EUKARYOTIC TRANSLATION INITIATION FACTORS (EIFS) AS NOVEL BIOMARKERS IN BLADDER CANCER

Abstract

The present invention relates to a method of diagnosing bladder cancer in an individual. Further, the present invention relates to a method of determining the course of bladder cancer in an individual. Furthermore, the present invention relates to a kit for diagnosing bladder cancer in an individual or determining the course of bladder cancer in an individual.

Claims

1. A method of diagnosing bladder cancer in an individual (suspected of having bladder cancer) comprising the step of: determining the level of at least one eukaryotic Initiation Factor (eIF) in a biological sample from an individual, wherein the at least one eIF is selected from the group consisting of eIF1, eIF5A, eIF4B, eIF4G, eIF5B, and eIF6.

2. The method of claim 1, wherein the level of the at least one eIF is compared to a reference level of said at least one eIF.

3. The method of claim 2, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual.

4. The method of claim 2, wherein the level of the at least one eIF selected from the group consisting of eIF1 and eIF5A which is below the reference level indicates that the individual suffers from bladder cancer, and/or the level of the at least one eIF selected from the group consisting of eIF4B, eIF4G, eIF5B, and eIF6 which is above the reference level indicates that the individual suffers from bladder cancer.

5. A method of determining the course of bladder cancer in an individual (suffering from bladder cancer) comprising the step of: determining the level of at least one eukaryotic Initiation Factor (eIF) in a biological sample from an individual, wherein the at least one eIF is selected from the group consisting of eIF1, eIF5A, eIF4B, eIF4G, eIF5B, and eIF6.

6. The method of claim 5, wherein the level of the at least one eIF is compared to a reference level of said at least one eIF.

7. The method of claim 6, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual, or at least one patient having bladder cancer.

8. The method of claim 5, wherein said determining comprises determining the level of the at least one eIF in a biological sample at a first point in time and in at least one further biological sample at a later point in time and comparing said levels determined at the different time points.

9. The method of claim 8, wherein the level of the at least one eIF selected from the group consisting of eIF1 and eIF5A which (i) decreases over time indicates that bladder cancer worsens in the individual, (ii) does not change over time indicates that bladder cancer does not worsen/is stable in the individual, or (iii) increases over time indicates that bladder cancer improves in the individual.

10. The method of claim 8, wherein the level of the at least one eIF selected from the group consisting of eIF4B, eIF4G, eIF5B, and eIF6 which (i) increases over time indicates that bladder cancer worsens in the individual, (ii) does not change over time indicates that bladder cancer does not worsen/is stable in the individual, or (iii) decreases over time indicates that bladder cancer improves in the individual.

11. The method of claim 5, wherein the individual receives, has received, or had received a therapeutic treatment of bladder cancer.

12. The method of claim 1, wherein the bladder cancer is selected from the group consisting of urothelial bladder cancer (also designated as transitional cell bladder cancer), squamous cell bladder cancer, adenocarcinoma, sarcoma, and small cell bladder cancer.

13. Use of at least one eIF for diagnosing bladder cancer in an individual (suspected of having bladder cancer) or determining the course of bladder cancer in an individual (suffering from bladder cancer), wherein the at least one eIF is selected from the group consisting of eIF1, eIF5A, eIF4B, eIF4G, eIF5B, and eIF6.

14. A kit for diagnosing bladder cancer in an individual (suspected of having bladder cancer) or determining the course of bladder cancer in an individual (suffering from bladder cancer) comprising means for determining the level of at least one eIF in a biological sample from an individual, wherein the at least one eIF is selected from the group consisting of eIF1, eIF5A, eIF4B, eIF4G, eIF5B, and eIF6.

15. The kit of claim 14, wherein the kit is useful for conducting the methods according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0166] The following Figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

[0167] FIG. 1: Manual production of a Tissue Microarray-Block (TMA-Block). 1) The Acceptor-Tube (left) is used to punch out paraffin of a pre-selected region of the recipient TMA-Block. 2) The Donor-Tube (right) is used to punch out a pre-selected region of a donor block with tumor tissue. 3) The tissue core is transferred into the pre-made hole of the recipient block.

[0168] FIG. 2: Tissue microarray (TMA). Tissue cores of 0.6 mm in diameter were punched out from the chosen tumor areas and embedded as TMA in a fresh paraffin block according to a specific 6 x 13 pattern. The distance between two cores is 1.7 mm. This specific coordinate system was established internally and showed the best results.

[0169] FIG. 3: TMA-Blocks containing patient sample after construction. A) The blocks in this row contain the tumor tissue to be analyzed and the diameter of a tissue cylinder is 0.6 mm. The distance between two cylinders is 1.7 mm A 613 coordinate grid was used as template. Three tumor tissue cylinders were embedded per patient (107 patients, tumor group). This results in 321 tissue cylinders in total. B) The cylinders contain the urothelial tissue to be analyzed. Here the diameter of a cylinder is sized with 1.0 mm larger in comparison to 0.6 mm for the tumor tissue. The distance between two cylinders is 2.5 mm. Two urothelial tissue cylinders were embedded per patient (76 patients, comparison group). This results in 152 embedded urothelial tissue cylinders.

[0170] FIG. 4: Evaluation of the immunohistochemical staining for eIF1. A) NPar Tests, Descriptive statistics, B) Outcome of the immunohistochemical staining, left graphic=cancerous tissue, right graphic=normal tissue, C) Wilcoxon Signed Rank Tests, test statistics.

[0171] FIG. 5: Evaluation of the immunohistochemical staining for eIF4B. A) NPar Tests, Descriptive statistics, B) Outcome of the immunohistochemical staining, left graphic=cancerous tissue, right graphic=normal tissue, C) Wilcoxon Signed Rank Tests, test statistics.

[0172] FIG. 6: Evaluation of the immunohistochemical staining for eIF4G. A) NPar Tests, Descriptive statistics, B) Outcome of the immunohistochemical staining, left graphic=cancerous tissue, right graphic=normal tissue, C) Wilcoxon Signed Rank Tests, test statistics.

[0173] FIG. 7: Evaluation of the immunohistochemical staining for eIF5A. A) NPar Tests, Descriptive statistics, B) Outcome of the immunohistochemical staining, left graphic=cancerous tissue, right graphic=normal tissue, C) Wilcoxon Signed Rank Tests, test statistics.

[0174] FIG. 8: Evaluation of the immunohistochemical staining for eIF5B. A) NPar Tests, Descriptive statistics, B) Outcome of the immunohistochemical staining, left graphic=cancerous tissue, right graphic=normal tissue, C) Wilcoxon Signed Rank Tests, test statistics.

[0175] FIG. 9: Evaluation of the immunohistochemical staining for eIF6. A) NPar Tests, Descriptive statistics, B) Outcome of the immunohistochemical staining, left graphic=cancerous tissue, right graphic=normal tissue, C) Wilcoxon Signed Rank Tests, test statistics.

[0176] FIG. 10: Preferred eIF combinations of eIF1, eIF5A, eIF4B eIF4G, eIF5B, and/or eIF6.

EXAMPLES

[0177] The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Material and Methods

1. Patient Samples

[0178] The study comprised 107 patients with the diagnosis UBC which underwent a TUR-B treatment or radical cystectomy. Tumor material was obtained from the department of Pathology at the Medical University of Magdeburg. Patients who were diagnosed with urothelial bladder cancer (UBC) were considered as suitable for the project. 107 formalin-fixed, paraffin-embedded patient samples were retrospectively collected from the University Hospital in Magdeburg. Hematoxylin-eosin-stained (H/E) slides were reviewed by two experienced, board-certified pathologist, who confirmed the diagnoses and identified the areas of tumor and non-neoplastic tissue for each tissue microarray core. 107 of the 107 patient samples contained tumor tissue and were considered as suitable for the manufacturing of a Tumor-TMA (tumor group). 76 of the 107 patient samples contained non-neoplastic urothelial tissue and were used to manufacture a Non-Tumor-TMA (comparison group).
Clinical patient data was stored in a Microsoft Excel 2016 table. The following data was determined: Forename, family name, sex, date of birth, case number, time of first diagnosis, histopathological diagnosis of tumor tissue, t-stage, tumor-grade.

2. Tissue Microarray (TMAs)

[0179] Tissue Microarrays (TMAs) are a unique method which allows to embed over 1000 tissue cores in a paraffin block. This technique gives opportunity to perform immunohistochemistry and analyze specified regions of various tumors.

3. Establishing the Arraying Technique

[0180] For the Production of the TMAs a Manual-Tissue-Arraying Instrument (MTA Booster, Version 01, Alphelys, France) was used. The manual production of a TMA-block is described in FIG. 1. A tissue microarray is shown in FIG. 2.
4. TMAs with UBC Samples
To prepare for the cut, the MIAs were placed in a warming cabinet for four hours (40 C.). The TMAs were cut into 4 m thick slices. After smoothing in a water bath, the sections were transferred to Polysine Slides Adhesion Slide (Thermo Scientific). 12 cuts were made per TMA (9 TMAs12 cuts=108), In the next step, the immunohistochemical staining took place. FIG. 3 shows the TMA-blocks containing patient sample after construction.

5. Immunohistochemical Staining

[0181] The protein expression of eIF1, eIF1AY, eIF2A, eIF3A, eIF3B, eIF3H, eIF4B, eIF4E, eIF4G, eIF5A, eIF5B, eIF6 was analyzed by immunohistochemical staining. The staining was carried out under standardized conditions in the BenchMark Ultra stainer (Ventana Medical Systems, Tucsin, USA). For antigen unmasking, sections were treated with Cell Conditioning Solution (CC1-mild, Ventana). The incubation time per primary antibody used was 32 minutes. The detailed information on the antibodies, manufacturer names and established dilutions can be found in Table 1.

TABLE-US-00001 TABLE 1 Detailed overview of the antibodies that were used for IHC order- established antibody manufacturer Nr. dilution method eIF1 Thermo MA1- 1:3000 DAB Monocl. Fischer 077 CC1mild BenchmarkUltra AB (2B9) eIF1AY Thermo PA5- 1:500 DAB Fisher 31198 CC1mild BenchmarkUltra (Invitrogen) eIF2a Cell Signalling #5324P 1:2000 DAB (D7D3) CC1mild BenchmarkUltra XP eIF3A Thermo PA5- 1:100 DAB Fisher 31296 CC1mild BenchmarkUltra (Invitrogen) eIF3B Santa Cruz sc- 1:50 DAB (eIF3 137215 CC1mild BenchmarkUltra D-9) eIF3H Cell Signalling #3413 1:1600 DAB (D9C1) CC1mild BenchmarkUltra XP eIF4B GeneTex GTX33 1:500 DAB 175 CC1mild BenchmarkUltra eIF4E Cell Signalling #9742 1:100 DAB CC1mild BenchmarkUltra eIF4G Cell Signalling #2498 1:50 DAB CC1mild BenchmarkUltra eIF5A Thermo PA5- 1:250 DAB Fisher 29204 CC1mild BenchmarkUltra (Invitrogen) eIF5B Thermo PA5- 1:50 DAB Fisher 36456 CC1mild BenchmarkUltra (Invitrogen) eIF6 biomol/BETHYL A303- 1:100 DAB 030A/M CC1mild BenchmarkUltra

6. Evaluation of the Immunohistochemical Staining

[0182] The 108 TMA sequences were scanned in a NanoZoomer 360S Whole Slide Imaging Scanner (Hamamatsu). The evaluation was done semiquantitatively using the digital pathology program NanoZoomerDigitalPathology (NDP.View2).
In the evaluation, the staining intensity I (Intensity=0-3) and the percentage of the stained tumor area D (Density=0-100%) were separately analyzed for each spot and the medians were formed. It was also differentiated per spot whether it was a cytoplasmic, nuclear or mixed cytoplasmic-nuclear staining.
The following values were used for the intensity I of the staining:

[0183] 0negative color reaction

[0184] 1slightly positive staining

[0185] 2medium positive staining

[0186] 3strong positive staining

The median of staining intensity I (min 0-max 3) was then multiplied by the median of the percentage of stained area D (min 0-max 100). The product was then divided by 10 to obtain the immunoreactive score (IRS score: min 0-max 30) per patient case. Both staining intensity and IRS score formed the basis for further statistical data processing.

[00001]$I.Math..Math.R.Math..Math.S=\frac{I*D}{1.Math.0}$

7. Statistical Analysis

[0187] The statistical evaluation was carried out using the Microsoft Excel program. The data was then exported to IBM SPSS Statistics (version 22) and statistically evaluated. Differences in eIF expression between tumor and non-tumor groups were assessed using Wilcoxon non-parametric test. A p value <0.05 was considered as statistically significant. Statistical analysis was performed with the statistic program IBM SPSS Statistics Version 22. Graph generation was performed using Microsoft Word and Excel.

Results

[0188] In this study, the performance of eukaryotic translation initiation factors (eIFs) in bladder cancer was examined. It was found that eIFs are deregulated between patients suffering from UBC and healthy individuals. With eIF1, eIF5A, eIF4B eIF4G, eIF5B, eIF6, and eIF3H seven new diagnostic biomarkers for UBC were identified. These new diagnostic biomarkers allow the diagnosis and monitoring of UBC. In particular, immunohistochemical data from tissue microarray (n=107) demonstrated significantly higher expression levels of eIF4B eIF4G, eIF5B, and eIF6 in patients suffering from UBC compared to non-neoplastic tissue (healthy controls). In contrast thereto, eIF1 and eIF5A were significantly downregulated in patients suffering from UBC compared to non-neoplastic tissue (healthy controls). The results are shown in FIGS. 4 to 9. eIF3H was also downregulated in patients suffering from UBC compared to non-neoplastic tissue (healthy controls). FIG. 10 lists preferred eIF combinations of eIF1, eIF5A, eIF4B eIF4G, eIF5B, and/or eIF6.