WO2023054851A1 - Novel biomarker for predicting prognosis of peripheral t cell lymphoma, and use thereof - Google Patents

Abstract

The present invention relates to a novel biomarker for predicting the prognosis of peripheral T-cell lymphoma patients and a use thereof, and more specifically, to a composition and a kit for predicting the prognosis of peripheral T-cell lymphoma and a method for providing information for predicting the prognosis of peripheral T-cell lymphoma. The present invention provides a composition and a kit for predicting the prognosis of peripheral T-cell lymphoma by suggesting a combination of novel biomarkers capable of predicting the prognosis of peripheral T-cell lymphoma, thus making it possible to quickly, accurately, and conveniently determine the prognosis of peripheral T-cell lymphoma in a non-invasive manner.

Description

Novel biomarkers for predicting prognosis of peripheral T-cell lymphoma and their uses

The present invention relates to a novel biomarker for predicting the prognosis of patients with peripheral T-cell lymphoma and its use, and more specifically, to a method for providing information for predicting the prognosis of peripheral T-cell lymphoma, and a prognosis of peripheral T-cell lymphoma using the same. It relates to predictive systems, compositions and kits.

Lymphoma can occur in the lymphatic vessels, spleen, bone marrow, blood or other organs and is more common in men than women. Typically, lymphomas represent solid tumors of lymphoid cells. Most treatments use chemotherapy, but in some cases, radiation therapy and/or bone marrow transplantation are used. Generally, lymphoma is treatable depending on the history, type and stage.

Among lymphomas, lymphoma originating from T cells is called peripheral T-cell lymphoma (PTCL), and it is a disease encompassing various types of lymphomas of the T cell lineage arising from lymph nodes or extralymph nodes. In particular, in Korea, unclassifiable peripheral T-cell lymphoma (PTCL, not otherwise specified), angioimmunoblastic T-cell lymphoma, and anaplastic large cell lymphoma are relatively common. Unclassifiable peripheral T-cell lymphoma is the most common, accounting for 6.3% of all lymphomas. Angioimmunoblastic T-cell lymphoma occurs mainly in the 60s, and anaplastic large cell lymphoma is diagnosed in the 20s in the case of benign cases and between the ages of 40 and 65 in the case of negative cases depending on the presence of anaplastic lymphoma kinase (ALK). It happens frequently.

Since lymphoma tends to recur as it progresses to a later stage, treatment of lymphoma should be attempted at an early stage, and it is important to predict recurrence after treatment and establish a follow-up treatment plan. However, although biomarkers for diagnosis and prognosis of B-cell-derived lymphoma or central nervous system lymphoma have been developed, research and development of biomarkers for T-cell lymphoma are still lacking.

[Prior art literature]

[Patent Literature]

Korean Patent Registration No. 10-1801980

Korean Patent Registration No. 10-1182974

An object of the present invention is to provide a method for providing information for predicting the prognosis of peripheral T-cell lymphoma by measuring the expression level of a novel biomarker capable of predicting the prognosis of peripheral T-cell lymphoma.

In addition, an object of the present invention is to provide a peripheral T-cell lymphoma prognosis prediction system capable of analyzing novel biomarkers that can predict the prognosis of patients with peripheral T-cell lymphoma.

In addition, an object of the present invention is to provide a composition and kit for predicting the prognosis of peripheral T-cell lymphoma, which can detect or measure novel biomarkers that can predict the prognosis of patients with peripheral T-cell lymphoma.

The present inventors found that mutations in one or more of RHOA, CREBBP, KMT2D, TP53, and IDH2 genes through sequencing of cell-free DNA in the plasma of patients with peripheral T-cell lymphoma were significantly better than those without mutations. Mutations in RHOA, CREBBP, KMT2D, TP53 and IDH2 by confirming that the survival rate is remarkably low and that the accuracy of prognosis prediction increases when combining the mutated CREBBP gene with one or more genes of RHOA, KMT2D, TP53 and IDH2 A gene is presented as a biomarker to predict the prognosis of peripheral T-cell lymphoma, and a method for providing information for predicting the prognosis of peripheral T-cell lymphoma using the biomarker, and a system, composition, and kit for predicting peripheral T-cell lymphoma prognosis using the same The present invention was completed by providing.

One aspect of the present invention comprises the steps of a) taking a biological sample from a subject; b) measuring the mutation level of the CREBBP gene and at least one gene selected from the group consisting of RHOA, KMT2D, TP53 and IDH2 in the biological sample; And c) provides a method for providing information for predicting the prognosis of peripheral T-cell lymphoma, comprising comparing the mutation level of the measured gene with a control group.

Steps a) to c) will be described in detail below.

Step a) is a process of collecting a biological sample from an individual in need of an examination on the survival prognosis of a patient with peripheral T-cell lymphoma.

According to one embodiment of the present invention, the subject in step a) may be a peripheral T-cell lymphoma patient before or after treatment.

The peripheral T-cell lymphoma is a type of non-Hodgkin's lymphoma that occurs in T lymphocytes and occurs in external lymphoid tissues such as lymph nodes, spleen, stomach, and skin, and has slow growth in some, such as cutaneous T-cell lymphoma. Except for lymphoma, most are aggressive lymphomas, which are cancerous tumors with a high rate of progression and a high recurrence rate. In the treatment of peripheral T-cell lymphoma, chemotherapy is mainly prescribed. In many cases, the disease is not alleviated even with the current standard treatment, first-line combination chemotherapy (CHOP; Cyclophosphamide/Doxorubicin hydrochloride/Vincristine sulfate/Prednisolon). If primary treatment fails or relapses, the survival time is about 5.8 months (median value), and the prognosis is poor. Other adjuvant treatments for chemotherapy include radiation therapy and autologous stem cell transplantation.

As used herein, “prognosis” refers to determining recurrence, metastasis, drug responsiveness, resistance, etc. of an individual before/after treatment for an individual who has not yet been diagnosed or has been diagnosed. In the present invention, the CREBBP gene of peripheral T-cell lymphoma patients and the mutation level of one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2 are checked to predict whether the future survival prognosis of peripheral T-cell lymphoma patients will be good means to do

According to one embodiment of the present invention, the peripheral T-cell lymphoma is unclassifiable peripheral T-cell lymphoma (not otherwise specified, PTCL-NOS), angioimmunoblastic T-cell lymphoma, AITL), anaplastic large cell lymphoma (ALCL), nodal peripheral T-cell lymphoma (PTFH) with T follicular helper cell (TFH) phenotype , follicular T-cell lymphoma (FTCL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), subcutaneous panniculitis-like T-cell lymphoma , SPTCL) and transformed mycosis fungoides (transformed MF).

As used in the present invention, "biomarker" is a substance that is generally detectable in biological samples, and organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, sugars, etc. All inclusive. In the present invention, mutated RHOA, CREBBP, KMT2D, TP53 and IDH2 genes can be used as biomarkers.

According to one embodiment of the present invention, the biological sample in step a) may be at least one selected from the group consisting of blood, plasma, serum, lymph, saliva, urine, and tissue.

In one embodiment of the present invention, a biological sample is prepared so that the prognosis of peripheral T-cell lymphoma can be predicted non-invasively, quickly, accurately and simply by confirming that the plasma cell-free DNA obtained from a patient with peripheral T-cell lymphoma matches the somatic mutation in the tumor tissue. It is preferably blood, plasma or serum.

Step b) is a process of measuring the mutation level of each gene together with the presence or absence of the biomarkers RHOA, CREBBP, KMT2D, TP53 and IDH2 in the biological sample.

The RHOA gene is represented by the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. The CREBB gene is represented by the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4. The KMT2D gene is represented by the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6. The TP53 gene is represented by the nucleotide sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8. The IDH2 gene is represented by the nucleotide sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10.

According to one embodiment of the present invention, the gene mutation in step b) is one or more selected from the group consisting of single nucleotide sequence mutation, deletion or insertion of a nucleotide sequence of 1 to 50 nucleotides, and copy number mutation. can

As used herein, “mutation” means an alteration of bases, nucleotides, polynucleotides or nucleic acids in a genome. The mutation may include substitution, insertion, or deletion of a base, nucleotide, polynucleotide, or nucleic acid. Here, substitution means a change in which a base, nucleotide, polynucleotide or nucleic acid is replaced with another base, nucleotide, polynucleotide or nucleic acid. An insertion is a change in which another base, nucleotide, polynucleotide or nucleic acid is added. Deletion refers to an alteration in which a base, nucleotide, polynucleotide or nucleic acid is removed.

The single nucleotide variant (SNV) refers to a change or mutation of a sequence showing a difference of one base or nucleotide on the genome, and one specific base is changed to another base at the same location in the genome of several people It can be used interchangeably with single nucleotide polymorphism (SNP), which means that it is expressed in other traits.

The 1 to 50 nucleotide sequence deletion or insertion refers to a sequence change or mutation showing a difference of 1 to 50 or more consecutive or discontinuous bases, nucleotides, polynucleotides or nucleic acids on the genome.

Such nucleotide sequence variations can affect even one amino acid composed of three nucleotide sequences, and base differences can contribute to individual differences in susceptibility to specific diseases, disease expression patterns, and therapeutic response.

The copy number mutation refers to a mutation section of 1 kb or more in length showing a difference in the number of repeated sequences when compared to a reference sequence, and susceptibility to a specific disease and expression of a disease due to the difference in copy number It can contribute to the expression of individual differences in aspects, treatment responsiveness, etc.

In order to measure the mutation level of these genes, sequencing or amplification reactions can be used.

According to one embodiment of the present invention, the mutation level of the gene in step b) may be measured using next-generation sequencing (NGS).

The NGS has a multiplexing ability to simultaneously perform hundreds of thousands of reactions, and sequencing is possible even with a small amount of sample. NGS has slightly different specific application techniques depending on the commercialized technology, but in general, clonal amplification, massively parallel sequencing, and a new sequencing method with a different mechanism of action from the Sanger method are used. Commercialized technologies include the 454 GS improved FLX model sequencer released by Roche in 2007, Genome Analyzer HiSeq released by Illumina in 2006, and SOLiD released by Applied Biosystems in 2007. In common, these three platforms abandoned complex library construction and cloning processes and adopted clonal amplification technology, chose massively parallel sequencing technology that can process a large amount at once, and adopted cyclic sequencing. The nucleotide sequence was determined by sequencing by synthesis, thereby eliminating the cumbersome electrophoresis process. It also uses an algorithm that arranges the short reads read using the shotgun method into a computer to find overlapping parts and complete the whole.

In addition, according to one embodiment of the present invention, the mutation levels of the RHOA, CREBBP, KMT2D, TP53 and IDH2 genes are polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, It may be measured by one or more methods selected from the group consisting of real-time RT-PCR, nuclease protection assay, in situ hybridization, DNA microarray, and Northern blot.

In one embodiment of the present invention, targeted ultra-deep sequencing was performed to measure the mutation levels of RHOA, CREBBP, KMT2D, TP53 and IDH2 genes from patients with peripheral T-cell lymphoma, and the nucleotide sequence of the gene All included panels were used. As a result of analyzing the mutation profile of 53 patients with cell-free DNA mutations detected in plasma among a total of 94 patients with peripheral T-cell lymphoma enrolled in the prospective cohort study, the top 5 genes with somatic mutations in patients with peripheral T-cell lymphoma were RHOA, CREBBP, KMT2D, TP53 and IDH2 were identified.

More specifically, the mutation of RHOA was mainly substituted from G to V at the 17th residue of the amino acid sequence (SEQ ID NO: 2) in lymphoma originating from TFH, and substituted from T to I at the 19th residue of the amino acid sequence in PTCL-NOS.

The mutation of CREBBP is mainly a substitution of the 2384th residue of the amino acid sequence (SEQ ID NO: 4), for example, H to T or P substitution, and in some cases, the 72nd residue of the amino acid sequence from E to G and the 238th residue to S to V, the 1118th residue was substituted from Q to a stop codon (*), and the 1894th residue from T to H. In patients with AITL, CREBBP p.S2382V mutation increased and relapsed after chemotherapy, and in relapsed patients with ALK-positive ALCL, CREBBP p.H2384P mutation increased and disease progression was observed.

The mutation of KM2TD was confirmed in the overall amino acid sequence, for example, the 1712th residue of the amino acid sequence (SEQ ID NO: 6) from K to R, the 2120th residue from A to P, and the 2215th residue from S to T , residue 2466 from L to F or C, residue 2494 from F to S, residue 2599 from P to A, residue 2681 from R to Q, residue 3099 from R to C, residue 3435 Residues I to S, residue 4341 from P to Q, residue 4540 from R to W, and residue 4995 from T to I. In patients with PTCL-NOS , KM2TD p.R2681Q and p.R1524C mutations were removed after chemotherapy and survived.

The mutation of TP53 was confirmed in the overall amino acid sequence, for example, the 11th residue of the amino acid sequence (SEQ ID NO: 8) from E to Q, the 72nd residue from P to R, and the 126th residue from Y to C , residue 134 from F to V, residue 181 from R to C or H, residue 214 from H to R, residue 224 from E to D, residue 237 from M to I, residue 241 Residue 273 is substituted from S to F, residue 273 is substituted from R to C, and residue 287 is substituted from P to S. After chemotherapy, patients with PTCL-NOS survived if the TP53 p.Y126C mutation was eliminated, but relapsed if the TP53 p.F134V mutation was increased again. In addition, AITL recurrence patients showed disease progression with an increase in the TP53 p.H214R mutation.

Mutations in IDH2 were mainly substituted at the 172nd residue of the amino acid sequence (SEQ ID NO: 10), for example, substitution of K, G or S from R, and substitution of 146th residue from T to P was also confirmed.

Amino acid mutations in the RHOA, CREBBP, KMT2D, TP53, and IDH2 genes are common in patients with peripheral T-cell lymphoma, and in particular, the CREBBP gene or its mutations have not been known to be associated with peripheral T-cell lymphoma in previous studies.

According to one embodiment of the present invention, when a cell-free DNA mutation is detected among patients with peripheral T-cell lymphoma, the progression-free survival rate (PFS) and overall survival rate (OS) are significantly higher than those when the cell-free DNA mutation is not detected. It was low. In addition, the accuracy of predicting prognosis for survival rate increased in the case of combining one or more genes of RHOA, KMT2D, TP53, and IDH2 compared to the case of having only the CREBBP gene as a biomarker. More specifically, when comparing the prognosis prediction accuracy according to the combination of genes, the CREBBP gene and RHOA, KMT2D, TP53 and IDH2 were 30 to 70% when one gene was combined, and the CREBBP gene and RHOA, KMT2D, TP53 and IDH2 at a level of 50 to 80%, and at a level of 70 to 90% when combining the CREBBP gene and three genes among RHOA, KMT2D, TP53 and IDH2, at a level of 70 to 90%, CREBBP, RHOA, KMT2D, In the case of including both TP53 and IDH2 genes, it was more than 90%.

Step c) is a process of determining the survival prognosis of patients with peripheral T-cell lymphoma by comparing the mutation levels of RHOA, CREBBP, KMT2D, TP53, and IDH2 genes measured in the biological sample with a control group that does not show mutations in the corresponding genes.

According to one embodiment of the present invention, the control group may be a normal person or an individual without mutations in the RHOA, CREBBP, KMT2D, TP53 and IDH2 genes among patients with peripheral T-cell lymphoma.

According to one embodiment of the present invention, in step c), when the measured CREBBP gene and the mutation level of one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2 are increased compared to the control group, peripheral T-cell lymphoma It may be that the prognosis is determined to be poor.

Another aspect of the present invention is an input unit for receiving sequence data for a subject's CREBBP gene, and one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2; and an analyzer configured to perform a prognosis of peripheral T-cell lymphoma of the subject by measuring a mutation level of each gene based on the sequence data of the gene.

Here, the description of the contents common to the foregoing is omitted in order to avoid excessive complexity.

The input unit corresponds to a device that provides data on the subject, and transmits the entire nucleotide sequence or amino acid sequence of each gene of the subject to the analysis unit.

According to one embodiment of the present invention, sequence data for the gene may be obtained from next-generation sequencing (NGS).

The analysis unit measures the mutation levels of the RHOA, CREBBP, KMT2D, TP53 and IDH2 genes based on the sequence data, and predicts the prognosis of peripheral T-cell lymphoma through a previously prepared algorithm.

According to one embodiment of the present invention, the analysis unit may calculate the accuracy of peripheral T-cell lymphoma prognosis according to a combination of the mutated CREBBP gene and one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2 .

The peripheral T-cell lymphoma prognosis prediction system including the input unit and the analysis unit may further include a display unit capable of outputting information on the prognosis in a certain form.

In addition, the system for predicting the prognosis of peripheral T-cell lymphoma including the input unit and the analysis unit may be implemented as a computer device for predicting the prognosis of peripheral T-cell lymphoma.

Another aspect of the present invention provides a composition for predicting the prognosis of peripheral T-cell lymphoma comprising an agent for measuring the mutation level of the CREBBP gene and one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2.

Here, the description of the contents common to the foregoing is omitted in order to avoid excessive complexity.

According to one embodiment of the present invention, the peripheral T-cell lymphoma is unclassifiable peripheral T-cell lymphoma (not otherwise specified, PTCL-NOS), angioimmunoblastic T-cell lymphoma, AITL), anaplastic large cell lymphoma (ALCL), nodal peripheral T-cell lymphoma (PTFH) with T follicular helper cell (TFH) phenotype , follicular T-cell lymphoma (FTCL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), subcutaneous panniculitis-like T-cell lymphoma , SPTCL) and transformed mycosis fungoides (transformed MF).

According to one embodiment of the present invention, the mutation of the gene may be at least one selected from the group consisting of single nucleotide sequence mutation, 1 to 50 nucleotide sequence deletion or insertion, and copy number mutation.

According to one embodiment of the present invention, the agent may specifically bind to each gene to measure the gene or its mutation and its expression level.

According to one embodiment of the present invention, the agent may include one or more selected from the group consisting of primers, probes, antibodies, aptamers, oligopeptides, and PNAs.

More specifically, the agent is preferably a primer or probe that specifically binds to each gene.

As used herein, “specifically binding” means that the binding ability to a target substance is superior to other substances to the extent that the presence or absence of the target substance can be detected by binding.

As used herein, "primer" refers to a primer capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerases) at a suitable temperature and buffer. It refers to a single stranded oligonucleotide. The suitable length of a primer varies depending on various factors, such as temperature and use of the primer, but typically consists of 15 to 30 nucleotides.

As used herein, “probe” refers to a linear oligomer of natural or modified monomers or linkages, comprising deoxyribonucleotides and ribonucleotides, and specifically hybridizing to a target nucleotide sequence It can exist naturally, or it can be artificially synthesized.

In addition, one aspect of the present invention provides a kit for predicting the prognosis of peripheral T-cell lymphoma comprising the composition.

The "kit for predicting prognosis of peripheral T-cell lymphoma" used in the present invention refers to a substance that can predict whether or not a future survival prognosis will be good through a biological sample collected from a test subject or a patient with peripheral T-cell lymphoma. The prognosis of survival can be diagnosed quickly, accurately and simply. In the present invention, an agent for measuring the mutation level of RHOA, CREBBP, KMT2D, TP53 and IDH2 genes is included.

The kit may include, without limitation, a diagnostic kit based on conventional genetic quantitative analysis.

According to one embodiment of the present invention, the kit may be at least one selected from the group consisting of a PCR kit, an RT-PCR kit, and a DNA chip kit.

For example, when the kit is applied to a PCR amplification process, the kit of the present invention may optionally include reagents necessary for PCR amplification, such as a buffer, DNA polymerase, DNA polymerase cofactor, and dNTPs, When the kit is applied to an immunoassay, the kit of the present invention may optionally include a substrate of a secondary antibody and a label. In addition, the kit according to the present invention may be manufactured in a plurality of separate packaging or compartments including the reagent components described above, and the kit of the present invention may be a diagnostic kit including essential elements necessary for performing a DNA chip. there is. A DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, reagents, enzymes, and the like for producing a fluorescently labeled probe. In addition, the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

The present invention provides a prognostic system, composition, and kit for peripheral T-cell lymphoma prognosis by presenting a combination of novel biomarkers capable of predicting peripheral T-cell lymphoma prognosis, which can non-invasively, quickly, accurately, and conveniently discriminate peripheral T-cell lymphoma prognosis. can

Figure 1 shows the mutation profile of plasma cell-free DNA of 53 patients with peripheral T-cell lymphoma obtained from a reference sample from a prospective cohort. Somatic mutations of RHOA , CREBBP , KMT2D , TP53 and IDH2 are most commonly found in various subtypes, and the red boxes indicate mutations detected in each gene.

Figure 2 shows the mutation site of the most common cell-free DNA mutation. Most contain a RHOA mutation encoding p.Gly17Val, while the IDH2 mutation primarily affects residue R172. The dominant mutation of CREBBP exists at position 2384 including p.His2384Thr, and the mutation sites of TP53 and KM2TD do not overlap.

Figure 3 compares plasma cell-free DNA mutations and tumor tissue DNA mutations. A paired analysis using 21 cases showed concordance between plasma cell-free DNA and tumor tissue somatic mutations.

Figure 4 shows the correlation of longitudinal analysis of plasma cell-free DNA mutations with treatment outcomes. (a) A 61-year-old male with PTCL-NOS maintains a complete response as the TP53 mutation disappears. (b) A 65-year-old woman with PTCL-NOS showed an increase in TP53 mutations at the end of treatment and eventually relapsed. (c) A 59-year-old male with PTCL-NOS maintains a complete response with a decrease in initially detected mutations including EZH2 . (d) In a 58-year-old male with AITL, CREBBP and MEF2B mutations increased after ASCT and recurred during the follow-up period. (E) A 50-year-old male with relapsed AITL had increased TP53 and SOCS1 mutations after rescue treatment during disease progression. (f) A 34-year-old woman with ALK-positive ALCL had increased CREBBP and STAT6 mutations after rescue treatment.

Figure 5 shows the emergence of new somatic mutations during disease relapse or progression in the monitoring of plasma cell-free DNA containing somatic mutations. (a) In a 56-year-old male with ALK-negative ALCL, CREBBP , SOCS1 , MAPK1 , and MAP2K1 mutations increased during disease progression even after specific treatment with brentuximab vedotin (top), whereas KMT2D and BRAF mutations Various patterns are shown depending on the mutation site (middle). Mutations in PIM1 , MTOR and CARD11 increase as the disease progresses, reflecting the emergence of new mutations, whereas mutations in KRAS and PIK3CA decrease (bottom). (b) A 57-year-old male with PTCL-NOS with elevated mutational volume in multiple genes such as CREBBP , KMT2D , GATA3 and MEF2B upon complete response finally shows disease relapse with a further increase in mutations after ASCT (top and middle) . In relapsed patients, new mutations such as BRAF and IRK1 appear (bottom). (c) A 48-year-old male with PTCL-NOS treated in combination with copanlisib and gemcitabine has multiple mutations including CREBBP , MEF2B , MAPK3 , KRAS , STAT3 and STAT6 .

Figure 6 shows the prediction of survival outcome based on plasma cell-free DNA. (A, B) Patients with detected mutations in cell-free DNA have poorer progression-free and overall survival rates than patients with undetected mutations. (C, D) Patients with reduced mutation volume (log change in GE ≥ 1.5) at the end of treatment were compared with patients with increased mutation volume (log change in GE < 0) or patients with a slight decrease (log change in GE < 1.5). Progression-free survival and overall survival are better.

Figure 7 is a graph showing the prognosis prediction accuracy according to the combination of biomarkers RHOA , CREBBP , KMT2D , TP53 and IDH2 for predicting prognosis of peripheral T-cell lymphoma (R; RHOA, C; CREBB, K; KMT2D, T; TP53 and I ;IDH2).

Hereinafter, the present invention will be described in more detail. However, these descriptions are merely presented as examples to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

1. Materials and Methods

1-1. patient

It was performed for the purpose of exploring the usefulness of ctDNA genotyping and monitoring as a biomarker for PTCL patients excluding extranodal NK/T-cell lymphoma.

All patients were enrolled in a prospective cohort study for lymphoma (NCT03117036) approved by the Institutional Review Board of the Samsung Medical Center (approval number 2016-11-040). It became. Germline DNA and plasma cell-free DNA were collected and stored at -80 °C after obtaining written informed consent from patients admitted to the hospital according to approved guidelines. Plasma samples were collected at prospective cohort enrollment (baseline), at interim assessment (interim), at final response assessment after first-line treatment (end of treatment), and/or at relapse or progression (progression). Staging work during clinical practice included computed tomography (CT), ¹⁸ F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT), and bone marrow biopsy. Tumor pathology was confirmed by two lymphoma pathologists (KYH and CH). Newly diagnosed patients received primary treatment such as CHOP, and relapsed or refractory patients received salvage treatment. Response evaluation was performed using CT and PET/CT according to the Lugano Classification (Cheson BD, et al. J Clin Oncol. 2014;32(27):3059-3068). Patients were followed up every 3 to 6 months after completion of the planned treatment, and CT and PET/CT scans were used to confirm the occurrence of recurrence or disease progression. The survival status of all enrolled patients was updated regularly, and the last update on survival and disease status was done in May 2021.

1-2. sample preparation

A total of 206 peripheral blood samples were obtained from 94 patients with T-cell lymphoma at the time of examination for diagnosis and evaluation of treatment response. Tissue samples (n = 19) were obtained from paraffin-embedded tissues fixed in formalin. Whole blood samples were collected in Cell-Free DNA™ BCT tubes (Streck Inc., USA). Plasma was prepared using a three-step centrifugation at room temperature with increasing centrifugal force (840 xg for 10 min, 1,040 xg for 10 min, and 5,000 xg for 10 min). Peripheral blood leukocyte (PBL) was collected from initial centrifugation. Plasma was separated in the initial centrifugation, followed by Ficoll gradient centrifugation, and granulocytes were separated from precipitated lymphocytes using lysis buffer (Qiagen, USA). Also, for comparison, 16 males and 10 females (median age: 38 years old, range: 25-50 years old) were selected with the approval (SMC 2016-04-107) of the Institutional Review Board of Samsung Medical Center. Plasma samples were collected from a control group consisting of 26 healthy volunteers.

1-2-1. DNA extraction

Circulating DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, USA). An AllPrep DNA/RNA Mini Kit (Qiagen) was used to purify gDNA from paraffin-embedded tissues fixed in formalin. DNA concentration and purity were quantified using a Picogreen fluorescence assay on a Nanodrop 8000 UV-Vis spectrometer (Thermo Fisher Scientific, USA) and a Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA). Fragment size distribution was measured using a 2200 TapeStation Instrument (Agilent Technologies, USA).

1-2-2. library creation

The purified gDNA was sonicated into 150-200 bp fragments using Covaris S2 (Covaris Inc., USA). For diagnosis, a reference library was constructed using previously obtained tissue samples and subjected to targeted sequencing. Libraries for tumor biopsy samples were constructed using the SureSelect XT reagent kit, HSQ (Agilent Technologies) according to the manufacturer's instructions. gDNA and cell-free DNA libraries were generated using the KAPA Hyper Prep Kit (Kapa Biosystems, USA).

1-3. Ultra-deep targeted sequencing and data processing

Hybrid selection was performed using custom probes for 426 lymphoma-related genes. In addition, capture probes for 66 genes selected from the 426 gene panel were prepared in-house, used for cell-free DNA sequencing, matched with normal samples, and analyzed on the HiSeq 2500 system (Illumina, USA). . All sequencing data were aligned to the hg19 reference using the BWA-mem (v0.7.17; Wellcome Trust Sanger Institute, UK) algorithm. The SAM (sequence alignment map) format was converted to a BAM (binary alignment map) file using SAMTOOLS software (v1.9; Wellcome Trust Sanger Institute). Polymerase chain reaction (PCR) duplicates were identified using the MarkDuplicates software from the Picard package (v2.19.1; Broad Institute, UK). A home-built Python (v3.6.4) script was used to implement the integrated design environment (IDE) method and process duplicate reads (Newman AM, et al. Nat Biotechnol. 2016;34(5):547- 555). GATK (v4.1.0.0), Picard (v2.19.1) and SAMTOOLS (v1.9) were used for basic quality recalibration and classification of SAM and BAM files. The filtering step and sequencing metrics were performed in the following way. ctDNA levels were quantified by genome equivalent (GE) determined as the product of total cell-free DNA concentration and maximum variant allele frequency (VAF) of somatic mutations.

1-3-1. Sequencing data processing

The basic recalibration process was performed using the BaseRecalibrator and ApplyBQSR functions in GATK software. Picard was used to identify a unique identifier (UID) family in each group of PCR replicates. After identifying the UID family, an in-house built Python (v. 2.7.9; Python Software Foundation, USA) script was applied to handle duplicate leads. A modification of the integrated digital error suppression (iDES) method described by Newman et al. and a molecular barcoding strategy for the sensitive detection of circulating tumor DNA (ctDNA) to reduce stereotypical background error. A script was created to perform the combined method with in silico removal (Vose J, et al. J Clin Oncol. 2008;26(25):4124-4130). Parallel sequencing of matched leukocytes was performed to exclude mutations associated with clonal hematopoiesis and to allow for proper mutation calling. During processing, discordant pairs and off-target reads were filtered out.

1-3-2. Detection of somatic mutations

Somatic mutations were identified with a targeted deep sequencing method. Prior to analysis, base sequences with low quality scores were removed, and only positions with a sequencing depth of 500x or higher were used for mutation detection. Somatic mutations were identified using a digital error suppression method to minimize sequencing background errors with minor corrections (Vose J, et al. J Clin Oncol. 2008;26(25):4124-4130). For a detailed process for identifying somatic mutations, refer to (Briski R, et al. Blood Cancer J. 2014;4:e214). Briefly, the PBL gDNA of all patients was used as a matched normal sample to remove patient-specific germline mutations. Error distributions generated from matched normal samples were used to discriminate low variant allele frequency (VAF) mutations from background noise and eliminate false positives. Mutations were annotated for their effect and synonymous mutations were excluded from analysis. Mutants with a VAF of 0.15% or more were selected and used for analysis. A Z-test was performed (Bonferroni corrected p-value < 0.05) to identify variants present at significantly higher frequencies than the corresponding background errors in normal samples. In addition, a threshold of allele frequency ≥ 0.5% and alternative allele number ≥ 20 was applied. For biopsy specimens, single nucleotide variation (SNV) profiling was performed with varying thresholds of total core ≥ 100x, allele frequency ≥ 2% and number of alternative alleles ≥ 10. Indels (meaning insertions and deletions of nucleotides) were called in all samples using Somatic Strelka2 and MANTA with default parameters (Ellin F, et al. Blood. 2014;124(10) :1570-1577;Phan A, et al. Curr Hematol Malig Rep. 2016;11(6):492-503). Variants that passed the filter in Strelka2 were further reviewed.

1-4. statistical analysis

Descriptive statistics were determined as proportions and medians, and comparisons between groups for categorical variables were assessed using Fisher's exact test. Differences in cell-free DNA concentration were compared by the Wilcoxon rank sum test. Progression-free survival (PFS) was calculated as the period from the date of enrollment to the date of PFS designated as disease recurrence, or progression or death from any cause. Overall survival (OS) was defined as the period from the date of enrollment until death or the last date of follow-up. Survival curves were described using the Kaplan-Meier estimate and compared between groups using the log-rank test. Statistical analysis was performed using the IBM PASW version 24.0 software program (IBM SPSS Inc., USA).

2. Results

2-1. patient

Peripheral blood samples were obtained from 94 patients with peripheral T-cell lymphoma (PTCL) consecutively enrolled in a prospective cohort study conducted between March 2017 and November 2019. Referring to Table 1 below, there were 73 newly diagnosed/primary treatment patients and 21 patients with relapse or treatment resistance at the time of enrollment, and the average age of the patients was 58 years (range: 19 to 82 years). According to World Health Organization (WHO) classification, 39 patients had angioimmunoblastic T-cell lymphoma (AITL) (n = 31), follicular T-cell lymphoma (FTCL) (n = 4), and TFH phenotype. Nodular lymphoma of TFH origin, including nodular peripheral T-cell lymphoma (PTFH) (n = 4). Excluding FTCL and PTFH, 33 patients had unclassifiable peripheral T-cell lymphoma (PTCL-NOS). The 14 patients with anaplastic large cell lymphoma (ALCL) included patients with systemic ALK-negative ALCL (n = 6), systemic ALK-positive ALCL (n = 5), and cutaneous ALK-negative ALCL (n = 3). Other disease subtypes include monomorphic epithelial intestinal T-cell lymphoma (MEITL) (n = 4), subcutaneous panniculitis-like T-cell lymphoma (SPTCL) (n = 3), and transformed MF ( n = 1). As of the last update on 31 May 2021, the median follow-up was 31.8 months (95% confidence interval: 26.9 to 36.7 months), and PFS had occurred in 56 patients. Of these, 36 patients died at the time of analysis, and the cause of death of all patients except for one treatment-related toxicity was lymphoma.

	Sum	cell-free DNA mutation		P value
		not detected	detection
	n = 94	n = 41	n = 53
disease state
Primary Treatment New Diagnosis	73	36	37	0.047
relapse or treatment resistance	21	5	16
Diagnosis
PTCL-NOS	33	15	18	0.079
AITL	31	7	24
ALK negative ALCL	6	3	3
ALK-positive ALCL	5	4	One
Cutaneous ALK Negative ALCL	3	3	0
Follicular helper T-cell lymphoma	4	2	2
T follicle-assisted TCL	4	2	2
MEITL	4	3	One
SPTCL	3	2	One
Deformation MF	One	0	One
Age (n, %)
≤ 60 years	56	25	31	0.835
> 60 years old	38	16	22
Gender (n, %)
male	61	26	35	0.792
female	33	15	18
Serum LDH (n, %)
normal	41	19	22	0.679
increase	53	22	31
BMI (n, %)
absence	71	31	40	0.988
existence	23	10	13
stage (n, %)
I/II	25	16	9	0.020
III/IV	69	25	44
IPI (n, %)
low/low-medium risk	63	31	32	0.119
high/high risk	31	10	21
cell free DNA
low (≤ median)	47	25	22	0.061
High (> Median)	47	16	31
LDH: lactate dehydrogenase BMI: bone marrow involvement IPI: International Prognostic Index

2-2. Baseline cell-free DNA genotyping

Cell-free DNA was extracted and measured from reference samples obtained at enrollment, and the median baseline level of plasma cell-free DNA in 94 patients (12.0 ng/dL, range: 2.3 to 648.0 ng/dL) was found in healthy volunteers. (7.4 ng/dL, range: 3.7 to 14.4 ng/dL, p < 0.001). Targeted sequencing using reference cell-free DNA samples identified 53 patients (56%, 53/94) with cell-free DNA containing somatic mutations representing the tumor-derived fraction (FIG. 1). Lymphoma of TFH origin (28/39, 71.8%) than PTCL-NOS (18/33, 54.5%) because somatic mutations are frequently detected in AITL (24/31), FTCL (2/4) and PTFH (2/4) ), somatic mutations were identified more frequently. In addition, ALK-negative ALCL, although fewer in number, had a higher mutation frequency (3/6) than ALK-positive ALCL (1/5) and cutaneous ALCL (0/3). However, the median plasma cell-free DNA concentration (13.3 ng/dL, range: 2.4 to 648.0 ng/dL) of patients with lymphoma of TFH origin was significantly higher than PTCL-NOS (10.5 ng/dL, range: 2.5 to 299.6 ng/dL) and ALCL (15.4 ng/dL). ng/dL, range: 3.1 to 44.1 ng/dL, p > 0.05). Clinically stage III/IV patients (44/69, 63.8%) and relapsed/treatment resistant patients (16/21, 76.2%) were compared with stage I/II patients (9/25, 36.0%) and newly diagnosed patients. (37/73, 50.7%) showed higher somatic mutations (see Table 1 above). Somatic mutations were detected in 51 of 66 genes sequenced, and the top 10 genes were: RHOA , CREBBP , KMT2D , TP53 , IDH2 , ALK , MEF2B , SOCS1 , CARD11 , KRAS (FIG. 1). The most commonly identified mutations were detected in RHOA , primarily in lymphomas of TFH origin, such as AITL (n = 16), FTCL (n = 1) and PTFH (n = 1), and rarely in PTCL-NOS (n = 3). It became. Most cases (n = 20) contained the RHOA mutation encoding p.Gly17Val, and only one case (PTFH) contained the RHOA mutation encoding p.Thr19Ile (FIG. 2). All IDH2 mutations affecting the R172 residue (e.g. p.Arg172Ser) were found more frequently in lymphomas of TFH origin including AITL (n = 8) and FTCL (n = 1) than in PTCL-NOS (n = 1) ( Fig. 2). Somatic mutations in CREBBP , TP53 and KM2TD were identified in all subtypes (Fig. 2). A dominant mutation of CREBBP was detected at position 2384 including p.His2384Thr, but the mutation sites of TP53 and KM2TD did not overlap (FIG. 2). The genotyping results of baseline cell-free DNA were compared with mutational profiles of primary tumor tissue in 21 patients for whom tumor and plasma samples were available in pairs. For the above-mentioned top 10 genes (FIG. 3), it was found that somatic mutations in plasma cell-free DNA and tumor tissue were consistent. For example, concordance of RHOA mutations was observed in plasma cell-free DNA and tumor tissues of lymphomas of FTC origin AITL (n = 7), FTCL (n = 1) and PTFH (n = 1), whereas concordance of TP53 mutations was were identified in PTCL-NOS (N = 4), AITL (n = 1) and ALCL (n = 1) (Fig. 3).

2-3. Longitudinal assessment of cell-free DNA mutations

Longitudinal analyzes using serially drawn plasma samples from baseline to relapse or progression showed a correlation between mutation and clinical outcome. One out of two patients with newly diagnosed PTCL-NOS who received CHOP chemotherapy showed rapid clearance of the TP53 mutation at interim evaluation and survived with a complete response, while the other patient had a recurrence due to persistence of the TP53 mutation. was seen (Fig. 4a, b). Among patients who underwent upfront autologous stem cell transplantation (ASCT), one PTCL-NOS patient, who initially had a high GE ctDNA mutation in EZH2 , had a mutation level of 1.5 logs at the end of treatment. decreased to an excess level, and full response was maintained (Fig. 4c). On the other hand, patients with AITL failed to show clearance of baseline mutations in CREBBP and MEF2B , and developed additional mutations in MEF2B even though radiation showed a complete response after ASCT. It eventually relapsed with persistent mutation detection (Fig. 4d). An association between disease progression and increased somatic mutations was also found in relapsed patients. For example, TP53 and SOCS1 mutations were increased in one patient with AITL relapse who received salvage chemotherapy (FIG. 4e), and in another patient with ALK-positive ALCL relapse after treatment with brentuximab vedotin. Increased CREBP and STAT6 mutations showed disease progression (Fig. 4f).

2-4. Emergence of novel somatic mutations during disease progression

Termination genotyping of cell-free DNA confirmed the emergence during disease progression of new somatic mutations with an increase in mutations detected earlier. During disease progression, a 56-year-old male with ALK-negative ALCL had an increase in initially detected SOCS1 , MAPK1 , MAP2K1i and KM2TD mutations. In addition, the appearance of new mutations in genes including PIM1 , MTOR and CARD11 was also observed, although the levels of other mutations in genes including PIK3CA decreased. In patients with BRAF mutations, BRAF p.D594G increased, whereas BRAF p.V600G and p.V600M decreased, and this patient developed treatment resistance in subsequent treatment with brentuximab vedotin (FIG. 5a). A 57-year-old man with PTCL-NOS underwent ASCT after achieving a complete response to six cycles of divided ifosfamide, carboplatin, and etoposide chemotherapy. However, mutations related to CREBBP , KMT2D , MEF2B , BRAF , IRK1 and GATA3 were detected at the time of the patient's complete response, and as the appearance of these mutations further increased, they eventually recurred (FIG. 5b). A 48-year-old man with relapsed PTCL-NOS was treated with the PI3K inhibitor copanlisib in combination with gemcitabine as part of a clinical trial. The patient showed development of new mutations including KRAS and STAT3 as the disease progressed during treatment (Fig. 5c).

2-5. Cell-free DNA and survival results

Comparing survival results according to baseline detection of cell-free DNA mutations, 53 patients with PFS and OS with cell-free DNA mutations were significantly worse than 41 patients with no mutations (Fig. 6A, B). In addition, survival outcomes depended on longitudinal differences in GE, which indicated differences in mutation volume of cell-free DNA between baseline and end of treatment. Thus, patients with more than 1.5 log reduction in mutation volume at the end of treatment showed better PFS and OS than patients with less than 1.5 log reduction. In particular, patients with increased GE after treatment with a log change less than zero had the worst PFS and OS (Fig. 6C,D).

2-6. Prognostic prediction accuracy according to the combination of biomarkers

For 53 patients with peripheral T-cell lymphoma with somatic mutations used in the above-mentioned targeted deep sequencing, the proportion of patients corresponding to individual genes or gene combinations among RHOA, CREBBP, KMT2D, TP53, and IDH2 genes was identified.

As a result, as shown in FIG. 7, RHOA, CREBBP, KMT2D, TP53 or IDH2 genes each contain only 20 to 40% of peripheral T-cell lymphoma patients (FIG. 7A), but combining two or more genes The scope of inclusion has increased. In particular, when two genes of RHOA, KMT2D, TP53, and IDH2 were combined with the CREBBP gene, coverage increased to about 50% or more, and when combined with three genes, coverage increased to about 70% or more (Fig. 7B and C ). In the case of including the RHOA, CREBBP, KMT2D, TP53, and IDH2 genes, the level was about 90%, but in the case of including the four genes except CREBBP, the level was only 70% (FIG. 7D).

These results suggest that the prognosis of peripheral T-cell lymphoma can be predicted with high accuracy through a combination of the subject's mutated gene, particularly the CREBBP gene and any one or more genes of RHOA, KMT2D, TP53, and IDH2.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (15)

1. a) taking a biological sample from the subject;

   b) measuring the mutation level of the CREBBP gene and at least one gene selected from the group consisting of RHOA, KMT2D, TP53 and IDH2 in the biological sample; and

   c) comparing the mutation level of the measured gene with a control group

   A method of providing information for predicting a prognosis of peripheral T-cell lymphoma comprising a.
2. The method of claim 1,

   Wherein the biological sample of step a) is at least one selected from the group consisting of blood, plasma, serum, lymph, saliva, urine and tissue.
3. The method of claim 1,

   Wherein the gene mutation in step b) is at least one selected from the group consisting of single nucleotide sequence mutation, deletion or insertion of a nucleotide sequence of 1 to 50 nucleotides, and copy number mutation.
4. The method of claim 1,

   Wherein the mutation level of the gene in step b) is measured using next-generation sequencing.
5. The method of claim 1,

   The mutation level of the gene in step b) is determined by polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, nuclease protection assay, A method that is measured by at least one method selected from the group consisting of in situ hybridization, DNA microarray, and Northern blot.
6. The method of claim 1,

   In step c), when the measured CREBBP gene and the mutation level of one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2 are increased compared to the control group, peripheral T-cell lymphoma prognosis is determined as poor. Method .
7. The method of claim 1,

   The peripheral T-cell lymphoma is unspecified peripheral T-cell lymphoma (not otherwise specified, PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (anaplastic large cell lymphoma, ALCL), nodal peripheral T-cell lymphoma (PTFH), and follicular T-cell lymphoma with a T follicular helper cell (TFH) phenotype. -cell lymphoma (FTCL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), subcutaneous panniculitis-like T-cell lymphoma (SPTCL) and transforming myoclonus (transformed mycosis fungoides, transformed MF) method of one or more selected from the group consisting of.
8. an input unit for receiving sequence data for the subject's CREBBP gene and at least one gene selected from the group consisting of RHOA, KMT2D, TP53 and IDH2; and

   An analysis unit that measures the level of mutation of each gene based on the sequence data of the gene and performs the prognosis of the subject's peripheral T-cell lymphoma.

   Peripheral T-cell lymphoma prognosis prediction system comprising a.
9. The method of claim 8,

   The system wherein the sequence data is obtained from next-generation sequencing.
10. The method of claim 8,

    The analysis unit calculates the accuracy of peripheral T-cell lymphoma prognosis according to a combination of the mutated CREBBP gene and one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2.
11. CREBBP gene, and

    A composition for predicting the prognosis of peripheral T-cell lymphoma, comprising an agent for measuring the level of mutation of one or more genes selected from the group consisting of RHOA, KMT2D, TP53 and IDH2.
12. The method of claim 11,

    The peripheral T-cell lymphoma is unclassifiable peripheral T-cell lymphoma (not otherwise specified, PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma ( anaplastic large cell lymphoma (ALCL), nodal peripheral T-cell lymphoma (PTFH), and follicular T-cell lymphoma with a T follicular helper cell (TFH) phenotype. cell lymphoma (FTCL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), subcutaneous panniculitis-like T-cell lymphoma (SPTCL) and transforming A composition that is one or more selected from the group consisting of transformed mycosis fungoides and transformed MF).
13. The method of claim 11,

    The mutation of the gene is a composition in which the mutation of the gene is at least one selected from the group consisting of a single nucleotide sequence mutation, 1 to 50 nucleotide sequence deletion or insertion, and copy number mutation.
14. The method of claim 11,

    The agent is a composition comprising at least one selected from the group consisting of primers, probes, antibodies, aptamers, oligopeptides and PNA.
15. A kit for predicting the prognosis of peripheral T-cell lymphoma comprising the composition of any one of claims 11 to 14.

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