  • specific primers for the T cell receptor nucleotide sequence are used to accurately amplify the T cell receptor nucleotide sequence and establish an unbiased overall picture of the T cell receptor gene (see Figure 2 shown).
  • non-completely recombined T cell receptor nucleotide sequences (herein, also referred to as non-regular sequences) can be found, for example, sequences without V segments.
  • digital PCR refers to a nucleotide quantification technology that can directly calculate the number of DNA molecules and is an absolute quantification of the starting sample.
  • each reaction space hole, droplet
  • the presence or absence of the endpoint signal is used as the result of the quantitative method.
  • the droplet containing the nucleic acid molecule will The fluorescence signal is released, and finally based on the principle of Poisson distribution and the proportion of positive droplets, the concentration or copy number of the original molecule is calculated through analysis software.
  • Digital PCR can directly calculate the copy number of the target sequence, so it can perform accurate absolute quantitative detection without relying on control samples and standard curves.
  • the present disclosure can also be used as a quantitative detection method for incomplete recombination through real-time quantitative PCR technology.
  • Quantitative real time PCR quantitative real time PCR
  • quantitative real time PCR also known as qPCR
  • the primers are used for PCR amplification, and the fluorescent probes improve specificity. Fluorescence changes were detected and recorded using real-time polymerase chain reaction.
  • the PCR product increases in a positive correlation with the number of PCR cycles, and the amount of the PCR product can be detected in real time through the optical system.
  • the RNA extraction kit is QIAamp RNA Blood Mini (QIAGEN), which can be used to extract RNA from blood, tissues and cells.
  • the extraction method uses centrifugation to selectively attach total RNA to a silica-based membrane, and then uses a high-salt buffering system to attach RNA with more than 200 bases to the membrane. On the film, 5.8S RNA, 5S RNA, and tRNA with less than 200 bases will be removed.
  • This extraction method does not require the treatment steps of traditional toxic substances (such as phenol, chloroform, etc.), and also includes a denaturation step to lyse leukocytes and avoid activation of ribonucleic acid hydrolase (RNase) to retain intact RNA.
  • This reagent kit can avoid heme contamination and remove interference from heparin anticoagulant.
  • the extracted RNA can be used for subsequent experiments such as reverse transcription-PCR (RT-PCR) and cDNA synthesis.
  • RT-PCR reverse transcription-PCR
  • Original sample type RNA extracted from bone marrow, blood, and tissue.
  • the extracted RNA is reverse transcribed into cDNA using reverse transcriptase, and the obtained cDNA is then subjected to subsequent PCR amplification.
  • the extracted total RNA was reverse transcribed into cDNA using specific primers for the T cell receptor constant region (C), and then amplified by the 5' end universal primer (SMARTer oligo).
  • C T cell receptor constant region
  • SMARTer oligo 5' end universal primer
  • Perform the second PCR (nested PCR).
  • the second PCR product (approximately 520 bp) was then used for next-generation sequencing to build a library.
  • the reagent kit used for the RACE method is Clontech SMARTer TM RACE cDNA Amplification Kit; detailed experimental procedures can be found in Motomura M, et al. Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.Appl.Environ.Microbiol.1999Nov;65(11):4987-94.
  • dmtA O-methyltransferase I gene
  • Clarity digital PCR system JN MEDSYS
  • chip in tube technology the special chip is divided into tens of thousands of individual reaction spaces (partitions), and DNA can be distributed to Each reaction space contains only 0 or 1 DNA. Then, the DNA is PCR amplified in a 0.2 mL reaction tube, and then used with a Clarity digital PCR detector (reader) to detect the fluorescence signal, convert the Poisson distribution calculation, and obtain the copy number to achieve the absolute quantitative result of a single molecule.
  • Clarity digital PCR detector reader
  • Step 1 Use clean scissors to cut an appropriate number of 200 ⁇ L SnapStrip II PCR reaction tubes according to the number of samples.
  • Step 2 According to the number of samples, configure the reagents required for the PCR reaction according to Table 5 below.
  • Step 3 Place the required reagents into a 1.5mL test tube.
  • Step 4 Use a vortex mixer to mix the above prepared PCR reaction reagents evenly.
  • Step 5 Centrifuge the above mixed PCR reaction reagents in a microcentrifuge (fixed speed 6,600 rpm) for 5 seconds.
  • Step 6 Add 9 ⁇ L of PCR reaction reagent to each reaction space, then add 1.0 ⁇ L of the sample to be tested (reverse transcribed cDNA), and adjust the final volume to 15 ⁇ L. Add 1 ⁇ L of nuclease-free water to the blank control group.
  • Step 7 Use a thermal cycler to perform the PCR reaction under the following program conditions:
  • Step 8 Close the lid tightly, mix the reagent and the sample to be tested thoroughly, and then centrifuge.
  • Step 9 Take out the tube strip and sample loading kit (including slider and platform) from the JN Clarity consumable set, open the tube strip cover and confirm the chip position and shape, put the stage into the serial tube, close to the upper edge of the chip, and install it on the automatic loader.
  • the tube strip and sample loading kit including slider and platform
  • Step 10 Place the slider on the stage, press the start button and try to push it once. If the chip gets stuck while the machine is pushing, adjust the angle and try again. Add 15 ⁇ L of the thoroughly mixed reagent sample mixture to the triangle of the slider, add the samples in order, and press the start button to evenly distribute the reagent sample mixture onto the chip. If there is still liquid remaining on the stage, press the start button again to push the liquid completely into the chip and clear the remaining liquid on the tube wall.
  • Step 11 Place the connecting tube into the sealing enhancer to seal the sample up to 2 times.
  • Step 12 Add 245 ⁇ L sealing fluid to each tube.
  • Step 13 Close the drain pipe cover and remove excess or leaked sealing fluid.
  • Step 14 Use a gradient polymerization reactor (VWR Peqlab) to perform the PCR reaction under the following program conditions:
  • the upper cover temperature is set to 90°C;
  • Step 15 After PCR, wipe the wall of the serial tube clean with lens paper and alcohol, and put it into the viewing jig of the Clarity detector. Make sure that the chip is completely immersed in the sealing liquid, fasten the upper cover and Add 6 mL of sterilized water and remove excess air bubbles on the tube wall.
  • Step 16 Open the Clarity software and connect the detector to set the experimental parameters.
  • Step 17 Place the observation fixture into the detector and press the RUN key.
  • Step 18 Result judgment (including data calculation, result interpretation or reportable range):
  • Dye1 is set to J2-3C (FAM fluorescence)
  • Dye2 is set to J2-2P (HEX fluorescence)
  • the positive rate is calculated as (Dye2DNA copy number/Dye1DNA copy number) ⁇ 100%, which can be calculated The proportion of incomplete recombination of T cell receptor nucleotides.
  • Example 1 Expression of T cell receptor nucleotide incomplete recombination in healthy individuals and lymphoma patients
  • the ratio of J2-2P genotype in complete sequences to incomplete recombinant sequences was highest in blood and bone marrow cells of patients with I11 lymphoma. Importantly, a high proportion of non-complete recombinant sequences (88.2%) could be identified in the bone marrow cells of one of the patients with confirmed lymphoma, with the J2-2P genotype also having the highest proportion of non-complete recombinant sequences (80%). .
  • Figure 6 shows the results of digital PCR quantification of J2-2P and J2-3 segment sequences of bone marrow cells.
  • the incomplete recombination ratio was shown to have potential as a biomarker for T-cell lymphoma.
  • Bone marrow cells were detected in both T-cell lymphoma patients (24 cases) and non-lymphoma patients (6 cases). The blood cells of healthy controls (11 cases) were detected.
  • Figure 7 shows the relative expression of J2-3 genotype between J2-2P genotype (left) and intD1 genotype (right) in 6 lymphoma patients (including angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, mature T-cell lymphoma, and extranodal NK/T-cell lymphoma) to tissue from a person without lymphoma.
  • Example 4 Expression of J2-2P ⁇ J2-3 segment sequences in T cell lines and healthy individuals

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Abstract

提供一种诊断个体中T细胞淋巴瘤的方法，包括检测该个体的生物样本中的非完整重组的T细胞受体核苷酸序列的存在量；以及比较该非完整重组的T细胞受体核苷酸序列的存在量与正常参考个体或标准的存在量。还提供一种用于该方法的试剂盒，包括用于检测该非完整重组的T细胞受体核苷酸序列的引物对及探针。

Description

利用非完整重组的T细胞受体核苷酸序列诊断T细胞淋巴瘤的方法及试剂盒 技术领域
本公开涉及一种诊断T细胞淋巴瘤的方法及试剂盒。更具体地，本公开通过数字聚合酶链反应(digital PCR)或实时PCR(real-time PCR)技术定量非完整重组的T细胞受体核苷酸序列作为生物标记，用以诊断T细胞淋巴瘤的方法及试剂盒。
背景技术
T细胞具有识别多种抗原的能力，因此在适应性免疫(adaptive immunity)中扮演重要角色。T细胞识别抗原的能力源自于细胞发育成熟过程中，V基因、J基因或D基因发生基因间的重组，以形成编码各式各样T细胞受体(T cell receptor，TCR)的基因(Schatz D.G.et al.,Recombination centres and the orchestration of V(D)J recombination.Nat.Rev.Immunol.2011；11(4):251-63)，如图1A至图1C所示。例如，TCRβ基因具有67个V、2个D和13个J编码片段，在每个T细胞中的V(D)J重组过程中，通过核苷酸缺失和在V-D和D-J连接处的核苷酸添加，产生了互补决定区(complementary determining region，CDR)的变异，因此增强了抗原识别的多样性。研究个体的免疫图谱(immune repertoire)通常以重组VJ和互补决定区3(CDR3)序列为特征，区分出多种T细胞克隆物(clones)。
早期免疫的研究可能因技术未尽完善，无法探究非完整重组的T细胞受体(non-completely recombined TCR)基因的全貌，随着分子生物学技术的精进，许多团队通过更完整的技术，陆续发现免疫系统中非完整重组的T细胞受体基因的存在，包括健康人外周血液中V(DD)J重组的发现(Briney B.S.et al.,Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.Immunology 2012；137: 56-64)。此外，2003年Gonzalez团队以不完整重组(incomplete rearrangement)的序列与疾病关联的应用，发现不完整的重组序列D-J-H在多发性骨髓瘤中的表达量增加(González D.et al.,Incomplete DJH rearrangements of the IgH gene are frequent in multiple myeloma patients:immunobiological characteristics and clinical implications.Leukemia 2003；17(7):1398-403)。该团队发现利用与完整的重组序列互比，非完整的重组序列可作为肿瘤的标志物(González D.et al.,DJH rearrangements.Methods Mol.Med.2005；113:165-73)，且可使用实时定量聚合酶链反应法(RQ-PCR)量身订制不完整的重组序列，用于监控残存癌细胞(González D.et al.,Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.Leukemia 2003；17(6):1051-7；Catherwood M.A.et al.,Improved clonality assessment in germinal centre/post-germinal centre non-Hodgkin’s lymphomas with high rates of somatic hypermutation.J.Clin.Pathol.2007；60(5):524-528；Nishana M.et al.,A non-B DNA can replace heptamer of V(D)J recombination when present along with a nonamer:implications in chromosomal translocations and cancer.Biochem.J.2012；448:115-125)。
当某个T细胞变成恶性而异常扩增时(即T细胞淋巴瘤)，其独特的T细胞受体基因频率也会异常提升，因此可以作为T细胞淋巴瘤的生物标志物(Gazzola A.et al.,The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies.Ther.Adv.Hematol.2014；5(2):35-47；Scheijen B.et al.,Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment:a technical feasibility study by EuroClonality-NGS.Leukemia 2019；33(9):2227-2240)。异常扩增代表相应克隆的高频率，即克隆性(clonality)，T细胞淋巴瘤的诊断通过寻找高频率的T细胞受体重组基因来进行，称为克隆性评估(clonality assessment)。目前，克隆性评估的标准方法 是采用EuroClonality联合会议所提出的多对引物(即BIOMED-2引物)，针对T细胞受体重组进行检测。多重T细胞受体分析会采用多对引物的方法(即多重聚合酶链反应)，然而，即使多对引物仍无法涵盖所有V和J的使用频率，因而不一定能真实反映各种不同V和J的使用频率，且在引物相互竞争的情形下，仍可能会有假阴性的情形(He L.et al.,Toward a more accurate view of human B-cell repertoire by next-generation sequencing,unbiased repertoire capture and single-molecule barcoding.Sci.Rep.2014；27(4):6778；Liu X.et al.,Systematic comparative evaluation of methods for investigating the TCRβrepertoire.PLoS One 2016；11(3):e0152464)。尽管已有文献提出减少引物偏差的方法(Carlson C.S.et al.,Using synthetic templates to design an unbiased multiplex PCR assay.Nat.Commun.2013；4:2680)，但仍无法保证能完全避免假阴性的产生。
对比于使用BIOMED-2引物检测B细胞淋巴瘤的临床检出率可高达95.7％，目前使用BIOMED-2引物检测T细胞淋巴瘤的临床检出率约为76％，显见T细胞淋巴瘤的临床检测仍有很大的进步空间(Chen YL,et al.,Leuk Lymphoma.2010 Apr；51(4):650-5.doi:10.3109/10428191003660631.PMID:20233058.)。此外，当T细胞淋巴瘤患者的骨髓或组织样本存在品质不佳的情形时，则无法提供适当的检测，进而影响临床诊断与治疗的建议。
因此，临床上仍需要一种新的检测方法，用于改进并提升T细胞淋巴瘤的临床诊断，以提供后续正确治疗及残存癌细胞的追踪。
发明内容
在一些具体实施例中，本公开提供一种诊断个体中T细胞淋巴瘤的方法，包括：提供取自该个体的生物样本；检测该生物样本中的非完整重组的T细胞受体核苷酸序列中第一靶序列的表达量、第二靶序列、以及参考序列的表达量；比较该第一靶序列的表达量与该参考序列的表达量的比值，或该第二靶序列的表达量与该参考序列的表达量的比值，以诊断该个体患有T细胞淋巴瘤；其中，该非完整重组的T细胞 受体核苷酸序列位于T细胞受体基因的外显子上游节段间非编码区；并且该非完整重组的T细胞受体核苷酸序列包括下列所组成的组中的至少一者：J基因内含子序列、假基因(pseudogene)、D基因上游内含子序列和D1基因前的基因间片段序列。在另一些具体实施例中，该个体的生物样本包含外周血细胞、骨髓细胞或组织。
在一些具体实施例中，该非完整重组的T细胞受体核苷酸序列进一步包括下列所组成的组中的至少一者：J基因片段序列、假基因(pseudogene)、D基因片段序列和C基因片段序列。
在一些具体实施例中，该非完整重组的T细胞受体核苷酸序列包括多个J基因片段序列、假基因(pseudogene)、J基因内含子序列和C基因片段序列。在另一些具体实施例中，该非完整重组的T细胞受体核苷酸序列包括D基因上游内含子序列、D基因片段序列、J基因片段序列、假基因(pseudogene)、和C基因片段序列。在另一些具体实施例中，该非完整重组的T细胞受体核苷酸序列包括D1基因前的基因间片段序列和C基因片段序列。
在一些具体实施例中，该非完整重组的T细胞受体核苷酸序列包括SEQ ID NO：4至6所示核苷酸序列中的至少一者，和SEQ ID NO：8至11所示核苷酸序列中的至少一者。在另一些具体实施例中，该非完整重组的T细胞受体核苷酸序列由SEQ ID NO：1至3中的任一者表示。
在一些具体实施例中，该第一靶序列为J2-2P基因序列(SEQ ID NO：8)，且该参考序列为J2-3基因序列(SEQ ID NO：9)。在另一些具体实施例中，该第二靶序列为D1基因前的基因间片段序列(SEQ ID NO：6)，且该参考序列为J2-3基因序列(SEQ ID NO：9)。在一些具体实施例中，该第一靶序列的表达量与该参考序列的表达量的比值大于15％，或该第二靶序列的表达量与该参考序列的表达量的比值大于50％时，表示该个体患有T细胞淋巴瘤。在另一些具体实施例中，该J2-2P基因的表达量与该J2-3基因的表达量的比值大于15％，或该D1基因前的基因间片段的表达量与该J2-3基因的表达量的比值大于50％时，表示该个体患有T细胞淋巴瘤。
在一些具体实施例中，本公开还提供一种用于诊断个体中T细胞淋巴瘤的试剂盒，包括用于检测第一靶序列的第一引物对及探针、用于检测第二靶序列的第二引物对及探针以及用于检测参考序列的参考引物对及探针，其中，该第一靶序列和该第二靶序列位于该个体的非完整重组的T细胞受体核苷酸序列中，且该第一靶序列不同于该第二靶序列。
在一些具体实施例中，该第一靶序列和该第二靶序列分别选自由下列所组成的组：J基因内含子序列、假基因(pseudogene)、D基因上游内含子序列、D1基因前的基因间片段序列、J基因片段序列、D基因片段序列和C基因片段序列。
在一些具体实施例中，该第一靶序列为J2-2P基因序列，第二靶序列为D1基因前的基因间片段序列，且该参考序列为J2-3基因序列。
本公开提供在非完整重组的T细胞受体核苷酸序列中用于检测T细胞淋巴瘤的生物标志物，通过测量各类非完整重组的T细胞受体核苷酸序列的存在量(例如J2-2P基因/J2-3基因的表达量比值或D1基因前的基因间片段/J2-3基因的表达量比值)进行T细胞淋巴瘤的诊断评估，并可与已知用于诊断T细胞淋巴瘤的额外过程(例如通过流式细胞仪分辨细胞标志物、病理切片、免疫化学染色或BIOMED-2多重PCR检测)合并使用，以提供完整的检测平台，并辅助T细胞淋巴瘤的诊断及后续治疗。
附图说明
图1A为抗原(Ag)-T细胞-组织相容抗原(MHC)之间互动关系的示意图(引用自Woodsworth et al.Genome Medicine 2013,5:98)；图1B为T细胞呈递外来抗原的示意图，其中V区包含V α和V β；J区包含J α和J β；D区包含C α和C β以及CDR3β区；图1C为T细胞中的V(D)J重组过程，TCR-βVDJ基因重组产生TCR多样性(diversity)。
图2为利用cDNA末端快速扩增的RACE方法(rapid amplification of cDNA ends)结合二代测序技术的实验流程示意图。
图3显示非完整重组的T细胞受体核苷酸序列在21位健康个体和2位淋巴瘤患者(I11a和I13)外周血液细胞以及一位淋巴瘤患者的骨 髓细胞(I11b)中TCRβ的RACE测序都有非完整重组的TCR序列(non-completely recombined sequences)，而其中J2-2P基因型占非完整重组序列的多数。J2-2P基因型在全部序列与非完整重组序列中的比例在I11淋巴瘤患者的血液与骨髓细胞是最高的。也由淋巴瘤患者(I11)的结果显示骨髓细胞与血液细胞在TCR基因型组成上的相似度。
图4显示淋巴瘤患者(I11)的BIOMED-2克隆性评估两次结果均为阴性。
图5显示J2-2P～J2-3区段序列具有T细胞特异性，仅在T细胞相关的细胞(系)上表达。利用数字PCR确认J2-2P只在T细胞类(系)中表达。
图6为骨髓细胞J2-2P及J2-3区段序列经数字PCR定量的结果。24位淋巴瘤患者(TCL)的骨髓细胞与11位健康对照组(healthy control)的血液细胞中J2-2P基因型(左)与intD1基因型(右)相对J2-3基因型的表达量比值。LP：淋巴瘤患者。non-LP：非淋巴瘤患者。
图7为J2-2P基因型(左)与intD1基因型(右)相对J2-3基因型表达量在6位淋巴瘤患者(左，包括血管免疫母细胞T细胞淋巴瘤、外周T细胞淋巴瘤、成熟T细胞淋巴瘤及结节外NK/T细胞淋巴瘤等类型的T细胞淋巴瘤)与1位非淋巴瘤者(右)的组织的比值。
图8显示以Sanger测序法针对健康个体的外周血细胞中J2-2P～J2-3区段序列的检测结果。
图9显示以Sanger测序法验证T细胞系有J2-2P～J2-3序列，中间包含内含子序列。
图10为利用数字PCR(digital PCR)检测J2-2P型别相对J2-3的占比。
具体实施方式
以下的具体实施方案用以说明本公开的技术内容，在阅读本说明书的公开内容后，所属技术领域技术人员能轻易地理解其优点及功效。本公开还可通过其他不同的实施方式加以施行或应用，本说明书中的 各项细节也可基于不同观点与应用，在不悖离本公开所描述的范围下赋予不同的修饰与变更。
除非本文中另有说明，否则说明书及所权利要求书中所使用的单数形式“一”及“该”包括多数个体，并且术语“或”包括“及/或”的含义。如本文中所使用，“样本”、“生物样本”、及“试验样本”是指自个体中取出，并于活体外进行实验的细胞或组织，即体外(in vitro)或间接体内(ex vivo)的细胞或组织。在本公开的一些具体实施例中，该个体为动物，例如哺乳动物。在本公开的一些具体实施例中，该个体为人类。
如本文中所使用，“靶序列”是指被扩增、检测、或扩增并检测的序列，其或与本文中提供的序列互补，或于其自然状态下具有至少一个内含子，即作为基因组DNA(genomic DNA)或染色体外DNA(Extrachromosomal DNA)。在本公开中，可包含多个靶序列，且该多个靶序列中的每一个可具有编号(例如第一靶序列、第二靶序列)。靶序列的编号仅作为标示不同的靶序列使用，不具有顺序关系。举例而言，在本公开的实施例中，第一靶序列、第二靶序列可以先于、后于、或同时与参考序列被扩增、检测、或扩增并检测。
如本文中所使用，术语“外显子上游节段间非编码区”(intersegment non-coding region)是指T细胞受体基因(例如TCRβ基因)中介于各个V、D和J编码节段之间未被用于编码氨基酸的序列区段，正常情况下，位于“外显子上游节段间非编码区”内的核苷酸序列在V(D)J重组过程中会被裁剪，不会用来编码形成互补决定区的氨基酸。换言之，“外显子上游节段间非编码区”也就是用于V(D)J重组的正常外显子以外的序列，包括:内含子、假基因(pseudogenes)、基因前的基因间节段等。如本文中所使用，当用语“上游”置于参考位置之前或后时，其是指自该参考位置朝核酸中(正向链，若为双链核酸)5’端方向的序列段。例如，D基因上游是指核酸中自D基因往正向链5'端方向的序列区段。在本公开的实施例中，“上游”可以指自参考位置朝核酸中5'端方向1至20个核苷酸、1至50个核苷酸、1至100个核苷酸、或1至200个核苷酸处的序列区段。若该参考位置具有超过1个核苷酸，则“上游”可自该参考位置的任一位置起算。例如，J2-3基 因上游可以指J2-3基因中任一位置的核苷酸朝核酸中5'端方向1至20个核苷酸、1至50个核苷酸、1至100个核苷酸、或1至200个核苷酸处的序列区段。
本公开采用cDNA末端快速扩增方法(rapid amplification of cDNA ends，RACE)来避免PCR方法的偏差(bias)，并搭配二代测序及分析来进行检测。RACE方法可避免引物的偏差，过程中利用T细胞受体核苷酸序列的特异性引物确切地扩增T细胞受体核苷酸序列，建立无偏差的T细胞受体基因全貌(参见图2所示)。通过RACE方法，可发现非完整重组的T细胞受体核苷酸序列(本文中，也称为非完整重组(non-regular sequence))，例如，没有V区段的序列。
在本公开的一些具体实施例中，利用RACE方法搭配二代测序与分析，21位健康个体和2位淋巴瘤患者(I11a和I13)外周液细胞与一位淋巴瘤患者的骨髓细胞(I11b)中TCRβ的RACE测序都有非完整重组的TCR序列(non-completely recombined sequences)，而其中J2-2P基因型占非完整重组序列的多数。J2-2P基因型在全部序列与非完整重组序列中的比例在I11淋巴瘤患者的血液与骨髓细胞是最高的(参见图3)。然而淋巴瘤患者的BIOMED-2克隆性评估两次结果(外周血液及骨髓))均为阴性(参见图4)。以上结果显示，非完整重组的T细胞受体核苷酸序列可作为T细胞淋巴瘤的生物标志物，也可用以辅助BIOMED-2克隆性评估的TCRβ检验的临床假阴性的不足。
在本公开的一些具体实施例中，T细胞受体的非完整重组类型例示于下表1，包括：第一类，具有上游J基因-J基因内含子(intron)-J基因-C基因的序列区段，即，J基因中的内含子序列未经裁剪(splicing)；第二类，具有D基因上游内含子-D基因-J基因-C基因的序列区段，即，D基因上游的内含子序列未经裁剪；以及第三类，具有D1基因前的基因间节段(intergenic segment)-C基因的序列区段。第四类，有假基因(pseudogene)的序列。此外，表1所载的例示区段的核苷酸序列显示于表2，其中例示的各基因节段序列显示于表3。
表1、主要的T细胞受体非完整重组种类
Figure PCTCN2022098132-appb-000001
表2、T细胞受体非完整重组的核苷酸序列
Figure PCTCN2022098132-appb-000002
表3、T细胞受体中各基因节段的核苷酸序列
Figure PCTCN2022098132-appb-000003
在本公开中，“数字PCR”是指一种核苷酸定量技术，可直接计算出DNA分子的个数，是对起始样本的绝对定量。在实验过程中，每个反应空间(孔洞、微滴)只含0或1条的DNA，利用终点信号的有或无作为定量方法的结果，当扩增结束后，含有核酸分子的微滴会释放出荧光信号，最终根据泊松分布(Poisson distribution)原理以及阳性微滴的比例，通过分析软件计算出原始分子的浓度或拷贝(copy)数。数字PCR可以直接计算目标序列的拷贝数，因此无需依赖对照样本和标准曲线即可进行精确的绝对定量检测。此外，由于数字PCR在进行结果判读时仅判断有或无两种扩增状态，因此不需要检测荧光信号与设定阈值线的交点，可完全毋须依赖Ct值的鉴定，使得数字PCR的反应不受扩增效率的影响，对PCR反应抑制物的耐受能力大幅提高。数字PCR实验中标准反应体系的分配过程可以极大程度上降低与目标序列有竞争性作用的背景序列浓度。
本公开还可通过即时定量PCR技术作为非完整重组的定量检测方法。“实时定量PCR”(quantitative real time PCR)又称为qPCR，涉及特异性的荧光探针(TaqMan probe)和引物的使用，其中该引物用于 进行PCR扩增，荧光探针则提高特异性，利用实时聚合酶链反应来检测并记录荧光变化。PCR产物随着PCR循环数呈正相关增加，经由光学系统可实时检测PCR产物的量。
以下通过特定的具体实施例进一步说明本公开的特点及功效，但非用于限制本公开的范围。
实施例
研究方法与材料：
(一)T细胞淋巴瘤患者样本的RNA核酸提取
原始样本种类：血液(含ACD(acid-citrate-dextrose)抗凝剂)、骨髓(含K2EDTA抗凝剂)、组织(FFPE)。
样本量：血液8至10毫升(mL)、骨髓3至5毫升、组织(FFPE 3-5片，5uM/片)。
提取RNA试剂试剂盒为QIAamp RNA Blood Mini(QIAGEN)，借此提取血液、组织与细胞中的RNA。提取方法为利用离心方式使总RNA选择性附着在二氧化硅基薄膜(silica-based membrane)上，再以高盐缓冲系统(high-salt buffering system)使具有超过200个碱基的RNA附着在薄膜上，而少于200个碱基的5.8S RNA、5S RNA、tRNA则会被去除。该提取方式无须传统有毒物质(例如酚、氯仿等)的处理步骤，另包含变性步骤使白细胞溶解，并避免核糖核酸水解酶(RNase)活化，以保留完整的RNA。该试剂试剂盒可避免血红素污染并移除肝素(heparin)抗凝剂干扰，所提取的RNA可以继续进行逆转录PCR(reverse transcription-PCR，RT-PCR)、cDNA合成等后续实验。
(二)RNA逆转录cDNA
原始样本种类：自骨髓、血液、组织提取的RNA。
样本量：浓度500ng RNA转cDNA。
将经提取的RNA利用逆转录酶(reverse transcriptase)逆转录为cDNA，再以获得的cDNA进行后续PCR扩增作用。
(三)RACE方法结合二代测序技术
如图2所示，经提取的总RNA以T细胞受体恒定区(constant region，C)的特异性引物逆转录为cDNA，接着通过5'端的通用引物(SMARTer oligo)进行扩增。第一次PCR完成后，加入等比例的三种引 物(AP1、TCRβ-C1、TCRβ-C2，参见Freeman JD,et al.Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing.Genome Res.2009 Oct；19(10):1817-24.)进行第二次PCR(巢式PCR，nested PCR)。随后将第二次的PCR产物(大约520bp)进行二代测序建库。用于RACE方法的试剂试剂盒为Clontech SMARTer TM RACE cDNA Amplification Kit；详细的实验程序可参见Motomura M,et al.Cloning and characterization of the O-methyltransferase I gene(dmtA)from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.Appl.Environ.Microbiol.1999Nov；65(11):4987-94.
(四)以数字PCR技术定量非完整重组节段
采用Clarity数字PCR系统(JN MEDSYS)及管式芯片(chip in tube)技术，其中的特殊芯片划分有上万个单独反应空间(partitions)，可通过Clarity自动装载器(auto loader)将DNA分布至每个反应空间，使每个反应空间只含0或1条的DNA。接着使DNA在0.2mL反应管中进行PCR扩增，后续搭配Clarity数字PCR检测仪(reader)检测荧光信号，转换泊松分布计算后求得拷贝数，达到单分子绝对定量的结果。
在此实验中，本公开通过数字PCR检测J2-2P～J2-3区段及intD1:C1区段所使用的引物和探针的序列如下表4所示。
表4、检测J2-2P～J2-3区段及intD1:C1区段的引物和探针序列
引物 5’→3’ SEQ ID NO.
J2-2P AGGCGCTGCTGGGCGTCT 12
TRBC2-1 GGGTGGGAACACGTTTTTCAGG 13
J2-3 GCACAGATACGCAGTATTTTGG 14
TRBC2-2 TCAGCTCCACGTGGTCGGGGT 15
intD-F1-d GGTACTGGAGAAGACCAGCC 16
Cβ1/2-1 GGTGTGGGAGATCTCTGCTTC 17
探针 5’→3’ SEQ ID NO.
J2-2P HEX-CTCTCCCAGCACCCAGAACCAGGA 18
J2-3 FAM-CTGACAGTGCTCGAGGACCTGAAAAACGT 19
Int-D FAM-GGACAGTGCCTGGAGAGGACCTG 20
数字PCR的具体反应步骤如下述。
步骤1：依照样本数目不同，使用干净的剪刀剪取适当数量的200μL SnapStrip II PCR反应管。
步骤2：再依照样本数目不同，依下表5配置PCR反应所需的试剂。
表5、PCR反应试剂
Figure PCTCN2022098132-appb-000004
步骤3：将所需要的试剂放入1.5mL的试管中。
步骤4：利用涡旋混合器，将上述配置好的PCR反应试剂混合均匀。
步骤5：将以上混合好的PCR反应试剂，以微量离心机(固定转速6,600rpm)离心5秒。
步骤6：在每个反应空间中加入9μL的PCR反应试剂，再加入待测样本1.0μL(逆转录的cDNA)，并调整最终体积为15μL。空白对照组加入1μL的无核酸酶水(nuclease-free water)。
步骤7：利用热循环仪(thermal cycler)，以下列程序条件进行PCR反应：
(1)上盖温度设定为105℃，体积为15μL进行聚合酶链反应扩增；
(2)37℃、30分钟；
(3)95℃、15分钟；
(4)维持12℃。
步骤8：盖紧盖子，将试剂与待测样本充分混合后离心。
步骤9：取出JN Clarity耗材组的连排管(tube strip)与样品荷载试剂盒(sample loading kit，包含滑件(slider)和载物台(platform))，打开连排管盖子确认芯片位置与形状，将载物台放入连排管中，靠紧芯片上缘，装到自动装载器上。
步骤10：将滑件置于载物台上，按下启动键试推一次，若机器推的过程中有卡住芯片的情况则调整角度后再试一次。将经充分混合的15μL试剂样本混合液加到滑件的三角处，样本依照顺序加入，按下启动键使试剂样本混合液均匀分布至芯片上。若载物台仍有液体残留，则再按一次启动键将液体完整推入芯片，并清除管壁的残留液体。
步骤11：将连排管放入密封增强器(sealing enhancer)进行样本密封至多2次。
步骤12：每一管加入245μL的密封液(sealing fluid)。
步骤13：盖上连排管盖，清除多余或渗出的密封液。
步骤14：利用梯度式聚合反应器(VWR Peqlab)，以下列程序条件进行PCR反应：
(1)上盖温度设定为90℃；
(2)95℃、5分钟，反应进行1次；
(3)95℃、50秒与58℃、90秒，反应进行50个循环；
(4)70℃、5分钟；
(5)维持70℃，2小时内进行判读。
步骤15：PCR结束后，以拭镜纸与酒精将连排管管壁擦拭干净，并放入Clarity检测仪的观测夹具(viewing jig)，确认芯片完整浸泡于密封液中，扣紧上盖并加入6mL灭菌水，移除管壁上多余气泡。
步骤16：开启Clarity软件并连线检测仪，设定实验参数。
步骤17：将观测夹具放入检测仪，并按下RUN键。
步骤18：结果判定(含数据计算、结果判读或可报告范围)：
(1)阳性反应：Dye1设定为J2-3C(FAM荧光)，Dye2设定为J2-2P(HEX荧光)，阳性率计算方法为(Dye2DNA拷贝数/Dye1DNA拷贝数)×100％，可计算出T细胞受体核苷酸非完整重组的比例。
(2)阴性反应：荧光信号需小于5颗以下，则该次实验可接受。
实施例1：T细胞受体核苷酸非完整重组在健康个体与淋巴瘤患者中的表达
如图3所示，通过TCRβ的RACE测序，可以发现21位健康个体和2位淋巴瘤患者(I11a and I13)外周血液细胞与一位淋巴瘤患者的骨髓细胞(I11b)中TCRβ的RACE测序都有非完整重组的TCR序列(non-completely recombined sequences)，而其中J2-2P基因型占非完整重组序列的多数。在非完整重组的序列中，J2-2P型的占比最大，在健康人中占所有非完整重组序列的22.9％～71.0％。J2-2P基因型在全部序列与非完整重组序列中的比例在I11淋巴瘤患者的血液和骨髓细胞是最高的。重要的是，其中一位确诊淋巴瘤患者的骨髓细胞可鉴定出高比例的非完整重组序列(88.2％)，其中J2-2P基因型在非完整重组序列中的比例(80％)也是最高的。
如图4所示，相较于BIOMED-2检测方法，临床确诊淋巴瘤患者I11在治疗前与治疗后均鉴定出有高比例的J2-2P～J2-3区段(>50％)，然而其BIOMED-2克隆性评估却为阴性(图4)。
为进一步验证非完整重组与T细胞淋巴瘤的相关性，细胞系的RT-PCR显示J2-2P型仅在T细胞系中表达，并不在B细胞或其他实体肿瘤细胞系中表达，显示其具有T细胞特异性(图5)。J2-2P～J2-3序列只在正常人外周血液的T细胞系(单核细胞)与T细胞相关的细胞系中表达，包括:Jurkat(急性T细胞白血病)、H9(T细胞淋巴瘤)、 MOLT-4(T成淋巴细胞；急性成淋巴细胞白血病)和健康人的T细胞。J2-2P～J2-3序列不在以下项中表达:(1)正常人外周血液的B细胞；(2)非T淋巴瘤类的细胞系：Bjab(伯基特淋巴瘤)；白血病细胞系：K562(CML；bcr-abl)、RS4；11(ALL；KMT2A‐AFF1)、REH(急性淋巴细胞白血病)、NB4(APL)、HL-60(APL)；和(3)实体肿瘤细胞系:A549(肺癌)、MKN45(胃腺癌)。
另一方面，图6为骨髓细胞J2-2P及J2-3区段序列经数字PCR定量的结果。21位淋巴瘤患者(TCL)的骨髓细胞与11位健康对照组(healthy control)的血液细胞中J2-2P基因型(左)与intD1基因型(右)相对J2-3基因型的表达量比值。21位淋巴瘤患者(TCL)的骨髓细胞与11位健康对照组(healthy control)的血液细胞中J2-2P基因型(左)(34.2比8.7；Wilcoxon p＝0.0039)与intD1基因型(右)(148.3比21.1；Wilcoxon p＝0.00014)相对J2-3基因型的表达量比值。显示非完整重组比值具有作为T细胞淋巴瘤生物标志物的潜力。
表6、健康个体与T细胞淋巴瘤患者中J2-2P/J2-3及intD1/J2-3的比值
Figure PCTCN2022098132-appb-000005
J2-2P/J2-3截止值(cutoff value)：15
intD1/J2-3截止值：50
说明：T细胞淋巴瘤患者(24例)，与非淋巴瘤患者(6例)，均检测其骨髓细胞。健康对照组(11例)检测其血液细胞。
又一方面，图7为J2-2P基因型(左)与intD1基因型(右)相对J2-3基因型表达量在6位淋巴瘤患者(包括血管免疫母细胞T细胞淋巴瘤、外周T细胞淋巴瘤、成熟T细胞淋巴瘤及结节外NK/T细胞淋巴瘤)与1位非淋巴瘤者的组织的比值。
综合上述结果，申请人发现高比例的J2-2P～J2-3区段及intD1区段等非完整重组可作为T细胞淋巴瘤诊断的辅助标志物，用以提升T细胞淋巴瘤的检出率。
实施例2：以Sanger测序法确认健康个体外周血细胞中具有J2-2P～J2-3区段序列(图8)
针对健康个体外周血细胞的cDNA序列，使用J2-2P基因的5'端引物(A引物，SEQ ID NO：21)和TCRβC2区引物(B引物，SEQ ID NO：22)进行扩增并测序(图8)，证实健康个体中也存在非常规的J2-2P～J2-3区段序列，表示其并非二代测序的伪像(artifacts)。
A引物:5'-ACCCTGTTCTTAGGGGAGTG-3'
B引物:5'-CACAGCGGCCGCGGGTGGGAACACGTTTTTCAGGT-3'
上述J2-2P～J2-3区段序列包含J2-2P外显子(exon)、未裁剪的J2-2P内含子(105bp)、J2-3外显子和TCRβC2外显子。正常的VDJ重组会经过裁剪序列(去除内含子)的过程，以J2-3为例，J2-3外显子到TCRβC2外显子中间的内含子(102bp)已经过裁剪。
实施例3：J2-2P～J2-3区段序列在T细胞系中表达
本实施例通过数字PCR技术检测J2-2P～J2-3区段序列分别在T细胞系及B细胞系中的表达。进行检测的细胞系如下所列：健康人类CD4 +T细胞、Jurkat细胞系(人类T细胞淋巴瘤)、H9细胞系(皮肤T细胞淋巴瘤)、Mol-4细胞系(人类急性T细胞淋巴瘤)、Bjab细胞系(伯基特淋巴瘤，Burkitt lymphoma)、K562细胞系(慢性骨髓性白血病，具有bcr-abl基因)、RS4；11细胞系(急性淋巴细胞白血病，具有KMT2A‐AFF1基因)、REH细胞系(急性淋巴细胞白血病)、NB4细胞系(急性髓系白血病)、HL-60细胞系(急性髓系白血病)以及实体肿瘤细胞系，包括A549细胞系(肺癌)、MKN45细胞系(胃腺癌)和G2细 胞系(肝癌)，其中健康人类CD4 +T细胞通过STEMCELL Technologies公司的CD4 +磁珠收集健康者的外周血中的CD4 +T细胞获得，其纯度达99.9％。
如图5所示，健康人类T细胞中J2-2P拷贝数占J2-2P与J2-3拷贝数总和的不到5％。相较之下，Jurkat、H9、Mol-4等T细胞系中J2-2P拷贝数占J2-2P与J2-3拷贝数总和的约为20％至40％，分别显示REH、Bjab、NB4、HL-60、K562、RS4；11等B细胞系以及A549、MKN45、G2细胞等实体肿瘤细胞系通过数字PCR确认J2-2P～J2-3区段序列的表达结果。由这些实验结果发现J2-2P～J2-3区段序列只在T细胞中表达，其并不在B细胞或实体肿瘤细胞中表达。
实施例4：J2-2P～J2-3区段序列在T细胞系和健康个体中表达
在本实施例中，先以STEMCELL Technologies公司的CD4 +磁珠收集健康人类外周血细胞中的CD4 +T细胞，所获得的CD4 +T细胞纯度达99.9％。对健康人类的CD4 +T细胞的TCR区域进行测序。由测序结果可发现CD4 +T细胞中均有J2-2P～J2-3区段序列，且这些J2-2P～J2-3区段序列包含J2-2P(45bp)-内含子(105bp)-J2-3(48bp)，如图9所示。
实施例5：通过数字PCR绝对定量技术检测J2-2P～J2-3区段序列
本实施例中采用管式芯片技术，其中0.2mL反应管中镶嵌有其上含有20,000个反应空间的芯片，并通过自动装载器将DNA样品涂抹于每个空间，使空间只含0或1条的DNA，并进行PCR扩增。进行数字PCR的反应试剂(每15μL)由下列各成分及其含量充分混合而得：2×主混合液7.5μL、10μM PCR引物2μL、10μM PCR探针0.5μL、20×JN溶液0.75μL、水3.25μL及cDNA 1μL(浓度500ng RNA转cDNA)，再由上述经充分混合的反应试剂中取出15μL加至反应管中，后续以Clarity数字PCR进行数字PCR扩增，检测荧光信号并转换泊松分布计算，求得拷贝数，达到绝对定量的结果。
图10显示本公开其中一个淋巴瘤病患组织样本经数字PCR扩增后进行定量的结果，其中J2-2P序列经数字PCR定量后得到9972颗信 号，换算后每微升中有4579.9个拷贝；J2-3序列经数字PCR定量后得到9742颗信号，换算后每微升中有2754.7个拷贝。经计算后可以得到该样本中J2-2P拷贝数占J2-2P与J2-3拷贝数总和的百分比：J2-2P(％)＝J2-2P/J2-3＝(4579.9/2754.7)x100＝166％。
综上可知，健康人类外周血细胞的cDNA中均可测得J2-2P～J2-3区段序列，证实此非完整重组也存在于健康个体中而并非二代测序的伪像。此外，针对各种细胞系的数字PCR结果显示J2-2P～J2-3区段序列仅在T细胞系中表达，并不在B细胞或其他实体肿瘤细胞系中表达，显示其具有T细胞特异性。进一步地，通过测序可确认J2-2P～J2-3区段序列，并通过绝对定量PCR方式验证其占比，结果显示由T细胞系(Jurkat、H9)和健康个体(以CD4 +磁珠自外周血细胞收集)的cDNA中都可发现J2-2P～J2-3区段序列，且其序列内容相同。
通过数字PCR检测J2-2P～J2-3区段序列，利用引物和探针更精准定量J2-2P～J2-3区段的表达，上述结果J2-2P～J2-3区段序列真实存在且具有T细胞特异性。还比较健康个体与T细胞淋巴瘤患者中非完整重组的表达，发现T细胞淋巴瘤患者中的J2-2P/J2-3和intD1/J2-3的比值均远高于健康个体，且其中包括BIOMED-2克隆性评估为阴性的患者。
因此，本公开提供检测T细胞淋巴瘤的生物标志物，可有效用于诊断T细胞淋巴瘤。鉴于BIOMED-2克隆性评估的临床假阴性，本公开的方法有利于成为替代的分析检测，也可搭配BIOMED-2评估成为完整的检测平台，辅助T细胞淋巴瘤的诊断及后续治疗。
上述实施例用以例示性说明本公开的原理及其功效，而非用于限制本公开。任何本领域技术人员均可在不违背本公开的范围下，对上述实施例进行修改。因此，本公开的权利保护范围，应如所附权利要求书中所列。

Claims (10)

  1. 一种诊断个体中T细胞淋巴瘤的方法，包括：
    提供所述个体的生物样本；
    检测所述生物样本中的非完整重组的T细胞受体核苷酸序列中第一靶序列、第二靶序列、以及参考序列的表达量；
    比较所述第一靶序列的表达量与所述参考序列的表达量的比值，或所述第二靶序列的表达量与所述参考序列的表达量的比值，以诊断所述个体患有T细胞淋巴瘤；
    其特征在于，所述非完整重组的T细胞受体核苷酸序列位于T细胞受体基因的外显子上游节段间非编码区，且所述非完整重组的T细胞受体核苷酸序列包括下列所组成的组中的至少一者：J基因内含子序列、假基因、D基因上游内含子序列和D1基因前的基因间片段序列。
  2. 根据权利要求1所述的方法，其特征在于，所述非完整重组的T细胞受体核苷酸序列进一步包括下列所组成的组中的至少一者：J基因片段序列、假基因、D基因片段序列和C基因片段序列。
  3. 根据权利要求2所述的方法，其特征在于，所述非完整重组的T细胞受体核苷酸序列包括SEQ ID NO：4至6所示核苷酸序列中的至少一者，和SEQ ID NO：8至11所示核苷酸序列中的至少一者。
  4. 根据权利要求2所述的方法，其特征在于，所述非完整重组的T细胞受体核苷酸序列包括：
    (1)多个J基因片段序列、假基因、J基因内含子序列和C基因片段序列；
    (2)D基因上游内含子序列、D基因片段序列、J基因片段序列、假基因和C基因片段序列；或
    (3)D1基因前的基因间片段序列、假基因和C基因片段序列。
  5. 根据权利要求4所述的方法，其特征在于，所述非完整重组的T细胞受体核苷酸序列由SEQ ID NO：1或SEQ ID NO：3表示。
  6. 根据权利要求1所述的方法，其特征在于，所述第一靶序列为J2-2P基因序列或所述第二靶序列为D1基因前的基因间片段序列，且所述参考序列为J2-3基因序列，其特征在于，所述J2-2P基因的表达量与所述J2-3基因的表达量的比值大于15％时，或所述D1基因前的基因间片段的表达量与J2-3基因的表达量的比值大于50％时，表示所述个体患有T细胞淋巴瘤。
  7. 根据权利要求1所述的方法，进一步包括进行至少一种额外的过程用于诊断所述T细胞淋巴瘤，其特征在于，所述额外过程为通过流式细胞仪分辨细胞标志物、病理切片、免疫化学染色或BIOMED-2多重PCR检测。
  8. 一种用于诊断个体中T细胞淋巴瘤的试剂盒，包括用于检测第一靶序列的第一引物对和探针，以及用于检测第二靶序列的第二引物对和探针，以及用于检测参考序列的参考引物对和探针，其中，所述第一靶序列和所述第二靶序列位于所述个体的非完整重组的T细胞受体核苷酸序列中。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述非完整重组的T细胞受体核苷酸序列由SEQ ID NO：1或SEQ ID NO：3表示。
  10. 根据权利要求9所述的试剂盒，其特征在于，所述第一靶序列为J2-2P基因序列或第二靶序列为D1基因前的基因间片段序列，且所述参考序列为J2-3基因序列。
PCT/CN2022/098132 2022-06-10 2022-06-10 利用非完整重组的t细胞受体核苷酸序列诊断t细胞淋巴瘤的方法及试剂盒 WO2023236189A1 (zh)

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