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10031 - MCM7, Bladder Cancer

INTRODUCTION

The DNA replication licensing factor MCM7 is part of the MCM-complex which ensures that DNA undergoes a single round of replication per cell cycle [See REFERENCES: 3]. It has also been shown to unwind double-stranded DNA at the replication forks [See REFERENCES: 4]. The MCM7 gene has been shown to be up-regulated in a number of cancers compared to normal tissue. Of special interest is that the gene has been shown to be overexpressed in prostate cancer compared with the normal prostate, and this overexpression has been associated with prostate cancer progression [See REFERENCES: 5]. This relation of overexpression and progression has also been shown for bladder cancer [See REFERENCES: 6]. SiRNA mediated knockdown of the MCM7 gene in bladder cancer cell lines has been shown to diminish proliferation [See REFERENCES: 7].

This APP can be used for quantifying the nuclear expression of MCM7. The approach used here to calculate expression, have in a recent study by Fristrup et al [See REFERENCES: 8] been shown to be highly prognostic in both a test cohort (n = 283) and a validation cohort (n = 576) of non-muscle invasive bladder cancer with long-term follow-up. The MCM7 APP was in the study shown to prognosticate patients very similar to manual scoring of the protein.

KEYWORDS

MCM7, bladder cancer, metastasis, immunohistochemistry, image analysis

METHODS

The method used for computing the MCM7 expression starts by identifying nuclei using a novel pattern recognition method adapted from Kårsnäs et al [See REFERENCES: 1], which is followed by a step that separates adjacent nuclei [See REFERENCES: 2].

The identified nuclei are classified as either positive or negative based on a computation of DAB intensity, obtained using color deconvolution. This is followed by a calculation of the positive area fraction as the area of positive nuclei divided by the total area of nuclei (within the outlined ROI). The average DAB intensity across all positive nuclei is measured and subtracted from 255, to have an association between large numbers and a high staining intensity, which is more intuitive. The final expression is calculated by multiplying the intensity with the area fraction.

The actual implementation of the pattern recognition allows the user to choose settings for nuclear detection sensitivity and classification into positive and negative, in order to account for differences in local staining protocols. Optimal settings were determined for the specific tissue preparation protocol, and all tissue sections in the study were batch-processed with those settings.
It should be noted that this approach to quantifying biomarker expression is vulnerable to variations in protocols for tissue preparation (fixation time, tissue thickness, reagents, auto-stainer type and settings, etc.). Using this APP for TMA study designs, however, is robust for the cores within the TMA.
This APP requires manual outlining of tumor regions.

QUANTITATIVE OUTPUT VARIABLES

The output variables obtained from this protocol include:

- Nuclear Area : Total area of all nuclei within tumor ROI

- Positive Area : Total area of positive nuclei within tumor ROI

- Positive Area Fraction = Positive area / Nuclear area

- INT = 255 - average DAB intensity for positive nuclei

- Expression = Positive Area Fraction * INT

AUXILIARY APPS (included)

Auxiliary APPs are used for additional process steps, e.g. finding Region of Interest (ROI).

There are no Auxiliary APPs available.

STAINING PROTOCOL

There is no staining protocol available

ADDITIONAL INFORMATION

This APP was developed for, and validated by, Niels Fristrup MD and Dr. Lars Dyrskjøt Andersen, Center for Molecular Clinical Cancer Research Department of Molecular Medicine (MOMA) Aarhus University Hospital.

The APP utilizes the EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. Viewer gives a fast review together with several types of image adjustment properties ex. outlining of regions, annotations and direct measures of distance, curve length, radius, etc. 
By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTMoffers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.

REFERENCES

USER

This APP was developed for, and validated by, Niels Fristrup MD and Dr. Lars Dyrskjøt Andersen, Center for Molecular Clinical Cancer Research Department of Molecular Medicine (MOMA) Aarhus University Hospital.

LITERATURE

1. Learning Dictionaries of Discriminative Image Patches, A. L. Dahl et al., British Machine Vision Conference 2011.

2. Segmenting Clustered Nuclei Using H-minima Transform-Based Marker Extraction and Contour Parameterization, C. Jung et al., IEEE Transactions on Biomedical Engineering, 57(10): 2600-2604, 2010

3. Purification of an MCM-containing complex as a component of the DNA replication licensing system, J. P. Chong, H. M. Mahbubani, C. Y. Khoo and J. J. Blow, Nature 375:418-421, 1995

4. A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex, Y. Ishimi, J Biol. Chem. 272: 24508-24513, 1997.

5. MCM7 amplification and overexpression are associated with prostate cancer progression, B. Ren, G. Yu, G. C. Tseng, K. Cieply, T. Gavel, J. Nelson, G. Michalopoulos, Y. P. Yu and J. H. Luo, Oncogene 25: 1090-1098, 2006.

6. Gene expression signatures predict outcome in non-muscle-invasive bladder carcinoma: a multicenter validation study, L. Dyrskjot, K. Zieger, F. X. Real, N. Malats, A. Carrato, C. Hurst, S. Kotwal, M. Knowles, P. U. Malmstrom, M. de la Torre, K. Wester, Y. Allory, D. Vordos, A. Caillault, F. Radvanyi, A. M. Hein, J. L. Jensen, K. M. Jensen, N. Marcussen and T. F. Orntoft, Clin. Cancer Res. 13: 3545-3551, 2007.

7. Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer, G. Toyokawa, K. Masuda, Y. Daigo, H. S. Cho, M.Yoshimatsu, M. Takawa, S. Hayami, K. Maejima, M. Chino, H. I. Field, D. E. Neal, E. Tsuchiya, B. A. Ponder, Y. Maehara, Y. Nakamura and R. Hamamoto, Mol. Cancer 10: 65, 2011.

8. Multicenter validation of Cyclin D1, MCM7, TRIM29, and UBE2C as prognostic protein markers in non-muscle invasive bladder cancer (in review), N. Fristrup, K. Birkenkamp-Demtröder, T. Reinert, M. Sanchez-Carbayo, U. Segersten, P.-U. Malmström, J. Palou, M. Alvarez-Múgica, C.-C. Pan, B. P. Ulhøi, M. Borre, T. F. Ørntoft and L. Dyrskjøt.009.

RUO
FIGURE 1
FIGURE 1
Overview of a single MCM7 stained core.
FIGURE 2
FIGURE 2
Same core as in Figure 1. The core has been automatically outlined, followed by manual editing to exclude stromal tissue.
FIGURE 3
FIGURE 3
Same core as in Figure 1 and 2. The outlined area has been analyzed, where red denotes positive nuclei and green negative nuclei.
FIGURE 4
FIGURE 4
One field of view of the original image at 20X (scaled down to fit this space).
FIGURE 5
FIGURE 5
Same field of view as in Figure 4, but with labels denoting identified positive (red) and negative (green) nuclei.