Predicting Cancer Recurrence

Abstract

The present invention relates to predicting cancer recurrence or predicting the survival time of a patient diagnosed with blood cancer by measuring the level of serum thymidine kinase 1 (STK1) substance in a body sample of the patient using an antibody or a fragment thereof that specifically binds to a serotype of human TK1. The measured level of STK1 substance is compared with a threshold value selected based on the age of the patient. Cancer recurrence is then predicted based on the comparison.

Description

[Technical field]

[0001] The present invention relates generally to the measurement of thymidine kinase 1 (TK1), and in particular to the prediction of cancer recurrence based on measured serum TK1 levels. [Background technology]

[0002] Thymidine kinase 1 (TK1) (EC 2.7.1.21), also called 2'-deoxythymidine kinase or ATP-thymidine 5'-phosphotransferase, is an enzyme involved in deoxyribonucleic acid (DNA) precursor synthesis. TK1 phosphorylates thymidine, allowing its incorporation into DNA. Expression of TK1 is a marker of active cell proliferation, with intracellular concentrations low during the G0 / G1 phase of the cell cycle and increasing during the S / G2 phase.

[0003] A form of TK1 is present at high levels in the serum and plasma of humans and animals with malignancies, and therefore measurement of serum TK1 activity has been used for monitoring and predictive purposes in several different malignancies, but so far mainly in leukemias and lymphomas.

[0004] Furthermore, TK1 is the only proliferation marker that can be measured in blood, which could provide great clinical benefit if available as a routine test.

[0005] Serum TK1 activity was measured using a radioactive substrate 125 Although it has been measured for decades using I-dUrd (PROLIFIGEN® TK-REA, DiaSorin Inc.), this radioenzymatic assay is of limited use and is preferred in cases of aggressive hematological malignancies. Since 2000, a non-radiometric TK1 activity assay (TK LIAISON® assay, DiaSorin Inc.) has been available. It is a sensitive and robust assay that provides clinically valuable information in humans and dogs, especially for monitoring treatment and predicting recurrence.

[0006] During the past 15 years, antibodies against human TK1 have become available, making it possible to measure TK1 protein levels, as opposed to TK1 activity, in both hematological and solid tumor diseases, e.g., breast cancer, as well as several other forms of solid and hematological tumors.

[0007] One type of TK1 protein measurement relies on the dot blot method based on anti-TK1 antibodies generated against the C-terminal part of TK1. The main reason for choosing this antibody production strategy is that the C-terminal region is involved in cell cycle regulation of TK1. It is assumed that it contains a recognition sequence to initiate the degradation of TK1 during mitosis and that this is an exposed region to which antibodies may be generated. Although the dot blot assay has been used successfully in many studies, a major limitation is that it is not routine in clinical laboratory practice.

[0008] AroCell TK 210 ELISA is a quantitative immunoassay kit for the measurement of TK1 in human blood. This ELISA configuration is simple and robust, does not require specialized instrumentation to perform, and can be easily incorporated into standard laboratory processes. AroCell TK 210 ELISA is not only a fast and simple method for monitoring TK1 from blood samples, but also produces reliable and reproducible results using standard equipment in clinical chemistry. Summary of the Invention

[0009] A general object of the present invention is to accurately predict cancer survival and / or cancer recurrence in patients.

[0010] This and other objectives are achieved by the embodiments disclosed herein.

[0011] An embodiment of the present invention relates to a method for predicting cancer recurrence, comprising measuring the level of serum thymidine kinase 1 (STK1) substance in a body sample of a patient diagnosed with hematological cancer using an antibody or fragment thereof that specifically binds to a serotype of human TK1. The method also comprises comparing the measured level of STK1 substance to a threshold value selected based on the age of the patient. The method further comprises predicting the recurrence of cancer in the patient based on the comparison.

[0012] The present invention predicts cancer recurrence based on a comparison of measured STK1 levels to age-dependent thresholds. Experimental data presented herein demonstrate that the accuracy of using STK1 as a biomarker for hematological cancers is significantly improved by using age-related criteria or threshold ranges for different age patient populations.

[0013] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0014] [Figure 1a] Pretreatment serum TK1 concentrations in relation to Ann Arbor stage. [Figure 1b] Pretreatment serum TK1 concentrations in relation to the age-adjusted International Prognostic Index. [Figure 2a] Pre-treatment serum TK1 concentrations in relation to time to disease recurrence. [Figure 2b] Pretreatment serum TK1 concentrations in relation to survival. [Figure 3a] Difference in overall survival between patients with high and low TK1 concentrations in serum taken before treatment. The dichotomous cutoff value was set at the upper limit for healthy subjects, i.e., 0.45 μg / L. [Figure 3b] Difference in disease-free survival between patients with high and low TK1 concentrations in serum taken before treatment. The dichotomous cutoff value was set at the upper limit for healthy subjects, i.e., 0.45 μg / L. [Figure 3c]Difference in disease-specific survival between patients with high and low TK1 concentrations in serum taken before treatment. The dichotomous cutoff value was set at the upper limit for healthy subjects, i.e., 0.45 μg / L. [Figure 4a] Multivariate analysis of pretreatment dichotomized TK1 (cutoff was 0.45 μg / L, the upper limit of normal) and aaIPI scores. [Figure 4b] Multivariate analysis of pretreatment dichotomized TK1 (cutoff was 0.45 μg / L, the upper limit of normal) and aaIPI scores. [Figure 4c] Multivariate analysis of pretreatment dichotomized TK1 (cutoff was 0.45 μg / L, the upper limit of normal) and IPI scores. [Figure 4d] Multivariate analysis of pretreatment dichotomized TK1 (cutoff was 0.45 μg / L, the upper limit of normal) and IPI scores. [Diagram 5] TK1 concentrations measured in blood samples collected within the U-CAN research project. On-treatment blood samples (after the third chemotherapy cycle) were not included at the inception of the U-CAN project, but have been added to the U-CAN protocol very recently. [Figure 6] Comparison of TK1 concentrations in serum before and after treatment for living and affected patients. [Figure 7a] TK1 concentrations in serum for individuals with age-adjusted international prognostic index (aaIPI) 0 to 1. The analyzed blood samples were taken within the U-CAN research project presenting blood samples before the start of treatment, during treatment (i.e. after three chemotherapy cycles) and after the end of treatment. [Figure 7b] TK1 concentrations in serum for individuals with aaIPI 2 to 3. The analyzed blood samples were taken within a U-CAN research project presenting blood samples before the start of treatment, during treatment (i.e. after three chemotherapy cycles) and after the end of treatment. [Figure 8]On-treatment blood samples were not taken at start-up but have only recently been added to the protocol (samples after the third chemotherapy cycle). Therefore, the number of patients with on-treatment TK1 values ​​available is limited, which is reflected in the available pre / post-treatment TK1 ratios. [Figure 9a] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma with a cutoff of 0.25 μg / L in relation to time to recurrence, days. [Figure 9b] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma with a cutoff of 0.35 μg / L in relation to time to recurrence, days. [Figure 9c] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma with a cutoff of 0.45 μg / L in relation to time to recurrence, days. [Figure 9d] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma with a cutoff of 0.75 μg / L in relation to time to recurrence, days. [Figure 9e] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma with a cutoff of 1.05 μg / L in relation to time to recurrence, days. [Figure 9f] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma with a cutoff of 2.5 μg / L in relation to time to recurrence, days. [Figure 10a] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pretreatment in patients with diffuse large B-cell lymphoma at a cutoff of 0.45 μg / L in relation to time to recurrence, days. All patients. [Figure 10b] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pretreatment in patients with diffuse large B-cell lymphoma with a cutoff of 0.45 μg / L in relation to time to recurrence, days. Patients aged 66 years or older. [Figure 10c] Kaplan-Meier plot of the probability of disease recurrence for serum concentrations of TK1 at diagnosis and pretreatment in patients with diffuse large B-cell lymphoma with a cutoff of 0.45 μg / L in relation to time to recurrence, days. Patients < 67 years of age. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention relates generally to the measurement of thymidine kinase 1 (TK1), and in particular to the prediction of cancer recurrence based on measured serum TK1 (STK1) levels.

[0016] The present invention predicts cancer recurrence based on the measured STK1 levels and comparison with age-dependent thresholds. Experimental data presented herein show that higher STK1 concentrations correlated with further progression of the stage of diffuse large B-cell lymphoma (DLBCL) as an example of hematological cancer, and that for the entire patient cohort, elevated pre-treatment STK1 levels correlated with shorter disease-free and disease-specific survival. However, when this patient cohort was divided into groups according to the age of the patient at diagnosis, a different story emerged. For elderly patients, there was no proportional correlation between the measured STK1 levels and the risk of cancer recurrence. In clear contrast, a relatively low increase in STK1 levels above the mean level of healthy individuals was already associated with a high risk of cancer recurrence. Moreover, there was no significant difference in the risk of cancer recurrence between an elderly patient with STK1 levels slightly above the defined threshold and another elderly patient with STK1 levels significantly above the defined threshold. However, this situation was different in younger DLBCL patients, where higher STK1 levels correlated with a higher risk of cancer recurrence compared with older DLBCL patients.

[0017] Therefore, the accuracy of using STK1 as a biomarker for hematological cancers is significantly improved by using age-related criteria or threshold ranges for patient groups of different ages.

[0018] An embodiment of the present invention relates to a method for predicting cancer recurrence, comprising measuring the level of serum thymidine kinase 1 (STK1) substance in a body sample of a patient diagnosed with hematological cancer using an antibody or fragment thereof that specifically binds to a serotype of human TK1. The method also comprises comparing the measured level of STK1 substance to a threshold value selected based on the age of the patient. The method further comprises predicting the recurrence of cancer in the patient based on the comparison.

[0019] Human TK1 protein exists in various forms depending on the presence of certain molecules, e.g., the presence or absence of adenosine triphosphate (ATP), depending on the concentration of the protein, i.e., high or low, depending on the type of the protein, e.g., native or recombinant TK1, and depending on the location of the protein, i.e., serum or cytoplasm.

[0020] Generally, cytoplasmic and recombinant human TK1 occur as tetramers in the presence or high concentration of ATP, and as dimers in the absence or low concentration of ATP. The tetrameric forms of cytoplasmic and recombinant human TK1 have high TK1 activity, while the dimeric forms have lower TK1 activity. Cytoplasmic TK1, also called cellular TK1, is the TK1 present inside cells and can be isolated from such cells.

[0021] In stark contrast, human STK1 may have TK1 activity in the form of high molecular weight complexes, such as oligomers, or in the form containing such oligomers, and dimer and tetramer forms may have very low or no TK1 activity.This oligomerization is thought to be associated with the formation of disulfide bridges that occur in blood.STK1 is found in the blood of patients, and can therefore be measured in blood samples, plasma samples, or serum samples, among others.

[0022] As used herein, STK1 substance refers to various forms of STK1, such as dimer, tetramer, oligomer and complex containing STK1.The STK1 substance is the STK1 substance present in the blood, plasma or serum of a patient.The STK1 substance may further include STK1 in the above-mentioned forms, such as dimer, tetramer, oligomer and complex containing STK1.The STK1 substance also includes at least one TK1 protein unit and complex with other molecules and / or macromolecules.

[0023] In the art, various gene expression arrays have been proposed for measuring TK1 messenger ribonucleic acid (mRNA) transcripts in cancer cell samples and biopsies, including lymph node biopsy samples.As mentioned above, TK1 is available in various forms in subjects, including cytoplasmic TK1 and serum TK1.The gene expression arrays that measure TK1 mRNA transcripts from such biopsy samples mainly measure the TK1 mRNA transcripts of cytoplasmic TK1 present in cancer cells.Therefore, such gene expression arrays cannot be used to measure the level of STK1 substance in subjects.

[0024] In an embodiment, measuring the level of the STK1 agent comprises contacting the body sample with the antibody or fragment thereof. This embodiment also comprises measuring the amount of antibody or fragment thereof bound to the STK1 agent.

[0025] Contacting the body sample with the antibody or fragment thereof can be accomplished by adding the antibody or fragment thereof to the body sample and incubating the body sample with the antibody or fragment thereof, whereby the antibody or fragment thereof binds to the STK1 substance and a complex is formed between the antibody or fragment thereof and the STK1 substance. In such an embodiment, measuring the amount of the antibody or fragment thereof bound to the STK1 substance can include measuring or quantifying the amount of the antibody or fragment thereof bound to the STK1 substance by measuring or quantifying the complex between the antibody or fragment thereof and the STK1 substance.

[0026] In embodiments, the method also includes correlating the measured amount of the antibody or fragment thereof bound to the STK1 agent to the level of the STK1 agent. This can be done using a predetermined correlation between the measured amount of the antibody or fragment thereof bound to a reference TK1 agent and the concentration of the reference TK1 agent. An exemplary reference TK1 agent that can be used in generating such a predetermined correlation is recombinant human TK1.

[0027] Thus, the predetermined correlation can be generated by adding the antibody or fragment thereof to different samples containing different concentrations of the reference TK1 substance, preferably recombinant human TK1, and then measuring the amount of the antibody or fragment thereof bound to the reference TK1 substance, preferably recombinant human TK1, in the different samples to obtain a standard curve, function or relationship between the concentration of the reference TK1 substance, preferably recombinant human TK1, and the measured amount of the antibody or fragment thereof bound to the reference TK1 substance, preferably recombinant human TK1.

[0028] This predetermined correlation, e.g., a standard curve, function or relationship, can then be used to map or convert the measured amount of the antibody or fragment thereof bound to the STK1 substance in the body sample to the concentration of the STK1 substance contained in the body sample.

[0029] For measuring the level of STK1 substance in the body sample of a patient diagnosed with hematological cancer, it is generally preferred to use the same type of antibody or its fragment to generate the predetermined correlation.Therefore, in a preferred embodiment, the antibody or its fragment can specifically bind not only to the serotype of human TK1, but also to the reference TK1 substance, preferably recombinant human TK1.

[0030] In embodiments, the body sample is treated prior to or during incubation of the body sample with the antibody or fragment thereof, which sample treatment may be used to stabilize selected STK1 forms contained in the body sample and / or to split larger STK1 complexes or oligomers into smaller complexes or multimers.

[0031] Thus, in embodiments, a sample dilution or pretreatment buffer is added to the body sample prior to or in conjunction with adding the antibody or fragment thereof to the body sample, preferably prior to adding the antibody or fragment thereof to the body sample.

[0032] In an embodiment, the sample dilution buffer comprises ATP, preferably at a concentration selected within the interval of 0.5 mM to a maximum of 50 mM, for example 0.5 mM to a maximum of 20 mM or 1.5 mM to a maximum of 50 mM. As described herein above, ATP stabilizes the tetrameric form of TK1, which has a high enzymatic TK1 activity.

[0033] In another embodiment, the dilution buffer of the sample contains a reducing agent, which may then cleave disulfide bridges contained in larger STK1 complexes and oligomers, resulting in smaller STK1 forms, such as tetramers. Various reducing agents capable of cleaving disulfide bonds, including but not limited to dithioerythritol (DTE), dithiothreitol (DTT), dithiobutylamine (DTBA), tris(2-carboxyethyl)phosphine (TCEP), and combinations thereof, may be used according to this embodiment. The amount of the reducing agent is typically selected within the interval of 0.1 mM to a maximum of 10 mM.

[0034] The sample dilution buffer, in embodiments, contains both ATP and a reducing agent.

[0035] In an embodiment, the level of the STK1 substance is measured in association with the diagnosis of the patient with blood cancer, or at least before the start of the treatment of the blood cancer, by using the antibody or its fragment that specifically binds to the serotype of human TK1 contained in the body sample taken from the patient.Therefore, in a preferred embodiment, the body sample is preferably taken at the time of diagnosis, or at least immediately after the time when the patient is diagnosed with blood cancer or at least suspected to suffer from blood cancer.

[0036] In embodiments, the method includes estimating a hazard ratio (HR) for the patient based on a comparison of the measured level of STK1 agent to a selected threshold. In such embodiments, predicting recurrence of cancer in the patient includes predicting recurrence of cancer in the patient based on the estimated HR.

[0037] In embodiments, predicting cancer recurrence includes predicting a high risk of cancer recurrence for the patient when the level of STK1 substance measured in the body sample exceeds a selected threshold, and predicting a low risk of cancer recurrence for the patient in other cases.

[0038] In an embodiment, the method also includes selecting a threshold value based on an agent of the patient.

[0039] In certain embodiments, the method includes selecting a first threshold value if the patient's age is greater than or equal to a specified age, and selecting a second, different threshold value if the patient's age is less than a specified age.

[0040] Thus, in this particular embodiment, two different age-optimized thresholds are employed, with the particular age-optimized threshold used depending on whether the patient's age is equal to or greater than a specified age, or less than a specified age.

[0041] In embodiments, the second different threshold is higher than the first threshold, thus in embodiments a higher threshold is used for younger patients compared to older patients.

[0042] In an exemplary but currently preferred embodiment, the predefined age is 67 years of age. This means that if the patient is 67 years of age or older, a first threshold value is used for the comparison with the measured level of STK1 substance, whereas if the patient is younger than 67 years of age, a second, preferably higher, threshold value is used for the comparison.

[0043] As shown in Table 5, the choice of a threshold in the range of 0.35 μg / L to 0.60 μg / L, and in particular 0.45 μg / L, allowed for the differentiation of patients with a high hazard ratio for recurrence and therefore a high risk for cancer recurrence from those with a low hazard ratio for recurrence for patients aged 67 years or older. In fact, the use of a higher threshold actually reduced the p-value for distinguishing between high and low hazard ratios. This is in clear contrast to Table 6, which shows the corresponding hazard ratios for recurrence for patients younger than 67 years. For such younger patients, a higher threshold, at least up to 1.05 μg / L, was associated with a reduced p-value for distinguishing between high and low hazard ratios. Thus, in older patient groups (age ≧67 years), the optimal threshold for predicting cancer recurrence is in the range of 0.35 μg / L to 0.60 μg / L, in particular 0.45 μg / L, whereas in younger patient groups (age <67 years), the optimal threshold is greater than 0.75 μg / L and less than 2.5 μg / L, e.g., about 1 μg / L.

[0044] The experimental data presented herein shows that in elderly patients, the increase in STK1 level is almost two-fold, and the hazard ratio is significantly increased.Furthermore, in elderly patients, the hazard ratio is only moderately increased at higher STK1 levels.This means that in this elderly patient group, higher STK1 levels are not proportionally associated with the high risk of cancer recurrence in elderly patients.This discovery requires that the STK1 assay, and in particular the sensitivity and robustness of the STK1 assay, be able to detect relatively low STK1 levels in biological samples and distinguish such low STK1 levels from the corresponding STK1 levels in healthy individuals.

[0045] The antibody or fragment thereof specifically binds to the STK1 agent, and in particular to the serotype of the STK1 protein.

[0046] The specificity of an antibody or fragment thereof can be determined based on affinity and / or avidity. The equilibrium constant for the dissociation of an antibody or fragment thereof with an antigen (Kd Affinity, expressed as K, is a measure of the binding strength between an antigenic determinant and the antigen-binding site of the antibody or fragment thereof. d The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antibody or fragment thereof. Alternatively, the affinity can be expressed as 1 / K d The affinity constant (K a As will be apparent to those skilled in the art, affinity can be determined in a manner known per se depending on the particular antigen of interest.

[0047] Avidity is a measure of the binding strength between an antibody or fragment thereof and an associated antigen. Avidity is related to both the affinity between an antigenic determinant and the antigen-binding site of the antibody or fragment thereof, and the number of associated binding sites present on the antibody or fragment thereof.

[0048] Generally, antibodies have a dissociation constant (K d )10 -5 ~10 -12 moles / liter (M) or less, and preferably less than 10 -7 ~10 -12 M or less, and more preferably, 10 -8 ~10 -12 M, i.e., the binding constant (K a )10 5 ~10 12 M -1 More than 10, and preferably 7 ~10 12 M -1 More preferably, 10 8 ~10 12 M -1 Combine with.

[0049] In general, 10 -4 Any K greater than M d value (or 10 4 M -1 Any K less than aValues ​​below 500 nM are generally considered to indicate non-specific binding. Preferably, the antibody or fragment thereof binds to the STK1 substance with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, e.g., less than 5 nM or lower, e.g., less than 1 nM.

[0050] Specific binding of an antibody or fragment thereof to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as various modifications thereof known per se in the art.

[0051] In an embodiment, the antibody is a monoclonal antibody, i.e., a monoclonal anti-TK1 antibody. In another embodiment, the antibody is a polyclonal antibody, i.e., a polyclonal anti-TK1 antibody.

[0052] In an embodiment, the antibody or fragment thereof has specificity for an epitope or peptide consisting of an amino acid sequence from the C-terminal region of TK1, preferably human TK1.

[0053] The peptide is preferably selected from the portion of TK1 (SEQ ID NO: 28) extending from amino acid position 200 to the end of TK1, i.e., amino acid position 234 in humans. In a particular embodiment, the peptide is selected from the portion of the TK1 protein extending from amino acid position 205, preferably from 210 to amino acid position 230, preferably from 225.

[0054] The peptide is preferably an N-mer, where N is an integer in the range of 8 to 20, preferably in the range of 10 to a maximum of 15. The peptide preferably consists of N consecutive amino acids at the C-terminus of the TK1 protein.

[0055] In an embodiment, the peptide consists of the amino acid sequence: GEAVAARKLF (SEQ ID NO: 1). In another embodiment, the peptide consists of the amino acid sequence: NCPVPGKPGE (SEQ ID NO: 2). In a further embodiment, the peptide consists of the amino acid sequence: PVPGKPGEAV (SEQ ID NO: 3). In yet another embodiment, the peptide consists of the amino acid sequence: NCPVPGKPGEAV (SEQ ID NO: 4).

[0056] A monoclonal antibody having specificity for the epitope consisting of GEAVAARKLF (SEQ ID NO: 1) has a complementarity determining region 1 (CDR1) of a variable heavy (VH) domain having the amino acid sequence DYEMH (SEQ ID NO: 5), a CDR2 of a VH domain having the amino acid sequence AIHPGYGGTAYNQKFKG (SEQ ID NO: 6), a CDR3 of a VH domain having the amino acid sequence FITKFDY (SEQ ID NO: 7), a CDR1 of a variable light (VL) domain having the amino acid sequence KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a CDR2 of a VL domain having the amino acid sequence LVSKLDS (SEQ ID NO: 9), and a CDR3 of a VL domain having the amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0057] Monoclonal antibodies having specificity for the epitopes NCPVPGKPGE (sequence number 2), PVPGKPGEAV (sequence number 3) and NCPVPGKPGEAV (sequence number 4) have a VH domain CDR1 having the amino acid sequence DYEMH (sequence number 5), a VH domain CDR2 having the amino acid sequence AILPGSGGTAYNQKFKG (sequence number 11), a VH domain CDR3 having the amino acid sequence LITTFDY (sequence number 12), a VL domain CDR1 having the amino acid sequence KSSQSLLDSDGKTYLN (sequence number 13), a VL domain CDR2 having the amino acid sequence LVSKLDS (sequence number 9), and a VL domain CDR3 having the amino acid sequence WQGTHFPWT (sequence number 10).

[0058] In another embodiment, the antibody or fragment thereof has specificity for a conformation-dependent epitope of human TK1. The monoclonal antibody having specificity for such a conformation-dependent epitope has a VH domain CDR1 having the amino acid sequence SGYSWH (SEQ ID NO: 14), a VH domain CDR2 having the amino acid sequence YIHYSGSTTYNPSLKG (SEQ ID NO: 15), a VH domain CDR3 having the amino acid sequence WGTGHWYFDV (SEQ ID NO: 16), a VL domain CDR1 having the amino acid sequence RSSTGAVTTTNYAN (SEQ ID NO: 17), a VL domain CDR2 having the amino acid sequence GTNNRVP (SEQ ID NO: 18), and a VL domain CDR3 having the amino acid sequence ALWYSNHWV (SEQ ID NO: 19).

[0059] The above-presented three examples of monoclonal anti-TK1 antibodies that can be used according to the embodiments are further disclosed in WO2015 / 094106, the teachings of which regarding monoclonal anti-TK1 antibodies are incorporated herein by reference.

[0060] Thus, in an embodiment, the monoclonal antibody or fragment thereof is selected from the group consisting of a monoclonal antibody or fragment thereof having specificity for GEAVAARKLF (sequence number 1) of human TK1, a monoclonal antibody or fragment thereof having specificity for at least one of NCPVPGKPGE (sequence number 2), PVPGKPGEAV (sequence number 3) and NCPVPGKPGEAV (sequence number 4) of human TK1, and a monoclonal antibody or fragment thereof having specificity for a conformation-dependent epitope of human TK1.

[0061] In another embodiment, the antibody or fragment thereof has specificity for an epitope or peptide consisting of KPGEAVAARKLFAPQ (SEQ ID NO: 20). At least one additional amino acid, e.g., a cysteine ​​residue, may be added to the N-terminus or C-terminus of the peptide, preferably the N-terminus, for use as coupling to other molecules, e.g., carrier proteins.

[0062] Antibodies with specificity for this epitope are further disclosed in WO95 / 29192, the teachings of which regarding anti-TK1 antibodies are incorporated herein by reference.

[0063] In a further embodiment, the antibody or fragment thereof has specificity for an epitope or peptide consisting of an amino acid sequence from the active site of TK1. The peptide is preferably selected from a portion of TK1 spanning from amino acid position 150 to amino acid position 190 of human TK1. In a particular embodiment, the peptide is selected from a portion of TK1 spanning from amino acid position 155, preferably 160 and more preferably 161, to amino acid position 185, preferably 183.

[0064] The peptide is preferably an M-mer, where M is an integer ranging from 10 to a maximum of 40, preferably ranging from 20 to a maximum of 30, and more preferably 23 or 24. The peptide preferably consists of M consecutive amino acids in the active site of the TK1 protein.

[0065] At least one additional amino acid, eg, a cysteine ​​residue, may be added to the N-terminus or C-terminus of the peptide, preferably the N-terminus, for use as coupling to other molecules, eg, carrier proteins.

[0066] In an embodiment, the peptide consisting of an amino acid sequence from the active site of TK1 has an amino acid sequence corresponding to amino acid positions 161 to 183 of human TK1, that is, the amino acid sequence of AYTKRLGTEKEVEVIGGADKYHS (SEQ ID NO: 21).

[0067] Antibodies with specificity for this epitope are further disclosed in WO2008 / 142664, the teachings of which regarding anti-TK1 antibodies are incorporated herein by reference.

[0068] In a further embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof disclosed in WO2019 / 201901, the teachings of which regarding monoclonal anti-TK1 antibodies are incorporated herein by reference.

[0069] For example, the monoclonal antibody can be mAb 6C6, mAb 4H4, or mAb 23C11.

[0070] mAb 6C6 VH Domain (SEQ ID NO:22): METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCTASRFFSSSSYWICWVRQAPGKGLEWIACIYAGDSGSSYYASWAKGRFTVSKTSSTTVTLQTTSLTAADTATYFCARASVGAAYDYFALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG

[0071] mAb 6C6 VL Domain (SEQ ID NO:23): MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAMGGTVTIKCQASEDVSSHLAWYQQRPGQPPKLLIYGASDLASGVPSRFTGSGSGTQFTLAISDLECADAATYYCQGYYYISD SPYVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0072] mAb 4H4 VH Domain (SEQ ID NO:24): METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSGYDMCWVRQTPGKGLEWIACISVDSDGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGYESSSGVYIPYFTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSG

[0073] mAb 4H4 VL Domain (SEQ ID NO:25): MDMRAPTQLLGLLLLWLPGARCADIVLTQTPASVEAAVGGTVTIKCQASQSIYSYLAWYQHKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQHYYYSST SGGGVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0074] mAb 23C11 VH Domain (SEQ ID NO:26): METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGIIYGDDNTYCANWTKGRFTISKTSTTVDLTITSPTTEDTATYFCARGPDYIAAKMDIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSG

[0075] mAv 23C11 VL Domain (SEQ ID NO:27): MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSISGYLSWYQQKPGQRPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGSSTF SSYGNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0076] In an embodiment, the level of STK1 substance contained in the body sample is measured using a kit. The kit preferably comprises a first antibody or a first fragment thereof and a second antibody or a second fragment thereof. The first and second antibodies can be selected from the above-mentioned exemplary monoclonal and polyclonal anti-TK1 antibody examples.

[0077] In a particular embodiment, the kit comprises a first monoclonal antibody or a first fragment thereof having specificity for an epitope selected from the group consisting of i) GEAVAARKLF (SEQ ID NO:1) of human TK1, ii) at least one of NCPVPGKPGE (SEQ ID NO:2), PVPGKPGEAV (SEQ ID NO:3), and NCPVPGKPGEAV (SEQ ID NO:4) of human TK1, and iii) a conformation-dependent epitope of human TK1. The kit also comprises a second monoclonal antibody or a second fragment thereof having specificity for an epitope selected from the group consisting of i) GEAVAARKLF (SEQ ID NO:1) of human TK1, ii) at least one of NCPVPGKPGE (SEQ ID NO:2), PVPGKPGEAV (SEQ ID NO:3), and NCPVPGKPGEAV (SEQ ID NO:4) of human TK1, and iii) a conformation-dependent epitope of human TK1.

[0078] In an embodiment, the first antibody or a first fragment thereof is a so-called capture antibody immobilized or intended to be immobilized on a support, and the second antibody or a second fragment thereof is a so-called detection antibody. In another embodiment, the second antibody or a second fragment thereof is a capture antibody immobilized or intended to be immobilized on a support, and the first antibody or a first fragment thereof is used as a detection antibody.

[0079] In an embodiment, the first and second antibodies or the first and second fragments have specificity for different epitopes in the STK1 agent.

[0080] In another embodiment, the first and second antibodies or the first and second fragments have specificity for the same epitope in the STK1 substance.This can occur because the same epitope can be present in multiple copies in a high molecular weight complex of multiple TK1 protein units.Therefore, the STK1 substance can be a multivalent complex of multiple, i.e. at least two, TK1 protein units.In practice, the same type of antibody or fragment thereof can be used as the first and second antibodies or the first and second fragments.

[0081] In an embodiment, one of the first and second antibodies or the first and second fragments has specificity for a peptide consisting of an amino acid sequence from the active site of TK1, and the other of the first and second antibodies or the first and second fragments has specificity for a peptide consisting of an amino acid sequence from the C-terminal region of TK1.

[0082] In another embodiment, one of the first and second antibodies or the first and second fragments has specificity for a peptide consisting of a first amino acid sequence from the C-terminal region of TK1, and the other of the first and second antibodies or the first and second fragments has specificity for a peptide consisting of the first amino acid sequence from the C-terminal region of TK1 or a second, different amino acid sequence from the C-terminal region of TK1.

[0083] In a further embodiment, one of the first and second antibodies or the first and second fragments has specificity for a peptide consisting of a first amino acid sequence from the C-terminal region of TK1, and the other of the first and second antibodies or the first and second fragments has specificity for a conformation-dependent epitope of human TK1.

[0084] In yet another embodiment, one of the first and second antibodies or the first and second fragments has specificity for a peptide consisting of an amino acid sequence from the active site of TK1, and the other of the first and second antibodies or the first and second fragments has specificity for a conformation-dependent epitope of human TK1.

[0085] As used herein, an antibody fragment includes a single chain antibody, an Fv fragment, an scFv fragment, an Fab fragment, an F(ab') 2 The fragment may be selected from the group consisting of a Fab' fragment, a Fd fragment, a single domain antibody (sdAb), a scFv-Fc fragment, a di-scFv fragment and a CDR region.

[0086] In an embodiment, the kit is a sandwich assay kit. In a particular embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit, preferably a sandwich ELISA.

[0087] In the discussion that follows, the first antibody or first fragment is assumed to be a capture antibody and the second antibody or second fragment acts as a detection antibody, although the embodiment is not so limited and the capture and detection antibodies can be interchanged.

[0088] Sandwich ELISA can be used to detect STK1 substances contained in body samples by preparing a support, for example a surface of a solid support, and binding the first antibody or the first fragment thereto as a so-called capture antibody. In a preferred embodiment, a known amount of the first antibody or the first fragment is bound to the surface of the support. Any non-specific binding sites on the surface are optionally, but preferably, blocked. The body sample is then placed on the surface, and any STK1 substances present therein are captured by the immobilized first antibody or first fragment. Unbound substances are preferably removed by one or more washing steps. Then, a second antibody or a second fragment, usually called a detection antibody, is added and bound to any STK1 substances captured by the first antibody or the first fragment.

[0089] Then, the amount of the second antibody or the second fragment bound is measured by direct or indirect detection method.For example, label or enzyme can be attached to the second antibody or the second fragment directly or indirectly through binding, for example, biotin-streptavidin or biotin-avidin binding.Alternatively, it is also possible to use the second antibody or the second fragment that is labeled or bound to enzyme and specifically binds to the second antibody or the second fragment.

[0090] Thus, in embodiments, the second antibody or second fragment has a covalently attached biotin. Alternatively, the second antibody or second fragment has a covalently attached streptavidin or avidin.

[0091] The kit preferably also comprises horseradish peroxidase (HRP)-labeled streptavidin or HRP-labeled avidin. Alternatively, the kit also comprises HRP-labeled biotin. The kit also comprises a substrate for HRP, such as 3,3',5,5'-tetramethylbenzidine (TMB) substrate, 3,3'-diaminobenzidine (DAB) substrate or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. In this case, the level of STK1 substance contained in the sample can be measured by spectrophotometry, which detects the conversion of the chromogenic substrate by HRP into a detectable colored product.

[0092] In embodiments, the kit also comprises a microtiter plate (MCP) as a support on which the first antibody or first fragment is immobilized or intended to be immobilized.

[0093] In an embodiment, one of the first antibody or first fragment and the second antibody or second fragment has specificity for an epitope consisting of GEAVAARKLF (SEQ ID NO: 1) and has a CDR1 of a VH domain having the amino acid sequence DYEMH (SEQ ID NO: 5), a CDR2 of a VH domain having the amino acid sequence AIHPGYGGTAYNQKFKG (SEQ ID NO: 6), a CDR3 of a VH domain having the amino acid sequence FITKFDY (SEQ ID NO: 7), a CDR1 of a VL domain having the amino acid sequence KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a CDR2 of a VL domain having the amino acid sequence LVSKLDS (SEQ ID NO: 9), and a CDR3 of a VL domain having the amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0094] In a particular embodiment, the other of the first antibody or first fragment and the second antibody or second fragment has specificity for the epitopes NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3) and NCPVPGKPGEAV (SEQ ID NO: 4), and has a CDR1 of a VH domain having the amino acid sequence DYEMH (SEQ ID NO: 5), a CDR2 of a VH domain having the amino acid sequence AILPGSGGTAYNQKFKG (SEQ ID NO: 11), a CDR3 of a VH domain having the amino acid sequence LITTFDY (SEQ ID NO: 12), a CDR1 of a VL domain having the amino acid sequence KSSQSLLDSDGKTYLN (SEQ ID NO: 13), a CDR2 of a VL domain having the amino acid sequence LVSKLDS (SEQ ID NO: 9), and a CDR3 of a VL domain having the amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0095] The kit does not necessarily have to be an ELISA kit. In another embodiment, the kit uses affinity chromatography, in which the first antibody is bound to the stationary phase, for example, a gel matrix or beads contained in a column. For example, the gel matrix or beads can be made of agarose, for example, SEPHAROSE®.

[0096] In this case, the STK1 substance present in the body sample is captured on the column through binding to the immobilized first antibody or first fragment.After washing, the bound STK1 substance can be eluted and detected using the second antibody or second fragment.For example, the amount of eluted STK1 substance can be measured using Western blotting, and using direct or indirect detection methods with the second antibody or second fragment for detecting STK1.

[0097] The support may alternatively be a magnetic bead, for example, DYNABEADS® magnetic beads.

[0098] In a further embodiment, the kit is a chemiluminescence immunoassay (CLIA) kit. CLIA is an immunoassay technique in which the label is a luminescent molecule. CLIA methods can be direct, using luminophore markers, or indirect, using enzyme markers. Both methods can be competitive or non-competitive. In direct CLIA methods, the luminophore markers used are usually acridinium and ruthenium esters, while the enzyme markers used in indirect methods are usually alkaline phosphatase with adamantyl 1,2-dioxetane aryl phosphate (AMPPD) as substrate, and HRP with luminol or its derivatives as substrate.

[0099] The kit does not necessarily have to include two antibodies or fragments, but instead may include only one type of antibody or fragment.

[0100] Moreover, the kit does not necessarily have to include a so-called capture antibody or fragment. In clear contrast, multiple, i.e. at least two different, antibodies or fragments can be used to measure the level of the STK1 substance without the need for immobilization of at least one of the antibodies or fragments.

[0101] In an embodiment, the method also includes selecting an anti-cancer treatment for the patient based on the predicted risk of cancer recurrence. Thus, an optimal or at least suitable anti-cancer treatment is selected for a blood cancer patient based on the level of STK1 substance contained in the measured body sample, and thus based on the predicted risk of cancer recurrence estimated for the patient based on the comparison of the level of STK1 substance contained in the measured body sample with a selected threshold value. This means that a more aggressive anti-cancer treatment can be selected for a patient whose measured STK1 substance level is higher than a selected threshold value, and thus a high risk of cancer recurrence is predicted, compared to a patient whose STK1 substance level is lower than the threshold value, and thus a low risk of cancer recurrence is predicted. Examples of anti-cancer treatments that can be selected include one or more of symptomatic treatments, such as blood transfusion, chemotherapy, radiation therapy, immunotherapy, and bone marrow transplantation.

[0102] For example, a first anti-cancer treatment may be selected for patients having measured levels of STK1 substance that exceed the selected threshold referenced above, while a second, different anti-cancer treatment is selected for other patients having measured levels of STK1 substance that are below the selected threshold.

[0103] In an embodiment, the method comprises selecting a patient surveillance schedule for the patient based on the predicted risk of cancer recurrence of the patient. Thus, an optimal or at least suitable patient surveillance schedule or scheme is selected for the patient based on the comparison of the level of STK1 substance contained in the measured body sample with the selected threshold value, and thus based on the predicted risk of cancer recurrence estimated for the patient based on the level of STK1 substance contained in the measured body sample. This means that for patients whose measured STK1 substance level is higher than the selected threshold value and thus predicted to have a high risk of cancer recurrence, more frequent surveillance and follow-up (first surveillance schedule) can be selected, whereas patients whose measured STK1 substance level is lower than the selected threshold value and thus predicted to have a low risk of cancer recurrence can instead follow less frequent surveillance and follow-up (second surveillance schedule). EXAMPLES

[0104] Serum levels of thymidine kinase 1 (TK1) from 146 patients (64 females, 82 males) with diffuse large B-cell lymphoma (DLBCL) and treated with R-CHOP were measured by immunoassay before, during, and after treatment. Serum TK1 concentrations were found to be significantly elevated in lymphoma patients compared to TK1 concentrations in healthy controls (p<0.00001). Higher concentrations correlated with more advanced disease stage and higher International Prognostic Index (IPI) (p<0.00001). Elevated TK1 before treatment correlated with shorter survival (p<0.001). Overall, TK1 concentrations increased approximately 10-fold during treatment and then declined. This change was most pronounced in stage 1 and stage 2 patients. In conclusion, TK1 measured by immunoassay may correlate with DLBCL characteristics and outcomes and may provide an important reference value for treatment evaluation.

[0105] Materials and Methods Study design and setup Serum and clinical data of 146 patients diagnosed with DLBCL between 2010 and 2016 were obtained from the Uppsala-Umea Comprehensive Cancer Consortium (U-CAN). The core of the U-CAN project is to establish a longitudinal collection of data, blood and tissue samples with informed consent from cancer patients for future research. Within the framework of U-CAN, blood samples are taken at several defined time points during the course of cancer diagnosis and subsequent treatment for biobanking. For patients with DLBCL, these time points are defined as at diagnosis (or at least before the start of treatment), during treatment (i.e. after three treatment cycles), 3 months and 1 year after the end of treatment, and at the time of relapse. Blood samples during treatment were not included at the launch of U-CAN, but were added to the protocol very recently. All patients included in the study were treated with the standard chemotherapy regimen R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or equivalent with curative intent, administered at 14- or 21-day intervals.

[0106] Baseline data were obtained via U-CAN and consisted of age at diagnosis, sex, Ann Arbor stage, B-symptoms (fever >38°C, night sweats, weight loss >10% in the last 6 months), bulky disease (defined as tumor diameter >7.5 cm), Eastern Cooperative Oncology Group performance status (ECOG-PS), International Prognostic Index (IPI) score, age-adjusted IPI (aaIPI), type of treatment, and treatment outcomes defined according to the International Response Criteria (Cheson et al., Revised Response Criteria for Malignant Lymphoma. J Clin Oncol. 2007;25:579-586).

[0107] TK1 was measured in serum by immunoassay (AroCell TK 210 ELISA) provided by AroCell AB, Uppsala, Sweden. The assay was performed according to the manufacturer's instructions. The imprecision of the assay was less than 10% standard deviation (CV) as determined by measuring duplicate samples. The lower limit of quantification, i.e., CV% exceeded 20% for duplicates, was 0.20 μg / L (Kumar et al., AroCell TK 210 ELISA for determination of TK1 protein: age-related reference ranges and comparison with other TK1 assays. BioTechniques. 2020; btn-2019-0148.41).

[0108] Uninfected, apparently healthy controls were included in the study. Fifty-seven men (median age 42 years, IQ range 29.7-52.2 years) and 88 women (median age 45.5 years, IQ range 34.5-57.5 years).

[0109] Statistics Disease-free survival (DFS) was calculated from the date of diagnosis to documented recurrence or latest follow-up. Patients who did not achieve complete remission were considered to have DFS day zero. Disease-specific survival (DSS) was calculated from the date of diagnosis to the date of death with residual lymphoma or latest follow-up. Overall survival (OS) was calculated from the date of diagnosis to the date of death or latest follow-up.

[0110] Non-parametric statistics were used throughout the examples unless otherwise stated. The Mann-Whitney U test was used for comparison of two groups, and the Kruskal-Wallis analysis of variance was used for comparisons of more than two groups. Paired samples were compared using the Wilcoxon test. p values ​​are indicated in the text or figures, and p<0.05 was considered significant. Correlations between variables were calculated by Spearman rank. Estimates of DFS, DSS and OS were calculated by Kaplan-Meier statistics and associated log-rank tests. The Cox proportional hazards test was used for multivariate time-dependent analyses. The statistical program Medcalc (MedCalc Statistical Software version 19.2.6 (MedCalc Software Ltd, Ostend, Belgium; https: / / www.medcalc.org; 2020) and the statistical program R version 3.4.3 (R Foundation for Statistical Computing; https: / / www.R-project.org / ) were used for all analyses.

[0111] ethics The study was approved by the Institutional Ethics Committee in Uppsala, Sweden (Dnr 2014 / 233). The study was also approved by the U-CAN Study Selection Committee. Written informed consent was therefore previously obtained within the U-CAN program.

[0112] result Patients and Treatment Patient characteristics are summarized in Table 1. Forty-four patients died during follow-up, including 31 who died of residual lymphoma. [Table 1] DLBCL: diffuse large B-cell lymphoma, TK1: thymidine kinase 1, n: number, ECOG: Eastern Cooperative Oncology Group, NA: not applicable, IPI: International Prognostic Index, aaIPI: age-adjusted IPI, GC: germinal centre

[0113] Relationship between serum TK1 and disease Pretreatment serum TK1 concentrations were significantly higher in lymphoma patients versus those in healthy controls, median 0.44 μg / L (IQ range 0.22-1.06 μg / L) versus median 0.22 μg / L (IQ range 0.18-0.29 μg / L), p<0.00001. Figure 1a shows the relationship between TK1 concentration and disease stage, with TK1 concentrations increasing with more advanced disease. The highest concentrations were found in stage 4 patients (p<0.00001). Figure 1b also shows a strong relationship with the International Prognostic Index (IPI); p<0.00001.

[0114] Relationship of TK1 to outcomes Time to disease recurrence (Fig. 2a) and time to death (Fig. 2b) were both significantly correlated with pretreatment serum TK1 concentrations (rs=-0.30 and rs=-0.29, respectively, p<0.001 for both correlations) and were shortest in patients with the highest concentrations. By dichotomizing pretreatment TK1 concentrations at the upper limit of normal, i.e., 0.45 μg / L, OS, DFS and DSS (Fig. 3a-3c), estimated by Kaplan-Meier and associated log-rank tests, became most favorable for patients with normal concentrations (p=0.03, p=0.0006 and p=0.0009, respectively). In multivariate analysis, dichotomized TK1 was also found to be a significant prognostic factor for DFS and DSS together with aaIPI and IPI, respectively (p<0.01 and p<0.025, respectively) (Fig. 4a-4d). Table 2 shows that patients with B-symptoms had significantly higher (p<0.0001) serum TK1 concentrations. [Table 2]

[0115] TK1 on treatment In a portion of patients (40 / 146, 27%), TK1 concentrations were measured during active treatment after three treatment cycles. Overall, concentrations increased approximately 10-fold during treatment (p<0.00001) (Figure 5) and then decreased from initial values ​​(Table 3). Patients whose disease relapsed (n=18) or who died from lymphoma (n=12) had significantly higher TK1 concentrations after treatment compared to patients who did not relapse or die (p=0.002 and p=0.0001, respectively) (Figure 6). Changes in TK1 concentrations during treatment for aaIPIs are shown in Table 3 and Figures 7a and 7b. The relative variation in TK1 concentrations between measurements at diagnosis compared to measurements after the third treatment cycle was 3832% in stages 1 and 2, compared to 302% in stages 3 and 4 (p=0.0008). The relative variation with respect to stage is also shown in FIG. [Table 3]

[0116] According to the protocol, blood samples were 1 At the time of diagnosis (or at least before treatment begins), 2 After three chemotherapy cycles (whether the treatment cycle was 14 days or 21 days long), and 3 Blood samples were taken 3 months and 1 year after the end of treatment and TK1 was analyzed.

[0117] Table 4 below summarizes the hazard ratios and calculated p-values ​​for recurrence in DLBCL patients using various cut-off values ​​ranging from 0.25 μg / L up to 2.5 μg / L for all patients. [Table 4]

[0118] Tables 5 and 6 list the corresponding hazard ratios and calculated p-values ​​for recurrence in DLBCL patients over 66 years of age (Table 5) or under 67 years of age (Table 6) using various cut-off values ​​ranging from 0.25 μg / L up to 2.5 μg / L for all patients. [Table 5] [Table 6]

[0119] Figure 9 shows Kaplan-Meier plots of the probability of disease recurrence at cut-offs of (Figure 9a) 0.25 μg / L, (Figure 9b) 0.35 μg / L, (Figure 9c) 0.45 μg / L, (Figure 9d) 0.75 μg / L, (Figure 9e) 1.05 μg / L and (Figure 9f) 2.5 μg / L for serum concentrations of TK1 at diagnosis and pre-treatment in patients with diffuse large B-cell lymphoma in relation to time to recurrence.

[0120] Figure 10 shows Kaplan-Meier plots of the probability of disease recurrence in patients with diffuse large B-cell lymphoma with a cutoff of 0.45 μg / L for serum concentrations of TK1 at diagnosis and pretreatment in relation to time to recurrence. Figure 10a shows the results for all patients, Figure 10b shows the results for patients over 66 years old, and Figure 10c shows the results for patients under 67 years old.

[0121] Consideration This study demonstrated the utility of TK1 measurement in patients with lymphoma, with a highly robust association with disease severity and outcome as evidenced by Kaplan-Meier curves and associated log-rank tests as well as multivariate analysis.

[0122] A finding in this study was an increase in serum TK1 during treatment. This increase was significantly higher in patients who experienced disease recurrence or death, but was also somewhat more pronounced in patients with low-stage lymphoma. In contrast, no clear differences were seen when comparing aaIPI 0-1 with aaIPI 2-3.

[0123] In this study, we observed an increase in serum TK1 and a clear complete remission of the disease in the majority of treated DLBCL patients. In this study, we did not observe a difference in negative events in the low TK1 response group, but we associated this with a lower overall number of events (relapse and / or death) among patients with available TK samples.

[0124] The results from Tables 5 and 6 show that in elderly patients, an almost two-fold increase in STK1 levels correlated with a large increase in the hazard ratio. Moreover, in elderly patients, the hazard ratio only increased moderately further at higher STK1 levels. This means that in this patient group, higher STK1 levels are not proportionally associated with a higher risk of cancer recurrence in elderly patients.

[0125] The above-described embodiments should be understood as some illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations, if technically possible.

Claims

1. A method for predicting cancer recurrence, comprising: measuring the level of serum thymidine kinase 1 (STK1) substance contained in a body sample of a patient diagnosed with blood cancer using an antibody or a fragment thereof that specifically binds to the serum type of human TK1; comparing the measured level of the STK1 substance with a threshold value selected based on the age of the patient; and predicting the recurrence of cancer in the patient based on the comparison. The method as described above.

2. Further comprising estimating a hazard ratio (HR) for the patient based on the comparison, and predicting the recurrence of cancer comprises predicting the recurrence of cancer in the patient based on the estimated HR. The method according to claim 1.

3. Predicting the recurrence of cancer comprises predicting a high risk of recurrence of cancer in the patient when the level of the STK1 substance measured in the body sample exceeds the selected threshold value, and predicting a low risk of recurrence of cancer in the patient in other cases. The method according to claim 1.

4. Further comprising selecting the threshold value based on the age of the patient. The method according to any one of claims 1 to 3.

5. Further, selecting a first threshold value when the age of the patient is above a specified age; and selecting a second different threshold value when the age of the patient is below the specified age The method according to any one of claims 1 to 3.

6. The method according to claim 5, wherein the second different threshold value is higher than the first threshold value.

7. The method according to claim 5, wherein the specified age is 67 years old.

8. The method according to any one of claims 1 to 3, wherein the blood cancer is selected from the group consisting of lymphoma, leukemia, and multiple myeloma, preferably, the blood cancer is lymphoma, and more preferably, the blood cancer is diffuse large B-cell lymphoma (DLBCL).

9. Measuring the level of the STK1 substance includes measuring the level of the STK1 substance contained in a serum sample or a plasma sample using an antibody or a fragment thereof that specifically binds to the serum type of the human TK1. The method according to any one of claims 1 to 3.

10. Measuring the level of the STK1 substance contained in the body sample contacting the body sample with the antibody or a fragment thereof, and measuring the amount of the antibody or a fragment thereof bound to the STK1 substance The method according to any one of claims 1 to 3, comprising:

11. The method according to claim 10, further comprising correlating the measured amount of the antibody or a fragment thereof bound to the STK1 substance with the level of the STK1 substance.

12. Correlating the measured amount of the antibody or fragment includes correlating the measured amount of the antibody or a fragment thereof with the level of the STK1 substance using a predetermined correlation between the measured amount of the antibody or a fragment thereof bound to recombinant human TK1 and the concentration of recombinant human TK1. The method according to claim 11.

13. Measuring the level of the STK1 substance includes measuring the level of the STK1 substance contained in a body sample collected from the patient using an antibody or a fragment thereof that specifically binds to the serum type of the human TK1, in relation to the diagnosis of a patient with the blood cancer or before the start of treatment of the blood cancer. The method according to any one of claims 1 to 3.

14. The method according to any one of claims 1 to 3, wherein the antibody or fragment thereof is a monoclonal antibody or fragment thereof that specifically binds to the serotype of the human TK1.

15. The monoclonal antibody or fragment thereof is a monoclonal antibody or fragment thereof having specificity for GEAVAARKLF (SEQ ID NO: 1) of human TK1, a monoclonal antibody or fragment thereof having specificity for at least one of NCPVPGKPGGE (SEQ ID NO: 2), PVPKPGGEAV (SEQ ID NO: 3), and NCPVPGKPGGEAV (SEQ ID NO: 4) of human TK1, and a monoclonal antibody or fragment thereof having specificity for a conformation-dependent epitope of human TK1 The method according to claim 14, which is selected from the group consisting of

16. The monoclonal antibody or fragment thereof is complementary determining region 1 (CDR1) of the variable heavy (VH) domain having the amino acid sequence of SEQ ID NO: 5, CDR2 of the VH domain having the amino acid sequence of SEQ ID NO: 6, CDR3 of the VH domain having the amino acid sequence of SEQ ID NO: 7, CDR1 of the variable light (VL) domain having the amino acid sequence of SEQ ID NO: 8, CDR2 of the VL domain having the amino acid sequence of SEQ ID NO: 9, and CDR3 of the VL domain having the amino acid sequence of SEQ ID NO: 10 The method according to claim 15, which has

17. The monoclonal antibody or fragment thereof is complementary determining region 1 (CDR1) of the variable heavy (VH) domain having the amino acid sequence of SEQ ID NO: 5, CDR2 of the VH domain having the amino acid sequence of SEQ ID NO: 11, CDR3 of the VH domain having the amino acid sequence of SEQ ID NO: 12, CDR1 of the variable light (VL) domain having the amino acid sequence of SEQ ID NO: 13, CDR2 of the VL domain having the amino acid sequence of SEQ ID NO: 9, and CDR3 of the VL domain having the amino acid sequence of SEQ ID NO: 10 The method according to claim 15, comprising:

18. The monoclonal antibody or fragment thereof, Complementary determining region 1 (CDR1) of the variable heavy (VH) domain having the amino acid sequence of SEQ ID NO: 14, CDR2 of the VH domain having the amino acid sequence of SEQ ID NO: 15, CDR3 of the VH domain having the amino acid sequence of SEQ ID NO: 16, CDR1 of the variable light (VL) domain having the amino acid sequence of SEQ ID NO: 17, CDR2 of the VL domain having the amino acid sequence of SEQ ID NO: 18, and CDR3 of the VL domain having the amino acid sequence of SEQ ID NO: 19 The method according to claim 15, comprising:

19. Measuring the level of the STK1 substance includes measuring the level of the STK1 substance contained in the body sample using a kit for measuring the level of the STK1 substance contained in the body sample, and the kit includes GEAVARKLF of human TK1 (SEQ ID NO: 1), At least one of NCPVPGKPGGE (SEQ ID NO: 2), PVPGKPGGEAV (SEQ ID NO: 3) and NCPVPGKPGGEAV (SEQ ID NO: 4) of human TK1, and Conformation-dependent epitope of human TK1 A first monoclonal antibody or a first fragment thereof having specificity for an epitope selected from the group consisting of GEAVARKLF of human TK1 (SEQ ID NO: 1), At least one of NCPVPGKPG (SEQ ID NO: 2), PVPKPGKGEAV (SEQ ID NO: 3), and NCPVPGKPGKGEAV (SEQ ID NO: 4) of human TK1, and a conformation-dependent epitope of human TK1 The method according to claim 15, comprising a second monoclonal antibody or a second fragment thereof having specificity for an epitope selected from the group consisting of.

20. The method according to claim 19, wherein one of the first monoclonal antibody or its first fragment and the second monoclonal antibody or its second fragment is immobilized on a solid support or is intended to be immobilized on the solid support.

21. The method according to claim 19, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

22. The method according to any one of claims 1 to 3, further comprising selecting an anti-cancer treatment for the patient based on the predicted cancer recurrence of the patient.

23. The method according to any one of claims 1 to 3, further comprising selecting a patient surveillance schedule for the patient based on the predicted cancer recurrence of the patient.

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