CZ31584U1 - A diagnostic kit for assessing relapse risk after nephrectomy in patients with non-metastatic renal cell carcinoma - Google Patents

Description

Technical field

The present invention relates to a diagnostic set for the detection of tissue microRNAs allowing the determination of the risk of relapse after nephrectomy in patients with renal cancer.

BACKGROUND OF THE INVENTION

Renal cell carcinoma (RCC) is the most common adult renal cancer, accounting for approximately 3% of solid tumors in the adult population, with mortality slightly above 40%. Several large retrospective studies have shown that the current TNM system (Tumor, Node, Metastasis) for classifying non-metastatic renal cancer (Stage I-III) is not accurate enough to stratify patients according to their prognosis. Although the TNM system has been continually improved to improve accuracy, it does not sufficiently reflect the molecular-biological aspects of tumor behavior. This fact significantly influences in particular correct indication of adjuvant treatment and follow-up programs after curative surgery (nephrectomy). As the adjuvant therapy is not indicated in the vast majority of patients with non-metastatic renal cell carcinoma, one of the so-called unmet medical needs is a prognostic biomarker enabling the identification of patients at high risk of relapse after nephrectomy and individualization of treatment and follow-up.

The essence of the technical solution

Within the scope of the present invention, a specific miRNA profile in tumor tissue associated with the risk of renal cancer relapse after nephrectomy has been identified and a prognostic set based on miRNA expression for renal cancer patients (RCC) has been developed to identify patients at high risk of nephrectomy would benefit from adjuvant therapy, eg targeted therapy, and a more intensive monitoring regime.

The present invention provides a diagnostic kit for determining the risk of renal cell relapse after nephrectomy based on the detection of tissue miRNAs by real-time PCR, which comprises:

- miR-429 expression level kit containing primers, a TaqMan fluorescent probe and a stem-coop primer

Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACGGTT - 3 ’ Universal reverse primer 5 ’- GTGCAGGGTCCGAGGT - 3’ Forvard primer 5 '- GTTGGGTAATACTGTCTGGTAA - 3' TaqMan probe 5 ’- AGCCAACACGGTTTTACCA - 3’

- miR-30a-5p expression level kit containing primers, TaqMan fluorescent probe and stem-loop primer:

Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTTCCA - 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3'

-1 CZ 31584 Ul

Forvard primer 5 '- GGGTGTAAACATCCTCGAC - 3' TaqMan probe 5 '- AGCCAACCTTCCAGTCGAG - 3'

- miR-30e-5p expression level kit containing primers, a TaqMan fluorescent probe and a stem-loop primer:

Stem-loop primer 5 ’- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTTCCA - 3’ Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GTTTGGTGTAAACATCCTTGAC - 3' TaqMan probe 5 ’- AGCCAACCTTCCAGTCAAG - 3’

- miR-30e-3p expression level kit containing primers, a TaqMan fluorescent probe and a stem-loop primer:

Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGCTGTA - 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GGGCTTTCAGTCGGATGTT - 3' TaqMan probe 5 '- AGCCAACGCTGTAAACATC - 3'

- miR-200b expression level kit containing primers, a TaqMan fluorescent probe and a stem-loop primer:

Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCATCA - 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3 ’ Forvard primer 5 '- GTTTGGTAATACTGCCTGGTAA - 3' TaqMan probe 5 '- AGCCAACTCATCATTACCA - 3'

- a kit for determining the expression level of RNU44 RNA as a reference gene, containing primers, a TaqMan fluorescent probe and a stem-loop primer:

Stem-loop primer 5 ’- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTTAGTC - 3’

- 7 CZ 31584 Ul

Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GTCCTGGATGATGATAAGCAAA - 3' TaqMan probe 5 '- AGCCAACTTAGTCAGTTAG - 3 ’

The kits are based on the stem-loop RT-qPCR method. Typically, this method involves the step of selectively capturing the detected small RNA by stem-loop primers and reverse transcription, and quantifying by a sequence-specific TaqMan fluorescent probe, forward primers and reverse primers, which is usually universal.

The diagnostic set according to the invention allows to specifically and sensitively differentiate renal cancer patients based on the risk of relapse after nephrectomy. This kit and the diagnostic procedure using this kit are based on a quantitative analysis of the expression levels of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p, miR-200b in tumor tissue of renal cancer patients using the method qRT-PCR. Determination of prognostic microRNA levels miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p, and miR-200b makes it possible to distinguish patients with a low and high risk of relapse after nephrectomy. The expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p, miR-200b is determined in RNA isolated from tumor tissue of renal cancer patients using the kit of the present invention.

The nucleotide sequences of the subject microRNAs are as follows:

miR-429: 5 'UAAUACUGUCUGGUAAAACCGU 3';

miR-30a-5p: 5 'UGUAAACAUCCUCGACUGGAAG 3';

miR-30e-5p: 5'UGUAAACAUCCUUGACUGGAAG 3 ';

miR-30e-3p: 5 'CUUUCAGUCGGAUGUUUACAGC 3';

miR-200b: 5 'UAAUACUGCCUGGUAAUGAUGA 3'.

The nucleotide sequence of RNU44 is as follows:

5 'CCTGGATGATGATAAGCAAATGCTGACTGAACATGAAGGTCTTAATTAGCTCTAACTGACTAA 3'.

The prognostic score calculated based on the expression levels of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b measured in renal cancer tissue using a diagnostic set was found to be significantly reduced in patients, who developed disease relapse within 36 months after nephrectomy, compared to patients who had not relapsed at this time. The determination of the expression levels of these miRNAs provides significant information about the prognosis of the disease, enabling the identification of patients who would benefit from adjuvant therapy and should be monitored more intensively after surgery.

The kit may also preferably contain deionized water serving as a negative control, and / or a mixed qRT-PCR standard corresponding to the expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b in the sample. a renal cancer patient relapsed earlier than 36 months after nephrectomy, and a standard corresponding to the expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p, and miR-200b in a patient sample renal carcinoma that did not relapse or relapsed more than 36 months after nephrectomy.

Clarification of drawings

Figure 1 shows the difference in the level of prognostic miRNA score (PMS) combining the expression of said miRNAs between RCC patients who relapsed within 36 months of nephrectomy and RCC patients who did not relapse or relapsed up to more than

Ul of months from nephrectomy (P <0.0001). The significance of the difference in PMS values was monitored by Mann-Whitney test.

Figure 2 shows a combined ROC curve for PMS that indicates with which sensitivity and specificity the prognostic set is able to distinguish renal cancer patients based on the risk of disease relapse (AUC = 0.8333, specificity 90%, sensitivity 77%).

Example of technical solution implementation

To test the diagnostic ability of miRNAs according to the present invention, a qRT-PCR set with the following composition was prepared:

Component Amount Purpose Mix RT - reverse transcription ¹ 1 x 4.67 μΐ (1 rxn) Transcription of miRNA into cDNA Mix PCR ² 1 x 17.67 μΐ (1 rxn) Amplification of miRNA Detection assay for miR-429 (RT primer; qPCR primers, TaqMan probe) 2 μΐ; 1 μΐ (1 rxn) Reverse transcription and amplification of miR-429 Detection assay for miR-30a-5p (RT primer; qPCR primers, TaqMan probe) 2 μΐ; 1 μΐ (1 rxn) Reverse transcription and amplification of miR-30a-5p Detection assay for miR-30e-5p (RT primer; qPCR primers, TaqMan probe) 2 μΐ; 1 μΐ (1 rxn) Reverse transcription and amplification of miR-30e-5p Detection assay for miR-30e-3p (RT primer; qPCR primers, TaqMan probe) 2 μΐ; 1 μΐ (1 rxn) Reverse transcription and amplification of miR-30e-3β Detection assay for miR-200b (RT primer; qPCR primers, TaqMan probe) 2 μΐ; 1 μΐ (1 rxn) Reverse transcription and amplification of miR-200b Detection assay for RNU44 (RT primer; qPCR primers, TaqMan probe) 2 μΐ; 1 μΐ (1 rxn) Reverse transcription and amplification of RNU44 Deionized water RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Negative control Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 1 for miR-429 Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 2 for miR-429 Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 1 for miR-30a-5p Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 2 for miR-30a-5p Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 1 for miR-30e-5p Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 2 for miR-30e-5p Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 1 for miR-30e-3p Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 2 for miR-30e-3p Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 1 for miR-200b Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 2 for miR-200b Positive control RT, qPCR 3,33 μΐ; 1.33 μΐ (1 rxn) Positive Control 1 for RNU44

Λ

CZ 31584 Ul

Positive control RT, qPCR 3,33 μϊ; 1.33 μϊ (1 rxn) Positive Control 2 for RNU44

Note * Mix RT contains 10X RT buffer, dNTP mix w / dTTp (100M total), RNase inhibitor (20U / pL), and MultiScribe ™ RT enzyme (50U / pL). ² PCR mix contains AmpliTaq Gold® DNA Polymerase, UP (Ultra Pure), dNTPs with dUTP / dTTP mixture, ROX ™ Passive Reference and optimized buffer. The stated volumes of components apply to one miRNA / RNU within a 10 μΐ RT reaction and a 20 μϊ qPCR reaction. Positive Control 1 contains a mixed qRT-PCR standard corresponding to the expression of the miRNA in the patient's sample of renal cell carcinoma that had developed before 36 months after nephrectomy. Positive Control 2 contains a mixed qRT-PCR standard corresponding to the expression of the miRNA in a renal cancer patient sample who has not relapsed or relapsed more than 36 months after nephrectomy.

To isolate RNA, tumor tissue from renal cancer patients collected in a tube containing RNA later was used to stabilize RNA. Tissue was homogenized with FastPrep-24 5G and FastPrep Lysing Matrix (MP Biomedicals) and total RNA enriched for the short RNA fraction was isolated using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific). The quality and amount of RNA obtained was determined spectrophotometrically (NanoDrop-2000).

The above-described detection kits containing both specific stem-loop reverse transcription (RT) primers were used to detect miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p, miR-200b and RNU44 expression, by which RNA is transcribed into cDNA as well as primers and fluorescent probes of the TaqMan type for qPCR. QPCR amplification was performed using a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). The quantity of miRNAs determined is expressed by the so-called Cq (quantitation cycle), ie the cycle number at which the fluorescent signal reached the set threshold value. The detected expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b was normalized to the expression level of the short nuclear RNA RNU44, commonly used as a reference gene for relative miRNA quantitation.

The relative expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b was determined by the 2 ' ^ACq method, where the ACq value represents the difference between the Cq of the miRNAs determined and the Cq reference gene (RNU44) within the sample.

All patients whose RNA was used for analysis were pre-diagnosed by histopathological examination of the collected tissue. Only patients in whom non-metastatic renal cancer was confirmed were included in the study.

In RCC patients with srelapsed disease up to 36 months after nephrectomy, a significantly reduced PMS value was calculated based on a combination of normalized expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b compared to patients who relapsed more than 36 months after nephrectomy (P <0.0001). The expression difference between the observed cohorts was tested using the Mann-Whitney test.

The specificity and sensitivity with which PMS can distinguish renal cancer patients based on the risk of disease relapse was determined using a receiver operating characteristic (ROC) analysis.

To calculate the risk of relapse, normalized expressions of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b were combined into a so-called PMS (prognostic miRNA score), calculated according to the formula:

PMS = 1.1004 + 116.823 * miR-429 - 2.519 * miR-30a-5p + 3.831 * miR-30e-5p 34.4007 * miR-30e-3p - 11.4517 * miR-200b

Note miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b represent the decimal logarithm of the relative expression of the respective miRNA calculated according to said formula 2 ' ^ACq , where ACq represents the difference between the Cq of the microRNAs determined and Cq of the reference gene (RNU44) within a given sample.

Based on the ROC analysis performed from the PMS values, an evaluation threshold (cut-off = -0.2231) was obtained according to which RCC patients with a low and high risk of relapse after nephrectomy can be distinguished. In patients with PMS higher than the threshold, the risk of relapse is considered low, while in patients with PMS lower than the threshold, the risk is considered high.

-5GB 31584 Ul

The results of the documented figures show that the PMS value combining expression levels of miR429, miR-30a-5p, miR-30e-5p, miR-30e-3p, and miR-200b is significantly higher in tumor tissue of patients who have not relapsed / developed. to relapse the disease for more than 36 months after nephrectomy. Thus, the level of these microRNAs makes it possible to predict the risk of renal cancer relapse. Figure 2 shows a combined ROC curve for prognostic microRNA score (PMS), which indicates the sensitivity and specificity the prognostic set is able to distinguish patients with renal carcinoma based on the risk of disease relapse.

Claims (3)

PROTECTION REQUIREMENTS 1. Diagnostic set for determining the risk of relapse after nephrectomy in patients with non-metastatic renal cell carcinoma based on real-time quantitative PCR detection of tissue microRNAs, comprising: - miR-429 expression level kit containing primers, a TaqMan fluorescent probe and a stem-coop primer: Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACGGTT - 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GTTGGGTAATACTGTCTGGTAA - 3' TaqMan probe 5 '- AGCCAACACGGTTTTACCA - 3' - miR-30a-5p expression level kit containing primers, a 15 TaqMan fluorescent probe and a stem-loop primer: Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTTCCA - 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GGGTGTAAACATCCTCGAC - 3' TaqMan probe 5 '- AGCCAACCTTCCAGTCGAG - 3' - miR-30e-5p expression level kit containing primers, a TaqMan fluorescent probe and a stem-loop primer: Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTTCCA- 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' CZ 31584 Ul Forvard primer 5 '- GTTTGGTGTAAACATCCTTGAC - 3' TaqMan probe 5 '- AGCCAACCTTCCAGTCAAG - 3' - miR-30e-3p expression level kit containing primers, a TaqMan fluorescent probe and a stem-loop primer: Stem-loop primer 5 ’- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGCTGTA - 3 ' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3 ’ Forvard primer 5 '- GGGCTTTCAGTCGGATGTT - 3' TaqMan probe 5 '- AGCCAACGCTGTAAACATC - 3 ’ - miR-200b expression level kit containing primers, a TaqMan fluorescent probe and a stem-loop primer: Stem-loop primer 5 ’- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCATCA - 3’ Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GTTTGGTAATACTGCCTGGTAA - 3' TaqMan probe 5 '- AGCCAACTCATCATTACCA - 3' 5 - kit for determining the expression level of RNU44 RNA as a reference gene, containing primers, a TaqMan fluorescent probe and a stem-loop primer: Stem-loop primer 5 '- GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTTAGTC - 3' Universal reverse primer 5 '- GTGCAGGGTCCGAGGT - 3' Forvard primer 5 '- GTCCTGGATGATGATAAGCAAA - 3' TaqMan probe 5 '- AGCCAACTTAGTCAGTTAG - 3' 2. The set of claim 1 further comprising deionized water as a negative control. Set according to any one of the preceding claims, characterized in that it further comprises a mixed qRT-PCR standard corresponding to the expression of miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p and miR-200b in the sample. a patient with renal cancer who relapsed more than 36 months after nephrectomy and a standard corresponding to miR-429, miR-30a-5p, miR-30e-5p, miR-30e-3p 31584 U1 and miR-200b in a renal cancer patient sample who did not relapse or relapse more than 36 months after nephrectomy.