ANALYSIS OF DNA PLOIDY HETEROGENEITY IN PROSTATE CANCER
ROBERTO KIEHL, EDUARDO DE TOLEDO CORRÊA, FILADELFO EUCLIDES VENÇO, VALDEMAR ORTIZ
Department of Urology, Professor Edmundo Vasconcelos Hospital, Diagnóstika, Diagnosis in Surgical Pathology, São Paulo, SP
ABSTRACT
The
objectives of this study were to assess DNA ploidy heterogeneity and thus
the specificity of digital cytometry for prostate adenocarcinoma, as intratumoral
variation of DNA content is believed to exist among these cells, as occurs
with histological differentiation.
Fifteen prostate adenocarcinoma patients
were selected. They underwent radical prostatectomy and the tissue obtained
had 3 distinct areas analyzed to determine cellular ploidy and tumor grade
(Gleason score). Statistical analysis has described the values in the
Gleason score in the different areas, both in the diploid and aneuploid
groups, in relation to average, standard deviation, median, and maximal
and minimum values.
The prostatic tissue studied showed only
diploid tumors in some patients and only aneuploid tumors or an association
between diploid and aneuploid tumors in others. There were 11 tumors where
the areas were diploid (73.3%), 3 aneuploid tumors (20%) and a tumor with
2 diploid areas and an aneuploid one (6.7%). There was thus homogeneity
among the areas studied in 93.3% of the prostatic tumors. The values in
the Gleason score were compared with the ploidy of each area in the several
tumors. The average of values in the Gleason score in the tumor areas
of the diploid group was significantly smaller (p < 0.05) than the
observed average in the areas of aneuploid group of tumors. This relationship
occurred globally in the areas of tumors.
In conclusion, ploidy analysis of the cellular
DNA in prostate adenocarcinoma through digital cytometry shows high specificity
(93,3%) and cellular differentiation rate was significantly lower in diploid
tumors.
Key words:
prostate, prostatic neoplasms, DNA ploidy, heterogeneity
Braz J Urol, 26: 29-31, 2000
INTRODUCTION
Neoplasias
are widely recognized by inter and intratumoral heterogeneity characterized
by several aspects: morphological, phenotypic and genetic. This diversity
does not, however, imply necessarily in chaotic behavior. If there is
any structure in a system, and if anybody understands the rules and interactions
of this system, they may understand the ways of its evolution and its
potential behavior (1). The natural history of the prostatic adenocarcinoma
is still poor known. The factors that trigger prostate cancer, that make
it develop, penetrate the prostatic capsule, send metastases or become
refractory to hormone therapy are still hardly known. Some of these tumors
evolve rapidly, while others stay quiescent (2). It is early to assert
that all prostate cancers begin with the same rate of cellular multiplication
and become more aggressive with the passage of time or whether they begin
already at different growth rates (3). The forecast of the potential malignity
of the tumors is thus one of the greatest problems in handling prostate
cancer. The clinical and pathological stage, the tumor grade, volume and
PSA levels are the principal methods used. However, they cannot be considered
to be consistently objective and trustworthy. This is the context in which
DNA ploidy analysis has emerged as a promising technique for the classification
of the malignity potential of the tumor. Tumor cells with a nucleic acid
content similar to normal cells are believed to have a less aggressive
behavior.
The objectives of this study were to assess
DNA ploidy heterogeneity and thus, the specificity of digital cytometry
for prostate adenocarcinoma, besides verifying the existence of a corresponding
of this with the variation of cellular differentiation.
MATERIAL AND METHODS
The
group studied consisted of 15 patients with prostate adenocarcinoma, verified
by transrectal biopsies, who underwent radical prostatectomy. The tissue
obtained in these surgeries was submitted to histopathological routine
preparation and kept in paraffin.
In order to evaluate the heterogeneity of
prostate tumors, the cellular ploidy and the tumor grade (Gleason score)
were determined in 3 distinct areas of each tumor, area A being the most
expressive and area C the least frequent.
The quantitative DNA analysis was made in
5-m sections of prostatic material by Feulgen stoichiometric method. The
new slides were observed in an optic videomicroscopy system joined to
an IBM-PC compatible microcomputer. The tumor images generated by the
camera were frozen and fed into software specifically designed for nucleic
tracing, named W.ING.
The total optical density of the nucleus
represents its DNA content. In this manner, when measuring the amount
of DNA of a certain cellular population, a chart is obtained, called a
DNA histogram, representing the ploidy pattern of this cellular population.
The determining of the control histograms was made by the analysis of
DNA content of the cells of either normal prostatic tissue or tissue with
benign hiperplasia in the sample of each patient. Comparing the histograms
of the DNA of neoplasic cells to those of normal cells we obtained its
DNA index, meaning the proportion between the DNA of cells at rest (G0/G1)
in neoplasia and in the normal population. So, a DNA index equal to one
marks a diploid pattern, while a DNA index markedly different from one
determines an aneuploid pattern.
Statistical analysis has described the values
in the Gleason score in the different areas, both in the diploid and aneuploid
groups, in relation to average, standard deviation, median, and maximal
and minimum values. The non-parametric test of Mann-Whitney was used for
determining the significance, with the adoption of “p” values
smaller than 0.05 as statistically significant.
RESULTS
The
prostatic tissue studied showed only diploid tumors in some patients and
only aneuploid tumors or an association between diploid and aneuploid
tumors in others. There were 11 tumors where the areas were diploid (73.3%),
3 aneuploid tumors (20%) and a tumor with 2 diploid areas and an aneuploid
one (6.7%). There was thus homogeneity among the areas studied in 93.3%
of the prostatic tumors.
The values in the Gleason score were compared
with the ploidy of each area in the several tumors. The average of values
in the Gleason score in the tumor areas of the diploid group was significantly
smaller (p < 0.05) than the observed average in the areas of aneuploid
group of tumors. This relationship occurred globally in the areas of tumors
(Table-1).
DISCUSSION
The
real value of ploidy in estimating the potential malignity of prostate
tumors and the survival of patients remains controversial. Some studies
show a strong forecast value while others failed in attempt of proving
the significance of this relationship.
As a group, diploid tumors present a disease-free
period after treatment greater than shown by the group of patients with
aneuploid tumors. Determination of the ploidy of a given tumor cannot,
however, be useful to determine the survival chance of a particular patient
(4).
Some 30% of the tumors restricted to the
gland are “pure” non-diploid (4). In this study, there were
73.3%, which were homogeneously diploid tumors. This predominance is probably
due to our having studied the tumors localized in the prostate, for which
surgery was indicated.
In a recent consensus meeting on ploidy
of prostate tumors, the conclusion was reached that there is a significant
relationship between DNA content and the evolution, survival and response
to hormone therapy. It was also established at the meeting that there
were tumors heterogeneous as to ploidy, with diploid and aneuploid areas,
and that this variation could compromise the results of ploidy determination
in prostatic tumors (5). Studies of ploidy heterogeneity of prostate tumors
through digital cytometry have shown that patients with a localized disease,
restricted to the prostate, aneuploid tumors are rare (13%) and that ploidy
heterogeneity occurs in less than 10% of these tumors (2,6).
We demonstrated that, as far as the correspondence
of ploidy with the Gleason score is concerned, the values of Gleason score
of diploid tumors are in average significantly smaller than the values
for aneuploid tumors. As the degree of cellular differentiation is a well-established
parameter in determining the aggressiveness of tumor cells, despite its
subjective component, the association of ploidy gives probably greater
objectivity to the forecast of the potential malignity of the tumor.
CONCLUSIONS
We conclude that ploidy analysis of cellular DNA in prostate adenocarcinoma through digital cytometry shows high specificity (93.3%). This permits cellular ploidy to be determined by simple transrectal biopsy, while yet in the pre-operative phase. Biopsy cannot determine the degree of histological differentiation as reliably (6). We have also concluded that the cellular differentiation rate was significantly higher in diploid tumors, which probably represent diseases that are really less aggressive than aneuploid tumors.
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Received: February 12, 1999
Accepted after revision: December 16, 1999
_______________________
Correspondence address:
Roberto Kiehl
Al. Jurupis, 455, cj 76 / 77, Moema
São Paulo, SP, 04088-001
Fax: (11) 573-6818
E-mail: kiehl@uol.com.br