Method for predicting prognosis after chemotherapy in dogs affected by lymphoma

Abstract

The present invention is a method for predicting the post-chemotherapy prognosis of a canine with lymphoma, said method comprising a measurement step for measuring the methylation rate of one or more types of CpG site selected from the group consisting of Chr4:29559893 and Chr1:14232692 in DNA which has been collected from biological sample taken from a canine that is subject to the prediction; and a prediction step for predicting, on the basis of the methylation rate measured in the measurement step and a reference value set in advance, the post-chemotherapy prognosis of the canine with lymphoma, wherein the reference value is for differentiating between a canine with a good post-chemotherapy prognosis and a canine with a poor post-chemotherapy prognosis and is set for the respective methylation rate of each CpG site.

Description

Technical Field

[0001] The present invention relates to a method for predicting the prognosis after chemotherapy in dogs suffering from lymphoma. This application claims priority based on U.S. Application No. 63 / 287,534, filed on December 9, 2021 in the United States of America, the content of which is incorporated herein by reference.

Background Art

[0002] In veterinary clinical practice, canine lymphoma is an important disease with a high incidence. Specifically, the incidence rate of canine lymphoma is 107 / 100,000 dog-years, which is more than five times higher than that in humans. Representative dog breeds with a high incidence include Golden Retrievers and Boxers. Canine lymphoma accounts for 24% of all tumors and >80% of hematological tumors. In the case of canine lymphoma, if left untreated, most dogs will die within three months, but most can achieve clinical remission due to the development of current multi-drug combination chemotherapy.

[0003] A typical chemotherapy for canine malignant lymphoma is CHOP (Chop) therapy. CHOP therapy is a treatment that combines three types of anticancer drugs (cyclophosphamide, doxorubicin, vincristine) with a corticosteroid (prednisolone). In particular, the currently most standard protocol for lymphoma, called the University of Wisconsin (UW) protocol, is used. In this protocol, treatment is performed weekly for the first two months and then once every two weeks for the next four months, that is, a total of six months of treatment. Canine lymphoma responds well to CHOP therapy. However, even if clinical remission is achieved with chemotherapy, the disease will ultimately relapse and lead to death. The remission rate is >85%, and the median survival period is one year. Usually, relapse occurs 3-4 months after the end of treatment, with a one-year survival rate of 50%, a two-year survival rate of 20%, and a three-year survival rate of less than 10%.

[0004] Currently, as prognostic factors in canine lymphoma, reports mainly rely on broad classifications such as anatomical site of origin, lymphocyte phenotype, and clinical stage. Table 1 shows the prognostic factors reported in canine lymphoma.

[0005]

Table 1

[0006] These prognostic factors may be prognostic factors for all types of canine lymphoma, and there is a lack of prognostic parameters for further subdividing each type of lymphoma. For example, prognostic factors in the homogeneous group of multicentric lymphoma have not yet been established. As a background for this, it can be cited that the mechanisms of occurrence and treatment response of multicentric lymphoma have not been elucidated.

[0007] CpG sequences (base sequences in which C (cytosine) and G (guanine) are consecutive) have a lower occurrence frequency than their simple probability, and most of them are methylated. DNA methylation is a modification of the transfer of a methyl group to the 5th position of cytosine. Locations where CpGs are densely present are called CpG islands, and it is thought that about half of mammalian genes have CpG islands in their promoter regions. Methylation of cytosine in CpG islands in the promoter region correlates with a decrease in gene expression of neighboring genes by causing changes in the chromatin structure of the promoter region. In the genome of normal cells, methylation often does not occur in CpG islands in the promoter region, but in tumor cells, these properties change dramatically, and an increase in methylation in CpG islands and a decrease in methylation (a decrease in the methylation rate) in non-CpG islands are often observed. That is, DNA methylation can be a biomarker for tumor development and its malignancy.

[0008] Abnormal DNA methylation has also been reported in canine lymphoma. For example, it has been reported that the quantitative value of the amount of methylation across the genome is less than that in healthy cells (Non-Patent Document 1). In addition, the DNA methylation rates of the FHIT gene (Non-Patent Document 2), DLC1 gene (Non-Patent Document 3), TFPI-2 gene (Non-Patent Document 4), ABCB1 gene (Non-Patent Document 5), p16 gene (Non-Patent Document 6), and DAPK1 gene (Non-Patent Document 7) have also been reported to have different quantitative methylation amounts in lymphoma compared to healthy cells. However, there is no clear indication of the relationship between these DNA methylations and the prognosis of lymphoma chemotherapy.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a method for predicting the possibility of whether the prognosis after chemotherapy for dogs suffering from lymphoma is good or not.

Means for Solving the Problems

[0011] As a result of intensive studies to solve the above problems, the present inventors quantitatively analyzed DNA methylation change sites in cases of canine multicentric lymphoma, and found two CpG sites with a significant difference in methylation rate between the group with a good prognosis and the group with a poor prognosis after chemotherapy, and completed the present invention.

[0012] That is, the present invention is as follows. [1] A method for predicting the prognosis after chemotherapy for a dog suffering from lymphoma, a measurement step of measuring the methylation rate of one or more CpG sites selected from the group consisting of Chr4:29559893 and Chr1:14232692 in DNA recovered from a biological sample collected from the dog to be predicted; a prediction step of predicting the prognosis after chemotherapy for the dog suffering from lymphoma based on the methylation rate measured in the measurement step and a preset reference value; and the reference value is a value set for the methylation rate of each CpG site for discriminating between dogs with a good prognosis and dogs with a poor prognosis after chemotherapy. Method for predicting prognosis after chemotherapy in dogs affected by lymphoma. [2] The method for predicting prognosis according to [1], wherein the lymphoma is multicentric. [3] The method for predicting prognosis according to [2], wherein the sub-stage of the lymphoma is a or b. [4] The method for predicting prognosis according to any one of [1] to [3], wherein the chemotherapy is CHOP therapy. [5] In the prediction step, when the methylation rate of Chr4:29559893 is equal to or lower than a preset reference value, it is determined that the prognosis of the dog affected by the lymphoma after receiving the chemotherapy is likely to be good. The method for predicting prognosis according to any one of [1] to [4]. [6] The method for predicting prognosis according to [5], wherein the preset reference value is 45%. [7] In the prediction step, when the methylation rate of Chr1:14232692 is equal to or lower than a preset reference value, it is determined that the prognosis of the dog affected by the lymphoma after receiving the chemotherapy is likely to be good. The method for predicting prognosis according to any one of [1] to [6]. [8] The method for predicting prognosis according to [7], wherein the preset reference value is 10%. [9] In the measurement step, the methylation rate of the CpG site is measured by the bisulfite pyrosequencing method. The method for predicting prognosis according to any one of [1] to [8]. [Advantages of the Invention]

[0013] According to the method for predicting prognosis after chemotherapy in dogs affected by lymphoma according to the present invention, for a biological sample collected from a dog affected by lymphoma, which is a test animal, by examining the methylation rate of a specific CpG site in genomic DNA, the prognosis after chemotherapy can be predicted. [Brief Description of the Drawings]

[0014]

Figure 1

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Figure 2B

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Figure 4A

Figure 4B

[0015] The cytosine base of the CpG site in genomic DNA can be methylated at the 5th carbon. In the present invention and this specification, the methylation rate of the CpG site refers to the ratio (%) of the amount of methylated cytosine bases (methylated cytosine) to the amount of non-methylated cytosine bases (non-methylated cytosine) among the CpG sites in a biological sample collected from a single biological individual, where the amounts of methylated and non-methylated cytosine bases are measured and the ratio is calculated with respect to the sum of both.

[0016] The method for predicting the prognosis after chemotherapy for dogs suffering from lymphoma according to the present invention (hereinafter sometimes referred to as "the prognosis prediction method according to the present invention") is a method for predicting the prognosis after chemotherapy for dogs suffering from lymphoma, and has the following measurement step and prediction step. A measurement step of measuring the methylation rate of one or more CpG sites selected from the group consisting of Chr4:29559893 and Chr1:14232692 in DNA recovered from a biological sample collected from a dog to be predicted; A prediction step of predicting the prognosis after chemotherapy for the dog suffering from lymphoma based on the methylation rate measured in the measurement step and a preset reference value.

[0017] The prognosis prediction method according to the present invention identifies DNA methylation change sites that correlate with the prognosis of canine multicentric lymphoma cases, and uses these as biomarkers to predict the prognosis of chemotherapy by quantitative analysis of the methylation rate of these DNA methylation change sites. By implementing this method at the time of lymphoma diagnosis or before the start of chemotherapy after diagnosis, it is possible to identify a group with a good prognosis for chemotherapy at the time of diagnosis.

[0018] In the prognosis prediction method according to the present invention, the lymphoma to be predicted for prognosis may be any disease in which lymphocytes are tumorized, and is not particularly limited. Examples of canine lymphoma include multicentric lymphoma in which lymph nodes on the body surface are enlarged, gastrointestinal lymphoma in which the digestive tract and associated lymph nodes are enlarged, thymic (mediastinal) lymphoma in which the thymus and mediastinum are enlarged, cutaneous lymphoma in which the skin and oral mucosa are enlarged, and extranodal lymphoma in which other biological sites are enlarged. In the prognosis prediction method according to the present invention, multicentric lymphoma, which accounts for 80% of canine lymphoma, is preferred as the lymphoma to be predicted for prognosis. Multicentric lymphoma mainly causes enlargement of the mandibular lymph nodes, superficial cervical lymph nodes, axillary lymph nodes, and popliteal lymph nodes. However, the multicentric lymphoma to be predicted in the prognosis prediction method according to the present invention may be one in which any lymph node is enlarged. Further, the lymphoma to be predicted in the prognosis prediction method according to the present invention may be primary or metastatic.

[0019] In the prognosis prediction method according to the present invention, the malignancy of the lymphoma to be predicted for prognosis is not particularly limited. For example, the prognosis prediction method according to the present invention may predict the prognosis of chemotherapy for a test dog suffering from lymphoma with a substage of a (a state in which no clinical signs are observed), or may predict the prognosis of chemotherapy for a test dog suffering from lymphoma with a substage of b (a state in which obvious clinical signs are observed).

[0020] In the prognosis prediction method according to the present invention, the chemotherapy to be predicted for prognosis is not particularly limited as long as it is chemotherapy performed on a dog suffering from lymphoma. In the present invention, since it is a standard chemotherapy for dogs suffering from lymphoma, it is preferable to predict the prognosis of CHOP therapy. CHOP therapy can be performed by a conventional method (<on line> https: / / www.cancer.gov / publications / dictionaries / cancer-terms / def / chop-regimen).

[0021] In the prognosis prediction method according to the present invention, the dog breed of the dog to be predicted (test dog) is not particularly limited. Examples of the dog breed of the test dog include American Cocker Spaniel, Welsh Corgi Pembroke, Airedale Terrier, Cavalier King Charles Spaniel, Golden Retriever, Shih Tzu, German Shepherd, Jack Russell Terrier, Standard Poodle, Spitz, Chinese Crested Dog, Chihuahua, Toy Poodle, Bernese Mountain Dog, Basenji, Beagle, Bull Terrier, French Bulldog, Boxer, Border Collie, Pomeranian, Mastiff, Maltese, Miniature Schnauzer, Miniature Dachshund, Miniature Pinscher, Yorkshire Terrier, Labrador Retriever, Wire Haired Fox Terrier, Shiba, etc. Also, a mixed breed may be used. As the test dog in the prognosis prediction method according to the present invention, Golden Retriever, Boxer, etc., which are dog breeds prone to lymphoma, are preferred.

[0022] In the measurement step, the methylation rate of one or more CpG sites selected from the group consisting of Chr4:29559893 and Chr1:14232692 in the DNA recovered from the biological sample collected from the test dog is measured. That is, in the present invention, the methylation rate of at least one CpG site of Chr4:29559893 and Chr1:14232692 is used as a biomarker for the prognosis of chemotherapy in dogs with lymphoma. The base sequences of each CpG site are shown in Table 2. In the base sequences in the table, the underlined cytosine is the target CpG site.

[0023]

Table 2

[0024] Both Chr4:29559893 and Chr1:14232692 are among the CpG sites in genomic DNA, and there was a significant difference in the methylation rate between the group of dogs with good prognosis after chemotherapy and the group of dogs with poor prognosis after chemotherapy among the dogs suffering from lymphoma and receiving chemotherapy.

[0025] The biological sample used in the prognosis prediction method according to the present invention is a biological sample collected from a test dog, and is not particularly limited as long as it contains the genomic DNA of the test dog, and may be lymph fluid, blood, plasma, serum, tear fluid, saliva, etc., or may be a tissue piece collected from other tissues such as the gastrointestinal mucosa and the liver. As the biological sample used in the prognosis prediction method according to the present invention, lymph fluid is preferable because it more strongly reflects the state of lymphoma, and lymph fluid collected from an enlarged lymph node is more preferable. In addition, from the viewpoint of being minimally invasive and reducing the burden on the test dog, blood, plasma, serum, tear fluid, saliva, etc. are also preferable. When collecting a biological sample such as the above-mentioned lymph fluid or a tissue piece, it may be collected using a collecting tool corresponding to each biological sample.

[0026] Further, the biological sample only needs to be in a state where DNA can be extracted, and may be subjected to various pretreatments. For example, it may be formalin-fixed paraffin-embedded (FFPE) tissue. Extraction of DNA from a biological sample can be performed by a conventional method, and various commercially available DNA extraction and purification kits can also be used.

[0027] As a method for measuring the methylation rate of a CpG site, it is not particularly limited as long as it is a method capable of distinguishing and quantifying methylated cytosine bases and unmethylated cytosine bases for a specific CpG site. The methylation rate of a CpG site can be measured by directly using a method known in the art or appropriately modifying it as necessary. Examples of the method for measuring the methylation rate of a CpG site include the bisulfite sequencing method and methylation-specific PCR (MSP).

[0028] In the prognosis prediction method according to the present invention, as a prediction step, based on the methylation rate measured in the measurement step and a preset reference value, the prognosis after chemotherapy of a dog suffering from the lymphoma is predicted. The reference value is a value for discriminating between a dog with a good prognosis after chemotherapy and a dog with a poor prognosis after chemotherapy, which are respectively set for the methylation rates of each CpG site of Chr4:29559893 and Chr1:14232692. A dog with a methylation rate of each CpG site below the reference value may be referred to as a hypomethylated group, and a dog with a methylation rate exceeding the reference value may be referred to as a hypermethylated group.

[0029] In the prognosis prediction method according to the present invention, in the prediction step, when the methylation rate of Chr4:29559893 is equal to or lower than a preset reference value, the test dog is determined to be in the hypomethylated group, and it is highly likely that the prognosis after receiving the chemotherapy is good. Similarly, in the prediction step, when the methylation rate of Chr1:14232692 is equal to or lower than a preset reference value, the test dog is determined to be in the hypomethylated group, and it is highly likely that the prognosis after receiving the chemotherapy is good.

[0030] The reference value of the methylation rate of each CpG site can be experimentally determined as a threshold value that can distinguish between two groups by comparing the methylation rate of a CpG site serving as a biomarker of a group that has at least once achieved remission and has a good prognosis and the methylation rate of the CpG site of a group that has never achieved remission and has a poor prognosis among dogs suffering from lymphoma and receiving chemotherapy.

[0031] For example, the reference value of the methylation rate of Chr4:29559893 can be set within the range of 25 to 55%. For example, when the reference value is 45%, when the methylation rate of Chr4:29559893 of the test dog is 45% or lower, it is determined that the prognosis is highly likely to be good when the test dog receives chemotherapy. When the methylation rate of Chr4:29559893 of the test dog exceeds 45%, it is determined that the prognosis is highly likely not to be good when the test dog receives chemotherapy.

[0032] For example, as the reference value of the methylation rate of Chr1:14232692, it can be set within the range of 5 to 30%. For example, when the reference value is 10%, if the methylation rate of Chr1:14232692 of the test dog is 10% or less, when the test dog undergoes chemotherapy, it is highly likely to be determined that the prognosis is good. When the methylation rate of Chr1:14232692 of the test dog exceeds 10%, when the test dog undergoes chemotherapy, it is highly likely to be determined that the prognosis is not good.

[0033] In the prediction step of the prognosis prediction method according to the present invention, regarding the prognosis of chemotherapy for a test dog, it may be predicted only from the methylation rate of Chr4:29559893, or it may be predicted only from the methylation rate of Chr1:14232692. However, predicting based on both the methylation rate of Chr4:29559893 and the methylation rate of Chr1:14232692 enables a more reliable prediction. For example, when the methylation rate of Chr4:29559893 exceeds the reference value and the methylation rate of Chr1:14232692 also exceeds the reference value, it is predicted that the prognosis of the test dog will be poor even if chemotherapy is performed. Also, when the methylation rate of Chr4:29559893 is below the reference value and the methylation rate of Chr1:14232692 is also below the reference value, it is predicted that the prognosis of the test dog will be good even if chemotherapy is performed.

[0034] The prognosis prediction method according to the present invention can provide important information for assisting a veterinarian in determining whether to perform chemotherapy on a dog suffering from lymphoma. Therefore, it is preferable to perform the prognosis prediction method according to the present invention on a dog suffering from lymphoma before performing chemotherapy. By performing chemotherapy on a test dog predicted to have a good prognosis by the prognosis prediction method according to the present invention and not performing chemotherapy on a test dog predicted to have a poor prognosis, the treatment efficiency can be further enhanced.

Example

[0035] Next, examples and the like will be shown to explain the present invention in more detail, but the present invention is not limited thereto.

[0036] [Example 1] Using Canine DREAM (Non-Patent Document 8), a genome-wide analysis method for DNA methylation established by the inventor of the present invention, an analysis of a comprehensive and large number (about 100,000) of CpG sequences in the canine genome, including not only promoter regions but also exons, introns within genes, and CpG sites in the extragenic region, was performed on 24 cases of canine multicentric lymphoma. As a result, two CpG sequences capable of discriminating between the good prognosis group and the poor prognosis group were identified (Test cohort).

[0037] (1) Test dogs Twenty-four cases of naturally occurring canine multicentric high-grade lymphoma (MHGL) were used as test dogs. MHGL was diagnosed by a pathology specialist based on the findings of cytological examination (high mitotic rate and medium to large cells), and graded according to the WHO domestic animal lymphoma clinical grading system. All cases were treated with at least one cycle of CHOP chemotherapy.

[0038] (2) Extraction of genomic DNA Genomic DNA was extracted from the cytological examination slides of each case using the DNA extraction kit "DNeasy Blood & Tissue Kit" (manufactured by Qiagen).

[0039] (3) Canine DREAM method Genome-wide DNA methylation was analyzed by next-generation sequencing. Genomic DNA (2 μg) extracted from the sample was mixed with 2 pg of artificial calibrator (methylation levels 0, 25, 50, 75, 100%). The resulting mixture was treated with 100 U of SmaI enzyme at 25 °C for 3 hours for cleavage, and then reacted with 50 U of XmaI enzyme at 37 °C for 16 hours. The cleaved DNA was purified with magnetic beads "Agencourt AMPure XP" (manufactured by Beckman Coulter). To the 3' overhanging ends of the DNA cleaved by XmaI, dCTP, dGTP, and dATP were added at 0.4 mM each, and 3'-dA ends were added to the restriction fragments with 3'-5' exonuclease-deficient Klenow DNA polymerase (manufactured by New England Biolabs).

[0040] Subsequently, the Illumina Paired-end sequencing adapter was ligated with T4 DNA ligase (manufactured by New England Biolabs). The size of the ligation mix was subjected to Dual-SPRI size selection using Agencourt AMPure XP to extract DNA fragments of 250 to 450 base pairs (bp). The purified DNA was amplified with Illumina pair-end PCR primers and KAPA Hifi Hotstart Ready Mix (Kapa Biosystems) for 11 cycles. The amplified sequencing library was purified with the magnetic beads and sequenced on an Illumina HiSeq 2000 (manufactured by Illumina). The sequenced reads were mapped to the SmaI / XmaI sites of the dog genome (canFam3.1), and the signatures of methylated and unmethylated CpGs at the SmaI / XmaI sites were calculated. Subsequently, the methylation frequency of individual SmaI / XmaI sites was calculated. The methylation ratio was obtained by dividing the number of tags starting with CCGGG by the total number of tags mapped to the SmaI / XmaI sites.

[0041] The methylation levels calculated by DREAM were corrected based on the standard spikes. For each standard, the log ratios ln(m / u) and ln(sm / u) were calculated, where m / u is the predicted methylation to unmethylation ratio, and sm and u are the observed methylation and unmethylation ratios, respectively. The log ratio of "predicted" to "observed" was calculated by subtracting the latter from the former for each standard. The correction factor (c) was calculated as the antilog of the mean of log([predicted] - [observed]), and the corrected methylation value for each individual CpG site was calculated as 100% × [c×sm / (c×[sm + u])]. The methylation levels of a single SmaI / XmaI site were analyzed using at least 20 sequencing reads.

[0042] (4) Statistical analysis In genome-wide DNA methylation analysis, the DNA methylation levels between groups were analyzed by t-test. For multiple testing correction, the Benjamini-Hochberg method was used to confirm methylation differences at FDR 10%. The survival curves of the Test cohort were plotted by the Kaplan-Meier method. The average survival periods of case groups with different DNA methylation patterns at CpG sites in MHGL cases were compared. The significant differences between survival curves were compared by the log rank test. Data of cases of death not related to MHGL, cases that could not be followed up, or living cases were truncated and excluded.

[0043] As a control, the CpG methylation rates of genomic DNA from three types of normal peripheral blood were also measured in the same way.

[0044] (6) Results In this experiment, 40,128 CpG sites where data were commonly obtained in all samples of 24 cases were used for analysis. When clustering analysis was performed using 16,992 CpG sites in CpG islands, it was found that the cases were divided into multiple groups.

[0045] For these multiple groups, when survival curves and remission periods were plotted, significant differences were obtained among the groups. From these results, it was found that a group with a favorable prognosis for chemotherapy could be extracted based on the genome-wide DNA methylation pattern of CpG islands.

[0046] Next, in order to search for CpG sites that define this group with a favorable prognosis, volcano plot analysis was performed, and a direct comparison was made between the group with a favorable prognosis and the group with an unfavorable prognosis. In the volcano plot analysis, in order to increase its statistical power, the means of 9 cases in the group with a favorable prognosis and 15 cases in the other group with an unfavorable prognosis were taken for 38,693 CpG island sites where comparative analysis was commonly possible in 20 or more cases, which was 80% or more of 24 cases, and the difference and p-value were shown.

[0047] The number of CpG sites showing a methylation difference of 20% or more and a statistical significance of 0.01 or less was identified as 520 CpG sites with high methylation in the group with an unfavorable prognosis and 414 CpG sites with high methylation in the group with a favorable prognosis, for a total of 934 CpG sites. Among these, when calculating the number of related genes from CpG sites where a gene was present in the vicinity and was considered to be present in its promoter region, the CpG sites with high methylation in the group with an unfavorable prognosis were 45 genes, while the CpG sites showing the opposite tendency were 5 genes, indicating that there were more genes with high methylation in the group with an unfavorable prognosis.

[0048] Next, analysis of these 45 genes and 45 CpG sites was performed. Figure 1 is a plot with the methylation rate (%) of these 45 CpG sites in normal blood on the X-axis and the methylation rate (%) of the group with a favorable prognosis and the group with an unfavorable prognosis on the Y-axis. As shown in Figure 1, most of these CpG sites had a low methylation rate of 20% or less in normal blood, and most were also 20% or less in the group with a favorable prognosis, but only the group with an unfavorable prognosis exceeded 20%, and some showed a methylation rate of 40 - 60%. Also, by Gene ontology analysis, it was found that many of these 45 genes were important genes for signal transduction and differentiation and maturation such as BMP and WNT (Table 3).

[0049]

Table 3

[0050] [Example 2] By performing DNA methylation analysis of the candidate CpG sites identified as prognostic markers for chemotherapy in Example 1, it was examined whether the prognostic status in a new case group could be predicted. Specifically, for 14 new DLBCL cases different from those in Example 1 and the cases, this time bisulfite pyrosequencing, which is highly reliable for local analysis and is cheaper, faster, and simpler to analyze than DREAM, was used for the analysis. As the target CpG site, the CpG site (Chr4:29559893) ranked at the top in terms of both the difference and significant difference in methylation rates between the good-prognosis group and the poor-prognosis group by the DREAM analysis in Example 1 was selected.

[0051] Figure 2A shows the methylation rate (%) obtained by bisulfite pyrosequencing of Chr4:29559893 in 14 cases. When these 14 cases were classified using 43%, which is the average methylation rate + SD value of this CpG site in the good-prognosis group in the DREAM analysis, as the boundary value, they were divided into 8 cases in the hypermethylation group and 6 cases in the hypomethylation group. Subsequently, survival curves were drawn for the hypermethylation group and the hypomethylation group in Figure 2B, and progression-free survival curves were drawn in Figure 2C. As a result, it was reconfirmed that both the survival period and the progression-free survival period were correlated with hypomethylation in the good-prognosis group, and it was confirmed that it was possible to identify CpG sites related to prognosis by DREAM analysis.

[0052] [Example 3] From the genome-wide DNA methylation analysis by the canine DREAM method of Example 1, for the two CpG sites at the top ranks in both the difference and significance of methylation rates between the favorable prognosis group and the poor prognosis group, namely Chr4:29559893 and Chr1:14232692, as a confirmation experiment, DNA was extracted from cell specimen slides of 96 cases diagnosed with canine multicentric lymphoma, and the DNA methylation level was quantified using the bisulfite pyrosequencing method (bisulfite treatment and pyrosequencing method) targeting the CpG sequences (Validation cohort).

[0053] (1) Test dogs 96 cases of MHGL different from Example 1 were used as test dogs. MHGL was diagnosed by a pathology specialist based on the findings of cytological examination (high mitotic rate and medium to large cells), and graded according to the WHO domestic animal lymphoma clinical grading system. All cases were treated with at least one cycle of CHOP chemotherapy.

[0054] Among the 96 cases, 67 cases (70%) were in sub-stage a (no clinical signs), and 29 cases (30%) were in sub-stage b (obvious clinical signs). Table 4 shows the dog breeds of the 96 test dogs.

[0055]

Table 4

[0056] (2) Extraction of genomic DNA The extraction of genomic DNA from the cytological examination slides of each case was performed in the same manner as in Example 1.

[0057] (3) Bisulfite treatment and pyrosequencing The DNA methylation rates of the identified target CpG sites (Chr4:29559893 and Chr1:14232692) were quantified and evaluated by bisulfite pyrosequencing. Genomic DNA was subjected to bisulfite treatment using the "EZ DNA Methylation-Lightning Kit" (manufactured by Zymo Research). Next, a region containing the CpG site was amplified by two-step PCR using primers specific to the target CpG site. The DNA methylation rate of the CpG site was measured by pyrosequencing to determine the percentage of DNA methylation level (CpG methylation rate) using the Pyro-Gold Reagent Kit of the PSQ24 system (manufactured by QIAGEN).

[0058] (4) Statistical analysis In genome-wide DNA methylation analysis, the DNA methylation levels between groups were analyzed by t-test. Correction for multiple testing was performed using the Benjamini-Hochberg method to confirm methylation differences at FDR 10%. The survival curves of the Validation cohort were plotted by the Kaplan-Meier method. In the MHGL cases of the Validation cohort, the mean survival periods of case groups with different DNA methylation patterns of the identified target CpG sites were compared. The significant differences between the survival curves were compared by the log rank test. Data of cases of death not related to MHGL, cases that could not be followed up, or living cases were truncated and excluded.

[0059] (5) Results Figure 3A shows the methylation rates (%) of Chr4:29559893 and Chr1:14232692 in 96 cases. As shown in Figure 3A, the DNA methylation levels of both CpG sites showed heterogeneity (showing various DNA methylation levels depending on the cases). Also, the success rate regarding the measurement of the DNA methylation levels of both CpG sites was 97% or more.

[0060] From the results in Fig. 3A, for convenience, the reference values for dividing the high methylation group and the low methylation group were set as 45% for Chr4:29559893 and 10% for Chr1:14232692. For the high methylation group and the low methylation group, survival curves were drawn in Fig. 3B. As a result, it was shown that for both CpG sites, the prognosis of the high methylation group was worse than that of the low methylation group. In other words, the prognosis of the low methylation group was better. These results were also consistent with the results in Examples 1 and 2.

[0061] Also, using the results in Fig. 3A, prognosis prediction was performed by combining the methylation rates of both Chr4:29559893 and Chr1:14232692. Among 96 cases of canine DLBCL, there were 17 cases in the group where both Chr4:29559893 and Chr1:14232692 were in the low methylation group (low methylation group 1), 74 cases in the group where either Chr4:29559893 or Chr1:14232692 was in the low methylation group and the other was in the high methylation group (low methylation group 2), and 31 cases in the group where both Chr4:29559893 and Chr1:14232692 were in the high methylation group (high methylation group). Among the groups where at least one of Chr4:29559893 and Chr1:14232692 was in the low methylation group, low methylation group 1 where both were in the low methylation group accounted for 18%, and 82% was low methylation group 2 where only one of them was in the low methylation group.

[0062] Fig. 4A shows the survival curves of low methylation group 1, low methylation group 2, and high methylation group. Low methylation group 1 showed a significantly better prognosis compared to low methylation group 2 and high methylation group. The 2-year survival rate of low methylation group 1 was 50%, and a significantly better prognosis was expected compared to the current standard prognosis.

[0063] Fig. 4B shows the proportion (%) of the cases in remission and the cases not in remission (non-remission) in low methylation group 1 and low methylation group 2 among the total number of cases (96 cases). As a result, in low methylation group 1, 14 cases (15%) out of 17 cases (18%) were in remission, showing a high remission rate of 82%.

[0064] This result suggests the usefulness as a biomarker for clinically applicable risk assessment, prognosis determination, prediction of treatment responsiveness, etc. At this time, by using the most reliable pyrosequencing method in the DNA methylation analysis of each site, the DNA methylation analysis of specific regions could be applied to a large number of case samples. Therefore, it is possible to examine disease-specific DNA methylation candidate regions at a reduced cost and to focus on verifying the universality for a large number of cases.

Claims

1. A method for predicting the prognosis of a dog suffering from lymphoma after chemotherapy, comprising: a measuring step of measuring the methylation rate of one or more CpG sites selected from the group consisting of Chr4:29559893 and Chr1:14232692 in DNA recovered from a biological sample collected from a dog to be predicted; a predicting step of predicting the prognosis of the dog suffering from lymphoma after chemotherapy based on the methylation rate measured in the measuring step and a preset reference value; and having wherein the reference value is a value for discriminating between dogs with good prognosis after chemotherapy and dogs with poor prognosis after chemotherapy, each set for the methylation rate of each CpG site, A method for predicting the prognosis of a dog suffering from lymphoma after chemotherapy.

2. The prognosis prediction method according to claim 1, wherein the lymphoma is of the multicentric type.

3. The prognosis prediction method according to claim 2, wherein the sub-stage of the lymphoma is a or b.

4. The prognosis prediction method according to claim 1, wherein the chemotherapy is CHOP therapy.

5. In the predicting step, when the methylation rate of Chr4:29559893 is equal to or lower than a preset reference value, it is determined that the dog suffering from lymphoma has a high possibility of having a good prognosis after receiving the chemotherapy. The prognosis prediction method according to claim 1.

6. The prognosis prediction method according to claim 5, wherein the preset reference value is 45%.

7. In the predicting step, when the methylation rate of Chr1:14232692 is equal to or lower than a preset reference value, it is determined that the dog suffering from lymphoma has a high possibility of having a good prognosis after receiving the chemotherapy. The prognosis prediction method according to claim 1.

8. The prognosis prediction method according to claim 7, wherein the preset reference value is 10%.

9. In the measuring step, the methylation rate of the CpG site is measured by the bisulfite pyrosequencing method. The prognosis prediction method according to claim 1.

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