CDKN2a companion diagnostic for interferon therapy for bladder cancer

Abstract

Providing a CDKN2a companion diagnostic for interferon therapy for bladder cancer. [Solution] A method for treating bladder cancer, comprising the steps of: diagnosing bladder cancer in a human; measuring the CDKN2A expression level in the human; and subsequently injecting an interferon expression-inducing agent into the human. Non-muscle-invasive bladder cancer (NMIBC) represents the most common disease state in patients with newly diagnosed bladder cancer. Those with high-grade (HG) tumors are at significant risk of both recurrence and progression.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 597473, filed December 12, 2017, the contents of which are incorporated herein by reference.

[0002] Federal government funding / rights none [Background technology]

[0003] background Non-muscle-invasive bladder cancer (NMIBC) represents the most common disease state in patients with newly diagnosed bladder cancer. Those with high-grade (HG) tumors are at significant risk of both recurrence and progression. Bacillus carcinoma (BCG) is currently representative of preferred management. Nevertheless, approximately 30% of patients do not respond to BCG; and of those who show an initial response, more than 50% experience recurrence and progression during long-term follow-up.

[0004] The optimal management of patients with persistent or recurrent tumors after BCG vaccination remains a subject of debate. While radical cystectomy offers a cure for the cancer, many patients are elderly, have significant comorbidities, and often lack the willingness to undergo radical surgery. Non-radical treatment options are available, but studies to date have included relatively small patient populations, and the definition of treatment success has been inconsistent. Indeed, the U.S. Food and Drug Administration (FDA) and the urogenital oncology community agree that the management of this disease has not progressed sufficiently since the initial approval of BCG. Therefore, effective alternatives to radical cystectomy for patients with disease recurrence after BCG treatment remain a significant and unmet clinical need.

[0005] Several drugs have been evaluated as second-line treatments after BCG; however, none have provided a strong and sustained response (to date). Valrubicin (Valstar; Endo Pharmaceuticals, Malvern, PA), the only drug currently approved by the FDA for the treatment of BCG-refractory CIS, provided a complete response rate of 18% at 6 months and a 1-year disease-free survival rate of approximately 10%. Promising results from early clinical trials have been reported for intravesical taxanes and gemcitabine. Joudi et al. reported the final results from a nationwide, multicenter Phase II trial of BCG + IFNα-2b, noting that 45% of patients who failed BCG were relapse-free at 2 years. However, only 44% were treated for HG relapse, and 61% had previously received only one course of BCG. A recent retrospective analysis of BCG and IFNα-2b reported a 38.6% RFS at 12 months. To reiterate, many of these patients (20 out of 44) had previously received only one course of BCG, and 16 patients experienced a relapse after 12 months. Overall, despite the limited number of patients studied in previous trials and the less strictly defined eligibility criteria, the relatively short RFS with treatment illustrates an unmet need for an effective, evidence-based second-line therapy for patients with BCG non-responsive disease that improves disease-specific patient outcomes and avoids cystectomy.

[0006] Recombinant intravesical interferon alpha-2b protein (IFNα-2b; Intron A; Merck, Kenilworth, NJ) showed promising initial clinical results in NMIBC.

[0007] Intravesical IFNα-2b gene delivery offers a novel approach that increases the duration of IFNα-2b exposure. Recombinant adenovirus (rAd)-IFNα-2b is a replication-deficient adenovirus-based gene transfer vector encoding the human IFNα-2b gene. Syn3 (a polyamide surfactant) is incorporated into the drug formulation to enhance adenoviral transduction of the bladder lining (INSTILADRIN® rAd-IFNα / Syn3). TM (FKD Therapies Oy, Kuopio, Finland). Dramatic enrichment of rAd-IFNα gene transfer and expression is observed in both normal urothelial cells and human urothelial carcinoma growing in mice, Syn3 TM As demonstrated, rAd-IFNα-2b gene therapy mimics physiological events associated with viral infection, which occur locally rather than systemically, leading to IFNα-2b production and subsequent tumor regression. [Overview of the Initiative] [Means for solving the problem]

[0008] We conducted a Phase I dose-escalation study of rAd-IFNα / Syn3 in patients with BCG-refractory and relapsing NMIBC. A first-generation replication-deficient serotype 5 adenovirus vector expressing human interferon alpha-2b (IFNα-2b) cDNA under a cytomegalovirus promoter was generated in 293 cells under the conditions of pharmaceutical manufacturing and quality control standards, as previously described, with minor modifications to the process. It was then tested for endotoxin, microbial contamination, and other impurities. The structure of the vector was verified by sequencing. Recombinant IFNα-2b generation was verified from each generation lot using immunological methods. The excipient Syn3 is a polyamide surfactant that enhances adenovirus gene transfer into bladder epithelium. Dose-dependent adenovirus gene transfer and IFNα-2b urinary concentrations were confirmed. Of the 14 patients treated with rAd-IFNα / Syn3 at dose levels that produced measurable urinary IFNα, 6 (43%) were recurrence-free at 3 months with no dose-limiting toxicity, and 2 patients remained disease-free at 29 and 39 months. The present invention provides, for example, the following items. (Item 1) A method for treating nonmuscle-invasive bladder cancer in humans, wherein the method is: a. A process for diagnosing nonmuscle-invasive bladder cancer in humans, and then, b. A step of measuring the human CDKN2A expression level, and then, c. The step of dripping interferon into the lumen of the human bladder. A method of including. (Item 2) The bladder cancer described above is of high grade, as described in item 1. (Item 3) The interferon is administered as a non-replicating vector, as described in item 1. (Item 4) The method according to item 3, wherein the non-replicating vector includes a replication-deficient viral vector carrying an interferon transgene. (Item 5) The method according to item 4, wherein the replication-deficient virus vector carrying the interferon-introducing gene comprises Nodfarazineene Filladenovec. (Item 8) The method according to item 1, wherein the step of measuring the CDKN2A expression level in the human requires a step of collecting a bladder tissue sample and a step of measuring the CDKN2A expression level in the tissue sample. (Item 9) The method according to item 1, wherein the step of measuring the CDKN2A expression level in the human requires a step of collecting a urine sample and a step of measuring the CDKN2A expression level in the urine sample. (Item 10) The method according to item 9, wherein the step of measuring the CDKN2A expression level includes a step of analyzing exosomes in the urine sample. (Item 11) The method according to item 9, wherein the step of measuring the CDKN2A expression level includes a step of analyzing free DNA in the urine sample. (Item 12) The method according to item 1, further including a step of administering a checkpoint inhibitor to the human after the step of measuring the CDKN2A expression level. (Item 1.) The method according to item 1, wherein the step of measuring the CDKN2A expression level in the human includes a step of using fluorescence in situ hybridization using a probe that can hybridize with a part of CDKN2A. (Item 14) The method according to item 13, wherein the probe includes a part that hybridizes with at least one exon contained in CDKN2A. (Item 15) The method according to item 13, wherein the probe is about 40 bp in length. (Item 16) The method according to item 14, wherein the probe is about 40 bp in length.

[0009] These exciting findings have mainly prompted us to continue the Phase II trial at the two highest doses, and to evaluate the efficacy and safety of intravesical rAd-IFNα / Syn3 in patients with BCG-unresponsive HG NMIBC or with recurrence after BCG TM This randomized open-label parallel-arm trial was conducted at 13 sites in the United States between November 5, 2012, and April 8, 2015. The protocol, management oversight, and accrual timelines were designed by the Society of Urologic Oncology Clinical Trials Consortium. The study protocol and informed consent form were reviewed and approved by the respective on-site institutional review boards and biosafety committees.

Example

[0010] We evaluated the efficacy and safety of recombinant adenovirus interferon alpha and Syn3 TM (rAd-IFNα / Syn3 TM ),a replication-deficient recombinant adenovirus gene transfer vector, in patients with high-grade (HG) BCG-unresponsive or recurrent NMIBC. In this open-label, multi-site (13 sites) parallel-arm Phase II trial, 43 patients with HG BCG-unresponsive or recurrent NMIBC received intravesical rAd-IFNα / Syn3 TM (randomly assigned 1:1 to 1×10 11 virus particles (vp) / mL or 3×10 11 vp / mL). Patients who responded at 3 months, 6 months, and 9 months were retreated at 4 months, 7 months, and 10 months. The primary endpoint was HG recurrence-free survival (RFS) at 12 months. All patients who received at least one dose were included in the efficacy and safety analyses.

[0011] In our trial, 40 patients received rAd-IFNα / Syn3 between November 5, 2012, and April 8, 2015TM (1×10 11 virus particles [vp] / mL, n = 21; 3×10 11 vp / mL, n = 19). This trial was designed to enroll 40 patients who were unable or unwilling to undergo radical cystectomy, with two dosing groups of 20 patients each. Eligible patients were 18 years of age or older and had high-grade BCG-unresponsive or recurrent NMIBC (including papillary NMIBC alone (Ta or T1), carcinoma in situ (CIS) alone, or a combination of CIS and papillary disease). BCG-unresponsive disease was defined as failure to achieve disease-free status at 6 months after adequate induction of BCG therapy, with either maintenance or re-induction at 3 months. "Adequate induction" was defined as a minimum of 5 out of 6 treatments, and "adequate maintenance" was defined as a minimum of 2 out of 3 treatments. BCG recurrence was defined as recurrence within 1 year after complete response to adequate BCG treatment (at least 5 and 2 instillations). Patients were required to have had a visual complete resection of papillary lesions by transurethral resection of bladder tumors. Patients could not have received intravesical therapy within 3 months prior to starting the study treatment, except for cytotoxic agents administered as a single instillation immediately after transurethral resection. All participants enrolled in this study provided written or oral informed consent.

[0012] Patients were assigned in a 1:1 continuous manner, restricted by computer-generated random assignment, to receive either low-dose (1×10 11 virus particles [vp] / mL) or high-dose (3×10 11 vp / mL) rAd-IFNα / Syn3. These doses were the most promising observed in the Phase I trial. The total dose administered was 7.5×10 12 vp in the low-dose group and 2.25×10 13The treatment was performed intensively in block size 2 on all patients who successfully completed screening, with the constraint that the first four patients at each site were balanced across cohorts.

[0013] rAd-IFNα / Syn3 in 75 mL TM The drug was administered intravesically via a urethral catheter with a planned indwelling time of 1 hour; anticholinergic treatment was permitted to relieve urgent urination and allow for adequate indwelling. Patients who were free from recurrence of HG disease at 3, 6, and 9 months were re-treated at 4, 7, and 10 months, if evaluated by cytology, cystoscopy, and biopsy (if clinically indicated). A final efficacy evaluation was performed at 12 months. This evaluation included protocol-mandated biopsies from the site of the indicator tumor, and at least five random biopsies (including the bladder fundus, bladder trigone, right and left lateral walls, posterior wall, and the prostatic urethra in men with cytology-positive or prior disease in this region).

[0014] During the trial, patients were contacted by telephone weekly for the first month after each treatment, on days 7, 14 (only at months 7 and 10), 21, and 28 (±1 day), to provide information on adverse events (AEs) and the use of associated drug therapies. Treatment failure was evaluated between 14 and 7 days prior to retreatment. Patients who discontinued treatment before the completion of the trial were evaluated for safety for at least 30 days after the last dose of the study drug. All patients were evaluated to (1) determine recurrence of HG disease in patients with complete response, and (2) rAd-IFNα / Syn3 TM To evaluate the long-term effects of the treatment, it is being monitored over a three-year long-term follow-up period.

[0015] The primary endpoint was freedom from HG disease recurrence at 12 months (defined by negative results for the cause or last exploratory biopsy). Secondary endpoints included no evidence of HG disease recurrence at 3, 6, and 9 months; incidence or time to cystectomy; and response to treatment, defined as urinary IFNα-2b concentration. Safety assessments included physical examination, vital sign monitoring, ECG, and evaluation of standard clinical chemistry, blood, and urine analyses (performed by local laboratories). Safety endpoints included type, incidence, relevance, and severity of AEs, as well as serious (≥grade 3) AEs (SAEs), as assessed by the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03).

[0016] We determined that a cohort of 20 patients was sufficient to give an 80% rejection rate of a 10% HG relapse-free survival (RFS) rate in a one-sided test with 5% precision, assuming a true HG RFS rate of 35%. The operational properties of this Fleming design were precisely calculated using the binomial distribution described by A'Hern. This hypothesis (i.e., the response rate was equal to or less than the reference rate) was rejected if 5 or more of the 20 patients achieved HG RFS at 12 months. The proportion of patients who achieved HG RFS at 3 months, 6 months, 9 months, and 12 months, along with their 90% confidence intervals (CIs), were reported for each dose group. Time to HG relapse or death was summarized using the Kaplan-Meier method. SAS TM (Version 9 or later; SAS Institute, Analysis was performed at Cary, NC. Both safety and efficacy (modified intention to treat) analysis sets included all patients who received at least one dose of rAd-IFNα / Syn3. A data monitoring committee oversaw the trial in accordance with the data monitoring plan. All analytical assays were developed and validated. Samples were tested according to the best practices methodology at Covance Laboratories Ltd (Harrogate, United Kingdom). Baseline patient characteristics are provided in Table 1: [Table 1]

[0017] The 12-month HG RFS rates were comparable between the two dose groups, with 33.3% (7 out of 21 patients; 90% CI, 16.8–53.6) in the low-dose group and 36.8% (7 out of 19 patients; CI, 18.8–58.2) in the high-dose group surviving and remaining HG-free at 12 months. Overall, 35.0% (14 out of 40 patients; 90% CI, 22.6%–49.2%) of patients remained HG-free at 12 months after initiation of rAd-IFNα / Syn3 treatment. Unplanned disease assessments did not affect the findings (see appendix, online only). The median time to HG recurrence or death was 6.5 months (90% CI, 3.52–12.78 months); the median time to HG recurrence was 3.52 months (90% CI, 3.02–12.78 months) in the low-dose group and 11.73 months (90% CI, 5.88 months–not quantifiable) in the high-dose group.

[0018] Fourteen patients (35.0%; 90% CI, 22.6%–49.2%) remained HG recurrence-free for 12 months after initial treatment. Comparable 12-month HG RFS were noted for both doses. Of these 14 patients, two experienced recurrence at 21 and 28 months after the start of treatment, and one died at 17 months without recurrence as a result of an upper tract tumor. rAd-IFNα / Syn3 was well-tolerated; no grade 4 or 5 adverse events (AEs) occurred, and no patients discontinued treatment due to adverse events. The most frequently reported drug-related AEs were urinary urgency (n=16; 40%), dysuria (n=16; 40%), fatigue (n=13; 32.5%), frequent urination (n=11; 28%), and hematuria and nocturia (n=10; 25%), respectively.

[0019] When patient subgroups and secondary endpoints were considered in exploratory analyses, the 12-month HG RFS rates were broadly similar across males and females, young and elderly patients, refractory NMIBC or relapsed NMIBC, CIS alone or papillary neoplasm and CIS, and patients with Ta and T1 disease alone. [Table 2]

[0020] Interestingly, of the 14 patients who did not experience a relapse in 12 months, 10 (71%) had an anti-adenovirus antibody response (defined as four times the pre-treatment titer), compared to 11 (24%) of the 25 patients who experienced a relapse.

[0021] In long-term follow-up, seven patients (18%) who discontinued the study due to a recurrence of HG disease within the 12-month trial period died median 16 months (range, 2–26 months) after the discontinuation date. There was no indication that these deaths were procedure-related. The cause of death was unknown in four patients, two died as a result of advanced bladder cancer, and one died 17 months after discontinuation of the trial as a result of procedure-unrelated liver failure. The four patients whose cause of death was unknown were locally observed after they completed their end-of-study evaluation. Fourteen patients (35%) who experienced HG recurrence within the first year underwent radical cystectomy from day 1 of month 1 to median 9 months (range, 4–28 months).

[0022] Patients were monitored for three years to collect long-term follow-up data. Of the 14 patients who remained disease-free at 12 months, further follow-up data was collected for 11; 3 discontinued the study. Of these 11 patients, 9 are alive, and 8 remained disease-free for a period of 15 to over 36 months. Two patients experienced HG recurrence at 21 and 28 months, respectively, from the start of treatment. One of these patients who experienced progression to muscle invasion underwent radical cystectomy 31 months after the start of treatment and later died at 41 months. The other patient who experienced recurrence at 21 months is still alive and has had no distant recurrence at 36 months. One patient who had no bladder recurrence at 12 months died at 17 months as a result of an upper urinary tract tumor. [Table 3]

[0023] Our results show that rAd-IFNα / Syn3 TM It demonstrated sufficient tolerability. This showed promising efficacy in patients with HG NMIBC after BCG therapy who are unable to undergo or unwilling to undergo radical cystectomy.

[0024] However, while potentially promising, these data indicate several failures in our treatment plan. Firstly, the treatment was ineffective in the majority of patients: completely, 65% of patients did not achieve 12-month HG RFS by intention-to-treat analysis of all treated patients. Similarly, the 12-month RFS rate in heavily pre-treated patients was 31%. Notably, the response was persistent: most remained disease-free for nearly 24 months. We noted that 70% of patients with any element of CIS did not achieve a sustained complete response. In fact, among patients with only papillary disease at trial enrollment, only 50% achieved RFS.

[0025] To address these failures, we pursued two approaches. First, we administered high doses of rAd-IFNα / Syn3 to a considerable number of patients. TM A larger clinical trial involving [the drug] has been designed and is currently underway. In this trial, if lower doses were not only ineffective but completely ineffective, higher doses of rAd-IFNα / Syn3 TM This could provide evidence that it is effective.

[0026] Secondly, we collected tissue samples from patients involved in the Phase II completed clinical trial discussed above. These samples allow us to analyze gene expression for each patient. This enables us to compare gene expression in patients who responded to the treatment with gene expression in patients who did not.

[0027] Gene expression analysis can be performed using tumor biopsy samples.

[0028] Alternatively, in the case of bladder cancer, urine samples, if properly preserved, may contain exosomes that enable the analysis of oncogene expression. Exosomes are small (30–100 nm) endocytic cell-derived vesicles. They are secreted by most human cell types, including cancer cells, and can be absorbed into recipient cells via endocytosis. Exosomes may contain functional biomolecules (e.g., dsdna) and can be found in human blood and urine. Exosomal DNA ("exoDNA") represents the entire genome and, if exosomes are produced from tumor cells, reflects the mutational state of the tumor cells. ExoDNA from tumor-derived exosomes found in urine samples taken from bladder cancer patients therefore provides a non-invasive circulating biomarker useful for highly sensitive detection of cancer and more accurate determination of potential responsiveness to interferon-based therapy.

[0029] ExoDNA contains both single-stranded and double-stranded DNA. ExoDNA is found to be encapsulated inside the exosome membrane and bound outside the exosome membrane. In internal exoDNA, dsDNA is dominant. Typically, exoDNA encapsulated inside the exosome membrane ranges from 0.1 to 2.5 kb, while exoDNA bound outside the exosome membrane is >2.5 kb. Much of the exoDNA associated with tumor exosomes is double-stranded DNA bound outside its exosome membrane. ExoDNA (including dsDNA) can provide a remarkably accurate diagnostic tool because it provides a complete sample of the entire genome in an easily assayable form in the urine of patients with bladder cancer.

[0030] Alternatively, microvesicles can be assayed. Microvesicles are another type of extracellular vesicle, found in many types of bodily fluids and in the interstitial space between cells, with a diameter between 50 and 1,000 nanometers (nm). Microvesicles are made from fragments of the plasma membrane. Therefore, unlike exosomes, they are smaller and produced intracellularly. In contrast to exosomes, which do not contain mitochondrial DNA, microvesicles derived from astrocytes and glioblastomas contain mitochondrial DNA. Microvesicles appear to be equivalent to exosomes for the purposes of this invention.

[0031] In human patients diagnosed with bladder cancer, exosomes can be collected by collecting urine samples. Sample storage techniques capable of preserving DNA and RNA are well known in the field. Similarly, the isolation of exosomes from urine can be performed using conventional separation techniques.

[0032] Alternatively, fluorescence in situ hybridization (FISH) may be used. FISH is a molecular cytogenetic technique that uses a fluorescent probe that binds to specific target regions of chromosomes, but with a high degree of sequence complementarity when sufficiently stringent hybridization conditions are used. FISH was developed in the early 1980s. See, for example, Langer-Safer, PR et al., Immunological Method For Mapping Genes On Drosophila Polytene Chromosomes, 79 Proceedings of the National Academy of Sciences 4381 (1982). FISH can detect and pinpoint the presence or absence of specific DNA sequences on chromosomes or in DNA fragments (e.g., free DNA found in urine).

[0033] For the use of FISH in our diagnostic method, a probe is constructed that can hybridize with a portion of CDKN2A, preferably one or more of the eight exons contained in CDKN2A. The probe must be large enough to specifically hybridize with its target, but not so large as to require non-stringent hybridization conditions (which can lead to the formation of false-positive hybridization to inappropriate target sequences). We prefer probes of about 40 bp, but longer or shorter probes may be used depending on the desired specific CDKN2A mutation. Once constructed, the probe is tagged with a fluorophore, a target for the antibody, or directly with biotin. Tagging can be performed by various methods taught in the art (e.g., nick translation or polymerase chain reaction using tagged nucleotides).

[0034] Fluorescence microscopy can be used to determine, for example, whether a fluorescent probe binds to target DNA in a urine sample, and where it binds. FISH can also be used to detect and locate specific RNA targets (e.g., mRNA) in cells, circulating tumor cells, and tissue samples.

[0035] As an alternative to sequencing exoDNA in a urine sample, free DNA can be isolated from the urine sample and then sequenced.

[0036] ExoDNA analysis reveals a crucial difference between bladder cancer patients who respond to interferon therapy and those who do not. Patients who respond to treatment have different CDKN2A expression levels than those who do not. These differing levels of expression appear to result from deletions or loss of heterozygosity in the gene.

[0037] In our view, the safety and efficacy of rAd-IFN gene therapy can be improved by allowing attending physicians to limit treatment to those patients who are most likely to respond. Furthermore, in our view, the safety and efficacy of other intravenous treatments that induce interferon expression (e.g., BCG vaccine) can similarly be improved. Similarly, in our view, the efficacy of cancer "checkpoint inhibitors" can be improved. Checkpoint inhibitors are thought to be ineffective in cancer patients with CDKN2A deletion. Identifying such patients will allow those skilled in the art to use interferon therapy and checkpoint inhibitor therapy in combination in such patients, using interferon to overcome the inhibition of checkpoint inhibitors.

[0038] Therefore, we intend hereby to encompass a method for treating bladder cancer, the method comprising the steps of diagnosing bladder cancer in a human, measuring the CDKN2A expression level in that human, and then injecting that human with an interferon-inducing agent.

[0039] Similarly, we intend this patent to cover a method for treating bladder cancer, wherein the bladder cancer is of a high grade.

[0040] We also intend this patent to cover methods for treating bladder cancer, wherein the agent is a non-replicating agent such as rAd-IFN or mycobacterial cell wall extract.

[0041] Similarly, we intend this patent to cover a method for treating bladder cancer, where the above-mentioned agent is a replicating agent such as the BCG vaccine.

[0042] Similarly, we intend that this patent encompasses a method for treating bladder cancer, wherein the step of measuring the human CDKN2A expression level comprises the steps of collecting a bladder tissue sample and measuring the CDKN2A expression level in the tissue sample. Alternatively, we intend that this patent encompasses a method for treating bladder cancer, wherein the step of measuring the human CDKN2A expression level comprises the steps of collecting a urine sample and measuring the CDKN2A expression level in the urine sample, preferably by analyzing the exosomes in the urine sample, but also, for example, by analyzing the free DNA in the urine sample.

[0043] Similarly, we intend this patent to cover a method for treating bladder cancer, in which a human is also treated with a cancer checkpoint inhibitor.

[0044] We also intend for our patent to cover companion diagnostic tests that can measure CDKN2A expression levels in bladder tissue samples.

[0045] Further embodiments and variations will be readily apparent to those skilled in the art after reviewing this disclosure. We therefore intend that the appended legal claims will encompass such embodiments.

[0046] The present invention provides, for example, the following items. (Item 1) A method for treating nonmuscle-invasive bladder cancer in humans, wherein the method is: a. A process for diagnosing nonmuscle-invasive bladder cancer in humans, and then, b. A step of measuring the human CDKN2A expression level, and then, c. A step of dripping a drug that induces interferon expression into the lumen of the human bladder. A method of including. (Item 2) The bladder cancer described above is of high grade, as described in item 1. (Item 3) The method described in item 1, wherein the aforementioned agent is a non-replicating agent. (Item 4) The method according to item 3, wherein the non-replicating agent includes a replication-deficient viral vector carrying an interferon transgene. (Item 5) The replication-deficient viral vector carrying the interferon transgene is the method described in item 4, comprising Nadfarajine Radenovec. (Item 6) The aforementioned drug is the method described in item 1, including a replication agent. (Item 7) The replicating agent is the method described in item 6, comprising a Calmette-Guérin vaccine. (Item 8) The method according to item 1, wherein the step of measuring the human CDKN2A expression level includes the steps of collecting a bladder tissue sample and measuring the CDKN2A expression level in the tissue sample. (Item 9) The method according to item 1, wherein the step of measuring the human CDKN2A expression level comprises the steps of collecting a urine sample and measuring the CDKN2A expression level in the urine sample. (Item 10) The method according to item 9, wherein the step of measuring the CDKN2A expression level includes the step of analyzing the exosomes in the urine sample. (Item 11) The method according to item 9, wherein the step of measuring the CDKN2A expression level includes the step of analyzing the free DNA in the urine sample. (Item 12) The method according to item 1, further comprising the step of administering a checkpoint inhibitor to the human after the step of measuring the CDKN2A expression level. (Item 13) The method according to item 1, wherein the step of measuring the human CDKN2A expression level includes the step of using fluorescence in situ hybridization with a probe that can hybridize with a portion of CDKN2A. (Item 14) The method according to item 13, wherein the probe comprises a portion that hybridizes with at least one exon contained in CDKN2A. (Item 15) The probe is approximately 40 bp in length, as described in item 13. (Item 16) The probe is approximately 40 bp in length, as described in item 14.

Claims

[Claim 1] The method described in the specification.

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