Contribution of the Microvessel Network to the Clonal and Kinetic Profiles of Adrenal Cortical Proliferative Lesions
SALVADOR J. DIAZ-CANO, MD, PHD, MANUEL DE MIGUEL, PHD, ALFREDO BLANES, MD, PHD, HUGO GALERA, MD, PHD, AND HUBERT J. WOLFE, MD
Monoclonal adrenocortical lesions have been characterized by an inverse correlation between proliferation and apoptosis, and poly- clonal lesions show a direct correlation. Their relationship with the vascular pattern remains unknown in adrenocortical nodular hyper- plasias (ACNHs), adenomas (ACAs), and carcinomas (ACCs). We studied 20 ACNHs, 25 ACAs, and 10 ACCs (World Health Organiza- tion classification criteria) from 55 women. The analysis included X-chromosome inactivation assay (on microdissected samples), slide and flow cytometry, and in situ end labeling. Endothelial cells were stained with anti-CD31, and the blood vessel area and density were quantified by image analysis in the same areas. Appropriate tissue controls were run in every case. Regression analyses between kinetic and vascular features were performed in both polyclonal and mono- clonal lesions. Polyclonal patterns were observed in 14 of 18 infor- mative ACNHs and 3 of 22 informative ACAs, and monoclonal pat- terns were seen in 4 of 18 ACNHs, 19 of 22 ACAs, and 9 of 9 ACCs. A progressive increase in microvessel area was observed in the AC- NH-ACA-ACC transition but was statistically significant between be-
Heterogeneous clonal profiles have been shown in adrenocortical proliferative lesions,1-3 with polyclonal lesions predominating in nodular hyperplasias (AC- NHs) and monoclonal lesions predominating in ade- nomas (ACAs) and carcinomas (ACCs). Similar results have also been reported in other endocrine hyperpla- sias2,4-6 and neoplasms,46 supporting the concept of multistep tumorigenesis.7 However, monoclonal hyper- plasias (parathyroid, in multiple endocrine neoplasia type 1 and uremic patients, and multinodular goiters) 6,8 and polyclonal adenomas (parathyroid and thyroid)2 have also been reported. Therefore, it has been con- cluded that clonality assay itself is of limited utility in differentiating hyperplastic from neoplastic conditions.
From the Department of Pathology, Tufts University-New En- gland Medical Center, Boston, MA; the Department of Pathology, Barts and The London Queen Mary’s School of Medicine and Den- tistry, London, England; the Department of Pathology, University Hospital of Seville, Seville, Spain; and the Department of Pathology, University Hospital of Malaga, Malaga, Spain. Accepted for publica- tion July 10, 2001.
Presented in part at the annual meeting of the United States and Canadian Academy of Pathology, New Orleans, LA, March 25-31, 2000; and the winter meeting of the Pathological Society of Great Britain and Ireland, London, England, January 18-20, 2000.
Address correspondence and reprint requests to Salvador J. Diaz- Cano, MD, PhD, Department of Histopathology & Morbid Anatomy, Barts and The London, The Royal London Hospital, Whitechapel, London E1 1BB, England.
Copyright @ 2001 by W.B. Saunders Company 0046-8177/01/3211-0013$35.00/0
doi:10.1053/hupa.2001.28949
nign and malignant lesions only (191.36 ± 168.32 v 958.07 ± 1279.86 um2; P < . 0001). In addition, case stratification by clonal pattern showed significant differences between polyclonal and monoclonal benign lesions; 6% of polyclonal and 57% of monoclonal lesions had microvessel area >186 um2 (P = . 0000008). Monoclonal lesions showed parallel trends (but with opposite signs) for microvessel area and density in comparison with proliferation and apoptosis, whereas polyclonal lesions showed inverse trends. In conclusion, the kinetic advantage of monoclonal adrenal cortical lesions (increased prolif- eration, decreased apoptosis) is maintained by parallel increases in microvessel area and density. HUM PATHOL 32:1232-1239. Copy- right @ 2001 by W.B. Saunders Company
Key words: adrenal cortex, nodular hyperplasia, adenoma, carci- noma, clonality, proliferation, apoptosis, microvessel density.
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocorti- cal carcinoma; ACNH, adrenocortical nodular hyperplasia; H&E, hematoxylin and eosin; PCR, polymerase chain reaction; HUMARA, human androgen receptor gene; ISEL, in situ end labeling.
A distinctive correlation between proliferation and apoptosis has been demonstrated in adrenocortical proliferative lesions by clonal patterns.3 Polyclonal le- sions have shown increasing apoptosis in response to increasing proliferative rates, whereas monoclonal le- sions had progressively lower apoptotic rates as prolif- eration increased. That inverted relationship between apoptosis and proliferation in monoclonal adrenal cor- tical lesions also provides a functional basis for clonal selection and segregates ACNHs from neoplastic ACAs. Cell kinetics represent the basic mechanisms leading to cellular selection, which results in clonal expansion and tumor growth.3,9,10 Downregulated apoptosis has also been reported in intraepithelial neoplasms at different locations and would allow both survival and replication of genetically damaged cells, giving rise to mutation accumulation and tumor promotion.11-14 Inversely re- lated proliferation and apoptosis in monoclonal lesions would then contribute to clonal progression in neo- plasms.3,9,15,16 Tumor cell growth has been related to the capacity to induce neoangiogenesis. However, the relationship among the vascular pattern, clonality, and cell kinetics remains unknown in ACNHs, ACAs, and ACCs.
MATERIALS AND METHODS
Case Selection and Sampling
Consecutive adrenocortical proliferative lesions (69) were selected and histologically evaluated.12 Of these lesions, 55 were detected in female patients, including 20 ACNHs, 25
ACAs, and 10 ACCs classified by World Health Organization criteria,18 although evidence of metastasis was the main cri- terion of malignancy. The mean follow-up time in this series was 159 months.
All surgical specimens were serially sectioned and em- bedded for routine histopathologic diagnosis (at least 1 block/cm). The most cellular areas from the biggest nodule in each case of ACNH and from every ACA and ACC were screened and selected for further analysis. The same areas were used in each analysis; hematoxylin and eosin (H&E)- stained sections taken before and after the specimen samples were used to check the cellular composition of each sample.
X-Chromosome Inactivation Assay for Clonality Analysis
Two 20-um unstained paraffin sections were used for microdissection under microscopic control. Adrenal cortical cells and controls (histologically normal adrenal cortex, ad- renal medulla, and periadrenal soft tissue from the same slide) underwent DNA extraction. At least 2 separate areas of 0.25 mm2, containing approximately 100 target cells each, were harvested from both peripheral and internal areas of the biggest nodule in ACNH, ACA, and ACC.
The samples were dewaxed with xylene, cleared with absolute ethanol, and digested with proteinase K; DNA was extracted using a modified phenol-chloroform protocol, as previously described.19 All samples were divided for restric- tion endonuclease digestion, using half of each sample for Hhal digestion (0.8 U/ul; New England Biolabs, Beverly, MA); the other half was kept as undigested control. Undi- gested and digested samples were processed equally, but Hhal was excluded in the reaction mixture in undigested samples. The samples were digested under appropriate buffer condi- tions (50 mmol/L potasium acetate, 20 mmol/L Tris acetate, 10 mmol/L magnesium acetate, 1 mmol/L dithiothreitol pH 8.0, 100 µg/mL bovine serum albumin, 100 µg/mL mussel glycogen) at 37℃ for 4 to 16 hours. A mimicker (0.3 µg of double-stranded and Xhol-linearized ¢X174-RII phage; Life Technologies, Inc, Gaithersburg, MD) was included in each reaction mixture. Complete digestion was checked by gel electrophoresis; incompletely digested samples were phenol chloroform-purified and redigested with higher Hhal con- centration.
HhaI was then inactivated by phenol chloroform extrac- tion as described.19 DNA was precipitated with ice-cold abso- lute ethanol in the presence of 0.3 mol/L sodium acetate (pH 5.2) and resuspended in 10 µL of polymerase chain reaction (PCR) buffer (10 mmol/L Tris-HCI pH 8.4, 50 mmol/L KCI, 1.5 mmol/L MgCl2, and 100 µg/mL bovine serum albumin). Both digested and undigested DNA were then used for PCR amplification of a region containing the CAG repeat in the first exon of the human androgen receptor gene (HUMARA) and a DNA sequence recognized by Hhal that is consistently methylated in the inactive HUMARA allele only.20-22 Primers and PCR cycling conditions were designed as previously de- scribed.21,23,24 The reactions were run in duplicate and opti- mized for a 10-pL reaction in a Perkin-Elmer thermal cycler model 480 (Perkin-Elmer, Norwalk, CT).
The whole PCR volume was electrophoresed into 0.75- mm-thick 8% nondenaturing polyacrylamide gel at 5 V/cm until a xylene cyanol band was located within the bottom inch of the gel. After fixation with 7% acetic acid (5 minutes), the gels were dried under vacuum (80℃, 40 minutes) and put inside a developing cassette containing 1 intensifying screen and preflashed films (Kodak XAR; Kodak Co, Rochester, NY) facing the intensifying screen (16 to 48 hours, -70℃). The
autoradiograms were developed using an automated proces- sor Kodak-Omat 100.
Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer; EC Apparatus Corp, St Pe- tersburg, FL), and evidence of monoclonal proliferation was considered to be allele ratios ≥4:1 with the normalized Hhal- digested samples. Sample normalization was done in relation to the corresponding undigested sample and tissue controls. Only informative cases (2 different alleles in Hhal-undigested and Hhal-digested samples) were included in the final analy- sis. 21,23-26
Slide Cytometric Analysis of DNA Content
Several Feulgen-stained 5-um sections were used for DNA quantification according to previously published proto- cols that have proven valid in such material.14,27,28 Densito- metric evaluation was performed using the cell analysis system model 200 and the quantitative DNA analysis package as software (Becton Dickinson, Franklin Lakes, NJ). At least 200 nuclei were measured in every case, beginning in the most cellular area through completion in consecutive microscopic high-power fields. Only complete, nonoverlapping, and fo- cused nuclei were quantified in each field.27
External staining calibration was carried out with com- plete rat hepatocytes (Becton Dickinson; 1 slide per staining holder) to normalize the internal controls; the latter included both lymphocytes and adrenocortical cells from histologically normal areas present in the same tissue section. The internal controls were used to set the Go/G1 cell limits and calculate the DNA index of each Go/G1 peak (>10% of measured cells with evidence of G2 + M cells).27,29 The proliferation rate (PR = S- + G2- + M-phase fraction) was calculated from the DNA histogram by subtracting the number of cells within Go/G1 limits from the total number of cells measured. The values were compared with total cell number and expressed as percentages.14,29
Nuclear DNA Quantification by Flow Cytometry
Serial 50-um-thick sections were microdissected, and nu- clear preparations were stained with propidium iodine after RNase A digestion to study DNA ploidy (by the technique of Hedley et al30). DNA quantification parameters included DNA indices and PRs as described,3,29 and the scatter analysis of nuclear area and DNA content allowed identification of apoptotic cells in each cell cycle phase (low nuclear area for a given DNA content in each cell cycle phase).31 Those results were additionally coupled with in situ end labeling (ISEL) to identify apoptotic cells in terms of DNA fragmentation (see below). External diploid controls from paraffin-embedded tissues (lymphocyte from reactive lymph nodes and adrenal cortical cells from histologically normal adrenal glands) were used to determine DNA indices and to standardize the nu- clear area/DNA content analysis (considering only adrenal cortical cells for the last purpose).3 Proliferation rate (PR) was calculated as described for slide cytometry, using the rectangular model for evaluation of the cell cycle histogram.29
ISEL of Fragmented DNA
Extensive DNA fragmentation associated with apoptosis was detected by ISEL as previously reported.13,14,32 After rou- tine dewaxing and hydration, the sections were incubated in 2x standard saline citrate (20 minutes at 80℃) and digested
with proteinase K (100 µg/mL in Tris-HCI, pH 7.6, for 30 minutes at room temperature) in a moist chamber.
DNA fragments were labeled on 5’-protuding termini by incubating the sections with the Klenow fragment of Esche- richia coli DNA polymerase I (20 U/mL in 50 mmol/L Tris- HCI, pH 7.5; 10 mmol/L MgCl2; 1 mmol/L dithiothreitol; 250 µg/mL bovine serum albimin; 5 um of each deoxyade- nosine triphosphate, deoxycytidine triphosphate, and de- oxyguanosine triphosphate; 3.25 umol/L deoxythymidine triphosphate; and 1.75 umol/L 11-digoxigenin-deoxyuri- dine triphosphate) at 37℃ in a moist chamber. The incorpo- rated digoxigenin-deoxyuridine monophosphates were im- munoenzymatically detected by using antidigoxigenin Fab fragments labeled with alkaline phosphatase (7.5 U/mL, in 100 mmol/L Tris-HCI pH 7.6, 150 mmol/L NaCl, 1% bovine serum albumin) for 4 hours at room temperature. The reac- tions were developed with the mixture nitroblue tetrazo- lium-X phosphate in 100 mmol/L Tris-HCI pH 9.5, 100 mmol/L NaCl, 50 mmol/L MgCl2 under microscopic con- trol. Appropriate controls were simultaneously run, including positive (reactive lymph node), negative (same conditions omitting DNA polymerase I), and enzymatic (DNase I diges- tion before the end labeling). The enzymatic controls were used to reliably establish the positivity threshold in each sample.
The ISEL index was expressed as percentages of positive nuclei referred to the total number of adrenal cortical cells present in the same high-power field.14,33,34 At least 50 con- secutive high-power fields were screened, beginning in the most cellular area (from the biggest nodule for ACNH).
Evaluation of Microvessel Pattern
The sections were mounted on positively charged micro- scope slides (Superfrost Plus; Fisher Scientific, Fair Lawn, NJ) and baked at 60℃ for 2 hours. The slides were routinely dewaxed and rehydrated. The endogenous peroxidase activ- ity was then quenched with 0.5% H2O2 in methanol, 10 minutes). A microwave antigen-retrieval method (20 minutes in 10 mmol/L citrate buffer, pH 6.0, at 600 W) was used, followed by incubation with polyclonal horse serum (20 min- utes, 1:100 dilution; Dako, Glostrup, Denmark) and with monoclonal anti-CD31 antibody (overnight at 4℃) at 2 ug/mL (Dako). Sections were then serially incubated with biotinylated antimouse antibody (30 minutes, 1:200 dilution; Dako) and peroxidase-labeled avidin-biotin complex (60 minutes, 1:100 dilution; Dako). All incubations were per- formed in a moist chamber at room temperature unless otherwise specified. The reaction was developed under mi- croscopic control, using 3,3’-diaminobenzidine tetrahydro- chloride with 0.3% H2O2 as chromogen (Sigma Chemical, St Louis, MO), and the sections were counterstained with he- matoxylin. Both positive (reactive lymph node) and negative
(omitting the primary antibody) controls were simultaneously run.
Blood vessel morphometry was evaluated in at least 20 medium-power fields (100x), using a computer-aided image analysis system (Kontron MOP Videoplan). The area and perimeter of each blood vessel and the number of blood vessels were measured per square millimeter, mean and SD were determined. The total vascular area results from the product of the number of microvessels times the microvessel average area. Therefore, an inverse correlation between mi- crovessel number and area per square millimeter should be expected for a given total vascular area. Imbalances of that correlation result in decreased or increased vascularization compared with the standard. Area/perimeter correlation per microvessel informs on the blood vessel shape.
Standard values for these microvessel variables were ob- tained from 10 histologically normal adrenal glands excised for extraadrenal pathology (ie, renal cell carcinoma). The upper limits of the 95% confidence intervals were area, 186 um2/mm2; perimeter, 56 um; and density, 50 microvessels/ mm2.
Statistical Analysis of Quantitative Variables
The results of microvessel variables were compared by diagnostic group (ACNH/ACA/ACC) and by clonality pat- tern in benign lesions (polyclonal v monoclonal). Variables showing normal distribution were analyzed using the Student t test, whereas analyses of variance were used for variables with nonparametric distribution. Normal distribution was previ- ously tested by the Kolmogorov-Smirnoff test. Microvessel variables were also categorized and analyzed by the Fisher exact test using the following thresholds: microvessel area, 186 um2; microvessel perimeter, 56 um; microvessel density, 50/mm2. The results were considered statistically significant at P < . 05 in 2-tailed distributions. Regression analyses were also performed to test the correlation between microvessel variables, proliferation, and apoptosis in both polyclonal and monoclonal lesions.
RESULTS
Polyclonal patterns were observed in 14 (78%) of 18 informative ACNHs and 3 (14%) of 22 informative ACAs, and monoclonal patterns were seen in 4 (22%) of 18 ACNHs, 19 (86%) of 22 ACAs, and 9 (100%) of 9 ACCs. The sequence ACNH-ACA-ACC was character- ized by a progressive increased in proliferation and apoptosis markers as well as in microvessel area and perimeter (Table 1, Fig 1), but differences were statis- tically significant only in comparisons between benign
| ACNH (Mean ± SD) | ACA (Mean ± SD) | ACC (Mean ± SD) | Statistical Significance | |
|---|---|---|---|---|
| Vessel area (um2/mm2) | 157.23 ± 123.61 | 216.32 ± 200.34 | 958.07 ± 1279.86 | P= . 0001 |
| Vessel perimeter (um/mm2) | 48.81 ± 10.15 | 55.04 ± 14.05 | 135.36 ± 108.58 | P <. 0001 |
| Vessel density (no./mm2) | 67.28 ± 45.90 | 59.50 ± 37.05 | 34.40 ± 19.61 | NS |
| Proliferation rate | ||||
| Slide Cytometry (%) | 16.67 ± 9.42 | 22.59 ± 11.03 | 34.17 ± 8.84 | P= . 0002 |
| Flow Cytometry (%) | 13.64 ± 5.73 | 13.69 ± 6.29 | 23.49 ± 5.60 | P= . 0002 |
| ISEL | 0.58 ± 0.19 | 1.03 ± 0.52 | 4.55 ± 1.52 | P <. 0001 |
Units/mm2
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100
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SV Area
SV Perim
SV Density
ACNH
ACA
ACC
(ACNH and ACA) and malignant (ACC) conditions (Fig 2). In contrast, microvessel density decreased pro- gressively in the same transition (Table 1). Case classi- fication of benign lesions by clonal pattern only re- vealed significant differences for the proliferation rate estimated by slide cytometry (Table 2, Fig 3). After case categorization, statistically significant differences were also revealed for microvessel area (6% of polyclonal and 57% of monoclonal lesions had microvessel area
| Polyclonal Lesions (Mean ± SD) | Monoclonal Lesions (Mean ± SD) | Statistical Significance | |
|---|---|---|---|
| Vessel area | 157.53 ± 128.42 | ||
| (um2/mm2) | 233.90 ± 204.43 | NS | |
| Vessel perimeter (um/mm2) | 49.98 ± 10.55 | 55.79 ± 14.32 | NS |
| Vessel density (no./mm2) | 65.74 ± 40.18 | 54.18 ± 37.92 | NS |
| Proliferation rate Slide cytometry (%) | 15.53 ± 7.85 | ||
| 22.73 ± 10.71 | P= . 0187 | ||
| Flow cytometry (%) | 13.54 ± 6.36 | 13.02 ± 5.36 | NS |
| ISEL | 0.68 ± 0.41 | 0.90 ± 0.31 | NS |
>186 pm2; P = . 0000008), whereas the distribution of microvessel perimeter and density was not significantly different. Using cytometric criteria, apoptotic cells were detected in 10 benign cases, 8 with microvessel area <186 pm2 (4 polyclonal and 4 monoclonal; P = . 0088) and 2 with microvessel density <50/mm2 (both cases monoclonal; P = . 0002; Fig 2). All malignant tumors also showed apoptotic cells in flow cytometry, but with no correlation with the microvessel pattern.
Figures 4 and 5 show the relationship between microvessel and kinetic features. Polyclonal lesions had an inverse correlation between microvessel area and both proliferation and apoptosis (Fig 4A, B), whereas
A
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Polyclonal ACA
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Distribution of DNA Mass
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Cells Displayed: 197 Cells Off Scale: 0
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the correlation was direct between microvessel density and both proliferation and apoptosis (Fig 4C, D). In contrast, monoclonal lesions had direct correlation be- tween proliferation and both area and density of mi- crovessels (Fig 5A, C) but an inverse correlation be- tween apoptosis and microvessel area and density (Fig 5B, D). A linear correlation was demonstrated in all these analyses.
DISCUSSION
The microvascular pattern of adrenocortical le- sions showed a significant increase in the vascular area in ACCs compared with benign adrenocortical lesions (ACNHs and ACAs) and a distinctive relationship be- tween proliferation and apoptosis in monoclonal be- nign proliferative lesions. The increase in both mi-
crovessel area and microvessel density helps maintain the inverted relationship between apoptosis and prolif- eration in monoclonal adrenocortical lesions, provid- ing a functional basis for clonal selection in ACNHs and ACAs.
Monoclonal lesions showed a distinctive correla- tion of microvessel area and density with proliferation (direct) and apoptosis (inverse), which would facilitate self-maintained growth. The microvessel pattern was shown to be significantly different for ACCs and for monoclonal benign adrenocortical lesions. These find- ings support the important role postulated for angio- genesis in tumor cell kinetic and progression of endo- crine tumors, including adrenocortical neoplasms. The mean microvessel area and the total vascular area was significantly higher in ACCs than in ACNHs and ACAs, similar to results previously reported,35 but no signifi- cant differences between benign adrenocortical lesions were seen. An inverse relationship between microvessel area and density must be expected under physiologic conditions. This scenario was proved in polyclonal le- sions: proliferation and apoptosis showed a direct cor- relation with each other and with microvessel density but an inverse correlation with microvessel area. The opposite situation (direct relationship between mi- crovessel area and density) was observed for monoclo- nal lesions: inverse correlation between proliferation and apoptosis, with greater microvessel area and den- sity in lesions with high proliferation rate and low ap- optosis index. These 2 microvessel factors (high area and density) help maintain the kinetic advantage re- lated to high proliferation and low apoptosis. The in- creased blood supply contributes to cell proliferation, providing oxygen and nutrients for cycling cells on 1 hand and, on the other hand, trophic/growth factors essential in most endocrine organs. The absence of these factors results in endocrine gland atrophy by apoptosis.9,36 A less efficient blood supply of growing glands helps increase their apoptotic rate, thus result- ing in parallel trends of proliferation and apoptosis. A better supply overrides apoptosis, explaining the in- verse proliferation/apoptosis correlation because of apoptosis downregulation in monoclonal lesions. Eventually, these 2 kinetic parameters will segregate monoclonal proliferation with ACAs rather than with ACNHs3
The distinctive microvessel pattern represents a key element to explain the kinetic abnormalities lead- ing to tumor promotion. Cell kinetics represent the basic mechanisms leading to clonal expansions and tumor growths.3,9,10 In terms of the correlation between apoptosis and proliferation, polyclonal lesions showed increasing apoptosis in response to increasing prolifer- ative rates. However, monoclonal lesions had progres- sively lower apoptotic rates as proliferation increased. That inverted relationship between apoptosis and pro- liferation in monoclonal adrenocortical lesions also provides a functional basis for clonal selection and segregates ACNHs from neoplastic ACAs. The correla- tion between proliferation and apoptosis provides rules for cellular selection, ie, clonal expansion or regres-
A
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y =- 1.0747x+ 142.67
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nal adrenocortical lesions is characterized by parallel increases in microvessel area and density that correlate directly with proliferation and inversely with apoptosis. This distinctive microvessel pattern certainly helps maintain the kinetic advantage (high proliferation and low apoptosis), clonal cell selection, and eventually cel- lular progression in those lesions.
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