Prognostic method for determining the risk of relapse in renal cancer patients with a clear cell type of renal carcinoma, stages I and II, and kit for same

Abstract

A prognostic method, and a kit, for determining the risk of relapse of renal cancer of the clear cell renal carcinoma type, stages I and II, in a human subject; comprised of: (a) determining, in a tumor sample (biopsy) from the human subject, the levels of expression of the microRNAs hsa-miR-223, hsa-miR-103, hsa-miR-107, hsa-miR-425, hsa-miR-340, hsa-miR-130b, hsa-miR-652, hsa-miR-214, and hsa-miR-204; (b) determining a value which depends on the levels of expression of the microRNAs; and (c) determining the risk of relapse in renal cancer of the clear cell renal carcinoma type, in stages I and II, in the said human subject, by comparing the value obtained in step (b) with a cut-off value.

Claims

1. A prognostic method for determining the risk of relapse of renal cancer of the clear cell renal carcinoma (CCRC) type, stages I and II, in a human subject; comprising: (a) determining, in a renal tumor sample biopsy from the human subject, the levels of expression of each of hsa-miR-223, hsa-miR-103, hsa-miR-107, hsa-miR-425, hsa-miR-340, hsa-miR-130b, hsa-miR-652, hsa-miR-214, and hsa-miR-204 with sequences identified by the sequences SEQ ID NOs: 1-9; (b) determining a value which depends on the levels of expression of said microRNAs; (c) determining the risk of relapse in renal cancer of the CCRC type, in stages I and II, in the said human subject, by comparing the value obtained in step (b) with a cut-off value.

2. The prognostic method according to claim 1, wherein the level of expression of said microRNAs is determined with the use of at least one microarray of microRNAs.

3. A prognostic and treatment method for determining the risk of relapse of renal cancer of the CCRC type, stages I and II, in a human subject and subsequently treating said subject, the method comprising: (a) determining, in a renal tumor sample biopsy from the human subject, the level of expression of each of the microRNAs: hsa-miR-223, hsa-miR-103, hsa-miR-107, hsa-miR-425, hsa-miR-340, hsa-miR-130b, hsa-miR-652, hsa-miR-214, and hsa-miR-204 with sequences identified by the sequences SEQ ID NOs: 1-9; (b) determining a value which depends on the levels of expression of said microRNAs; (c) determining the risk of relapse in renal cancer of the CCRC type, in stages I and II, in the said human subject, by comparing the value obtained in step (b) with a cut-off value, and (d) obtaining a value in step (b) that is greater than said cut-off value, classifying said human subject as being at high risk of relapse of renal cancer of the CCRC type and subsequently administering a therapeutically effective amount of sunitinib, sorafenib, everolimus, axitinib, and/or temsirolimus to said human subject as an adjuvant therapy.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Disease-free survival for various risk groups. The 5-year survival was 93.9% for patients at low risk, and 61.54% for patients at high risk (hazard ratio HR=12.1, p=0.0001) (confidence interval CI=3.012-37.92).

(2) FIG. 2. Cancer-specific survival (CSS) for various risk groups. The 5-year CSS was 95.7% for patients at low risk, and 86.4% for patients at high risk (HR=7.7, p=0.0084) (CI=1.687-35.14).

EXAMPLES

Example 1. Design of the Study, and Cohort of Patients

(3) An observational study was carried out which included all of the radical and partial nephrectomies performed at “October 12” University Hospital, of Madrid, between the year 1999 and 2008.

(4) All patients who underwent radical or partial nephrectomies, open or laparoscopic, between 1999 and 2008, were included in the study.

(5) For the staging, the TNM system was employed, which is the most commonly accepted classification. This classification was last modified in 2010.

(6) TABLE-US-00002 TABLE 1 TNM Classification T: Primary tumor: Tx: Primary tumor cannot be evaluated. T0: No evidence of a primary tumor. T1: Primary tumor <7 cm in major diameter, confined to kidney. T1a: Primary tumor <4 cm in major diameter, confined to kidney. T1b: Primary tumor >4 cm and <7 cm in major diameter, confined to kidney. T2: Primary tumor >7 cm in major diameter, confined to kidney. T2a: Primary tumor >7 cm and <10 cm in major diameter, confined to kidney. T2b: Primary tumor >10 cm in major diameter, confined to kidney. T3a: The tumor has invaded the renal vein or its segmental branches (with muscle), [and] has infiltrated the fat of the sinus; T3b: The tumor has invaded the vena cava below the diaphragm; T3c: The tumor has invaded the vena cava above the diaphragm or has infiltrated the wall of the vein; T4: The tumor has invaded beyond the Gerota's fascia. N: Regional lymphatic ganglia: Nx: Regional ganglia cannot be evaluated. N0: No evidence of regional ganglia metastasis. N1: Metastasis at the level of a single regional ganglion. N2: Metastasis in more than one regional ganglion. M0: No metastasis; M1: Distant metastasis.

(7) Grouping into stages TNM:

(8) TABLE-US-00003 Stage I T1 N0 M0 Stage II T2 N0 M0 Stage III T3 N0 M0 T1, T2, T3 N1 M0 Stage IV T4 any N M0 any T N2 M0 any T any N M1
Of a total of 164 patients, 71 were selected who met the following criteria: Cellular histology is clear Tumor stage is stage I (T1N0M0) or stage II (T2N0M0) Fuhrman grade: Any Tumor size: Any, provided that the tumor is confined to the kidneys (i.e. less than or equal to T2b) ECOG status (Eastern Cooperative Oncology Group Performance Status): Any. Patients who are asymptomatic or with symptoms which are related.
Any patient with any of the following characteristics was excluded: Tumor stage equal to or greater than T3 Any N+ Any M+ Any other histology (e.g. papillar, chromophobic, or with sarcomatoid differentiation) Time of monitoring less than 1 year Lack of sufficient data in the clinical history Lack of samples in paraffin for analysis of microRNA

Example 2. Analysis of the Expression of microRNAs

(9) The tumor pieces from the nephrectomy were fixed in formalin; in particular, after a piece from a partial or radical nephrectomy was received, it was weighed, measured, and fixed by immersion in formaldehyde (10% formalin) for 24-48 hours.

(10) Subsequently, the piece was included in paraffin, in an automatic tissue processor.

(11) Then the piece was cut, and RNA was extracted, from the tumor samples fixed in formalin and embedded in paraffin.

(12) The samples were hybridized with microarrays of human miRNA, version 14.0, 8×15K (Agilent Technologies), according to the manufacturer's protocol.

Example 3. Prognostication of the Risk of Relapse in CCRC Based on a Profile of miRNAs

(13) A level of statistical significance was calculated for each miRNA based on a Cox regression model (Shu, Y., Klein, J. P., and Zhang, M-J., 2007, Asymptotic theory for the Cox semi-Markov illness-death model. Lifetime Data Anal., March; 13(1):91-117), with the objective of ascertaining a profile of miRNAs the expression of which bears a significant relation to survival free from the disease. The miRNAs related to disease free survival were “filtered” based on their p values. Micro RNAs (miRNAs) showing a p value <0.01 were used to develop prediction models of the risk of relapse, using the method of supervised principal components. Additionally, the correlation between the miRNAs selected was evaluated, in order to establish correlation groups so as to be able find reduced profiles. Cross-validation was employed to evaluate the exactitude of prediction of the profiles. The cut-off point was established a priori; and, to test the statistical significance, the value of p in the log rank test for the risk groups was evaluated, using 10,000 aleatory permutations.

(14) A prediction model for progression in patients with CCRC diagnosed in stages I and II (RCC-9miR score) was generated. This model is comprised of the expression of the nine miRNAs (Table 2). The prediction score is calculated by the following formula:
Σ.sub.iw.sub.ix.sub.i−2,896,583,
where w.sub.i is the weight, and x.sub.i is the expression value of the microRNA i.

(15) The result of the formula is a value which depends on the levels of expression of the microRNAs hsa-miR-223, hsa-miR-103, hsa-miR-107, hsa-miR-425, hsa-miR-340, hsa-miR-130b, hsa-miR-652, hsa-miR-214, and hsa-miR-204, having sequences identified by the sequences SEQ ID NO: 1-9.

(16) TABLE-US-00004 TABLE 2 Prediction values of the 9 miRNAs: miRNA p w.sub.i (weight) hsa-miR-223 0.0013 0.098392 hsa-miR-103 0.0026 0.045806 hsa-miR-107 0.0076 0.045869 hsa-miR-425 0.0083 0.163188 hsa-miR-340* 0.0107 0.495063 hsa-miR-130b 0.0108 0.374902 hsa-miR-652 0.0152 0.311327 hsa-miR-214 0.0163 −0.064772 hsa-miR-204 0.0173 −0.220399
Table 3 illustrates the association between the miRNA profile and progression of the disease. The association of the miRNA profile with progression of the disease is statistically significant, with p=0.0001.

(17) TABLE-US-00005 TABLE 3 Contingency table, of miRNA profile versus progression of the disease: Progression Yes n (%) No n (%) P Low risk 3 (6%)   47 (94%).sup.  0.0001 High risk 9 (42.8%) 12 (57.2%)

(18) Patients with a score greater than 0.954 are regarded as high risk. The model assessed 30% of the patients as high risk. The 5-year disease-free survival (DFS) was 93.9% for low risk patients and 61.54 for high risk patients (hazard ratio HR=12.1, p=0.0001) (confidence interval CI=3.012-37.92). These difference in the long-rank test were validated with 10,000 permutations (p>0.0013). The 5-year cancer-specific survival was 95.7% for the low risk group and 86.4% for the high risk group ((HR=7.7, p=0.0084) (CI=1.687-35.14).

(19) The 9-miRNA predictor is an excellent predictor of progression of the disease. The 9-miRNA predictor has been used as a predictive factor, and is statistically significant (p=0.023, HR=6.55, 95% CI=1.29=33.165) in the analysis of cancer-specific survival (CSS).

(20) All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.