CIRCULATING MICROVESICLES EXPRESSING CARBONIC ANHYDRASE 9 FOR THE PROGNOSIS OF RENAL CELL CARCINOMA

Abstract

A method of predicting the risk of recurrence in a subject undergoing treatment for, or having undergone treatment for, clear cell renal cell carcinoma (ccRCC), by comparing the level of extracellular vesicles, preferably microvesicles, expressing carbonic anhydrase 9 (CA9.sup.+ MVs) in a sample from the subject with a reference level. Also, a method of diagnosing ccRCC or identifying a risk of developing ccRCC, by comparing the level of CA9.sup.+ MVs in a sample from the subject with a reference level.

Claims

1-14. (canceled)

15. A method of predicting the risk of recurrence in a subject undergoing treatment for, or having undergone treatment for, clear cell renal cell carcinoma (ccRCC), comprising: a) measuring the level of extracellular vesicles expressing carbonic anhydrase 9 (CA9.sup.+ EVs) in a sample previously obtained from the subject, b) comparing the level of CA9.sup.+ EVs with a reference level, c) assigning the subject to a high-risk group of ccRCC recurrence if the level of CA9.sup.+ EVs is substantially higher than the reference level, or assigning the subject to a low-risk group of ccRCC recurrence if the level of CA9.sup.+ EVs is substantially similar or lower than the reference level.

16. The method according to claim 15, wherein CA9.sup.+ EVs are microvesicles expressing carbonic anhydrase 9 (CA9.sup.+ MVs).

17. The method according to claim 15, wherein the level of CA9.sup.+ EVs is expressed as an absolute number of CA9.sup.+ EVs in a given volume of sample.

18. The method according to claim 17, wherein the absolute number of CA9.sup.+ EVs in a given volume of sample is determined by a method consisting of: a) centrifuging the sample previously obtained from the subject at about 260 g for about 15 minutes, b) centrifuging the supernatant retrieved after step a) at about 1500 g for about 20 minutes, and c) measuring the absolute number of CA9.sup.+ EVs in a given volume of the supernatant retrieved after step b).

19. The method according to claim 15, wherein the reference level is derived from the measurement of CA9.sup.+ EVs in a sample from a reference subject or in samples from a population of reference subjects, said reference subject(s) being known to have low risks of ccRCC recurrence.

20. The method according to claim 16, wherein the reference level is about 350 CA9.sup.+ MVs/μL of sample.

21. A method of diagnosing clear cell renal cell carcinoma (ccRCC) in a subject or of identifying a subject as being at risk of developing ccRCC, comprising: a) measuring the level of extracellular vesicles expressing carbonic anhydrase 9 (CA9.sup.+ EVs) in a sample previously obtained from the subject, b) comparing the level of CA9.sup.+ EVs with a reference level, c) concluding that the subject is affected with, or is at risk of developing, ccRCC if the level of CA9.sup.+ EVs is substantially higher than the reference level.

22. The method according to claim 21, wherein CA9.sup.+ EVs are microvesicles expressing carbonic anhydrase 9 (CA9.sup.+ MVs).

23. The method according to claim 21, wherein the level of CA9.sup.+ EVs is expressed as a percentage of CA9.sup.+ EVs out of the total extracellular vesicles in the sample.

24. The method according to claim 21, wherein the reference level is derived from the measurement of CA9.sup.+ EVs in a reference subject or in a population of reference subjects not suffering from and/or not diagnosed with ccRCC.

25. The method according to claim 22, wherein the reference level is 1.85% of CA9.sup.+ MVs out of the total microvesicles in the sample.

26. The method according to claim 21, wherein diagnosing ccRCC consists of determining the tumor size and/or grading of the ccRCC.

27. The method according to claim 15, wherein the sample is a blood sample.

28. The method according to claim 15, wherein measuring the level of CA9.sup.+ EVs is carried out by flow cytometry.

29. The method according to claim 21, wherein the sample is a blood sample.

30. The method according to claim 21, wherein measuring the level of CA9.sup.+ EVs is carried out by flow cytometry.

31. A method for treating clear cell renal cell carcinoma (ccRCC) in a subject in need thereof, said method comprising: a) diagnosing clear cell renal cell carcinoma (ccRCC) in said subject or identifying said subject as being at risk of developing ccRCC, by (1) measuring the level of extracellular vesicles expressing carbonic anhydrase 9 (CA9.sup.+ EVs) in a sample previously obtained from the subject, (2) comparing the level of CA9.sup.+ EVs with a reference level, (3) concluding that the subject is affected with, or is at risk of developing, ccRCC if the level of CA9.sup.+ EVs is substantially higher than the reference level; and b) treating said subject affected with ccRCC, or identified as being at risk of developing ccRCC, for ccRCC.

32. The method according to claim 31, wherein CA9+ EVs are microvesicles expressing carbonic anhydrase 9 (CA9+ MVs).

33. The method according to claim 31, wherein the level of CA9.sup.+ EVs is expressed as a percentage of CA9.sup.+ EVs out of the total extracellular vesicles in the sample.

34. The method according to claim 31, wherein the sample is a blood sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0134] FIGS. 1A-E are a set of flow cytometry plots and graphs showing the detection by flow cytometry of circulating MVs by staining for the cell surface marker CA9.

[0135] FIG. 1A: Representative flow cytometry plot for CA9-MVs from one control and one RCC patient after staining with an anti-CA9 antibody.

[0136] FIG. 1B: Graph showing the mean of the percentage of CA9.sup.+ MVs/μL of plasma from controls (n=16) and RCC patients (n=76). Data are shown as mean values±SEM. *P<0.05.

[0137] FIG. 1C: Representative flow cytometry plot for CA9-MVs from one control, one n-ccRCC patient and one ccRCC patient. The percentages show the number of positive events for staining of plasma circulating MVs visualized by plotting CA9 marker (x axis) vs FSlog properties (y axis) and gated based on isotype control.

[0138] FIG. 1D: Graph showing the mean of percentage of CA9.sup.+ MVs/μL of plasma in controls (n=16), n-ccRCC patients (n=12) and ccRCC (n=64) patients. Data are shown as mean values±SEM. *P<0.05.

[0139] FIG. 1E: Graph showing the spearman correlation between the percentage of MVs expressing CA9/μl of plasma detected by flow cytometry and the tumor size (cm).

[0140] FIGS. 2A-B are a set of flow cytometry plots and graph showing the comparison of the percentage of CA9.sup.+ MVs from plasma of ccRCC patients before and 1 month after surgical removal of tumor.

[0141] FIG. 2A: Representative flow cytometry plots for CA9.sup.+ MVs from one RCC patient before (day 0) and 1 month after (+1 month) nephrectomy.

[0142] FIG. 2B: Graph showing the mean of the percentage of CA9.sup.+ MVs from RCC patients before (ccRCC) and 1 month after (ccRCC 1M) nephrectomy (n=10). Wilcoxon test was used to determine statistical significance. *P<0.05.

[0143] FIGS. 3A-C are a set of graphs showing the concentration of s-CA9 in the serum in controls, RCC patients, n-ccRCC patients and ccRCC patients measured by ELISA.

[0144] FIG. 3A: Graph showing the mean of plasma s-CA9 concentration expressed in pg/mL in controls (n=16) and RCC patients (n=76). Data are shown as mean values±SEM. **P<0.01.

[0145] FIG. 3B: Graph showing the mean of plasma s-CA9 concentration expressed in pg/mL in controls (n=16), n-ccRCC patients (n=12) and ccRCC (n=64) patients. Data are shown as mean values±SEM. **P<0.01.

[0146] FIG. 3C: Graph showing the spearman correlation between plasma concentration of s-CA9 (pg/mL) observed by ELISA and tumor size (cm).

[0147] FIG. 4 is a receiver operating characteristic (ROC) curve analysis using the percentage of CA9.sup.+ MVs detected by flow cytometry.

[0148] FIGS. 5A-B are a set of graphs showing the progression free survival of all patients according to the number of circulating CA9.sup.+ MVs detected by flow cytometry (A) or s-CA9 concentration detected by ELISA (B).

[0149] FIG. 5A: RCC patients with low absolute number of CA9.sup.+ MVs (<350) measured by flow cytometry revealed a better progression-free survival than those with high value (≥350).

[0150] FIG. 5B: No correlation was observed between s-CA9 concentration measured by ELISA and the progression-free survival in these patients.

EXAMPLES

[0151] The present invention is further illustrated by the following examples.

Example 1: Detection of Microvesicles Carrying CA9 by Flow Cytometry

Materials and Methods

Patients Included in the Cohort

[0152] The clinical data reported for this study were collected within the framework of the UroCCR project (NCT03293563), CNIL authorization number DR-2013-206. This prospective monocentric study included all patients treated surgically for a localized renal tumor between May 2017-January 2019.

[0153] Pre-operative clinical data were collected and anonymized via a national renal cancer database (Réseau Français de Recherche sur le Cancer du Rein—Uro-CCR). They included age, sex and tumor size. Tumors were classified according to the TNM 2009 classification (Kidney (ICD-O C64), 2010. In Sobin et al. (Eds.), TNM classification of malignant tumours (7.sup.th Ed., pp. 255-257). Oxford, UK: Wiley-Blackwell), histological subtypes were recorded according to the 2015 WHO classification of kidney tumors (Moch et al., 2016. Eur Urol. 70(1):93-105). ISUP grade for renal cell carcinomas (Delahunt et al., 2019. Histopathology. 74(1):4-17) was analyzed. The date of nephrectomy was considered as the start of follow-up.

[0154] In total, 16 individuals served as controls; 8 of these had been admitted in hospital for urological conditions other than RCC (infections, urinary stones, etc.). Other plasma controls were obtained from 8 healthy blood donors.

[0155] 77 patients (52 male and 25 female) were included in the study. A patient with a carcinoma different from RCC was excluded. Among RCC patients, 71 had a localized or locally advanced renal cancer and 6 had a metastatic renal carcinoma. Patient characteristics are summarized in Table 1. At the end of the follow-up, 9 patients died with 5 deaths from cancer. Median follow-up for patient without metastasis was 13.5 (3-48) months. Among patients without metastasis at diagnosis, 11 experienced a local or a metastatic recurrence.

[0156] Lastly, the blood samples from 10 patients were collected 1 month after the tumor resection, and these samples were used to determine whether the levels of CA9.sup.+ MVs in blood were changed after the tumor resection.

TABLE-US-00001 TABLE 1 Patient and tumor characteristics Characteristics Controls n-ccRCC ccRCC Age at Median (IQR) 40 (24-66) 72 (52-80) 65 (38-85) diagnosis (years) Gender Male 10 9 42 Female 6 3 22 TNM stage T1-T2 — 10 32 T3-T4 — 1 32 ISUP grade I-II — 6 32 III-IV — 2 32 Size ≤4 cm — 4 27  >4 cm — 8 37 Recurrence — 2 13 Cancer-related death — 1 4 ccRCC, clear cell renal cell carcinoma; n-ccRCC, non-clear cell renal cell carcinoma (i.e., other types of renal cell carcinoma); IQR: interquartile range; TNM: (T) tumor, (N) node, (M) metastasis; ISUP: International society of urological pathology.

Sample Processing

[0157] Peripheral blood (8 mL) was collected in EDTA-treated tubes (Vacutainers, Becton Dickinson, Le Pont de Claix, France) from a peripheral vein using a 21-gauge needle to minimize platelet activation and was processed for assay within 2 hours from collection (Agouni et al., 2008. Am J Pathol. 173(4):1210-1219). Blood collection was carried out before surgery.

[0158] Briefly, blood was centrifuged at 260 g for 15 minutes and platelet-rich plasma was separated from whole blood. Then, platelet-rich plasma was further centrifuged at 1500 g for 20 minutes to obtain platelet-free plasma (PFP). PFP were frozen and stored at −80° C. until subsequent use.

Characterization of Microvesicles Harboring CA9

[0159] Characterization of plasma MVs was performed by flow cytometry using a specific antibody against Carbonic Anhydrase 9 (CA9)-PE, (Cat #130-110-057, Miltenyi Biotec, Bergisch Gladbach, Germany). The antibody was incubated for 30 minutes at 4° C. Irrelevant human IgG were used as isotype-matched negative control. Samples were analyzed in a flow cytometer 500 MPL system (Beckman Coulter, Villepinte, France). MV quantification was performed using calibrated 10 μm-sized Flowcount beads (Beckman Coulter) of known concentration on FC500 cytometer (Beckman Coulter, France).

Results

[0160] To determine whether MV quantification can be used as a diagnostic tool in clinical situation, we analyzed circulating MVs in the plasma without a purification step of MVs. The optimal configuration and settings for quantitative and qualitative flow cytometry analyses of MVs have been adapted from the study by Nolte-'T Hoen et al. (2013. J Leukoc Biol. 93(3):395-402). Therefore, using the optimal settings, 100 nm fluorescent polystyrene beads were efficiently detected above background noise.

[0161] CA9 was detected as circulating MV cargo component by flow cytometry. RCC patient samples exhibited a strong positive staining by anti-CA9 antibody, whereas the corresponding control sample showed only a weak fluorescence signal (FIG. 1A).

[0162] We assessed whether the percentage of MVs carrying CA9 (CA9.sup.+ MVs) in the plasma was associated with disease, stage or grade. Firstly, we found no significant association between levels of CA9 carried by circulating MVs and gender, age, or ccRCC TNM stage (Table 2). Next, we found that CA9.sup.+ MVs percentage was significantly higher in plasma from RCC patients than in plasma from controls (FIG. 1B). In subgroups of RCC, CA9.sup.+ MVs were significantly higher in plasma from ccRCC, when compared with healthy controls (FIGS. 1C and 1D). Moreover, the level of CA9.sup.+ MVs in ccRCC patients was markedly more elevated than in n-ccRCC patient samples (FIGS. 1C and 1D).

[0163] Interestingly, the percentage of CA9.sup.+ MVs in the plasma from ccRCC patients decreased after surgery (FIGS. 2A and 2B), suggesting that the signal of CA9 obtained by flow cytometry originates from tumor-derived MVs.

[0164] Moreover, in ccRCC patients, CA9.sup.+ MVs correlated with tumor size (FIG. 1E) and with ISUP grade I-II versus III-IV (Table 2).

TABLE-US-00002 TABLE 2 Relationship between clinical characteristics and levels of circulating CA9.sup.+ MVs (in % of the total circulating MVs). ccRCC CA9.sup.+ MVs Parameter patients (n) Mean p-value Gender 0.36 Male 42 7.3 Female 22 4.09 Age 0.72 ≥60 39 6.13  <60 25 6.93 TNM Stage 0.65 T1-T2 32 5.11 T3-T4 32 7.78 ISUP grade 0.05 I-II 32 4.3 III-IV 32 8.56 ccRCC: clear cell renal cell carcinoma; TNM: (T) tumor, (N) node, (M) metastasis; ISUP: International society of urological pathology

Example 2: Quantitative Analysis of Plasma CA9 Concentration

Materials and Methods

Measure by ELISA of CA9 Concentration in the Plasma

[0165] Soluble plasma CA9 (s-CA9) was quantified by human carbonic anhydrase 9 Quantikin ELISA kit (Cat #DCA900, R&D Systems, Minneapolis, MN, USA) according to the protocol of the manufacturer.

[0166] Briefly, plasma samples or standard control samples were incubated on microplates coated with a specific antibody to CA9 for 2 hours at room temperature. The plates were washed to remove unbound antibodies.

[0167] After incubation of a conjugate solution, a substrate solution was added. Color development was stopped after 30 minutes. A microplate reader (VICTOR Multilabel plate reader) was used to determine colorimetric densities at 450 nm.

[0168] Final results were calculated according to the standard curve. Results were expressed in pg/mL. The mean limit of quantification of this method was 2.28 pg/mL.

Results

[0169] Levels of s-CA9 detected by ELISA were significantly higher in plasma from RCC patients (n=70) than in plasma from healthy controls (FIG. 3A). In particular, the plasma s-CA9 levels in ccRCC patients were significantly higher than in healthy controls. The mean of plasma s-CA9 levels were 117.5 pg/mL (range 5-550.29) in ccRCC patients, 91.2 pg/mL in n-ccRCC patients, and 47.12 pg/mL in controls (FIG. 3B).

[0170] However, the plasma s-CA9 levels detected by ELISA did not correlate with the tumor size measured at pathologic examination in ccRCC patients (FIG. 3C), with the ISUP grade or the pathologic stage (Table 3). This contrasts with the data obtained in Example 1 where CA9.sup.+ MVs correlated with tumor size (FIG. 1E) and with ISUP grade I-II versus III-IV (Table 2).

TABLE-US-00003 TABLE 3 Relationship between s-CA9 levels measured by ELISA in plasma and clinico-pathological variables. ccRCC ccRCC ccRCC ccRCC TNM TNM ISUP ISUP stage stage p- grade grade p- T1-T2 T3-T4 value I-II II-III value Case 31 27 32 25 number Mean 112 135.6 0.47 140.2 103.9 0.41 (pg/mL) Range 3.82-357.5 16.18-550.3 3.82-550.3 5-410.8 (pg/mL) ccRCC: clear cell renal cell carcinoma; TNM: (T) tumor, (N) node, (M) metastasis; ISUP: international society of urological pathology

Example 3: Robustness of Circulating CA9.SUP.+ MVs as a Diagnostic Tool in ccRCC

Methods

Receiver Operating Characteristic (ROC) Curves

[0171] To evaluate the diagnostic performance of CA9.sup.+ MVs between ccRCC and healthy controls, receiver operating characteristic (ROC) curves were plotted and other diagnostic characteristics such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), Yule's Q coefficient, Youden's index and the Chi.sup.2 test of significant variables were calculated.

Results

[0172] In order to test the robustness of flow cytometry as diagnostic tool for detecting CA9.sup.+ MVs in ccRCC, we generated a ROC curve (FIG. 4). Cut-off values for CA9.sup.+ MVs detected by flow cytometry to predict ccRCC were derived from ROC curves in 64 patients. CA9.sup.+ MVs>1.85% showed an area under the curve (AUC) of 0.70 (95% CI: 0.57-0.84) and a sensitivity of 68.8%, specificity of 60.9%.

[0173] Of the 64 ccRCC patients, 88.6% (PPV) of individuals who achieve a higher cut-off of 1.85% on the flow cytometry are accurately diagnosed with ccRCC. Conversely, 30.7% (NPV) of individuals who achieve a cut-off of 1.85% or lower are accurately diagnosed as healthy. Despite Youden's index was 0.3, Yule's Q coefficient was calculated as 0.55, indicating a strong association between CA9.sup.+ MVs detected by flow cytometry and ccRCC. In addition, the Chi.sup.2 test result was ≤0.05 (Table 4), demonstrating that a threshold value of 1.85% could be considered predictive in these patients.

TABLE-US-00004 TABLE 4 Test characteristics of CA9.sup.+ MVs detected by flow cytometry for prediction of ccRCC CA9.sup.+ MVs True positives 39 True negatives 11 False positives 5 False negatives 25 Sensitivity (95% CI) 68.8 (41.3-88.9) Specificity (95% CI) 60.9 (47.9-72.9) Positive predictive value (%) 88.6 Negative predictive value (%) 30.7 Yule's Q coefficient 0.55 Youden's index 0.3 Chi.sup.2 test ≤0.05 Accuracy % 62.5

Example 4: Evaluation of Progression-Free Survival

Methods

Progression-Free Survival Prognosis

[0174] In addition to the percentage of CA9.sup.+ MVs, a numeration in absolute value per μL of plasma was also performed by flow cytometry on 76 patients using Flowcount beads. Association between clinical and pathological characteristics and the absolute number of CA9.sup.+ MVs detected by flow cytometry and s-CA9 concentration measured by ELISA is reported in Table 5. Here, the median values were used to divide the patients into high- and low-CA9 groups and to establish a threshold value. Progression-free survival (PFS) was estimated using the Kaplan-Meier method for patients without metastasis and comparison was performed by the log-rank test.

TABLE-US-00005 TABLE 5 RCC patient and tumor characteristics according to median levels of MVs carrying CA9/μL plasma and s-CA9 measured by flow cytometry and ELISA, respectively. The median was used to divide the patients into high- and low-CA9 groups. n = 76 n = 70 CA9-MVs CA9-MVs S-CA9 s-CA9 <350 ≥350 P <88 ≥88 P Gender 1 1 Female 13 11 11 10 Male 27 25 26 23 Median age (Year, SD) 62.8 (12) 63.8 (11) 0.717  62 (13)  65 (11) 0.275 T Stage 0.295 0.455 1 22 14 19 16 2 1 4 4 1 3 15 15 11 15 4 1 2 1 1 Histological subtype 1 0.23 Clear renal cell 33 30 28 29 carcinoma Others 7 6 37 33 ISUP grade 0.834 0.681 1 3 2 2 3 2 19 14 17 16 3 10 11 10 6 4 6 7 5 7 Metastasis 0.117 0 35 35 33 32 1 1 4 0 2 1 Median Tumor  4.95 (2.5)  6.26 (3.3) 0.05 5.42 (2.9) 5.42 (3)   0.99 size (cm, SD) Recurrence 1 11 0.006 3 7 0.139 For patient without metastasis at diagnosis SD: Standard deviation

Results

[0175] The median of the numbers of CA9.sup.+ MVs determined by flow cytometry was 350 MVs/μL of plasma (33-47328), whereas the median value of s-CA9 concentration quantified by ELISA was 88 pg/mL (4-550) (n=70). Tumor size (p=0.05) and tumor recurrence (p=0.006) were correlated with high values of CA9.sup.+ MVs/μL of plasma.

[0176] Patients with high number of CA9.sup.+ MVs (>350 CA9.sup.+ MVs/μL of plasma) had a lower progression-free survival (p=0.01) compared to patients with low CA9.sup.+ MV number (<350 CA9-MVs/μL of plasma) (FIG. 5A), whereas high concentrations of s-CA9 (>88 pg/mL) measured by ELISA did not significantly correlate with low progression-free survival (p=0.089) (FIG. 5B).

[0177] In conclusion, determining CA9.sup.+ MVs levels offers a robust prognostic of RCC recurrence, unlike s-CA9 concentration.