Multimodular biomarker analysis workflow for Diagnosis, PROgnosis and MONitoring of drug treatment response in bladder cancer.
Urinary bladder cancer (UBC) is a common disease worldwide. On average 2.7 million people have a history of UBC. The highest incidence of UBC is in developed countries, especially in North America and Europe. In USA alone, about 570.000 people suffered from bladder cancer in 2011. Bladder cancer prevalence is steadily increasing. The WHO estimates that there are 330,000 new cases annually worldwide. In Germany, about 26000 new cases of bladder cancer are diagnosed every year. The most common clinical presentation is blood in the urine or hematuria, which is the principle initial symptom in 90% of bladder cancer patients. Usually this is painless and the blood may be visible to the naked eye (gross hematuria) or can be seen only under the microscope (microscopic hematuria). Frequently, the diagnosis of bladder cancer is delayed because bleeding is intermittent or attributed to other causes, such as urinary tract infection or blood thinners. However, a substantial proportion of these patients will have a significant problem such as kidney stones or tumors, urinary tract obstruction and bladder cancer. Bladder cancer has high survival rates but also high recurrence rates, which leads to a requirement for an active surveillance regime for people with a history of bladder cancer. Even after full excision of superficial bladder tumors, there is up to a 75% chance of recurrence. Although most recurrences are also superficial, bladder cancer is a multi-focal disease and new tumors may not necessarily occur at the same location. Accordingly, a post-surgery monitoring program is always initiated. Guidelines from the American Urology Association recommend follow up is undertaken quarterly, reducing to annual checks at five years from first diagnosis. In most cases surveillance tests are performed by cystoscopy, a routine but unpleasant procedure for the patient.
Because of long-term survival and the need for lifelong routine monitoring and treatment, the cost per patient of bladder cancer from diagnosis to death is the highest of all cancers, ranging from $96000-187000 in the USA. Overall, bladder cancer is the fifth most expensive cancer in terms of total medical care expenditures, accounting for almost 3.7 billion $ in direct costs just in the USA.
Current laboratory diagnosis of bladder cancer is done by examination of the urine, vagina, or rectum. The following tests and procedures are mostly used:
Urine analysis: A test to check urine content including sugars, proteins, red/white blood cells
Urine cytology: Microscopic examination of urine to check for abnormal cells
Cystoscopy: A procedure to look inside the bladder and urethra to check for abnormal areas using a cystoscope
Despite its low sensitivity (35% to 40%) in the detection of urothelial carcinoma (UC) of all grades and stages (17% in grade 1, 61% in grade 2 and 90% in grade 3), urine cytology remains the most commonly used non-invasive test. If the urinary cytology is positive, then transitional cell cancer of the urothelium is almost certainly present. However, cytologic examinations may be negative in up to half of patients with bladder cancer; thus, a negative study does not rule out bladder cancer.
In this context the objectives of DIPROMON are to replace invasive cystoscopy in surveillance by non-invasive cytology in combination with biomarker tests. The set of biomarkers is aimed to give information on the probability of recurrence and invasiveness of the tumor and prognosis on the effectiveness of therapy. Especially the individual response to BCG immunotherapy in respect to chemotherapy should be assessed. Analysis of secreted biomarkers will be complimented by cytological analysis of tumor cells in urine characterized by surface biomarkers in an integrated platform.
- In total 249 transitional cell bladder cancer and 80 muscle invasive bladder patient samples have been collected for the Dipromon biobank to use in multimodular biomarker analysis. Dipromon has also collected samples from 114 healthy volunteers.
- In the first reporting period we derived molecular disease maps for non-muscle invasive bladder cancer and molecular drug mechanism of action models for BCG and mitomycin. This enabled us to select panels of biomarker adding information to cystoscopy in diagnosing recurrent disease as well as adding to the prediction of invasive disease. Furthermore, biomarker candidates for patient stratification regarding drug response to BCG and mitomycin standard treatment were selected.
- In the second reporting period a core set of biomarker assays was defined and passed on to first clinical validation. Seven markers demonstrated selectivity according to the criteria for marker selection and met the results of a second evaluation. A multiplexed assay of four biomarkers was derived, adding further biomarkers to the analysis kit. Further, transcriptome mapping was initiated for biomarker identification using clinical samples. Five new markers were purified which are commercially not available but are valuable biomarkers for our biomarker profile. In addition, using cells shed into urine by bladder cancer patients, we have developed a method for quantitative detection of biomarkers from the images of the cell probes.
- In the third and final reporting period the DIPROMON team completed a first validation study of a classifier aimed at diagnosing disease recurrence, reaching a classification performance as area under the curve of AUC = 0.86 (LOOC 0.76).
- Using a multiplexed assay established at P4-AXO, and an ELISA for EN2 the following molecular biomarkers were determined in the validation cohort:
|Marker gene symbol||Marker gene name|
|VEGFA||vascular endothelial growth factor A|
|MMP9||matrix metallopeptidase 9|
|EN2||Engrailed homeobox 2|
|Model||AUC||Leave one out cross validation AUC|
Clinical parameter model
(stage.diagnosis, number of BCG therapies, number of past recurrences)
Molecular marker model
(VEGFA, MMP9, IL8, CDH1, EN2)
The AUC value of the statistical model holding the three clinical parameters stage at diagnosis, number of past recurrences as well as number of past BCG treatments was 0.75 (corrected: 0.66).
The statistical model based on the measured molecular markers resulted in an AUC value of 0.71 (corrected: 0.58).
The combined model holding clinical parameters and molecular markers resulted in an AUC value of 0.86. Leave one out cross validation resulted in an AUC value of 0.76.
- Platform technology module integration was established including multiplexed assays together with a software component allowing generating classification functions/computing recurrence scores.
- A lateral flow device test prototype for point of care testing was established. For proof of principle, the test was evaluated with Urine samples from P1-SURREY. All tests values obtained using LIOcube reader. This small number of samples from Surrey show that the LIODetect®UBC works very well using the reader for obtaining quantitative result. The small cohort results revealed a promising sensitivity and specificity. Thus EN2 has shown potential value in improving diagnostic accuracy of patients with bladder cancer, which may facilitate the management of this cancer.
- Finally, further biomarker candidates were retrieved from an omics profiling approach (patent filing ongoing), and the link of biomarker readout and medication regimes (both given as well as alternative drug candidates) was retrieved.
- 5th ÖGMBT Annual Meeting, CCB Innsbruck, 2013: S. Gogalic, C. Preininger; Non-invasive multiplex detection of bladder cancer-associated protein markers in urinary and serum based samples
- 5th International Congress Bionanomed, Krems, 26-28.03.2014: S.Gogalic, U. Sauer, C. Preininger; Biomarker detection in urine- a challenge towards sample preparation and assay development
- 2nd Austrian Biomarker Symposium 2014-Early Diagnostics, Vienna, 31.03. – 01.04. 2014. S.Gogalic, U. Sauer, C. Preininger; Protein biomarker chip for bladder cancer
- 9th Deutsches Biosensor Symposium, München, 11.-13.3.2015: S. Gogalic, S. Doppler, U. Sauer, C. Preininger; A muliptlxed protein chip to detect BCa urine
- 4th Biosensors and Bioelectronics, Atlanta, 28.-30.9.2015: S. Gogalic, S. Doppler, U. Sauer, C. Preininger; A multiplexed protein based urine chip to distinguish recurrent from non-recurrent BCa 09/2015
- 8th Europtrode, Graz, 20.3.-23.3. 2016: S. Gogalic, U. Sauer, C. Preininger; Colorimetric multiplex protein array for detection of recurrence of bladder cancer
- Annual Congress of the European Society for Translational Medicine & Global Network Conference on Translational Medicine (EUSTM-2013) Mondorf, Luxembourg 14-16 October 2013. Gottfried Köhler ''Non-Invasive approach to urinary bladder cancer screening and recurrence', OnkoTec GmbH, Austria.
- 9th Deutsches Biosensor Symposium, München, 11.-13.3.2015: S. Gogalic, S. Doppler, U. Sauer, C. Preininger, A muliplexed protein chip to detect BCa urine
- 4th Biosensors and Bioelectronics, Atlanta, 28.-30.9.2015: S. Gogalic, S. Doppler, U. Sauer, C. Preininger, A multiplexed protein based urine chip to distinguish recurrent from non-recurrent BCa
- DIPROMON. G. Koehler, In New Horizons in Translational Medicine, (ISSN:2307-5023), Vol. 1, Issue 1, 2013.
- Bladder cancer microarray to detect aberrant levels of proteins in urine. Gogalic S, Sauer U, Doppler S, Preininger C. Analyst 140,724-35 (2014).
- Disease map-based biomarker selection and pre-validation for bladder cancer diagnostic. De Paoli M, Perco P, Mühlberger I, Lukas A, Pandha H, Morgan R, Feng GJ, Marquette C. Biomarkers. 2015;20(5):328-37.
- Protein Interactome of Muscle Invasive Bladder Cancer. A. Bhat, A. Heinzel, B. Mayer, P. Perco, I. Mühlberger, H. Husi, AS. Merseburger, J. Zoidakis, A. Vlahou, J. P. Schanstra, H. Mischak, V. Jankowski; PLoS ONE 10: e0116404 (2015).
- Multiplatform Biomarker Discovery for Bladder Cancer Recurrence Diagnosis. De Paoli M, Gogalic S, Sauer U, Preininger C, Pandha H, Simpson G, Horvath A, Marquette C, Dis Markers 2016, 4591910.
- Predictive Biomarkers for Linking Disease Pathology and Drug Effect. Mayer B, Heinzel A, Lukas A, Perco P, Curr Pharm Design 2016, 22, ahead of print.
- Validation of a protein panel for the non-invasive detection of recurrent non-muscle invasive bladder cancer. S. Gogalic, U. Sauer, S. Doppler, A. Heinzel, P. Perco, A. Lukas, G. Simpson, H. Pandha, A. Horvath, C. Preininger, submitted to Biomarkers 2016
Summary of Participants
|Particiant No.||Particiant Organisation name||Particiant Short name||Country||Contact|
|1 (Co-ordinator)||University of Surrey||SURREY||UK|
Prof Hardev Pandha
|2||Emergentec biodevelopment GmbH||EMTEC||Austria|
|3||AXO Science SAS||AXO||France|
Dr Christophe Marquette
|5||Austrian Institute of Technology GmbH||AIT||Austria|