DNA QUANTIFICATION AND PLOIDY PATTERNS IN HUMAN ADRENOCORTICAL NEOPLASMS.
Duarte Pignatelli, D. Leitão®, M. Maia and F. Schmidt®.
Dept. of Endocrinology and Dept. of Cellular Biology, Faculty of Medicine of Porto and *IPATIMUP, University of Porto. PORTO. PORTUGAL.
ABSTRACT
The determination of DNA ploidy and the study of the cell cycle in adrenocortical tumoral cells could help in the distinction between benign and malignant lesions and also in the prediction of the biological behaviour of these tumors. We analysed 32 cases of adrenal tissue (8 normal adrenals - N, 12 benign adenomas - A and 12 carcinomas - C). DNA was quantified by image analysis of Feulgen stained sections (Ahrens System) employing ACAS3 software. The DNA content was considered to be diploid in 70% of the N and in 67% of the A groups and in none of C. In this latter group nearly 90% were triploid or tetraploid while this did not occur in any of the A cases. The percentage of cases with a 5c- exceeding rate (5cER) above 5% was nil in the N and A groups and 100% in C. In what concerns the distribution in the cell cycle we found a very distinctive pattern between the groups as the percentage of cases in which the S-phase fraction exceeded 33% was nil in the normals, 8% in A and 83% in the C cases. In conclusion, there was a good correlation between the analysed parameters and the clinically defined groups of adrenal cortex tumors.
INTRODUCTION
Criteria for malignancy in human adrenocortical tumors, apart from the presence of metastases, include vascular invasion, tumor necrosis, diffuse growth patterns, high mitotic rates or the presence of atypical mitoses and, especially, tumor mass. None clearly predicts the biological behaviour of these tumors with absolute certainty.
Mitotic figures are rare and atypical mitotic figures exceptional. Even nuclear pleomorphism has also occasionally been seen in clinically benign tumors and so, long term follow-up will always be the final arbiter in the diagnosis of troublesome cases.
Adrenal cortical carcinomas are rare (1). The oncogenetic process involves modifications of the genetic structure of the nucleus by mutations, chromosomal translocations or amplifications (2). Studies of the clonal composition of these adrenocortical tumors have determined that malignant tumors are monoclonal while adenomas may be either monoclonal or polyclonal (3). This somewhat unexpected finding may imply that some benign tumors may arise from local paracrine influences or even systemic stimuli instead of resulting from genetic mutations. Monoclonal adenomas may represent transitional cases between benign and malignant tumors.
PCNA and other markers of proliferative activity have also been studied in adrenal cortical tumors. DNA ploidy, as determined by cytometric techniques, has also been considered a marker of neoplastic transformation (4). It can be studied by flow cytometric analysis of frozen specimens or material extracted from paraffin embedment or by static image analysis of Feulgen stained sections.
MATERIAL AND METHODS
We analysed 32 cases of adrenal tissue (8 normal adrenals - N, 12 adrenal cortex benign adenomas - A and 12 adrenal cortex carcinomas - C) using paraffin embedded sections stained with the Feulgen method (5) for DNA quantification. The DNA was quantified by an image analysis system (Ahrens System) employing ACAS3 software. Cerebellar tissue processed similarly was used as diploid control.
Briefly, this method involves a quantitative analysis of the cell nucleus DNA content by means of light absorption after Feulgen staining (at a wavelength of 546 nm). The DNA value that enters the histogram is calculated from the extinction integral over the nuclear area using a calibration factor determined by the measurement of the diploid reference cells. Image analysis techniques determine the nuclear area (size) and the amount of DNA (through the integrated optical density). We used an interactive measurement mode in which each cell to be measured is manually selected.
For the analysis of the DNA nuclear content, it is necessary to use stoichiometrical stains (i.e. molecule to molecule) in order to obtain accurate and reproducible results. The
Call Count
NOLENS ICH
2.30℃
C1:90.8%
:74.
8:9.24
CZ/11:0.0%
x:0.0%
20
fc
bc
8c
DNIA
Call Count
WIRLEMS ICH
2.50c
61:55.4%
$:44.6x
c2/11:0.0x
*: 0.0x
20
4c
60
8c
W
absorbance is directly proportional to the concentration of the stain as long as the wavelength, the light emitted and the thickness of the object through which the light is passed are kept constant. The DNA content of the nucleus is proportional to the nuclear area multiplied by the Integrated Optical Density. Between a larger group of cells corresponding to cells in the GO/G1 phases and another with a double DNA content (corresponding to the G2/M phases), there is an intermediate group that is considered to be in the S phase (DNA synthesis period).
RESULTS
The DNA content was considered to be diploid in 70% of the N (Fig.1) and 67% of the A groups (Fig. 2) and in none of the C. In this latter group nearly 90% were triploid or tetraploid (Fig.3 and 4) while this did not occur in any of the A cases (Fig. 2).
The percentage of cases with a 5c-exceeding rate (5cER) above 5% was nil in the N and A groups and 100% in C. In what concerns the distribution in the cell cycle we found a very distinctive pattern between the groups as the percentage of cases in which the S-phase fraction exceeded 33% was nil in the normals, 8% in A and 83% in the C cases.
DISCUSSION
The results of our study clearly point to a possible interest in determining the DNA
Col1 Count
MEINE ICH
3.13c
$1:15
5:00
cz/1:0 .**
.: 3
K:1.1x
20
40
&c
*c
w
I
Cell Count
NIEKS ICH
3.56c
81:36.2x
02/11:3.1x
D:22
2c
4c
€
Sc
MMM
| Normal Adrenals | p value | Adrenal Cortex Adenomas | p value | Adrenal Cortex Carcinomas | |
|---|---|---|---|---|---|
| Sex | F-6 M-2 | F-10 M-2 | F-5 M-7 | ||
| Age | 48.4±16.9 | NS | 46.2±18.7 | NS | 51.0±11.0 |
| Tumor weight | -- | Range: 8-103 g | p<0.05 | Range: 116-4770g | |
| DNA Ploidy ( c ) | 2.23±0.27 | NS | 2.28±0.27 | p<0.001 | 3.98±1.01 |
| S-Phase Fraction (%) | 16.6±8.1 | NS | 23.5±7.9 | p<0.002 | 53.7+20.2 |
| 5cER | 0.2±0.4 | NS | 0.3±0.9 | p<0.001 | 22.2±14.2 |
Results expressed as mean±SD
content, and calculating the 5cER in the diagnosis of adrenal cortex tumors as well as the evaluation of their prognosis.
Tissue was fixed immediately after surgical collection and the selection of the appropriate areas was also immediately performed. This minimized fixation artefacts and allowed the observers to be sure of what they were analysing thus improving the method’s reliability.
However, after having executed a reasonable amount of analyses we can point out several limitations of the method:
· In a sample with different cellular subpopulations, this method may not allow selective analysis of the cell cycle in the population of interest.
· It is impossible to be certain whether cells containing a DNA amount that corresponds to the S-phase are really synthesizing DNA. In fact, the S-phase fraction cannot be interpreted in absolute terms since the frequency of aneuploid populations leads to
appreciable overlapping that may increase the quantity of cells the software considers as being in this phase. Nevertheless, it may be useful in comparative terms.
There have been conflicting interpretations of the quantitative determinations of DNA cellular content and of the DNA content distribution patterns. Some previous studies reported that DNA aneuploid peaks contributed decisively to the distinction between benign and malignant adrenal cortical neoplasms (6-9). Others reported no statistically significant correlation between aneuploidy (or heterogeneous DNA content) and biological behaviour (10-13). Even the quantification of DNA content in the non-neoplastic adrenal gland has revealed considerable heterogeneity (14,15).
In conclusion, we found a good correlation between the studied parameters and the clinically defined groups of adrenal cortex tumors. However, some limitations of the method as well as a certain degree of overlapping between the results may hamper the use of this method with practical clinical purposes.
REFERENCES
1-Wooten M, King D. 1993 Cancer 72: 3145-3155.
2-Tomasi T. 1986 In Fenoglio-Beiser C, Weinstein R Kaufman N (eds.) New concepts in neoplasia as applied to diagnostic pathology. International Academy of Pathology Monographs, Vol. 27. Williams and Wilkins. Baltimore (USA). Pp 59-90.
3-Gicquel C, Francillard M, Bertagna X, Louvel A, Chapuis Y, Luton J, Girard F, LeBouc Y. 1994 Clin Endocrinol 40: 465-477.
4-Barlogie B. 1984 Eur J Cancer Clin Oncol 20: 1123-1125.
5-Schulte E. 1991 Analyt Cell Pathol 3: 167-182.
6-Suzuki T, Sasano H, Nisikawa T, Rhame J, Wilkinson D, Nagura H. 1992 Mod Pathol 5: 224-231.
7-Bowlby L, DeBault L, Abraham S. 1986 Cancer 58: 1499-1505.
8-Hosaka Y, Rainwater L, Grant C, et al 1987 Surgery 102: 1027-1034.
9-Taylor S, Roedever M, Murphy R. 1987 Cancer 59: 2059-2063.
10-Haak H, Corneliss C, Hermans J, Cobben L, Fleuren G. 1993 Br J Cancer 68: 151-155 .
11-Amberson J, Vaughn E, Gray G, Naus G. 1987. Cancer 59: 2091-2095.
12-Camuto P, Citrin D, Schinella R, Fredrickson G, Gilchrist K. 1991 Urology 37: 380-384. 13-Cibas E, Medeiros L, Weinberg D, Gelb A, Weiss L. 1990. Am J Surg Pathol 14: 948- 955.
14-Camuto P, Wolman S , Perle M, Grecco M. 1989 Pediatr Pathol 9: 551-558. 15-Favara B,Steele E, Grant J, Steele P. 1991 Pediatr Pathol 11: 521-536.
Endocr Res Downloaded from informahealthcare.com by Selcuk Universitesi on 12/27/14 For personal use only.