Methods and kits for identifying patients suitable for arginine deprivation therapy

Abstract

Disclosed herein is a method and a kit for identifying the responsiveness or non-responsiveness of a cancer patient to arginine deprivation therapy. The method comprises determining whether the G / G genotype of rs13338697 of a target nucleic acid exists in a biological sample derived from the patient by using a polymerase chain reaction (PCR)-based method, wherein the presence of the G / G genotype of rs13338697 of the target nucleic acid indicates that the patient will be responsive to arginine deprivation therapy.

Description

Technical Field

[0001] This disclosure relates to a method and kit for identifying tumor-associated single nucleotide polymorphisms (SNPs). Specifically, this disclosure relates to using the identified SNPs to predict whether patients with tumors will respond to arginine deprivation therapy. Background Technology

[0002] Some cancers may be unable to synthesize specific amino acids, leading to auxotrophic tumors. Therefore, one treatment for these tumors is to deprive them of specific exogenous amino acids, preventing them from replenishing the tumor's nutritional needs and thus causing tumor cell death. Arginine deprivation is a novel approach to treating tumors. Arginine can be degraded in the bloodstream by several enzymes, including arginine deiminase (ADI), achieving arginine deprivation. Although ADI is a microbial enzyme derived from mycoplasma, it still has a high affinity for arginine in the blood and can catalyze the synthesis of citrulline and ammonia from arginine. Citrulline can be recycled in normal cells expressing argininosuccinate synthase 1 (ASS1) to reform arginine, but tumor cells cannot synthesize arginine again, thus causing auxotrophic tumors and ultimately leading to tumor cell death. A PEGylated form of ADI (ADI-PEG 20) has been formulated, and clinical trials have shown that ADI-PEG20 can target arginine auxotrophic tumors via arginine deprivation therapy. Tumor resistance to arginine deprivation therapy is typically addressed through the reactivation of ASS1.

[0003] It is previously known that the expression of genes containing WW domain-containing oxidoreductase (WWOX) in tumors is associated with the inhibition of tumor growth and / or invasion. Furthermore, genetic variations in the human genome are associated with treatment responses and outcomes for specific cancers. For example, in patients with hepatocellular carcinoma (HCC), certain gene SNPs (such as the T / T genotype of rs9679162 in the GALNT14 gene and the A / G genotype of rs13338697 in the WWOX gene) are known to be associated with time-to-tumor progression (TTP) and overall survival (OS) after systemic chemotherapy (Lin et al., Asia Pac J. Clin. Oncol. 14(2):e54-e63 (2018)).

[0004] Therefore, there is an urgent need in the field of related technologies for a method and kit for identifying SNPs associated with the WWOX gene, where these SNPs are suitable predictive indicators for identifying cancer patients suitable for arginine deprivation therapy. Summary of the Invention

[0005] This disclosure is based, or at least in part, on the discovery that genotypes in the WWOX gene are associated with the responsiveness of tumors (e.g., HCC) in patients to pegylated forms of ADI (ADI-PEG20)-related therapies. Therefore, the genotypes of SNPs located at specific positions in the WWOX gene can predict whether patients with tumors will respond to arginine deprivation therapy.

[0006] In one aspect, this disclosure relates to a method for identifying patients with tumors (e.g., advanced HCC) based on their WWOX genotype, and whether they respond to arginine deprivation therapy.

[0007] Examples of tumors that respond to arginine deprivation therapy include, but are not limited to, breast cancer, brain tumors, colorectal cancer, squamous cell carcinoma of the head and neck, HCC, leukemia (e.g., acute myeloid leukemia (AML)), lung cancer, melanoma, mesothelioma (e.g., malignant pleural mesothelioma (MPM)), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma.

[0008] According to certain embodiments of this disclosure, the method includes determining whether a patient with HCC possesses the rs13338697 G / G genotype. Results provided by embodiments of the invention indicate that, among HCC patients, those carrying the rs13338697 G / G genotype are positively correlated with longer overall survival (OS) after receiving ADI-PEG 20 treatment; therefore, the presence of the rs13338697 G / G genotype suggests that the HCC patient will respond to arginine deprivation therapy.

[0009] The G / G genotype of rs13338697 can be determined by amplifying the target nucleic acid containing the aforementioned polymorphism sites using forward primers of sequence number 1 or 2 and reverse primer of sequence number 3.

[0010] According to embodiments of this disclosure, arginine deprivation therapy comprises administering to a patient an agent selected from the group consisting of difluoromethylornithine (DFMO), recombinant arginine deiminase (rADI), recombinant arginase (rArg), recombinant arginine decarboxylase (rADC), PEGylated rADI, rArg, or rADC, or combinations thereof. According to a preferred embodiment of this disclosure, HCC patients with the G / G genotype rs13338697 in the WWOX gene respond to arginine deprivation therapy, which comprises administering to the HCC patient a PEGylated rADI (e.g., ADI-PEG 20).

[0011] Alternatively, arginine deprivation therapy may further involve administering to patients arginine analogues, autophagy inhibitors, chemotherapy agents, MAPK (mitogen-activated protein kinase) kinase (MEK) inhibitors, tumor necrosis factor (TNF)-related apoptosis-inducing ligands (TRAIL), vitamins, or combinations thereof. Examples of autophagy inhibitors include, but are not limited to, bafilomycin A1, bortezomib, chloroquine (CQ), hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and quinacrine. Examples of chemotherapy agents include, but are not limited to, 5-fluororacil (5-FU), cisplatin, cytarabine, docetaxel, oxaliplatin, doxorubicin, methotrexate, and vincristine. Examples of arginine analogues include, but are not limited to, canavanine. Examples of MEK inhibitors include, but are not limited to, trametinib (GSK1120212), cobimetinib (XL518), binimetinib (MEK162), selumetinib, PD-325901, CI-1040, PD035901, TAK-733, and U0126. Examples of vitamins with similar effects include, but are not limited to, active folic acid.

[0012] Alternatively, the method may further include determining the T / T genotype of rs6025211, which is negatively correlated with TTP in HCC patients receiving arginine deprivation therapy. Therefore, the presence of the T / T genotype of rs6025211 indicates that the HCC patient will not respond to arginine deprivation therapy.

[0013] The T / T genotype of rs6025211 was determined by amplifying the target nucleic acid containing the aforementioned polymorphism site using the forward primer of sequence number 4 and the reverse primer of sequence number 5 or 6.

[0014] According to certain embodiments of this disclosure, arginine deprivation therapy comprises administering to a patient a combination of a PEGylated form of rADI (e.g., ADI-PEG 20), active folic acid, 5-FU, and oxaliplatin.

[0015] In another aspect, the present invention relates to a kit for predicting whether a patient with a tumor (e.g., HCC) will respond to or not respond to arginine deprivation therapy.

[0016] Examples of tumors that respond to arginine deprivation therapy include, but are not limited to, breast cancer, brain tumors, colorectal cancer, squamous cell carcinoma of the head and neck, HCC, leukemia (e.g., AML), lung cancer, melanoma, mesothelioma (e.g., MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma.

[0017] According to a preferred embodiment of this disclosure, the patient has HCC, and the kit contains a pair of primers for amplifying the target nucleic acid of an SNP containing the WWOX gene in a biosample taken from an HCC patient, wherein the SNP contains rs13338697. Specifically, the kit includes a first primer pair specifically for determining the G / G genotype of rs13338697, comprising a first forward primer with sequence number 1 or 2, and a first reverse primer with sequence number 3, wherein the presence of the G / G genotype of rs13338697 indicates that the HCC patient will respond to arginine deprivation therapy (e.g., PEGylated rADI).

[0018] Alternatively, the kit may further include additional primers suitable for amplifying the target nucleic acid containing the SNP rs6025211. Specifically, the kit further includes a second primer pair specifically for determining the T / T genotype of rs6025211, comprising a second forward primer with sequence number 4 and a second reverse primer with sequence number 5 or 6, wherein the presence of the T / T genotype of rs6025211 indicates that HCC patients will not respond to arginine deprivation therapy (e.g., PEGylated rADI or a combination of PEGylated rADI, active folic acid, 5-FU, and oxaliplatin).

[0019] The kit also includes a label or package insert located on or associated with the kit. Specifically, the label or package insert provides instructions on how to use the kit and indicates that the kit is based on the patient's WWOX genotype to identify whether cancer patients (e.g., patients with advanced HCC) will respond to arginine deprivation therapy.

[0020] Details of one or more embodiments of the present invention will be set forth in the following description. Other features and advantages of the present invention will become apparent from the following embodiments and the claims. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate various example systems, methods, and other illustrative embodiments of the present invention. The embodiments disclosed herein will be more clearly understood in conjunction with the accompanying drawings, wherein:

[0022] Figure 1 This study presents an analysis of clinicopathological factors and SNP genotypes associated with overall survival (OS) in patients receiving arginine deprivation therapy. Kaplan-Meyer analysis was performed on patient subgroups stratified by genotype (A) WWOX-rs13338697, (B) rs6025211, and (C) GALNT14-rs9679162. P-values ​​were obtained using the log-rank test, with a p-value < 0.05 considered significant.

[0023] Figure 2 This study presents an analysis of TTR-related SNP genotypes in patients receiving arginine deprivation therapy. Kaplan-Meier analysis was performed on patient subgroups stratified by genotype (A) WWOX-rs13338697, (B) rs6025211, and (C) GALNT14-rs9679162. P-values ​​were obtained using the log-rank test, with a p-value < 0.05 considered significant.

[0024] Figure 3 This study presents an analysis of clinicopathological factors and SNP genotypes associated with TTP in patients receiving arginine deprivation therapy. Kaplan-Meier analysis was performed on patient subgroups stratified according to genotypes (A) WWOX-rs13338697, (B) rs6025211, and (C) GALNT14-rs9679162. P-values ​​were obtained using the log-rank test, with a p-value < 0.05 considered significant.

[0025] Figure 4The effect of ADI-PEG 20 on cell viability in WWOX-rs13338697 G / G and non-G / G breast cancer cells was investigated. WWOX-rs13338697 G / G and non-G / G breast cancer cells were treated with different concentrations of ADI-PEG 20 (0 to 4.8 μg / mL) for 72 hours, and cell viability was assessed by MTT assay. Data represent mean ± standard error of the mean (SEM) (n = 3, *: p < 0.05; **: p < 0.01; ***: p < 0.001). P-values ​​were obtained using two-tailed analysis of variance (ANOVA).

[0026] Figure 5 The effect of ADI-PEG 20 on cell viability in G / G and non-G / G HCC cells of WWOX-rs13338697 was investigated. WWOX-rs13338697 G / G and non-G / G HCC cells were treated with different concentrations of ADI-PEG 20 (0 to 12 μg / mL) for 72 hours, and cell viability was assessed by cell viability analysis. Data represent mean ± SEM (n = 3, *: p < 0.05; **: p < 0.01; ***: p < 0.001). P-values ​​were obtained using two-tailed ANOVA.

[0027] Figure 6 In G / G HCC cells of WWOX-rs13338697, ADI-PEG20 significantly positively regulated WWOX expression and negatively regulated ASS1 expression. Representative immunoblots of WWOX, ASS1, and β-actin were obtained from WWOX-rs13338697 G / G and non-G / G HCC cells treated with different concentrations of ADI-PEG 20 (0, 0.75, 1.5, and 3 μg / mL) for 24 hours. For all independent experiments, at least three biological triplicate analyses were performed. Detailed Implementation

[0028] The embodiments described below in relation to the accompanying drawings are intended to illustrate this disclosure and are not intended to represent the only form in which this disclosure can be implemented or used.

[0029] 1. Definition

[0030] In this disclosure, "hepatocellular carcinoma" (HCC) refers to a malignant tumor originating from liver cells. HCC is a type of liver cancer. Liver cancer may cause bleeding and necrosis due to a lack of fibrous matrix. "Advanced hepatocellular carcinoma" refers to HCC that cannot be cured by local treatments (e.g., surgery or radiation therapy). Advanced HCC may refer to localized, late-stage HCC, or metastatic HCC. "Metastatic hepatocellular carcinoma" refers to HCC that has spread from the liver to another part of the body. Advanced HCC may also be unresectable, meaning it has spread to surrounding tissues and cannot be surgically removed.

[0031] In this disclosure, "nucleic acid" refers to single-stranded or double-stranded RNA, mRNA, and including cDNA and genomic DNA. Unless otherwise specified, the left end of a single-stranded nucleic acid sequence is the 5' end; the right end of a single-stranded nucleic acid sequence is the 3' end.

[0032] In this disclosure, "polymorphism" refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomic bodies or individuals. "Polymorphic" refers to the presence of two or more variants of a specific genomic sequence in a population. A "polymorphic site" is a locus where a variant occurs. In the context of this disclosure, "single nucleotide polymorphism" (SNP) refers to a variant that occurs when a single nucleic acid in the genome (or other shared sequence) differs from that of a species member. The SNPs listed in this disclosure are referenced to the SNP identification numbers assigned by the National Center for Biotechnology Information (USA) dbSNP (single nucleotide polymorphism database), and the variant nucleic acid at the polymorphic location is labeled with an SNP on the antisense (non-coding) strand.

[0033] In this disclosure, a "primer" refers to a single-stranded oligonucleotide that, in the presence of nucleic acids and reagents used for nucleic acid polymerization (e.g., DNA-dependent or RNA-dependent polymerases) and under appropriate conditions (e.g., buffer solutions, salts, temperature, and pH), can serve as the starting point for template-guided DNA synthesis. In this disclosure, the sequence to be amplified is referred to as the "target nucleic acid." To amplify double-stranded DNA, primer pairs (i.e., forward and reverse primers) are frequently used to amplify both the coding and non-coding strands. Generally, the "forward primer" has a sequence substantially identical to the upstream sequence of the coding strand of the target nucleic acid, allowing the forward primer to hybridize (or anneal) with the non-coding strand. Conversely, the "reverse primer" has a sequence substantially complementary to the downstream sequence of the coding strand of the target nucleic acid, allowing the reverse primer to hybridize (or anneal) with the coding strand.

[0034] In this disclosure, "arginine deprivation therapy" refers to compounds or reagents that remove the supply of exogenous arginine to cancer cells whose urea cycle has been disrupted, thereby inhibiting cancer cell growth and inducing cell death.

[0035] The term "prediction," used in connection with the method / kit for determining the responsiveness of cancer patients to treatment (e.g., arginine deprivation therapy) according to the present invention, refers to the ability to predict or infer whether a patient will respond to treatment in a positive (e.g., whether the patient will respond to treatment) or negatively. In one embodiment, prediction relates to the degree of response. In another embodiment, prediction concerns the probability of a patient's survival or improvement after treatment, and the probability of disease recurrence and / or relapse over a period of time. For example, the predictive methods of this disclosure can be used clinically to make treatment decisions by selecting the most appropriate treatment for a particular patient. The predictive methods of this disclosure are valuable tools in anticipating whether a patient will respond well to a treatment regimen, including, for example, administration of polyethylene glycol-modified ADI (e.g., ADI-PEG 20), or in anticipating the likelihood of long-term survival after a treatment regimen.

[0036] In the context of this disclosure, "responsiveness" refers to a measurable response, including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). "Complete response" means the complete disappearance of all target lesions without any residual lesions. It should be noted that complete response does not necessarily mean the disease is cured. "Partial response" means no progression of any target lesion and a reduction in total tumor mass of ≥30%, with no new lesions appearing. "Stable disease" is defined as a reduction in total tumor mass of target lesions of <30% or an increase of <20%. "Progressive disease" is defined as an increase in total tumor mass of target lesions of ≥20% or the appearance of new lesions. Patients who do not experience disease progression achieve disease control; therefore, patients with CR, PR, and SD are classified as "responsive." Conversely, patients exhibiting PD are referred to as "non-responsive."

[0037] In this disclosure, “survival” refers to the act or fact of surviving. The common term “overall survival” (OS) refers to the extended life expectancy of an individual or patient compared to someone who has not received any treatment or intervention.

[0038] In this disclosure, "time-to-tumor response" (TTR) is defined as the time from the start of arginine deprivation therapy (e.g., ADI-PEG 20) to the first objective tumor response (tumor shrinkage ≥30%) in patients who achieve CR or PR.

[0039] In this disclosure, "time-to-tumor progression" (TTP) is defined as the length of time from the date of diagnosis of a disease (e.g., HCC) or the start of treatment until the disease begins to worsen or spread to other parts of the body. Measuring progression time is a method used in clinical trials to understand the effectiveness of new treatments.

[0040] Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably in this disclosure and refer to any animal. This animal can be a human individual. An individual can be someone who has been identified as having HCC but has not yet received treatment. Alternatively, an individual can be someone who has not yet been identified as having HCC or is at risk of developing HCC and therefore has not yet received treatment.

[0041] Unless otherwise indicated, “treatment” is any action taken when a patient has a particular disease or condition in order to reduce the severity of the disease or condition, or the severity of one or more of its symptoms, or to prevent or slow the progression of the disease or condition.

[0042] Although the numerical ranges and parameters used to define the broader scope of this invention are approximate values, the values ​​presented in the specific examples have been presented as precisely as possible. However, any numerical value inevitably contains a certain degree of error, stemming from the standard deviation found in various testing methods. Furthermore, the term "about" as used herein generally refers to within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, when considered by one of ordinary skill in the art, the term "about" means within the acceptable standard error of the mean. Except for operational / operational examples, or unless explicitly stated otherwise, all numerical ranges, quantities, values, and percentages disclosed herein, such as those describing material quantities, durations, temperatures, operating conditions, quantity ratios, and the like, should be understood to be modified with "about" in all cases. Therefore, unless otherwise stated to the contrary, the numerical parameters presented in this disclosure and the appended claims are approximate values ​​and are subject to change as needed. In any case, each numerical parameter should be interpreted at least by the number of significant digits reflected and by the application of general rounding and decimal point calculation techniques.

[0043] Unless otherwise specified in the context, the singular form (a, an, and the) used here encompasses its plural form usage.

[0044] 2. A method for determining the responsiveness of cancer patients to arginine deprivation therapy.

[0045] The diversity of cancer treatment responses has long been recognized, primarily due to the underlying heterogeneity in cancer biology, the degree of variation in physiological function, and differences in patients' genetic profiles. Therefore, one objective of this disclosure is to provide molecular markers associated with objectively responsive or non-responsive cancer patients to arginine deprivation therapy. Once identified, these molecular markers can be used to provide prognostic information regarding whether a cancer patient is responsive or non-responsive to arginine deprivation therapy, allowing healthcare professionals to select appropriate treatment options based on prognostic outcomes.

[0046] Examples of tumors include, but are not limited to, breast cancer, brain tumors, colorectal cancer, squamous cell carcinoma of the head and neck, HCC, leukemia (e.g., AML), lung cancer, melanoma, mesothelioma (e.g., MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma.

[0047] According to an effective embodiment of this disclosure, the genomes of HCC patients who have undergone arginine deprivation therapy are screened. Specifically, rs13338697 and an intergenic SNP (rs6025211) are identified in the WWOX gene SNP. Analysis provided by an effective embodiment of this disclosure shows that the G / G genotype of rs13338697 is positively correlated with the responsiveness of HCC patients to arginine deprivation therapy; while the T / T genotype of rs6025211 is negatively correlated with the responsiveness of HCC patients to arginine deprivation therapy. Therefore, this invention, based at least on the discovery of one or more SNPs in the WWOX gene, is suitable as a molecular marker predicting the responsiveness of HCC patients to arginine deprivation therapy (e.g., ADI-PEG 20).

[0048] In view of the foregoing, one aspect of this disclosure is a method for identifying whether HCC patients (e.g., advanced HCC) are responsive to arginine deprivation therapy based on their WWOX genotype.

[0049] According to embodiments of this disclosure, the method includes obtaining a biological sample (e.g., a serum sample) from an individual; and screening the biological sample for the G / G genotype of the WWOX gene rs13338697. Results from effective embodiments show a positive correlation between the presence of the G / G genotype and better overall survival (OS) after receiving ADI-PEG 20 monotherapy; therefore, in HCC patients, the presence of the G / G genotype rs13338697 indicates that the HCC patient will respond to arginine deprivation therapy.

[0050] According to embodiments of this disclosure, the G / G genotype of rs13338697 can be determined using polymerase chain reaction (PCR)-related methods. This involves amplifying the target nucleic acid containing the aforementioned polymorphic site using a forward primer (sequence number 1 or 2) and a reverse primer (sequence number 3) in a PCR reaction. Specifically, the selected primer pair will hybridize with the amplified portion of the target nucleotide at the site containing the SNP (i.e., the G / G genotype) under hybridization conditions. Furthermore, the presence of the G / G genotype of rs13338697 indicates that HCC patients will exhibit a positive response to arginine deprivation therapy (i.e., better overall survival).

[0051] According to embodiments of this disclosure, arginine deprivation therapy includes administering a drug to a patient (e.g., an HCC patient) selected from DFMO, rADI, rArg, rADC, PEGylated rADI, rArg, or rADC, and combinations thereof. According to a preferred embodiment of this disclosure, HCC patients with the G / G genotype rs13338697 in the WWOX gene respond to arginine deprivation therapy, which includes administering 18 mg / m² weekly to the HCC patient. 2 Or 36mg / m 2 The dose of PEGylated rADI (e.g., ADI-PEG 20) was administered until the disease worsened or an unacceptable adverse event occurred or other conditions for withdrawal from the trial were met.

[0052] Alternatively, arginine deprivation therapy may further include administering arginine analogs, autophagy inhibitors, chemotherapeutic agents, MEK inhibitors, TRAIL, isoform vitamins, or combinations thereof to the patient. Examples of autophagy inhibitors include, but are not limited to, bafimycin A1, bortezomib, CQ, HCQ, 3-MA, and quinacrine. Examples of chemotherapeutic agents include, but are not limited to, 5-FU, cisplatin, cytarabine, paclitaxel, oxaliplatin, doxorubicin, methotrexate, and vincristine. Examples of arginine analogs include, but are not limited to, canavalialine. Examples of MEK inhibitors include, but are not limited to, trametinib (GSK1120212), cobimetinib (XL518), bimetinib (MEK162), selemetinib, PD-325901, CI-1040, PD035901, TAK-733, and U0126. Examples of isoform vitamins include, but are not limited to, active folic acid. In some embodiments, arginine deprivation therapy is a combination of ADI-PEG 20 and TRAIL. In other embodiments, arginine deprivation therapy is a combination of ADI-PEG 20 and cisplatin. In other embodiments, arginine deprivation therapy is a combination of ADI-PEG 20 and cytarabine. In other embodiments, arginine deprivation therapy is a combination of ADI-PEG 20 and U0126. In some embodiments, arginine deprivation therapy is a combination of ADI-PEG 20 and paclitaxel. In other embodiments, arginine deprivation therapy is a combination of ADI-PEG 20, active folic acid, 5-FU, and oxaliplatin. In other embodiments, arginine deprivation therapy is a combination of rArg and 3-MA. In other embodiments, arginine deprivation therapy is a combination of rArg and CQ.

[0053] Alternatively, the method may further include determining the T / T genotype of the target nucleic acid rs6025211 in the biological sample. Results from embodiments of this disclosure show a negative correlation between the T / T genotype of rs6025211 and ADI-PEG 20-related therapies, with unfavorable TTP associated with the presence of the T / T genotype of rs6025211 in HCC patients treated with arginine deprivation therapy.

[0054] According to the embodiments disclosed herein, the T / T genotype of rs6025211 is determined by amplifying the target nucleic acid containing the aforementioned polymorphism site using a forward primer of sequence number 4 and a reverse primer of sequence number 5 or 6. Specifically, the selected primer pair will hybridize with the amplified portion of the target nucleic acid at the site containing the SNP (i.e., the T / T genotype) under hybridization conditions. The presence of the rs6025211 T / T genotype indicates that HCC patients will not respond to arginine deprivation therapy. Furthermore, compared to patients without the rs6025211 T / T genotype, a shorter TTP was found to be positively correlated with the presence of the rs6025211 T / T genotype.

[0055] Alternatively, genotypes at specific sites in the WWOX gene can be determined using other known techniques and their equivalents. Examples of known techniques include, but are not limited to, restriction fragment length polymorphisms (RFLP) analysis, invader assay, single nucleotide primer extension, TaqMan analysis, dynamic allele-specific hybridization (DASH), molecular beaconassay, direct sequencing, electrophoresis, temperature gradient gel electrophoresis, and single-stranded conformation polymorphism (SSCP) analysis.

[0056] 3. Kit for determining the responsiveness of cancer patients to arginine deprivation therapy

[0057] In view of the foregoing, another aspect of this disclosure relates to a kit for identifying SNPs of target nucleic acids in biological samples from cancer patients (e.g., HCC patients) for prognostic purposes regarding whether the patient will respond to arginine deprivation therapy.

[0058] According to embodiments of this disclosure, the kit can be used to identify the presence or absence of one or more SNPs of a target nucleic acid in patients with tumors such as breast cancer, brain tumors, colorectal cancer, head and neck squamous cell carcinoma, HCC, leukemia (e.g., AML), lung cancer, melanoma, mesothelioma (e.g., MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma.

[0059] Generally, the target nucleic acid can be a clinical sample collected from a patient, such as a serum sample. The kit may contain materials for amplifying the target sequence containing at least one polymorphic site of the WWOX gene, and tools for identifying the genotype at that polymorphic site.

[0060] According to a preferred embodiment of this disclosure, the kit comprises a first primer pair for amplifying the target nucleic acid rs13338697, which contains the patient's WOXX gene. For example, in a PCR reaction, the target nucleic acid containing rs13338697 can be amplified using a forward primer of sequence number 1 or 2 and a reverse primer of sequence number 3, wherein the selected primer pair will hybridize with the amplified portion of the target nucleic acid at a site containing the SNP (i.e., the G / G genotype) under hybridization conditions. According to embodiments of this disclosure, the presence of the G / G genotype of rs13338697 indicates that the HCC patient will exhibit a positive response to arginine deprivation therapy (i.e., preferred overall survival).

[0061] In some embodiments, the target nucleic acid may contain one polymorphic site; in other embodiments, the target nucleic acid may contain two or more polymorphic sites. In this case, the kit may further include a second primer pair for amplifying the SNP rs6025211 in the target nucleic acid. Specifically, the second primer pair is for amplifying the target nucleic acid containing the T / T genotype of rs6025211 in the biological sample. The T / T genotype of rs6025211 is amplified by using a forward primer of sequence number 4 and a reverse primer of sequence number 5 or 6, wherein the selected primer pair will hybridize with the amplified portion of the target nucleic acid at the site containing the T / T genotype under hybridization conditions. The results of the embodiments disclosed herein indicate that the T / T genotype of rs6025211 is negatively correlated with arginine deprivation therapy, wherein the presence of the T / T genotype of rs6025211 in cancer patients who have received arginine deprivation therapy is associated with unfavorable TTP.

[0062] According to the embodiments of this disclosure, the presence of the rs6025211 T / T genotype indicates that HCC patients will not respond to arginine deprivation therapy, wherein shorter TTP was found to be positively correlated with the presence of the rs6025211 T / T genotype compared to patients without the rs6025211 T / T genotype.

[0063] Depending on the circumstances, the kit may include instructions explaining the results. These instructions may be included in the kit in printed or electronic form. Alternatively, the instructions may be provided to users via a link or URL accessing a website or external site. The website may be publicly accessible or protected. In particular, the instructions may explain the presence of a specific genotype at a particular location, which may predict CR or PR in a patient's tumor (e.g., HCC). In one embodiment, the specific genotype at a particular location includes the G / G genotype of rs13338697 in the WWOX gene. Depending on the circumstances, the specific genotype at a particular location may include the T / T genotype of rs6025211.

[0064] The invention will now be described in more detail with reference to the following embodiments, which are provided for illustrative purposes and not for limitation. Although conventional methods are used below, those skilled in the art will be able to carry out the invention using other alternative procedures, methods, or techniques.

[0065] Example

[0066] Materials and Methods

[0067] Cell culture

[0068] Human HCC cell lines, including J7, Huh7, HepG2, and Mahlavu cells, and human breast cancer cell lines, including BT20, MDA-MB-231, MDA-MB-435s, MDA-MB-468, and MCF-7, were obtained from the American Type Culture Collection (ATCC). Among these, J7, BT20, MDA-MB-231, MDA-MB-435s, and MDA-MB-468 cell lines belong to the G / G genotype of WWOX-rs13338697 (WWOX GG type); while Huh7, HepG2, Mahlavu, and MCF-7 cell lines belong to the non-G / G genotype of WWOX-rs13338697 (WWOX non-GG type).

[0069] J7, Huh7, Mahlavu, and MDA-MB-468 cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S), and incubated in a humidified incubator at 37°C with 5% CO2. The HepG2 cell line was cultured in Minimum Essential Medium α (MEMα) supplemented with 10% FBS and 1% P / S, and incubated in a humidified incubator at 37°C with 5% CO2. BT20 and MCF-7 cell lines were cultured in Roswell Park Memorial Institute-1640 (PRMI-1640) medium supplemented with 10% FBS and 1% P / S, and incubated in a humidified incubator at 37°C with 5% CO2. MDA-MB-231 and MDA-MB-435s cell lines were cultured in DMEM / F12 medium supplemented with 10% FBS and 1% P / S, and incubated in a humidified incubator at 37°C with 5% CO2.

[0070] MTT Analysis

[0071] MTT assay is a colorimetric assay that measures the enzyme activity in living cells that reduces 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow tetrazolium, to purple formazan. This reduction reaction occurs only in living cells; therefore, MTT assay is generally used to assess cell viability and proliferation. In short, at least 16 hours before proceeding with subsequent procedures, 3 × 10⁶ cells are seeded in each well of a 96-well plate. 3 Cells were treated with a specified dose of ADI-PEG 20 for one to three days. Subsequently, MTT dye (Bio Basic, USA) was added to each well of a 96-well disc, and the reaction was allowed to proceed for 3 hours. Relative cell viability was assessed by measuring the absorbance at 590 nm for each well, and normalized using the absorbance obtained from wells not treated with ADI-PEG 20.

[0072] Immunoblot (Western ink spot method)

[0073] Untreated and ADI-PEG 20-treated cells were collected and lysed to obtain total cellular protein extracts. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts). Protein extracts were injected into a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis system to separate proteins by molecular weight. The proteins in the gel were then transferred to a polyvinylidene fluoride (PVDF) membrane. Proteins on the PVDF membrane were labeled with a specific primary antibody and a secondary antibody binding to horseradish peroxidase (HRP). Specific protein signals were then detected using an enhanced chemiluminescence (ECL) kit (Millibert & Co., Bill Ricard, Massachusetts). The luminescent signal was imaged by exposure to X-ray film.

[0074] Recruiting patients

[0075] This study used serum samples from patients receiving arginine deprivation therapy. Patients included in this study were diagnosed with advanced HCC and had previously participated in a clinical trial of ADI-PEG 20 (NCT01287585). Specifically, patients received ADI-PEG 20 monotherapy (18 mg / m² weekly). 2 Post-hoc analysis was performed using de-linked samples and clinicopathological parameters until the disease worsened, an unacceptable adverse event occurred, or other conditions for withdrawal from the trial were met.

[0076] Treatment outcome assessment

[0077] OS (Overall Survival) is calculated from the date of randomization to the date of death, regardless of cause, or the date when regular follow-up is no longer possible. Objective tumor response is assessed using the Solid Tumor Scriteria according to the Response Evaluation Criteria (Eisenhauer et al, Eur J Cancer 2009, 45, 228-247). A response is defined as no progression in any target lesion, a tumor volume reduction of ≥30%, and no new lesions. TTR (Total Tumor Response) is calculated from the date of randomization to the date of tumor response, and is limited to the date of withdrawal from the trial. Disease progression is defined as an increase of ≥20% in total tumor volume in the target lesion or the appearance of new lesions. TTP (Total Tumor Response) is calculated from the date of randomization to the date of disease progression, and is limited to the date of the last radiological assessment.

[0078] SNP genotyping

[0079] Cell-free extracellular genomic DNA was isolated from serum samples using a small-volume DNA extraction kit (QIAamp DNA mini kit, QIAGEN, MD, USA) following the manufacturer's instructions. Semi-nested PCR was performed using the primers listed in Table 1. PCR products were purified using a PCR product purification kit (EasyPrepGel & PCR extraction kit, Tursys Biotechnology) according to the manufacturer's procedures. Genotyping of the purified PCR products was then performed using Sanger's sequencing.

[0080] Table 1 Primers used for SNP genotyping

[0081] Primer name Sequence (5'→3') Serial Number rs13338697-F1 5'-ACTTCTGACAGCCATCCAGA-3' 1 rs13338697-F2 5'-ATCCTGCTAGCATGTTGACT-3' 2 rs13338697-R2 5'-ACTGTAGATGCCTTCCATCT-3' 3 rs6025211-F1 5'-ACATTCACAGAGAACTTGGC-3' 4 rs6025211-R1 5'-CAAGCAGTCCTTCCACCTTG-3' 5 rs6025211-R2 5'-AAAGTGCTGGGATTACAGGT-3' 6 rs9679162-F1 5'-TCACGAGGCCAACATTCTAG-3' 7 rs9679162-R1 5'-TTAGATTCTGCATGGCTCAC-3' 8 rs9679162-R2 5'-TCCCTCCTACTGAACCTCTCC-3' 9

[0082] Statistical analysis

[0083] Dichotomized data were expressed as percentages (%) and compared using the chi-square test (χ² test) or Fisher's exact test. Parametric data were expressed as mean ± standard deviation (SD) and compared using the t-test for two samples. Nonparametric or abnormally distributed data were expressed as medians (ranges) and compared using the Mann-Whitney test. Univariate and multivariate Cox proportional hazard models were used to estimate survival rates associated with clinical and genotype variables. After classification, survival probabilities between groups were estimated using the Kaplan-Meyer (KM) method, and survival rates were compared using the log-rank test. p < 0.05 was considered statistically significant. Statistical analyses were performed using statistical software (SPSS (version 18.0) or Prism (version 8.0)).

[0084] Example

[0085] Example 1: Baseline characteristics of patients in this study

[0086] This study included 113 patients with advanced HCC who received ADI-PEG 20 treatment. Table 2 summarizes the baseline clinical parameters and genotypic characteristics of these patients.

[0087] Table 2. Baseline clinicopathological parameters and SNP genotypes of recruited patients.

[0088]

[0089]

[0090] AFP (alpha-fetoprotein): α-fetal protein; AST (aspartate aminotransferase): aspartate aminotransferase; ALT (alanine aminotransferase): alanine aminotransferase; OS: overall survival rate; TTR: tumor response time; TTP: time to tumor progression.

[0091] Example 2: Correlation between clinicopathological factors and SNP genotypes with OS, TTR, and TTP in advanced HCC patients receiving arginine deprivation therapy

[0092] After performing the analysis using the Cox proportional hazards model, a KM analysis was then performed to investigate whether any of the baseline characteristics and SNP genotypes listed in Table 2 were associated with OS, TTR, and TTP in the recruited patients.

[0093] Univariate analysis showed that patients with ≤4 tumors had longer overall survival (OS) compared to patients with >4 tumors (mean OS, 10.6 months [95% CI: 8.5 to 12.7] vs. 6.2 months [95% confidence interval (CI): 5 to 7.3]; P = 0.001). Furthermore, patients with the G / G genotype of WWOX-rs13338697 had longer OS compared to patients with the non-G / G genotype (mean OS, 15.1 months [95% CI: 6.7 to 23.4] vs. 8.1 months [95% CI: 6.8 to 9.3]; P = 0.025) (Table 3). In addition, these two factors were independent predictors of OS in patients receiving arginine deprivation therapy (adjusted P values ​​were 0.003 and 0.045, respectively). Subsequent KM analysis confirmed this finding, showing that in these patients, tumor number and the WWOX-rs13338697 genotype could serve as independent predictors of overall survival (OS). Figure 1 (Inset image (A)). However, the genotypes of rs6025211 and GALNT14-rs9679162 have no predictive value for OS ( Figure 1 Small figures (B) and (C)).

[0094] Similar analyses were performed to determine whether these factors were associated with TTR and TTP in the recruited patients. Unfortunately, neither the Cox proportional hazards model (Table 4) nor the KM analysis yielded satisfactory results. Figure 2 It was found that no parameter was related to TTR.

[0095] Interestingly, univariate analysis using the Cox proportional hazards model revealed that tumor number and the T / T genotype of rs6025211 were associated with TTP (Table 5, P values ​​0.011 and 0.021, respectively). Patients with >4 tumors were associated with shorter TTP when compared to patients with ≤4 tumors (mean TTP, 2.1 months [95% CI: 1.7 to 2.5] vs. 3.8 months [95% CI: 2.8 to 4.8]). Regarding the rs6025211 genotype, patients with the T / T genotype had shorter TTP when compared to patients without the T / T genotype (mean TTP, 2.0 months [95% CI: 1.3 to 2.6] vs. 3.3 months [95% CI: 2.6 to 4.0]). Multivariate analysis using a Cox proportional hazards model further showed that these two factors were independent predictors of TTP in patients (P values ​​were 0.007 and 0.012, respectively). Further analysis using tumor number (P = 0.0076) or the T / T genotype of rs6025211 (P = 0.0151 compared to non-T / T genotypes) in these patients... Figure 3 KM analysis revealed clear stratification among patients. No association with other SNP genotypes was found. Figure 3 ).

[0096] In conclusion, the G / G genotype of WWOX-rs13338697 and the T / T genotype of rs6025211 can predict the outcome of arginine deprivation therapy independently of clinicopathological parameters, such as tumor number.

[0097] Table 3 shows the univariate and multivariate analyses of the association between clinicopathological factors, SNP genotype, and overall survival (OS) in 113 HCC patients receiving arginine deprivation therapy.

[0098]

[0099]

[0100]

[0101]

[0102] The median was used as the critical value for the parameter data. OS: overall survival; HR (hazard ratio): risk ratio; CI: confidence interval; N: number of patients. *: P-value is derived from univariate analysis of the Cox proportional hazards model. # P-values ​​were derived from a multivariate analysis of a Cox proportional hazards model using tumor number and rs13338697-GG as covariates. All P-values ​​less than 0.05 were considered significant.

[0103] Table 4. Univariate and multivariate analyses of the association between clinicopathological factors, SNP genotype, and TTR in HCC patients receiving arginine deprivation therapy.

[0104]

[0105]

[0106]

[0107] The median was used as the critical value for the parameter data. TTR: time to tumor response; HR: hazard ratio; CI: confidence interval; N: number of patients. *: P-values ​​are derived from univariate analysis using the Cox proportional hazards model. A P-value < 0.05 is considered significant.

[0108] Table 5. Univariate and multivariate analyses of the association between clinicopathological factors, SNP genotype, and TTP in HCC patients receiving arginine deprivation therapy.

[0109]

[0110]

[0111]

[0112]

[0113] The median was used as the critical value for the parameter data. TTP: time to tumor progression; HR: hazard ratio; CI: confidence interval; N: number of patients. *: P-values ​​are derived from univariate analysis using the Cox proportional hazards model. # The p-value is determined by a multivariate analysis of a Cox proportional hazards model using tumor number and rs6025211-TT as covariates. A p-value < 0.05 is considered significant.

[0114] Example 3: ADI-PEG 20 inhibits the growth of G / G genotype cancer cells of WWOX-rs13338697.

[0115] In this embodiment, the effect of ADI-PEG 20 on the survival of cancer cells (including G / G and non-G / G genotypes of WWOX-rs13338697) was evaluated using MTT analysis. The results are presented as follows: Figure 4 and Figure 5 .

[0116] Compared to breast cancer cell lines with the non-G / G genotype WWOX-rs13338697 (such as the MCF-7 cell line), ADI-PEG 20 was found to have a strong inhibitory effect on the growth of breast cancer cell lines with the G / G genotype WWOX-rs13338697 (including BT20, MDA-MB-231, MDA-MB-435s, and MDA-MB-468 cell lines). Figure 4 Similar results were observed in HCC cells, where G / G genotype HCC cell lines (such as the J7 cell line) with WWOX-rs13338697 showed better responsiveness to ADI-PEG 20 administration than non-G / G genotype HCC cell lines (such as the Huh7, HepG2, and Mahlavu cell lines). Figure 5 Furthermore, the inventors unexpectedly discovered that the cell survival rate of Huh7 cell lines treated with high doses of ADI-PEG 20 (12 micrograms per milliliter) was higher than that of cell lines treated with low doses of ADI-PEG 20 (e.g., 0.75, 1.5, 3, or 6 micrograms per milliliter), suggesting that non-G / G genotype HCC cells may be resistant to ADI-PEG 20 treatment.

[0117] In summary, these results confirm that G / G genotype cancer cells of WWOX-rs13338697 respond better to ADI-PEG 20 treatment than non-G / G genotype cancer cells.

[0118] Example 4: In G / G genotype cancer cells of WWOX-rs13338697, ADI-PEG20 increased WWOX expression and decreased ASS1 expression.

[0119] In this embodiment, the effect of ADI-PEG 20 on the expression of WWOX and ASS1 proteins was examined in G / G and non-G / G genotype cancer cells of WWOX-rs13338697. The results are presented in... Figure 6 .

[0120] Compared to non-G / G genotype HCC cell lines (such as Huh7, HepG2, and Mahlavu cells), administration of ADI-PEG 20 significantly increased WWOX protein expression in G / G genotype HCC cell lines (such as J7 cells) with the WWOX-rs13338697 genotype. Conversely, compared to non-G / G genotype HCC cell lines (such as Huh7, HepG2, and Mahlavu cells), administration of ADI-PEG 20 inhibited ASS1 protein expression in G / G genotype HCC cell lines with the WWOX-rs13338697 genotype (such as J7 cells). Figure 6 Furthermore, the inventors discovered that administration of ADI-PEG 20 upregulates the expression of ASS1 in HepG2 and Mahlavu cell lines, clearly indicating that non-G / G genotype HCC cells are resistant to ADI-PEG 20 treatment. The results of this example confirm that in G / G genotype cancer cells (WWOX-rs13338697), ADI-PEG 20 inhibits cancer cell growth by increasing WWOX and decreasing ASS1 protein expression.

[0121] In summary, this disclosure confirms that WWOX-rs13338697 is an independent predictor of overall survival (OS) in patients receiving arginine deprivation therapy. Additionally, rs6025211 was found to be a predictor of treatment-to-progress (TTP) for arginine deprivation therapy.

[0122] The foregoing description should be understood as disclosing the embodiments by way of example only, and various modifications can be made by those skilled in the art. The foregoing description, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Although the foregoing description has described various embodiments of the present invention with a considerable degree of specificity or with reference to one or more individual implementations, those skilled in the art to which this invention pertains can make various modifications and alterations without departing from the principles and spirit of this disclosure. sequence list <110> Dirui Pharmaceutical (Chengdu) Co., Ltd. <120> Methods and kits for identifying patients suitable for arginine deprivation therapy <130> P4166-CN <150> US63174851 <151> 2021-04-14 <160> 9 <170> BiSSAP 1.3.6 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 1 acttctgaca gccatccaga 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 2 atcctgctag catgttgact 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 3 actgtagatg ccttccatct 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 4 acattcacag agaacttggc 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 5 caagcagtcc ttccaccttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 6 aaagtgctgg gattacaggt 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 7 tcacgaggcc aacattctag 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 8 ttagattctg catggctcac 20 <210> 9 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 9 tccctcctac tgaacctctc c 21

Claims

1. The use of primers in the preparation of kits for predicting whether individuals with tumors will respond to arginine deprivation therapy, wherein, The primers contain: The first forward primer is a nucleic acid sequence with sequence number 1 or 2; and The first reverse primer is the nucleic acid sequence with sequence number 3; in, The first forward primer and the first reverse primer are specifically designed to amplify target nucleic acids containing the rs13338697 G / G genotype in biological samples, and The presence of the G / G genotype of rs13338697 indicates that the individual with the tumor has responded to arginine deprivation therapy.

2. The application as described in claim 1, wherein, The tumors are selected from the group consisting of breast cancer, brain tumors, colorectal cancer, squamous cell carcinoma of the head and neck, HCC, leukemia, AML, lung cancer, melanoma, mesothelioma, MPM, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma.

3. The application as described in claim 1, wherein, The arginine deprivation therapy comprises administering an effective amount of an agent to the individual with the tumor, selected from one or more combinations of DFMO, rADI, rArg, rADC, PEGylated rADI, rArg, or rADC.

4. The application as described in claim 3, wherein, The arginine deprivation therapy comprises administering an effective amount of an autophagy inhibitor, a chemotherapy agent, a MEK inhibitor, TRAIL, an equivalent vitamin, or a combination thereof to the individual with the tumor.

5. The application as described in claim 4, wherein, The autophagy inhibitors are selected from the group consisting of bafimycin A1, bortezomib, CQ, HCQ, 3-MA and quinacrine; The chemotherapy agents are 5-FU, cisplatin, cytarabine, paclitaxel, oxaliplatin, doxorubicin, methotrexate, or vincristine; The MEK inhibitors are trametinib, cobimetinib, bimetinib, selemetinib, PD-325901, CI-1040, PD035901, TAK-733, or U0126; and The equivalent vitamin is active folic acid.

6. The application as described in claim 4, wherein, The arginine deprivation therapy involves administering an effective amount of canavanine to the individual suffering from the tumor.

7. The application as described in claim 1, wherein the primer further comprises, A second forward primer, which is the nucleic acid sequence with sequence number 4; and A second reverse primer, which is the nucleic acid sequence with sequence number 5 or 6.

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