Feature Article
Saturday, July 31st, 2010

 

Volume 32
February 2010
Number 1

 

   
Correlation of Centrosomal Aberrations with Cell Differentiation and DNA Ploidy in Prostate Cancer

Marieta I. Toma, M.D., Katrin Friedrich, M.D., Wolfdietrich Meyer, Ph.D., Michael Fröhner, M.D., Susanne Schneider, Ph.D., Manfred Wirth, M.D., and Gustavo B. Baretton, M.D.

   

OBJECTIVE: To analyze the centrosomal abnormalities in correlation with DNA ploidy and clinicopathologic data in prostate cancer.
STUDY DESIGN: Formalin-fixed, paraffin-embedded material from 63 prostate cancers (PCa) and 10 normal control cases were studied. Centrosomal features (number, area and shape) were assessed by immunohistochemistry with a gamma-tubulin monoclonal antibody. For each case centrosomal features were assessed in 100 cells, and the mean and median value was calculated. Statistical analysis was done by Student’s t test, Mann-Whitney U test and multivariate analysis. The colocalization of gamma-tubulin and pericentrin at the centrosome was proven by double immunofluorescence staining. The DNA ploidy status was analyzed on Feulgen-stained, disintegrated paraffin sections using the OPTIMAS-based work station (Media Cybernetics, Silver Spring, Maryland, U.S.A.).
RESULTS: PCa cells showed centrosomal aberrations when compared to normal tissue. Poorly differentiated PCa showed more centrosomal abnormalities than well differentiated PCa (p<0.05). Twenty-seven percent PCa were DNA nondiploid and 73% PCa were DNA diploid, respectively, just as all control specimens. DNA nondiploid status correlates with centrosomal abnormalities (p<0.05). pT4 tumors showed significantly more centrosomes than pT2 and pT3 tumors (p<0.05).
CONCLUSION: Changes in centrosome features indicate disturbed centrosome function and are significantly correlated with loss of differentiation in PCa. This is the first image analysis study of centrosome features in PCa, confirming that centrosome defects are involved in the acquisition of chromosomal aberrations in PCa. (Anal Quant Cytol Histol 2010;32:1–10)

Keywords: cell differentiation, centrosomal aberrations, DNA ploidy, prostate cancer.

The most commonly diagnosed cancer type in men is prostate cancer (PCa), with an estimated number of 186,320 new cases in the United States in 2008. PCa accounts for about 25% of incident cases and is the second leading cause of cancer death in men.1
    In patients with organ-confined prostate cancer, radical prostatectomy provides excellent cancer control. However, 15–17% of men will develop a biochemical recurrence (prostate specific antigen [PSA] serum elevation) 10 years following surgery.2,3 Among patients with biochemical recurrence, about 35% will develop metastatic disease.2 The aggressiveness of PCa correlates with known factors like preoperative PSA level and PSA velocity, histologic tumor differentiation (Gleason score), tumor stage and lymph node metastasis at the time of diagnosis.4 The use of validated nomograms like Partin tables is helpful for the estimation of PCa prognosis after radical prostatectomy, but this estimation gives only the statistical probability, not
certainty.5 
    Biomarkers predicting the prognosis after prostatectomy have been described often, including DNA ploidy,6 microvessel density, neuroendocrine differentiation7 and Her2-neu.8,9 
    Many molecular changes, like mutations, promoter hypermethylation, loss of heterozygosity and amplification of several genes (PTEN, CDKN1B, AR, AMACR), associated with prostate cancer also have been described.10 Other studies demonstrated that aberrations of chromosome 8 are correlated with poor prognosis in prostate cancer.11 
    Hence, improved understanding of PCa biology could lead to discovery of new prognostic markers and therapeutic targets.
    Carcinogenesis is a multistep process, transforming a diploid cell after sequential acquisition of genetic aberrations into a new, polymorphic and aneuploid cancer cell.
    Almost all solid tumors are characterized by chromosomal instability; the chromosomes have numerical and structural abnormalities, like inversions, deletions, duplications and translocations.12
    The new theory of carcinogenesis hypothesizes that cancer is initiated by a random aneuploidy, generated spontaneously or by a carcinogen. Cancer progress is sustained by alterations in chromosome structure and segregation by unbalancing mitosis and DNA synthesis and reparation.13,14
    Chromosomal stability is affected by changes in proteins of mitosis control.15 Chromosomes are segregated by attachment to the mitotic spindle, which has in a normal diploid cell a symmetric structure and 2 poles. In mammalian cells, a centrosome is localized at each pole. The centrosome contains a centriolar core composed of a pair of barrel-shaped, orthogonally arranged cylinders of microtubules. The pericentriolar material surrounds the centrosome, and this is the region were the microtubules are nucleated.16 gamma-Tubulin is an evolutionary, highly conserved member of the tubulin superfamily and was discovered in 1989 as a suppressor of gamma-tubulin mutations in Aspergillus nidulans.17 Cell fractionation experiments revealed 2 forms of gamma-tubulin in somatic animal cells: (a) bound at specific structures, including the centrosome, the mitotic spindle and the midbody,18,19 and (b) soluble in cytoplasm.20 gamma-Tubulin is involved in microtubule nucleation and organization, and at the centrosome it is colocalized with pericentrin within the centrosome matrix.21 The size of the gamma-tubulin complex and the number of associated proteins vary among organisms.22 However, the functional significance of these differences as well as the function of the cytoplasmatic gamma-tubulin complex and its relationship to the centrosomal form of gamma-tubulin is unclear so far.
    Centrosome abnormalities may result in formation of aberrant mitotic spindle and chromosome segregation errors,15 which have been described for many tumors. According to their relevance for genomic instability, the centrosome abnormalities are classified into 3 groups: (1) primary centrosome overduplication, (2) transient centrosome accumulation, and (3) permanent centrosome accumulation. Groups 1 and 2 may be associated with an increased risk for chromosomal missegregation. In contrast, it seems to be that the third group does not affect genomic stability.23
    Structural centrosomal aberrations often occur at a higher frequency in highly malignant non-Hodgkin lymphomas as compared to low malignant ones and correlate with DNA ploidy.24 Changes in centrosome numbers (from 1 or 2 copies to 3 or more copies per cell), shape (from uniform, round spots to an irregular shape and structure) and size have been found also in cancers of the breast, the lung and the head and neck region.25-27 and are correlated with atypical mitosis, DNA aneuploidy and loss of differentiation. Pihan et al,28 in 2003, showed centrosome abnormalities in 30–72% of preneoplastic lesions in breast, prostate and cervix. Aberrations of the centrosome structure already occur in early stages of dysplasia and are more prevalent in ductal carcinoma in situ of the breast and carcinoma in situ of the cervix than in prostate intraepithelial neoplasia. Ductal carcinoma in situ of the breast and carcinoma in situ of the cervix also showed genetic instability measured by fluorescence in situ hybridization, loss of cell polarity and cytologic disarray.28 
    Only one study analyzing centrosomes in PCa had been published at this writing.29 In PCa the centrosomes are structurally and numerically abnormal, and the extent of centrosome abnormalities correlates with the Gleason score.29 PCas with higher Gleason score have more extensive centrosome abnormalities than those with lower Gleason grade. The purpose of this study was to analyze the centrosome features in normal prostate cells compared to PCa cells and their association with tumor differentiation and DNA ploidy status. Furthermore, the prognostic impact of the centrosome features should be investigated in patients with PCa.
   
Materials
Formalin-fixed, paraffin-embedded tissue specimens from 63 patients with PCa were included. All patients were treated by radical prostatectomy in the department of urology (time frame: 2000–2001). Ten nonneoplastic prostate tissue samples of radical cystoprostatectomy explants of patients with bladder cancer but without PCa were used as controls. All procedures were reviewed by the Ethical Committee of the University of Technology, Dresden (No. 195092004 and EK59032007).
    The mean age was 66 years (range, 42–76) for the patients with PCa and 69 years (range, 50–75) for control patients with bladder cancer. Five years’ follow-up data are available for 47 patients showing no tumor specific death.
    The pathologic staging was performed according to the International Union Against Cancer 2003 classification, and the PCas were graded according to the Gleason score and divided into 3 groups: well (Gleason score 5 and 6), moderately (Gleason score 7) and poorly differentiated (Gleason score 8 and 9) carcinomas.
    Ten of the PCas showed lymph node metastases (pN1), whereas 53 cases had negative lymph node status (pN0) (Table I). The clinicopathologic data are shown in Table I.
   
   

   
   
Methods
Immunohistochemical Detection of Centrosomes by gamma-Tubulin
gamma-Tubulin immunostaining was performed on paraffin sections (5 µm thick) with gamma-tubulin antibody (clone GTU-88, Sigma; Sigma-Aldrich Biochemie GmBH, Hamburg, Germany) diluted 1: 1,000, overnight at 4°C. Before incubation the slides were pretreated by microwave heating at 600 W in citrate buffer (pH=6.0) for 20 minutes followed by incubation in 0.2% Triton X-100 (Merck, Darmstadt, Germany) in phosphate buffered saline (PBS) at 37°C for 10 minutes. For detection the ABC-PAP Kit (Vector Laboratories, Burlingame, California, U.S.A.) was used with corresponding diaminobenizidine (DAB) visualization. Negative and isotype controls were analyzed as well, avoiding unspecific structures.
  
gamma-Tubulin Staining Analysis
The slides were evaluated with a Spectracube-based workstation (DD 200 version 1.41, Digital Scanning Controller; Applied Spectral Imaging Ltd., Migdal Haemek, Israel). From each PCa tissue sample, the images of 100 isolated cells were acquired and analyzed by the SpectraView (Applied Spectral Imaging Ltd.) program. First of all, the reference spectrum for hematoxylin (nuclei) and DAB (gamma-tubulin) was defined, followed by the automatic analysis of number, shape (roundness factor [RF]) and size (µm2) of gamma-tubulin positive structures and cell nuclei. The RF was calculated with the formula:

RF = Diameter2/Surface 4  π.

The results of gamma-tubulin positive structures were considered representative of the centrosome.
    Finally, the data were transferred in an Excel (Microsoft, Redmond, Washington, U.S.A.) file for further statistical analysis.
    The intensity of the cytoplasmic gamma-tubulin staining was scored semiquantitatively as follows: negative, weak, and moderately and strongly positive.
   
Double Immunofluorescence Staining for gamma-Tubulin and Pericentrin
The double immunofluorescence staining was done on paraffin sections of 4 control and 6 PCa tissue specimens. Briefly, after deparaffinization, pretreatment with citrate buffer (pH=6.0) for 20 minutes was performed, followed by incubation in 0.2% Triton X-100 in PBS at 37° for 10 minutes. The sections were incubated with gamma-tubulin antibody diluted 1:200 (clone GTU-88, Sigma) for 1 hour at room temperature. Their consequent incubation with the secondary antibody labeled with Texas red was carried out in the dark. After that, the sections were incubated with pericentrin antibody (Covance, Berkeley, California, U.S.A.) diluted 1:500 for 1 hour at room temperature in the dark and then with the secondary antibody labeled with DTAF (green).
    The evaluation was performed by a laser scanning microscope (Zeiss LSM 510, version 3.2, with an Axioplan microscope [Carl Zeiss Jena GmBH, Jena, Germany]; 100 oil objective; laser 405 bright violet and 488 blue, data output format .lsm), and the image analysis was done with the Zeiss LSM Image Browser 3.5 program.
   
DNA Cytometry
DNA cytometry was performed on Feulgen-stained, disintegrated, 50-µm paraffin sections.
    The method for disintegrating the 50-µm paraffin sections was digestion with 1% pepsin for 30 minutes at 37°C (Hedley et al,31 modified according to Heiden et al32).
    Image analysis was appraised by an OPTIMAS- based cytometry workstation (Media Cybernetics, Silver Spring, Maryland, U.S.A.). Two hundred fifty tumor cells and 10 internal reference cells (lymphocytes) were measured for each PCa case. In the control cases, 250 prostate cells and 10 internal reference cells (lymphocytes) were analyzed. The DNA histograms were classified according to the recommendations of the fourth updated European Society for Analytical Cellular Pathology consensus report (DNA peridiploid cases with a stemline between 1.8c and 2.2c; DNA aneuploid cases with a DNA distribution different at a statistically significant level from those of normal cells—resting, proliferating or polyploidizing cells; DNA polyploidy with repeating doubling of chromosomal sets like 2c, 4c, 6c).32 
    For further analysis, the cases were categorized into peridiploid and nondiploid (aneuploid or polyploidy).
  
Statistical Analysis
In a univariate analysis we compared the measured centrosome features (number, size and shape) between the groups of interest (Gleason score: well differentiated, moderate differentiated, poorly differentiated PCas and controls; DNA ploidy status: diploid PCas, nondiploid PCas and controls; pN status: lymph node negative PCas, lymph node positive PCas and controls; pT status: pT2 tumors, pT3 tumors, pT4 tumors and controls).
First, using the Kolmogorof-Smirnov test, we showed the normal distribution of the measured centrosomal features in the analyzed groups.
    Centrosome area and number showed a normal distribution in all analyzed groups, so, for further statistical analysis, we were able to use the parametric Student’s t test.
    Centrosome shape (RF) showed inconstantly an abnormal distribution, so we used for further statistical analysis the nonparametric Mann-Whitney U test.
    We also performed a discriminant multivariate analysis with cross-validation, selection stepwise forward. We used SPSS software 15 (SPSS Inc., Chicago, Illinois, U.S.A.) for Windows (Microsoft, Redmond, Washington, U.S.A.).
    The significance threshold was p<0.05.
  
Results
Sixty-three cases of PCa as well as 10 control cases were analyzed. The numerical centrosomal feature is the mean number of centrosomes in 100 cells. The structural centrosomal features are the size and the shape (RF) (mean value in 100 cells).
    The results for the Kolmogorof-Smirnov test for the normal distribution for all analyzed groups are shown in Table II.
   
   

   
   
Correlation Between DNA Ploidy Status and Gleason Score
Normal prostate cells as well as 46 PCa (73%) were DNA diploid. In contrast, 17 PCas (27%) were DNA nondiploid (7 cases tetraploid and 10 cases aneuploid).
    DNA nondiploid status was shown in 10 cases of poorly, 6 cases of moderately and only 1 case of well-differentiated PCa.
   
Centrosome Features
The number of centrosomes was increased in PCa (mean, 1.3 centrosomes/cell) as compared with the nonneoplastic controls (mean, 1.01 centrosomes/ cell). Moreover, the centrosomes in PCa showed structural aberrations; they were significantly smaller (mean, 0.424 µm2) than in nonmalignant control tissue (mean, 1.22 µm2) (Table III and Figures 1–3).
   
   

   
   

   
   

   
   

   
   
    Poorly differentiated PCa showed significantly more centrosomal abnormalities than moderately or well-differentiated PCas. Significant differences for centrosome features were also observed depending on DNA ploidy and tumor stages.
    PCas with Gleason score 8 and 9 showed significantly enlarged centrosomes as compared with well-differentiated tumors (Gleason score 5 and 6, p=0.02). Poorly differentiated carcinomas (Gleason score 8 and 9) showed significantly more shape abnormalities than well-differentiated (Gleason score 5 and 6, p=0.009) and moderately differentiated PCas (Gleason score 7, p=0.02).
    DNA nondiploid PCa specimens showed a significantly higher centrosome number in opposition to DNA diploid ones (p=0.02).
    PCa specimens with tumor stage pT4 showed a significantly increased centrosome number (1.4 centrosomes/cell) in contrast to samples from pT2 and pT3 tumors (1.2 centrosomes/cell, p=0.01 and p=0.03, respectively). pT4 tumors showed significantly more shape aberrations of the centrosomes as compared with organ-confined tumors (pT2), p=0.03.
    The results of the corresponding t tests and U tests are summarized in Table IV.
   
   

   
Cytoplasmic gamma-Tubulin
Cytoplasmic staining for gamma-tubulin can be noticed only in prostate cancer cells and was observed in 95% of cases of poorly, 83% of moderately and 80% of well-differentiated PCa. In contrast, normal prostate cells showed no cytoplasmic staining (Figure 1).
   
Colocalization of gamma-Tubulin and Pericentrin at Centrosome
To confirm the gamma-tubulin results, the colocalization of gamma-tubulin and pericentrin at centrosomes was tested by double immunofluorescence in 10 cases. The results of laser scanning microscopy showed smaller centrosomes in PCa than in nonmalignant prostate cells once again, confirming the colocalization of gamma-tubulin and pericentrin at centrosomes (Figures 2 and 3).
   
Multivariate Discriminant Analysis and Cross-Validation Analysis
Based on multivariate analysis, the DNA ploidy status significantly correlated with the Gleason score (p=0.001) and the centrosome area (p=0.008).
    DNA nondiploid tumors were often poorly differentiated and showed larger centrosomes as compared with DNA diploid PCas.
    Centrosome area was enlarged when centrosomes had an abnormal form (p=0.01) and when tumor cells showed increased cytoplasmic expression of gamma-tubulin (p=0.02); centrosome area was also enlarged at advanced stages of PCa (p=0.02).
  
Discussion
In malignant tumors chromosomal instability (CIN) is a common phenomenon, which might result from abnormal or multipolar mitosis.15 Centrosomes are organelles responsible for regular chromosome segregation. For an accurate chromosome segregation each daughter cell must receive only 1 centrosome, and the centrosome must replicate only once per cell cycle.16 However, human tumors show aberrations in form and structure of the centrosomes.33 Although centrosomes do not lose the function of organizing the mitotic spindle in tumor initiation and progression, aberrations may be observed. This fact can support the theory of cancer depending on multistep aneuploidization.13,14
    If a single cell contains more than 2 centrosomes, these structures are able to form multipolar spindles. These spindles will separate the chromosomes in different positions, resulting in chromosomal imbalances.33 
    Centrosomal abnormalities are often described in various malignant tumors being associated with aneuploidy and CIN—e.g., in non–small cell lung cancer,27 colorectal cancer,15 prostate cancer,29 breast cancer,25 bladder cancer34 and non-Hodgkin lymphomas.24 
    In the present study, DNA nondiploid carcinomas also showed an increased centrosome number as compared with DNA diploid carcinomas (p= 0.02).
    In many studies the DNA ploidy status was associated with Gleason score and prognosis,35 with tumor stage10 and with metastasis and tumor-specific death in PCa.9 DNA ploidy status can also predict tumor recurrence in well-differentiated PCas.36 Loss of 8p22 is associated with a poor prognosis in organ confined PCa.11
    Our study showed concurrent results: 50% of poorly differentiated PCa with Gleason score 8 and 9 were DNA nondiploid, and only 5% of well-differentiated PCa (Gleason score 5 and 6) were DNA nondiploid.
    An association between CIN level and chromosomal abnormalities was observed in PCa as well, and the CIN level was dependent on the Gleason score.29
    The present study was the first one using image analysis to determine centrosome features like area, shape and number in PCa, and it showed centrosomal aberrations in correlation with PCa differentiation. So, an increased number of centrosomes, with higher areas and abnormal forms, could often be detected in tissue samples from PCa with poor differentiation. These results are similar to findings of Pihan et al.29 Therefore, it can be suggested that centrosomal abnormalities may play an important role in the progression of PCa.
    Besides in PCa, the correlation of increased centrosomal abnormalities with decreased tumor differentiation was also found in breast37 and bladder cancer,34 indicating the impact of centrosomal aberrations in the progression of other malignancies.
    In cervical carcinoma the centrosomal amplification increases from nearly zero in normal cells to 20% in grade 1 carcinomas, 50% in grade 2 carcinomas and 70% in grade 3 carcinomas. Also, aberrations in shape and size were increased in poorly differentiated cervical carcinomas as compared with well-differentiated tumors.28
    Centrosomal aberrations were often observed in pT4 tumors as compared to pT2 or pT3 PCas, but in comparison with the 5-year survival data on 47 patients that did not show any PCa specific deaths, no correlation of centrosomal abnormalities and survival was obvious.
    The centrosome is composed of a centriol pair and a surrounding matrix of protein aggregates—the pericentriolar material that contains gamma-tubulin. This unique tubulin superfamily member is not assembled into microtubules but is colocalized with other proteins, like dynein and pericentrin,38 in the pericentriolar material of the centrosome. We demonstrated the colocalization of gamma-tubulin and pericentrin with the help of double immunofluorescence staining. With confocal laser scanning microscopy we can differentiate real centrosomes from other organic structures (Figures 2 and 3), estimate the different centrosome sizes in PCa vs. normal prostate cells and confirm the colocalization of pericentrin and gamma-tubulin at the centrosome.21 
    In the present study, using an antibody against gamma-tubulin, the centrosome size in PCa was calculated as 0.42 µm2 and in nonmalignant prostate cells as 1.22 µm2. Besides gamma-tubulin as centrosome-localized protein, a further soluble form in the cytoplasm is described, and these 2 forms are continuously interacting.21,39
    In this study, tumor cells also showed cytoplasmic gamma-tubulin expression, which normal control cells did not.
    Smaller centrosomes and increased cytoplasmic gamma-tubulin expression may indicate an activated cell cycle and an increased interaction of both gamma-tubulin forms in malignant prostate cells. In addition, we observed increased cytoplasmic gamma-tubulin staining with the loss of PCa differentiation. This makes the cytoplasmic detection of gamma-tubulin a potential predictive marker for the discrimination of several differentiated PCas during the histopathologic examination of radical prostatectomy specimens.
    In a multivariate analysis, the cytoplasmic expression of gamma-tubulin also correlated with the cyclin A expression (data not shown), demonstrating the important role of centrosomes and centrosome-associated proteins in the cell cycle. For the verification of this observation, a larger cohort of PCa patients and longer-term follow-up data should be included in further studies.
    In conclusion, centrosomal aberrations are a common event in prostate cancer cells and corre-late with PCa differentiation. These abnormalities seem to be early events in carcinogenesis and may be involved in the acquisition of chromosomal aberrations.
    The prognostic and therapeutic value of centrosome features in PCa remains to be investigated.
   
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From the Institute for Pathology and Department of Urology, Technical University of Dresden, Dresden, Germany.

Drs. Toma and Friedrich are Pathologists, Institute for Pathology.

Dr. Meyer is Statistician, Institute for Pathology.

Dr. Fröhner is Urologist, Department of Urology.

Dr. Schneider is Researcher, Department of Urology.

Dr. Wirth is Professor, Department of Urology.

Dr. Baretton is Professor, Institute for Pathology.

This paper contains part of Dr. Toma’s doctoral thesis.

Address correspondence to: Marieta I. Toma, M.D., Institute for Pathology, Technical University of Dresden, Fetscherstrasse 74, 01307 Dresden, Germany (marieta.toma@uniklinikum-dresden.de).

Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.
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