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BIOMARKERS FOR PREDICTING A PATIENT'S RESPONSE TO BCG THERAPY, METHODS AND USES BASED THEREON

20230220483 · 2023-07-13

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to an in vitro method for predicting the response of a bladder cancer patient to Bacillus Calmette-Guérin (BCG) immunotherapy, comprising determining the expression level or ratio of expression levels of miRNAs or combinations of miRNAs in a sample and comparing said expression level, or ratio of expression levels, to a reference value, wherein a difference in said comparison is indicative of said patient’s response to BCG therapy. The present invention also relates to kits for carrying out such methods and to uses of miRNAs, or combinations of miRNAs, to predict whether or not a bladder cancer patient will respond to BCG therapy.

    Claims

    1. An in vitro method for predicting the response of a subject with bladder cancer to a therapy with Bacillus Calmette-Guerin (BCG), comprising determining in a sample obtained from said subject the expression level or ratio of expression levels of: (i) at least one pair of miRNAs selected from the group consisting of: miR-21/miR-182, miR-222/miR-182, miR-429/miR-182, miR223/miR-182, and miR-21/miR-106a; or combinations of said pairs; or (ii)at least one miRNA selected from the group consisting of: miR-21, miR-106a, miR-17, miR-222, miR-429, miR-223, let-7c, miR-29a, miR-199a, miR-199b, miR-23a, miR-218, miR-125b, miR-23b, miR-1, miR-483, miR-145, miR-4443, miR-874, miR-497, miR-143, miR-574, miR-579, miR-342, and miR-99a; or combinations thereof; and comparing said expression level, or ratio of expression levels, with a reference value, wherein a difference in said comparison identifies a subject as being a responder to BCG therapy.

    2. The method according to claim 1, wherein the sample is a tissue sample or a urine sample.

    3. The method according to claim 2, wherein the tissue sample is a bladder cancer tumour tissue sample.

    4. The method according to claim 1, wherein the sample comprises a tissue sample and the ratio of expression levels is determined for at least one pair of miRNAS selected from: miR-21/miR-182, miR-222/miR-182, miR-429/miR-182, and miR223/miR-182.

    5. The method according to claim 1, wherein the sample comprises urine and the ratio of expression levels is determined for at least one pair of miRNAs selected from: miR-21/miR-182, or miR-21/miR-106a.

    6. The method according to claim 1, wherein the sample comprises urine and the level of expression is determined for a miRNA selected from: miR-21 or miR-106a.

    7-12. (canceled)

    13. A kit for predicting the response of a subject with bladder cancer to a therapy with Bacillus Calmette-Guerin (BCG), comprising the reagents necessary to determine, in a sample obtained from said subject, the expression level or ratio of expression levels of: (i) at least one biomarker selected from the group of miRNA pairs consisting of: miR-21/miR-182, miR-222/miR-182, miR-429/miR-182, miR223/miR-182, and miR-21/miR-106a; or combinations of said pairs; or (ii)at least one biomarker selected from the group consisting of: miR-21, miR-106a, miR-17, miR-222, miR-429, miR-223, let-7c, miR-29a, miR-199a, miR-199b, miR-23a, miR-218, miR-125b, miR-23b, miR-1, miR-483, miR-145, miR-4443, miR-874, miR-497, miR-143, miR-574, miR-579, miR-342, and miR-99a; or combinations thereof.

    14. The kit according to claim 13, wherein the sample tested in said kit is a tissue sample or urine sample.

    15. The kit according to claim 14, wherein the tissue sample tested in said kit is a bladder cancer tumour tissue sample.

    16. The kit according to claim 13, wherein when the sample tested using said kit comprises a tissue sample, said biomarker is selected from the group of miRNA pairs consisting of: miR-21/miR-182, miR-222/miR-182, miR-429/miR-182 and miR223/miR-182.

    17. The kit according to claim 13, wherein when the sample tested using said kit comprises urine, said biomarker is selected from the miRNA pairs: miR-21/miR-182 or miR-21/miR-106a.

    18. The kit according to claim 13, wherein when the sample tested using said kit comprises urine, said biomarker is selected from: miR-106a or miR-21.

    19. A method for treating a subject with bladder cancer with Bacillus Calmette-Guerin (BCG) therapy, comprising predicting the response of the subject to the BCG therapy according to claim 1 and further comprising administering BCG therapy to the subject identified as being a responder to BCG therapy.

    20. The method according to claim 19, said method comprising administration of Bacillus Calmette-Guerin intravesical instillations, to the subject identified as being a responder to BCG therapy.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0068] FIG. 1. Representation of the Ct value ratios of the miR-21-5p/miR-182-5p (A), miR-222-3p/miR-182-5p (B), miR223-3p/miR-182-5p (C) and miR-429/miR-182-5p (D) pairs among BCG-responding (R) and non-responding (NR) patients. The mean of each group and its standard deviation are shown. ROC curves for the relationships of expression levels of the miR-21-5p/miR-182-5p (E), miR-222-3p/miR-182-5p (F), miR223-3p/miR-182-5p (G) and miR-429/miR-182-5p (H) pairs, obtained from the results shown in graphs A-D. The AUC values of the ROC curves were as follows: 0.90 (E), 0.88 (F), 0.84 (G) and 0.87 (H).

    [0069] FIG. 2. Representation of the Ct value ratios of the miR-21-5p/miR-182-5p (A) and miR-21-5p/miR-106a-5p (B) pairs among BCG-responding (R) and non-responding (NR) patients. The mean of each group and its standard deviation are shown. ROC curves for the relationships of expression levels of the miR-21-5p/miR-182-5p (C) and miR-21-5p/miR-106a-5p (D) pairs, obtained from the results shown in graphs A-B. The AUC values of the ROC curves obtained were 0.79 (C) and 0.76 (D).

    [0070] FIG. 3. Representation of Ct values of miR-182-5p (A), miR-106a-5p (B) and miR-21-5p (C) among BCG-responding (R) and non-responding (NR) patients. The mean of each group and its standard deviation are shown. Correlation between Ct values of miR-182-5p (D), miR-106a-5p (E) and miR-21-5p (F) in tissue and urine.

    DESCRIPTION OF EMBODIMENTS

    Patient Cohorts

    [0071] Samples from three patient cohorts from the “12 de Octubre” Hospital in Madrid, Spain, were used: discovery cohort, validation cohort and test cohort.

    [0072] Table 1 shows the clinical-pathological characteristics and parameters of patients in the discovery cohort, which consisted of 39 patients (25 responders and 14 non-responders to BCG therapy). Primary paraffinised tumour tissue samples from these patients were used, taken at the time of transurethral resection of the bladder tumour (TURBT) and subsequently subjected to BCG instillation therapy.

    TABLE-US-00001 Patients (Male; Female) 39 (M = 34; F = 5) Responders - Non-Responders 25 - 14 Mean Age and Range 69 (49-80). Tumour Stage Ta = 13; T1 = 25; Tx = 1 Tumour grade G1 = 8: G2 = 31 Tx = tumour stage not determined

    [0073] Table 2 shows the clinical-pathological characteristics and parameters of the patients in the validation cohort, which consisted of 43 patients (28 responders and 15 non-responders to BCG therapy). Primary paraffinised tumour tissue samples from these patients were used, taken at the time of transurethral resection of the bladder tumour (TURBT) and subsequently subjected to BCG instillation therapy.

    TABLE-US-00002 Patients (Male; Female) 43 (M = 36; F = 7) Responders - Non-Responders 28 - 15 Mean Aqe and Range 65 (53-80). Tumour Stage Ta = 19; T1 = 24 Tumour grade G1 = 8: G2 = 35

    [0074] Table 3 shows the clinical-pathological characteristics and parameters of the patients in the test cohort, which consisted of 20 patients (11 responders and 9 non-responders to BCG therapy). Urine samples from these patients were used, obtained prior to transurethral resection of the bladder tumour (TURBT), and who subsequently underwent BCG instillation therapy.

    TABLE-US-00003 Patients (Male; Female) 20 (M = 19; F = 1) Responders - Non-Responders 11 - 9 Mean Age and Range 59 (52-85). Tumour Stage CIS = 3; Ta = 7; T1 = 8; Tx = 2 Tumour grade G1= 1; G2 = 6; G3= 10; Gx= 3 Tx = tumour stage not determined; Gx = tumour grade not determined.

    Example 1. miRNAs Differentially Expressed in Tissue Samples From Responding and Non-Responding Patients to BCG Therapy

    [0075] In paraffinised tumour tissue samples from the 39 BCG-treated bladder cancer patients in the discovery cohort (25 responders and 14 non-responders to BCG therapy), miRNA expression was detected by the digital nCounter Human v3 miRNA panel assay (NanoString Technologies, Inc.). Said paraffinised primary tumour tissue samples from patients in the discovery cohort were taken at the time of transurethral resection of the bladder tumour (TURBT) and subsequently underwent BCG instillation therapy.

    [0076] A statistical analysis of the results of the digital assay was performed. In this statistical analysis, 26 differentially expressed miRNAs (7 induced and 19 repressed) were identified between responder and non-responder patients to BCG therapy (Table 4). These 26 miRNAs were differentially expressed in the tumours of the patients who showed a satisfactory response with respect to those who presented recurrence of the tumour in a period of less than 3 years.

    [0077] Relative increase expression values between responder and non-responder patients to BCG therapy are shown in Table 4. These relative increase expression values between responder and non-responder patients to BCG therapy range from +20.37 (for miR-579-3p) to -7.28 (for miR-1-3p).

    [0078] Additionally, the expression of the 26 miRNAs was detected by RT-qPCR, and a statistical analysis of the data obtained with both assays was performed. Table 4 shows the p-value obtained in the statistical analysis between responders (R) and non-responders (NR) obtained in the digital assay and in the RT-qPCR assay.

    TABLE-US-00004 miRNA Relative increase R vs NR P-value (NanoString digital assay) P-value (RT-qPCR assay) let-7c-5p -2.11 0.00109 0.013 miR-29a-3p -2.05 0.00230 0.050 miR-222-3p -1.74 0.00517 0.015 miR-199a-3p/miR-199b-3p * -2.28 0.00601 0.039 miR-23a-3p -1.72 0.00642 0.032 miR-223-3p -4.16 0.00942 0.006 miR-199b-5p -2.26 0.01039 0.033 miR-218-5p -3.20 0.01091 NS miR-125b-5p -3.34 0.01321 NS miR-23b-3p -1.77 0.01353 0.033 miR-199a-5p -1.84 0.01443 0.041 miR-1-3p -7.28 0.01735 0.048 miR-483-3p 6.72 0.01885 0.025 miR-145-5p -3.12 0.02327 0.030 miR-106a-5p ** 2.06 0.02724 0.036 miR-4443 1.79 0.02726 0.024 miR-874-5p 3.37 0.02841 0.025 miR-497-5p -2.24 0.03099 NS miR-143-3p -3.14 0.03452 0.046 miR-574-3p -2.91 0.03465 NS miR-579-3p 20.37 0.03616 0.027 miR-342-3p -1.65 0.03671 NS miR-182-5p 2.03 0.03870 0.042 miR-21-5p -2.34 0.03730 0.023 miR-429 2.24 0.04484 0.050 miR-99a-5p -5.30 0.04592 0.047 In bold, induced miRNAs, the remainder of miRNAs are repressed miRNAs. NS: not significant. * The sequences of said miRNAs are identical ** In the Nanostring assay, said miRNA and miR-17-5p, which only differs from miR-106a-5p in one nucleotide, are indistinguishable.

    [0079] Table 5 shows the correspondence between the sequences of the sequence listing and the mature sequences of the miRNAs.

    TABLE-US-00005 miRNA SEQ ID NO let-7c-5p 1 miR-29a-3p 2 miR-222-3p 3 miR-199a-3p/miR-199b-3p 4 miR-23a-3p 5 miR-223-3p 6 miR-199b-5p 7 miR-218-5p 8 miR-125b-5p 9 miR-23b-3p 10 miR-199a-5p 11 miR-1-3p 12 miR-483-3p 13 miR-145-5p 14 miR-106a-5p 15 miR-4443 16 miR-874-5p 17 miR-497-5p 18 miR-143-3p 19 miR-574-3p 20 miR-579-3p 21 miR-342-3p 22 miR-182-5p 23 miR-21-5p 24 miR-429 25 miR-99a-5p 26 miR-17-5p 27

    Example 2. Pairs of miRNAs in Predicting Response to BCG Therapy

    [0080] In paraffinised tumour tissue samples from the 43 BCG-treated bladder cancer patients in the validation cohort (28 responders and 15 non-responders to BCG therapy), Ct values were obtained by RT-qPCR from each of the 26 miRNAs in Table 4. Said primary paraffinised tumour tissue samples from patients in the validation cohort were taken at the time of transurethral resection of the bladder tumour (TURBT) and who subsequently underwent BCG instillation therapy.

    [0081] Statistical analysis was performed to evaluate the capability of pairs of the 26 miRNAs in Table 4 of differentiating between samples corresponding to BCG responder and non-responder patients. Surprisingly, the miR-21-5p/miR-182-5p, miR-222-3p/miR-182-5p, miR-429/miR-182-5p, and miR223-3p/miR-182-5p pairs differentiated between BCG-responder and non-responder patients with a very high statistical significance and very high AUC values. FIG. 1 shows the values of the Ct ratios, obtained by dividing the Ct value of the miRNA that decreases by the Ct value of the miRNA that increases. The Ct value is inversely proportional to the amount of miRNA detected in the sample and, consequently, the values of the Ct ratios obtained are positive. The AUC values obtained ranged from 0.84 for the miR223-3p/miR-182-5p pair to 0.90 for the miR-21-5p/miR-182-5p pair (FIG. 1).

    Example 3. Evaluation of miRNAs and miRNA Pairs in Predicting Response to BCG Therapy in Urine Samples

    [0082] An evaluation was made of the ability of the miR-21-5p, miR-182-5p and miR-106a-5p biomarkers and the miR-21-5p/miR-182-5p and miR-21-5p/miR-106a-5p pairs to differentiate between samples corresponding to BCG responders and non-responders in urine samples from the 20 test cohort patients (11 responders and 9 non-responders to BCG therapy), which had been obtained prior to transurethral resection of the bladder tumour (TURBT) in said patients, who subsequently underwent BCG instillation therapy.

    [0083] A statistical analysis was performed to assess the capability of said biomarkers to differentiate between samples corresponding to BCG-responder and non-responder patients, in urine samples.

    [0084] Surprisingly, the miR-21-5p/miR-106a-5p and miR-21-5p/miR-182-5p pairs differentiated responders from non-responders to BCG therapy with a high statistical significance and high AUC values. The AUC values obtained were 0.79 for the miR-21-5p/miR-106a-5p pair and 0.76 for the miR-21-5p/miR-182-5p pair (FIG. 2), indicating that said miRNA pairs are good predictors of bladder cancer patients’ response to BCG therapy.

    [0085] The Ct expression values of miR-21-5p, miR-182-5p and miR-106a-5p differentiated responders from non-responders to BCG therapy in urine samples, with a high statistical significance (FIGS. 3A-C). Therefore, miRNAs miR-21-5p and miR-106a-5p are good predictors of the response of bladder cancer patients to BCG therapy. The expression values of miR-21-5p and miR-106a-5p showed a good correlation with the levels obtained in tumour tissue (FIGS. 3D-F).

    REFERENCES

    [0086] Kiselyov et al. (2015). Treatment of non-muscle invasive bladder cancer with Bacillus Calmette-Guerin (BCG): Biological markers and simulation studies. BBA Clin., 10(4), 27-5 34.