WO2023090491A1 - Composition for diagnosis or prognosis prediction of philadelphia chromosome-like acute lymphoblastic leukemia - Google Patents

Abstract

A composition for the diagnosis or prognosis prediction of philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL), according to the present invention, can effectively measure the expression level of a fusion transcript consisting of ETV6-ABL1, NUP214-ABL1, EBF1-PDGFRB, P2RY8-CRLF2, EBF1-JAK2, ETV6-JAK2, and BCR-JAK2 in a patient group administered with induction or subsequent chemotherapy. Thus, the composition can accurately diagnose the difference in expression from subtypes showing expression patterns similar to Ph-like ALL, by comparing expression patterns in Ph-like ALL, Ph-positive ALL, B-other non-risk ALL and B-other standard-risk ALL patient groups. Therefore, since it has been confirmed that the composition is effective in the diagnosis and prognosis prediction of Ph-like ALL in patient groups, the composition can be effectively used in related industries.

Description

Composition for diagnosis or prognosis of Philadelphia chromosome-like acute lymphoblastic leukemia

The present invention relates to a composition for diagnosis or prognosis of Philadelphia chromosome-like acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a hematological cancer that mainly affects children, adolescents and young adults. Compared to pediatric BCP-ALL, the long-term disease-free survival (DFS) of adult BCP-ALL is 40% to 50%, resulting in a poorer overall treatment prognosis. The reasons for the negative prognosis of treatment in adults compared to children are the high frequency of genetic abnormalities with poor prognosis, such as BCR-ABL1 translocation, KMT2A rearrangement, or complex karyotypes, and resistance to chemotherapy. Based on the genome-wide analysis, there are various types of BCP-ALL, one of which has a gene expression profile similar to that of Philadelphia chromosome positive ALL (Ph-positive ALL), but BCR-ALL A new type of high-risk subtype called BCR-ABL1- like or Philadelphia chromosome-like ALL (Ph-like ALL) lacking the ABL1 fusion protein has been identified. . Ph-like ALL is known to have a low incidence, and CRLF2 rearrangements and mutations, ETV6-RUNX1 , TCF3-PBX1 and ABL-class rearrangements, JAK2 or EPOR rearrangements, JAK-STAT signaling and RAS signaling activating mutations and Genetic abnormalities, including uncommon kinase variants, have been discovered. Some genetic abnormalities can be targeted for treatment by tyrosine kinase inhibitors (TKIs), but research on Ph-like ALL is still insufficient, and in particular, research on Asians is lacking.

Ph-like ALL has a negative treatment prognosis at any age, and in a study of patients with genetic abnormalities related to Ph-like ALL, minimal residual disease after induction chemotherapy was found compared to other types of BCP-ALL. , MRD) was found to be higher, which can be interpreted as having a negative prognosis in conventional chemotherapy.

Here, the question arises whether allogeneic hematopoietic cell transplantation (HCT) contributes to improving the negative prognosis of adult Ph-like ALL against chemotherapy. The role of allogeneic HCT is unclear. Therefore, there is a need for genetic research to accurately predict treatment responsiveness and prognosis of Ph-like ALL.

An object of the present invention is to diagnose Ph-like ALL disease, including an agent for measuring the expression level of a fusion gene or protein related to Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) And to provide a composition for predicting prognosis.

Another object of the present invention is to provide a kit for diagnosing and predicting prognosis of Ph-like ALL, including the above composition.

Another object of the present invention is to separate a biological sample;

Measuring the expression level of a Ph-like ALL-related gene transcript or a peptide or protein expressed therefrom from the sample; and

It is to provide an information providing method for diagnosis and prognosis of Ph-like ALL, including the step of confirming the expression pattern of the fusion transcript in the expressed gene transcript.

In order to achieve the above object, the present invention, including a preparation for measuring the expression level of a fusion gene or protein related to Philadelphia chromosome-like acute lymphoblastic leukemia (Philadelphia chromosome-like acute lymphoblastic leukemia, Ph-like ALL), It provides a composition for diagnosing and predicting prognosis of -like ALL diseases.

The present invention provides a kit for diagnosing and predicting prognosis of Ph-like ALL, including the above composition.

The present invention comprises the steps of separating a biological sample;

Measuring the expression level of a Ph-like ALL-related gene transcript or a peptide or protein expressed therefrom from the sample; and

In the expressed gene transcript, confirming the expression pattern of the fusion transcript; It provides an information providing method for diagnosis and prognosis prediction of Ph-like ALL, including.

The composition for diagnosis or prognosis of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) of the present invention is ETV6-ABL1 , NUP214-ABL1 , EBF1-PDGFRB , P2RY8-CRLF2 , Expression levels of fusion transcripts consisting of EBF1-JAK2 , ETV6-JAK2 and BCR-JAK2 can be effectively measured. Therefore, by comparing the expression patterns of each patient group for Ph-like ALL, Ph-positive ALL, B-other high-risk ALL, and B-other standard-risk ALL, it was possible to accurately differentiate between subtypes showing similar expression patterns to Ph-like ALL. Since it can be diagnosed, it can be confirmed that it is effective in diagnosing and predicting the prognosis of Ph-like ALL for a patient group, and can be usefully used in related industries.

Figure 1 is a schematic diagram of the classification of the patient group of the present invention.

Figure 2 is a schematic diagram of the treatment schedule for the patient group of the present invention.

Figure 3 is a diagram comparing treatment prognosis according to disease subtypes in terms of overall survival (OS), complete remission (CR), and disease-free survival (DFS) in the present invention.

A: OS for all patient groups

B: OS of patients who received allogeneic hematopoietic cell transplantation (HCT) in CR

C: DFS for all patients,

D: DFS for patients receiving allogeneic HCT in CR

E: cumulative incidence of relapse for all patients

F: Cumulative incidence of relapse for patients who received allogeneic HCT in CR.

Figure 4 confirms the expression pattern of the fusion transcript in Ph-like ALL patients by multiplex RT-PCR using the primer set of the present invention.

Figure 5 is the result of comparing the expression of the fusion transcript in Ph-like ALL of the present invention with other subgroups by confirming multiplex RT-PCR.

6 to 11 are diagrams confirming kinase fusions targeted by multiplex RT-PCR in Ph-like ALL and identified by next-generation sequencing.

12 is a diagram comparing the prognosis of treatment according to the Ph-like ALL subtypes of the present invention by overall survival (OS), complete remission (CR), and disease-free survival (DFS).

A: OS for all patient groups

B: OS in the group of patients who received allogeneic HCT in CR

C: cumulative incidence of relapse for all patients

D: Cumulative incidence of relapse for patients who received allogeneic HCT in CR.

13 is a diagram comparing gene expression levels of B-other ALL and Ph-like ALL groups without genetic abnormalities in the present invention.

A: Differences between the Ph-like ALL patient group and other B-other ALL patient groups

B: Differences in subtypes of Ph-like ALL patient groups

The present invention diagnoses and predicts prognosis of Ph-like ALL disease, including an agent for measuring the expression level of a gene or protein related to Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) A composition for

According to one embodiment of the present invention, the measurement may be performed from a biological sample isolated from a patient, and the biological sample is tissue, cells, whole blood, serum, plasma, saliva, sputum, spinal fluid, or urine isolated from the patient. It may be selected from the group consisting of, preferably spinal fluid or blood.

According to one embodiment of the present invention, the patient is selected from the group selected from hyper-fractionated cyclophosphamide, vincristine, daunorubicin and dexamethasone for induction chemotherapy. It may be a patient who has received one or more selected anticancer drugs.

According to one embodiment of the present invention, the patient is treated with high-dose cytarabine, mitoxantrone, imatinib, dasatinib, methotrexate as follow-up chemotherapy , cytarabine (cytarabine) and hydrocortisone (hydrocortisone) may be a patient who has received one or more selected anticancer drugs from the selected group.

According to one embodiment of the present invention, the Ph-like ALL-related gene may be a fusion transcript.

According to one embodiment of the present invention, the fusion transcript may be selected from the group consisting of ETV6-ABL1 , NUP214-ABL1 , EBF1-PDGFRB , P2RY8-CRLF2 , EBF1-JAK2 , ETV6-JAK2 and BCR-JAK2 .

According to one embodiment of the present invention, the agent for measuring the expression level of the gene or protein is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the protein or peptide fragment ( aptamer).

According to one embodiment of the present invention, the agent for measuring the expression level is expressed by a nucleic acid primer binding to a nucleic acid sequence of a Ph-like ALL disease-related gene or a sequence corresponding thereto, or a Ph-like ALL-related gene It may include an antibody, aptamer, or probe that specifically binds to a transcript or protein, and is preferably a primer.

According to one embodiment of the present invention, the agent for measuring the expression level is a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 A primer pair consisting of SEQ ID NO: 7 and SEQ ID NO: 8, a primer pair consisting of SEQ ID NO: 9 and SEQ ID NO: 10, a primer pair consisting of SEQ ID NO: 11 and SEQ ID NO: 12, a primer consisting of SEQ ID NO: 13 and SEQ ID NO: 14 It may be one or more primer pairs selected from the group consisting of a pair and a primer pair consisting of SEQ ID NO: 15 and SEQ ID NO: 16.

According to one embodiment of the present invention, the agent for measuring the expression level is a probe consisting of SEQ ID NO: 17, a probe consisting of SEQ ID NO: 18, a probe consisting of SEQ ID NO: 19, a probe consisting of SEQ ID NO: 20, and a probe consisting of SEQ ID NO: 21 It may further include at least one probe selected from the group consisting of a probe consisting of SEQ ID NO: 22, a probe consisting of SEQ ID NO: 23, and a probe consisting of SEQ ID NO: 24.

Preparations for measuring the expression level of the present invention are primers or probes that specifically bind to fusion transcripts ETV6-ABL1 , NUP214-ABL1 , EBF1-PDGFRB , P2RY8-CRLF2 , EBF1-JAK2 , ETV6-JAK2 and BCR-JAK2 can include In addition, the composition may further include a reaction amplification mixture, wherein the reaction amplification mixture contains reagents necessary for performing the amplification reaction, a thermostable DNA polymerase, deoxynucleotide, nuclease-free sterile water, and divalent metal cations. It refers to a solution and the like, and may preferably include a reaction buffer, deoxynucleotide, and DNA polymerase.

A reporter such as a fluorescent substance may be labeled at the end of the probe.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a base pair with a template of a complementary nucleic acid and prevents strand copying of the nucleic acid template. A short nucleic acid sequence that serves as a starting point for Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

In the design of the primers, various restrictions are followed, such as the A, G, C, and T content ratio of the primers, prevention of primer dimer formation, and prohibition of repeating the same base sequence three or more times. (template) Conditions such as the amount of DNA, concentration of primer, concentration of dNTP, concentration of Mg2+, reaction temperature, and reaction time should be appropriate.

The above primers can incorporate additional features that do not change the basic properties. That is, nucleic acid sequences can be modified using a number of means known in the art. Examples of such modifications are methylation, capping, substitution of nucleotides with one or more homologues, and uncharged linkages such as phosphonates, phosphotriesters, phosphoroamidates or carbamates, or phosphorothioates or phosphorodithioates. Transformation of nucleotides into charged linkages such as Nucleic acids may also contain one or more nucleic acids, such as nucleases, toxins, antibodies, signal peptides, proteins such as poly-L-lysine, intercalating agents such as acridine or psoralen, chelating agents and alkylating agents such as metals, radioactive metals, and iron oxidizing metals. It may have additional covalently linked moieties.

In addition, the primer sequence of the present invention can be modified using a label that can directly or indirectly provide a detectable signal. The primer may include a label that can be detected using spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means. Useful labels include 32P, fluorescent dyes, electron dense reagents, enzymes (commonly used in ELISA), biotin or haptens, and proteins for which antiserum or monoclonal antibodies are available.

The primers of the present invention are suitable for cloning of appropriate sequences, digestion with restriction enzymes, and phosphotriester methods such as Narang (1979, Meth, Enzymol. 68:90-99), diethylphosphoramidase such as Beaucage. (1981, Tetrahedron Lett. 22: 1859-1862), and any other well-known method including direct chemical synthesis, such as the solid support method of US Pat. No. 4,458,066.

The method for diagnosing the prognosis of patients with Ph-like ALL of the present invention comprises a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 2 in a biological sample isolated from the patient. A primer pair consisting of SEQ ID NO: 7 and SEQ ID NO: 8, a primer pair consisting of SEQ ID NO: 9 and SEQ ID NO: 10, a primer pair consisting of SEQ ID NO: 11 and SEQ ID NO: 12, a primer pair consisting of SEQ ID NO: 13 and SEQ ID NO: 14 Fusion transcripts comprising ETV6-ABL1 , NUP214-ABL1 , EBF1-PDGFRB , P2RY8-CRLF2 , EBF1-JAK2 , ETV6-JAK2 and BCR-JAK2 containing the primer pair and the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16 It refers to a method for predicting the diagnosis and prognosis of patients with Ph-like ALL by confirming the presence of . More specifically , ETV6-ABL1 , NUP214-ABL1 , EBF1-PDGFRB , P2RY8-CRLF2 , EBF1-JAK2 , ETV6-JAK2 by performing an amplification reaction using primers for predicting the diagnosis and prognosis of Ph-like ALL patients of the present invention And it may be a method capable of specifically detecting a fusion transcript containing BCR-JAK2 .

The "amplification reaction" means a reaction to amplify a nucleic acid molecule, and is a polymerase chain reaction (PCR), multiplex real time-polymerase chain reaction (multiplex RT-PCR) , reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP) 439,182), transcription-mediated amplification (TMA) (WO88/10315), self sustained sequence replication (WO90/06995), selective amplification of target polynucleotide sequence polynucleotide sequences), consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), nucleic acid sequence-based amplification It may be an amplification method such as sequence based amplification (NASBA), strand displacement amplification, and loop-mediated isothermal amplification (LAMP), but is not limited thereto.

In the above amplification method, PCR may be performed using a PCR reaction mixture containing various components known in the art required for PCR reaction. The PCR reaction mixture may contain appropriate amounts of DNA polymerase, dNTP, PCR buffer, and water (dH2O) in addition to genomic DNA isolated from a biological sample isolated from a patient to be analyzed and the primer pair provided in the present invention. there is.

The PCR buffer is not limited thereto, but may include one or more of Tris-HCl, MgCl2, and KCl. At this time, the concentration of MgCl2 greatly affects the specificity and quantity of amplification, and may be preferably used in the range of 1.5 to 2.5 mM. In general, when Mg ²⁺ is excessive, non-specific PCR amplification products increase, and when Mg ²⁺ is insufficient, the yield of PCR products decreases. An appropriate amount of Triton X-100 may be further included in the PCR buffer.

In addition, in performing the PCR, the PCR reaction may be performed according to a known method. After pre-denaturation of the template DNA at 94 to 95 ° C., but not limited thereto, denaturation; annealing; And after going through a cycle of extension (extension), it can be carried out under general PCR reaction conditions that finally elongate at 70 to 75 ° C, for example, 72 ° C. In the above, denaturation may be performed at 90 to 99°C, 92 to 97°C, or 94 to 95°C, and amplification may be performed at 70 to 75°C, 71 to 74°C, or 72°C. The temperature at the time of binding may vary depending on the type of primer, and may be, for example, 50 to 59°C, 52 to 57°C, or 55°C. The time and number of cycles of each step may be determined according to conditions generally practiced in the art. The optimal reaction conditions for performing PCR using the SSR primer pair according to an embodiment of the present invention are as follows: pre-denature the template DNA at 95 ° C for 5 minutes, then 95 ° C for 30 seconds; 30 seconds at 55°C; And after repeating 30 cycles of 1 minute at 72 ° C, the reaction is finally performed at 72 ° C for 30 minutes.

In the "measurement of expression level", the DNA of the PCR product can be separated by size according to a method well known in the art. For example, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the label material may be a material that emits fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the labeling substance is 6-FAM, NEN, VIC or PET. When the target sequence is amplified, PCR is performed by labeling 6-FAM, NEN, VIC, or PET at the 5'-end of the primer, and the target sequence can be labeled with a detectable fluorescent labeling material. In addition, the labeling material may include Cy-5 or Cy-3. In addition, when a radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution during PCR, the radioactive material is incorporated into the amplification product as the amplification product is synthesized, and the amplification product can be radioactively labeled. Primer sets used to amplify the target sequence are as described above.

According to one embodiment of the present invention, the biological sample isolated from the patient may be selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum, spinal fluid or urine isolated from the patient.

In addition, the present invention provides a kit for diagnosing and predicting prognosis of Ph-like ALL, including the above composition.

In addition, the present invention, separating the biological sample;

Measuring the expression level of a Ph-like ALL-related gene transcript or a peptide or protein expressed therefrom from the sample;

In the expressed gene transcript, confirming the expression pattern of the fusion transcript; and

It provides a method for diagnosing and predicting the prognosis of Ph-like ALL, including the step of confirming the expression pattern of the fusion transcript.

According to one embodiment of the present invention, the method for confirming the expression pattern of the fusion transcript is multiplex real time-polymerase chain reaction (multiplex real time-polymerase chain reaction, multiplex RT-PCR), reverse transcriptase polymerization (RT- PCR), competitive RT-PCR, real time quantitative RT-PCR, quantitative RT-PCR, RNase protection method , it may be measured using Northern blotting or DNA chip technology, preferably multiplex real time-polymerase chain reaction (multiplex real time-polymerase chain reaction, multiplex RT-PCR) am.

According to one embodiment of the present invention, the step of measuring the expression level of the peptide or protein is Western blot (Western blot), ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorecence Activated Cell Sorter, FACS), or protein chip (protein chip) may be measured using a method selected from the group consisting of.

Hereinafter, the present invention will be described in more detail by examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1> Target patient group

Between December 2008 and March 2016, a cohort of 344 adults (median age 43 years, 15 to 65 years old) newly diagnosed with BCP-ALL who received the same front-line chemotherapy and had materials suitable for genomic analysis were included in this study. Recruited for diagnostic analysis of the invention. Among them, subjects who did not receive front-line chemotherapy or did not have analyzable RNA were excluded from the analysis. According to the WHO classification, bone marrow examination findings, immunophenotype, cytogenetic and molecular genetic diagnosis were used for analysis, and the patient groups were (1) Ph-like ALL, (2) Ph-positive ALL, and (3) B-other Non-risk ALL ( KMT2A rearrangement, hypodiploidy, and complex karyotype [5 chromosome abnormalities]) and (4) B-other standard risk ALL. All patients provided written informed consent approved by the Institutional Review Board of The Catholic University of Korea (KC17SESI0717) and performed in accordance with the Declaration of Helsinki. In addition, blood or bone marrow was collected from the patients and used for RNA analysis later. Ph-like ALL was classified by performing AMP-based targeted next-generation sequencing (NGS) on archived RNA.

As a result, 57 out of 344 patients had Ph-like ALL (16.6%), 197 patients had Ph-positive ALL (57.3%), and 90 patients had B-other ALL (26.1%, 63 patients: B-other standard). Risk ALL, 27 B-other non-risk ALL) were confirmed (FIG. 1). The baseline characteristics of the patients are shown in Table 1 below.

The Ph-like ALL patient group had a lower median age and lower white blood cell count at diagnosis than the Ph-positive ALL and B-other high-risk ALL patient groups, but there was no significant difference from B-other standard-risk ALL. In chemotherapy for Ph-like ALL, after the second cycle, the overall complete remission (CR) rate was 96.5%, similar to Ph-positive ALL (95.9%) and B-other ALL (92.1%), whereas B-other high-risk ALL showed the lowest 81.5%. Except for the B-other high-risk ALL patient group, the initial CR rate after the first induction cycle was relatively low in the patient group with Ph-like ALL. The actual allogeneic HCT progression rate in CR was 87.7% for Ph-like ALL, 84.3% for Ph-positive ALL, 71.4% for B-other standard risk ALL, and 70.4% for B-other high-risk ALL ( p=0.031).

<Example 2> Confirmation of chemotherapy and treatment prognosis

The treatment schedule is shown in FIG. 2 . In the patient group of Example 1, the induction chemotherapy was hyper-fractionated cyclophosphamide (300 mg/m2, 12 hours, 1 to 3 days), vincristine (1.4 mg/m2, maximum dose 2). mg/day, days 4 to 11), daunorubicin (45 mg/m2/day, days 4 to 11) and dexamethasone (40 mg/day, days 1 to 4 and days 11 to 14). received therapy. After the induction chemotherapy (every even cycle), high-dose cytarabine (2 g/m2, every 12 hours, 1-5 days) and mitoxantrone (12 mg/m2/day, 1-2 days) days) followed by chemotherapy (every odd cycle). In the Ph-positive ALL patient group, TKIs (imatinib or dasatinib) were added to the chemotherapy regimen. Central nervous system prophylaxis was performed by intrathecal administration of a triple agent (methorexate 12 mg, cytarabine 40 mg, and hydrocortisone 50 mg; total 6 times). The patient group received chemotherapy in a total of 4 cycles, based on donor availability in the case of a donor and based on patient tolerability in the absence of a donor. Patients with donors underwent hematopoietic cell transplantation (HCT) as quickly as possible under the same strategy for conditioning and prevention of graft-versus-host disease. Patients without suitable donors, especially those in the high-risk group, were offered cord blood transplantation.

As a result, after a median follow-up of 74.6 months, 169 of 344 patients (35 of 57 Ph-like ALL patients, 94 of 197 Ph-positive ALL patients, 32 of 63 B-other standard-risk ALL patients) , 8 of 27 B-other non-risk ALL patients) survived, and 165 of 169 patients had persistent CR. At the time of analysis, 175 patients had died (71 disease progression and 104 non-recurrence deaths). As a result, the 5-year DFS (53.6%) of Ph-like ALL patients was not significantly different from that of B-other standard-risk ALL (46.8%) patients, but compared with B-other high-risk ALL patient group (25.9%, P=0.014). When it was done, a significant difference was shown. Ph-like ALL (60.6%) patients had a significantly higher 5-year OS than the B-other non-risk ALL patient group (27.1%) (P=0.008), and significantly higher than the B-other standard-risk ALL patient group (53.1%). There was no difference. The 5-year cumulative relapse and non-recurrent mortality rates were 21.4% and 21.5% for Ph-like ALL, 32.2% and 19.5% for B-other standard-risk ALL, and 40.7% and 23.0% for B-other high-risk ALL, respectively. At this time, the P values were 0.125 and 0.971. When treatment results were individually analyzed for patients who received allogeneic HCT in CR, similar results were obtained for DFS, OS, and relapse rate (FIG. 3).

In addition, in multivariate analysis (Table 2), patients who did not receive allogeneic HCT in complete remission (CR) had long-term disease-free survival (DFS) (hazard ratio [HR], 6.540; 95 There was a higher risk of treatment failure in % confidence interval [CI], 2.586–16.540; P<0.001) and overall survival (OS) (HR, 4.123; 95% CI, 1.452–11.710; P = 0.007). We found that delayed CR was also associated with inferior DFS, (HR, 2.120; 95% CI, 1.160–3.873; P=0.014) and OS (HR, 2.088; 95% CI, 1.129–3.861; P = 0.019). Compared with the Ph-like ALL patient group, patients with B-other high-risk ALL had inferior DFS (HR, 2.101; 95% CI, 1.020–4.325; P=0.044) and OS (HR, 2.454; 95% CI, 1.168–50%). 5.158; P = 0.018), and there was no significant difference between Ph-like ALL and Ph-positive ALL or B-other standard-risk ALL patient groups.

<Example 3> AMP (Anchored Multiplex PCR)-based targeted next-generation sequencing

Ph-like ALL was screened using AMP-based targeted next-generation sequencing (NGS) to identify various fusions or mutations and expression levels in 81 key genes associated with ALL. Specifically, for the synthesis of cDNA, reverse transcription was performed using random primers, followed by end repair and adenylation. Purification of cDNA and ligation of molecular barcode adapters and universal primer sites were performed using Agencourt® AMPure® XP beads. The MBC-adapter-attached cDNA was amplified with gene-specific primer 1 (GSP1) and primers complementary to the universal primer site and a second PCR using gene-specific primer 2 (GSP2) was performed. Libraries were quantified using the KAPA Universal Library Quantification Kit (Kapa Biosystems, Woburn, MA), normalized and loaded onto NextSeq (Illumina, San Diego, CA) according to the manufacturer's guide. Data were analyzed with Archer® Analysis version 5.1.7 (ArcherDX). Normalized RNA expression values were calculated by dividing the unique RNA reads for each GSP2 by the arithmetic mean of the unique RNA reads for all control GSP2s included in the panel. Relative RNA expression values were reported in RNA_expression_visualization.tsv and visualized using heat maps, each heat map representing samples in columns and GSP2 RNA expression values (0–9) in rows.

<Example 4> Cytogenetic analysis and fluorescence in situ hybridization (FISH)

Cytogenetic G-splitting analysis was performed and genetic abnormalities were confirmed according to the 2016 International System for Human Cytogenetic Nomenclature guidelines. FISH was performed using appropriate probes ( BCR-ABL1 double fusion probe, PDGFRB break-a-part probe, PDGFRA break-a-part probe, JAK2 break-a-part probe, IGH break-apart probe, and P2RY8 deletion probe) according to the manufacturer's guidelines. (Cytocell, Cambridge, UK).

<Example 5> Reverse transcription (RT) PCR analysis

To confirm the fusion gene detected by AMP-based NGS, multiplex RT-PCR, RT-PCR and FISH were performed. First, the inventors used two multiplex RT-PCR primer sets to simultaneously screen four and three fusion transcripts, respectively: 1) ETV6-ABL1, NUP214-ABL1, EBF1-PDGFRB, P2RY8-CRLF2 and 2) EBF1-JAK2, ETV6-JAK2 and BCR-JAK2 primers were designed, and the ABL1 gene was used as an internal control. Multiplex RT-PCR was performed using CFX96 (Bio-Rad, Hercules, Canada). Primer sequences and probe sequences used for multiplex RT-PCR are shown in Tables 3 and 4 below. The amplification conditions were initial denaturation at 95 °C for 5 minutes, followed by 25 PCR cycles, denaturation at 95 °C for 30 seconds, annealing at 58 °C for 30 seconds, and 90 cycles at 72 °C. Seconds and held and stabilized at 4 °C.

As a result, the results of performing multiplex RT-PCR with the primers and probes in Tables 3 and 4 are shown in Figure 4, and all fusion transcripts and mutations identified for each patient are shown in Figure 5. Among 57 patients with Ph-like ALL, fusion transcripts were detected in 24 patients, CRLF2 rearrangements were confirmed in 13 patients, P2RY8-CRLF2 in 7, IGH-CRLF2 in 4, CLDN7-CRLF2 in 1, and HFM1-CRLF2 in 7. One CRLF2 was identified (two of them had CRLF2 mutations, and four had mutations in JAK2 and CRLF2 ). In addition, there were a total of 7 patients with JAK2 rearrangement, and the fusion transcripts included PAX5 (2 patients), BICD2 (1 patient), SMU1 (1 patient), ROCK (1 patient), ZCCHC7 (1 patient), and ZFP14 (1 patient). confirmed that it is ABL-class rearrangements were confirmed in 5 patients, including NUP214-ABL1 (2 patients), EBF1-PDGFRB (2 patients) and R CSD1-ABL2 (1 patient) (FIGS. 6 to 11). In 15 patients with no CRLF2 rearrangement or other kinase fusion, sequence mutations were identified in genes that activate JAK-STAT signaling, including IL7R (4 patients), FLT3 (7 patients), TYK2 (2 patients), JAK2 (1 person) and EPOR (1 person) were included. In addition, only genetic mutations related to the RAS pathway were observed in 17 patients, and sequence mutations of NRAS (7 patients), KRAS (5 patients), PTPN11 (4 patients), and NRAS/KRAS/PTPN11 (1 patient) were identified.

Based on the above results, patients with Ph-like ALL were classified into 5 subgroups. 1) CRLF2 abnormality (13 patients, 22.8%), 2) JAK2 rearrangement (7 patients, 12.3%), 3) ABL-class rearrangement (5 patients, 8.8%), 4) Other JAK-STAT pathway mutations (15 patients) , 26.3%) and 5) RAS pathway mutations (17 patients, 29.8%), and among the subgroups, patients with CRLF2 abnormality or ABL-class rearrangements had higher overall survival rates than patients with other genetic alterations. OS) was low, and in contrast, patients with RAS pathway mutations had a lower cumulative incidence of relapse and better OS (FIG. 12).

<Example 6> CRLF2 Expression measurement

CRLF2 gene expression was measured by quantitative RT-PCR (RT-qPCR) using TaqMan® Gene Expression Assays Hs00913509_s1 (Applied Biosystems, Foster City, CA) and was relative quantified by the expression of the internal control gene GUSB , the control was TaqMan® It was measured using Gene Expression Assays Hs00939627_m1 (Applied Biosystems). Primers and probes used for RT-PCR are shown in Table 5 below. 4 μg cDNA from each sample was identified using a 7500 Real Time PCR System (Applied Biosystems). The PCR amplification conditions were 50 cycles of 95 °C for 10 minutes, 95 °C for 15 seconds, and 60 °C for 1 minute, and the relative expression level was estimated using the 2 ^-ΔΔCt method, and the gene expression of AMP-based NGS data was compared.

As a result, the gene expression levels between Ph-like ALL (57 patients) and B-other ALL (40 patients) without known repetitive gene abnormalities were TLX1, TLX3, CRLF2, FBXW7, and DNTT in patients with Ph-like ALL. and SEMA6A were higher (FIG. 13A). Among patients with Ph-like ALL, the expression of CRLF2, P2RY8 , and FBXW7 was high in patients with CRLF2 abnormality, and the expression of SYNE1 gene was high in JAK2 rearrangement patients (FIG. 13B). In addition, KRF2 expression was increased in patients with JAK-STAT mutations. CRLF2 expression measured by RT-qPCR was confirmed to be correlated with AMP-based NGS data (r=0.476, P<0.001)

<Example 7> Sanger sequencing (Sanger sequence)

The identified mutations were sequenced by Sanger sequencing. Primers were designed using Primer 3, and the washed genome after the first PCR was subjected to a second PCR using the BigDye terminator. Subsequently, the nucleotide sequence was analyzed using a capillary electrophoresis sequencer, ABI Prism 3130xl Genetic Analyzer.

<Example 8> Statistical analysis

The end points of the present invention included complete remission (CR), long-term disease-free survival (DFS), overall survival (OS), and cumulative recurrence rate. For the 4 patient groups, patient characteristics were plotted using Fisher's exact test and test for categorical variables and the Kruskal-Wallis test for continuous variables and subgroups were compared with the log-rank test. Relapses were calculated using cumulative incidence estimates to accommodate competing death events, and subgroups were compared by Gray's test. The prognostic significance of covariates affecting response rate was analyzed using multiple logistic regression analysis, and covariates affecting OS were determined using Cox proportional hazards regression model. The prognostic significance of covariates affecting the cumulative incidence of relapse was determined by Fine-Gray proportional hazards regression analysis for competing events. Multivariate analysis was performed using variables with a p-value of 0.10 or greater in the previous univariate analysis. , All statistical analyzes were performed using 'R' software version 2.15.1 (R Foundation for Statistical Computing, 2012). Statistical significance was set when the p value was less than 0.05.

Based on the above study, rearrangements found in Ph-like ALL patients were confirmed, and it was confirmed that genetic characteristics and prognosis were different according to subgroups even within Ph-like ALL patients. Therefore, the composition for diagnosing Ph-like ALL of the present invention is composed of ETV6-ABL1, NUP214-ABL1, EBF1-PDGFRB, P2RY8-CRLF2, RT-PCR consisting of primers of EBF1-JAK2, ETV6-JAK2 and BCR-JAK2 can be used effectively to classify Ph-like ALL according to the expression pattern of the fusion transcript and predict treatment prognosis confirmed.

Claims (22)

1. A composition for diagnosing and predicting prognosis of Ph-like ALL disease, comprising an agent for measuring the expression level of a gene or protein related to Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL).
2. According to claim 1,

   The composition characterized in that the measurement of the expression level of the gene or protein is measured from a biological sample isolated from the patient.
3. According to claim 2,

   The biological sample isolated from the patient is at least one selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum, spinal fluid and urine isolated from the patient.
4. According to claim 2,

   The patient is a patient who has received at least one anticancer agent selected from the group consisting of hyper-fractionated cyclophosphamide, vincristine, daunorubicin and dexamethasone as induction chemotherapy. Composition characterized in that.
5. According to claim 2,

   The patient received high-dose cytarabine, mitoxantrone, imatinib, dasatinib, methotrexate, cytarabine, and hydrocortisone as follow-up chemotherapy. (Hydrocortisone) composition characterized in that the patient who received at least one anticancer agent selected from the group consisting of.
6. According to claim 1,

   The Ph-like ALL-related gene is a composition, characterized in that the fusion transcript.
7. According to claim 6,

   Wherein the fusion transcript is at least one selected from the group consisting of ETV6-ABL1, NUP214-ABL1, EBF1-PDGFRB, P2RY8-CRLF2, EBF1-JAK2, ETV6-JAK2 and BCR-JAK2 .
8. According to claim 1,

   The agent for measuring the expression level of the gene or protein is at least one selected from the group consisting of antibodies, interacting proteins, ligands, nanoparticles, and aptamers that specifically bind to the protein or peptide fragment. Composition characterized in that.
9. According to claim 1,

   The formulation is a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4, a primer pair consisting of SEQ ID NO: 5 and SEQ ID NO: 6, a primer pair consisting of SEQ ID NO: 7 and SEQ ID NO: 8 , a primer pair consisting of SEQ ID NO: 9 and SEQ ID NO: 10, a primer pair consisting of SEQ ID NO: 11 and SEQ ID NO: 12, a primer pair consisting of SEQ ID NO: 13 and SEQ ID NO: 14, and a primer pair consisting of SEQ ID NO: 15 and SEQ ID NO: 16 A composition characterized in that at least one primer pair selected from the group.
10. According to claim 1,

    The formulation comprises a probe consisting of SEQ ID NO: 17, a probe consisting of SEQ ID NO: 18, a probe consisting of SEQ ID NO: 19, a probe consisting of SEQ ID NO: 20, a probe consisting of SEQ ID NO: 21, a probe consisting of SEQ ID NO: 22, a probe consisting of SEQ ID NO: 23 A composition characterized in that it further comprises at least one probe selected from the group consisting of probes consisting of and probes consisting of SEQ ID NO: 24.
11. A kit for diagnosing and predicting prognosis of Ph-like ALL, comprising the composition of any one of claims 1 to 10.
12. isolating the biological sample;

    Measuring the expression level of a Ph-like ALL-related gene transcript or a peptide or protein expressed therefrom from the sample; and

    A method for providing information for diagnosis and prognosis of Ph-like ALL, comprising: confirming the expression pattern of the fusion transcript in the expressed gene transcript.
13. According to claim 12,

    Wherein the fusion transcript is selected from the group consisting of ETV6-ABL1, NUP214-ABL1, EBF1-PDGFRB, P2RY8-CRLF2, EBF1-JAK2, ETV6-JAK2 and BCR-JAK2 .
14. According to claim 12,

    Wherein the measurement is measured from a biological sample isolated from the patient.
15. According to claim 12,

    Wherein the biological sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, spinal fluid or urine isolated from a patient.
16. According to claim 14,

    The patient is a patient who has received at least one anticancer agent selected from the group consisting of hyper-fractionated cyclophosphamide, vincristine, daunorubicin, and dexamethasone as induction chemotherapy. An information providing method characterized in that.
17. According to claim 14,

    The patient received high-dose cytarabine, mitoxantrone, imatinib, dasatinib, methotrexate, cytarabine, and hydrocortisone as follow-up chemotherapy. (Hydrocortisone) information providing method characterized in that the patient is administered with at least one anticancer agent selected from the group consisting of.
18. According to claim 12,

    The step of confirming the expression pattern of the fusion transcript is at least one selected from the group consisting of antibodies, interacting proteins, ligands, nanoparticles, and aptamers that specifically bind to the protein or peptide fragment. Characterized information provision method.
19. According to claim 12,

    The step of confirming the expression pattern of the fusion transcript is a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4, a primer pair consisting of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: A primer pair consisting of SEQ ID NO: 7 and SEQ ID NO: 8, a primer pair consisting of SEQ ID NO: 9 and SEQ ID NO: 10, a primer pair consisting of SEQ ID NO: 11 and SEQ ID NO: 12, a primer pair consisting of SEQ ID NO: 13 and SEQ ID NO: 14, and a primer pair consisting of SEQ ID NO: 15 and An information providing method characterized in that at least one primer pair selected from the group consisting of primer pairs consisting of SEQ ID NO: 16.
20. According to claim 12,

    The step of confirming the expression pattern of the fusion transcript includes the probe consisting of SEQ ID NO: 17, the probe consisting of SEQ ID NO: 18, the probe consisting of SEQ ID NO: 19, the probe consisting of SEQ ID NO: 20, the probe consisting of SEQ ID NO: 21, and the SEQ ID NO: 22, a probe comprising SEQ ID NO: 23, and a probe comprising SEQ ID NO: 24.
21. According to claim 12,

    The gene transcript is multiplex real time-polymerase chain reaction (multiplex real time-polymerase chain reaction, multiplex RT-PCR), reverse transcriptase polymerization (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), Real time reverse transcriptase polymerase reaction (real time quantitative RT-PCR), quantitative polymerase reaction (quantitative RT-PCR), RNase protection method, Northern blotting and DNA chip technology ) To be measured using one or more methods selected from the group consisting of, information providing method.
22. According to claim 12,

    The step of measuring the expression level of the peptide or protein is Western blot, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorecence Activated Cell Sorter, FACS) and a protein chip (protein chip) that is measured using one or more methods selected from the group consisting of, an information providing method.

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