Annals of Surgical Oncology 10:426-431 (2003)
© 2003 Society of Surgical Oncology
Flow Cytometric DNA Analysis for Determination of Malignant Potential in Adrenal Pheochromocytoma or Paraganglioma: An Indian Experience
Manoj J. Shah, MD,
Nilkamal H. Karelia, PhD,
Shantibhai M. Patel, MD,
Nilima S. Desai, MSc,
Harshini V. Mehta, MSc and
Suman Singh, MSc
From the Department of Pathology, The Gujarat Cancer & Research Institute, New Civil Hospital Compound, Asarwa, Ahmedabad, India.
Correspondence: Address correspondence and reprint requests to: Manoj J. Shah, MD, Pathology Department, The Gujarat Cancer & Research Institute, New Civil Hospital Compound, Room 406, Asarwa, Ahmedabad 380 016, India; Fax: 079-2125490; E-mail: gcriad1{at}sancharnet.in
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ABSTRACT
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Background: We analyzed the histological features and DNA flow cytometric results in 34 patients with pheochromocytoma and paragangliomas and attempted correlation with the biological behavior for determination of the malignant potential of these tumors.
Methods: DNA analysis was done on a FACSort flow cytometer using paraffin-embedded tissues. Histopathological analysis was performed using parameters, i.e., cell size (large, medium, and small), cell size variation, mitotic rate, nuclear pleomorphism, golden yellow to brown pigment in the tumor, necrosis, and venous invasion.
Results: Six tumors had high (>5/10HPF) mitotic rate while venous invasion was seen in three tumors. Fifty percent (18/34) of patients had aneuploid tumors, and 68% (23/34) of patients had high (>10%) S-phase fraction tumors. Aneuploidy correlated with >5/10HPF mitotic rate (P < .05) and diploidy with golden yellow to brown pigment (P < .01). The patients with aneuploid tumor had a worse prognosis than patients with diploid tumors (P = .004). No such difference was observed with low and high S-phase fractions (P = .748), presence and absence of venous invasion (P = .927), and mitotic rate (P = .159). Nuclear pleomorphism and necrosis were not significant factors in prognosis.
Conclusions: Flow cytometric DNA analysis of paragangliomas and pheochromocytomas correlated with biological behavior in the patients with regard to metastasis and overall survival in the patients.
Key Words: DNA aneuploidy • Pheochromocytoma • Paraganglioma • Malignant behavior
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INTRODUCTION
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Adrenal pheochromocytoma and other paragangliomas are uncommon tumors composed predominantly of cells with presumptive evidence of differentiation toward paraganglionic chief cells. Although malignant pheochromocytoma is a life-threatening illness, there are no definite histological criteria predictive of malignancy. The biologic behavior of the majority of paragangliomas is normally benign, and metastatic potential cannot be predicted from their histologic appearance.1,2 Metastasis is the only bona fide criterion for a malignant paraganglioma. Features such as venous invasion, capsular invasion, extensive necrosis, and marked nuclear pleomorphism with one or more prominent nucleoli were considered to be particularly useful in assessing malignant potential.3,4 Further, mediastinal or retroperitoneal location, the above mentioned features, and decreased immunohistochemical reactivity to neuropeptides have been correlated with a clinical malignant behavior.5–7 Marked improvement on early diagnosis and anatomic localization of pheochromocytoma or paraganglioma have been achieved in recent years. This is mainly because of the introduction of new techniques for the detection of fractionated catecholamines and telomerase activity and the development of new imaging techniques including computerized axial tomography scanning and magnetic resonance imaging for detection.8–12
Analysis of DNA ploidy has been shown to be helpful in predicting the clinical course of pheochromocytoma, and some studies have found an association between aggressive clinical behavior and the presence of DNA aneuploidy.13–15
We analyzed the histological features and flow cytometric DNA analysis results of paragangliomas and pheochromocytomas and attempted correlation with the biological behavior of these tumors.
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MATERIALS AND METHODS
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In this retrospective study, 34 patients with adrenal pheochromocytoma or paraganglioma (1 cervical chemodectoma, 19 abdominal paragangliomas, 14 adrenal pheochromocytomas) who attended The Gujarat Cancer & Research Institute, Ahmedabad, India, for diagnosis and treatment between 1991 and 2000 were enrolled. Specimens came from the archives of the Pathology Department at The Gujarat Cancer & Research Institute. For microscopic study, representative samples of the materials were fixed in 10% buffered formalin and embedded in paraffin by routine processing procedure. Hematoxylin and eosin staining was performed for morphologic study. Histopathological analysis was performed with the following parameters in mind: cell size (large, medium, and small), cell size variation in the different tumor areas, mitotic rate (minimum 40 high power fields [HPF] counted using NikonTM (Shinagawa, Tokyo, Japan) Labophot microscope x40./65; 160./17 high power objective), venous invasion, golden yellow to brown pigment in the tumor, and presence or absence of necrosis. Clinical history was noted from the cases of the patients. Patients were followed for 2 years or more. Presence of metastasis or death due to tumor was particularly looked for in the follow-up of patients.
Flow Cytometric Analysis
The material for DNA analysis was obtained from archival paraffin blocks, and flow cytometric analysis was done on a FACSortTM flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The same paraffin-embedded tissue blocks from which histopathologic evaluation was done were used for flow cytometry. The cell nuclei were extracted from the paraffin blocks by a technique used by Hedley et al.16 Briefly a minimum of three 33-µm thick sections were obtained from the paraffin blocks. After deparaffinization in xylene and subsequent rehydration (graded ethanol and distilled water), the tissue was suspended in citrated buffer. The tissue was minced mechanically, filtered through a 35-µm nylon mesh, and incubated for 30 minutes at 37°C in .5% pepsin solution (Sigma Chemical Co., St. Louis, MO) pH 1.5 with intermittent vortexing. After incubation, the suspension was centrifuged for 10 minutes at 900 x g, the supernatant was removed, and the samples were resuspended in citrate buffer. They were filtered again through 35-µm nylon mesh, and subsequently, a simple staining procedure was performed with propidium iodide, partially using the cell cycle kit staining equipment (Becton Dickinson), which contained ribonuclease A.
Measurements were performed on a FACSort flow cytometer (Becton Dickinson) equipped with an argon laser set at an emission wavelength of 488 nm and calibrated daily with chicken erythrocytes nuclei and calf thymocyte nuclei (QC particles kit, Becton Dickinson). Acquisition was performed using CellQuestTM software (Becton Dickinson) and DNA statistical analysis was done using ModFit software (Becton Dickinson), which counted at least 10,000 nuclei per sample.
Mononuclear cells were used as a DNA diploid control. If there was only one peak in a tumor histogram, the cells of that tumor were classified as diploid. When two or more G0/G1 cell peaks were identified, the tumor was considered to be aneuploid. For DNA histogram peak coefficient variation (CV) (degree of resolution) was between 5% and 7%. If excessive tissue debris was detected (>20%) or CV of the G0/G1 peak was >7%, then another attempt was made to process the sample if sufficient material remained; if not, then it was excluded from the study. The proportion of cells in different cell cycle phases was calculated by using ModFitTM software (Becton Dickinson). If necessary, an electronic exclusion of doublets and cell aggregates using CellQuest software (Becton Dickinson) was performed. Deviation in cellular DNA content (aneuploidy) was expressed as the DNA index. The DNA index was calculated as the ratio of tumor G0/G1 peak channel divided by standard G0/G1 peak. DNA indices up to 1.0 were considered as diploid. When the mathematical model was unable to analyze the proportion of cells in different cell cycle regions, the cases were excluded. The prognostic cutoff for S-phase fraction was considered according to the results obtained from paraffin-embedded normal lymphocyte tissue DNA analysis. The S-phase fraction was considered uninterpretable if CV of the G0/G1 peak was >7% and excessive debris or background was observed on the side of G2M peak.
Statistical Analysis
Statistical analysis was performed using an SPSS statistical software program (SPSSTM Inc., Chicago, IL).17 The Kaplan-Meier life table analysis18 was used for overall survival. Differences in observed survival between groups were tested for statistical significance using log-rank test. P < .05 was considered significant.
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RESULTS
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Table 1 describes patient’s characteristics. Of 34 patients, follow-up history was available in 21 cases. Table 2 describes tumor characteristics (Figs. 1–4). Eighteen percent (06/34) of the tumors had high (>5/10HPF) mitotic rate, whereas venous invasion was seen in 9% (03/34) of the tumors. Fifty-three percent (18/34) of the patients had aneuploid tumors, whereas 68% (23/34) of the patients had high (>10%) S-phase fraction tumors (Table 2). When comparison of DNA analysis and pathologic parameters was done, aneuploidy was correlated with 
5/10HPF and >5/10HPF mitotic rate (P < .05; Table 3) with presence and absence of golden yellow to brown pigment (P < .01; Table 3). No such difference was noted with presence and absence of venous invasion, cell size, necrosis, nuclear pleomorphism, and cell size variation (Table 3). Twenty-four months follow-up was available in 21 cases. The patients with aneuploid tumor had a worse prognosis than patients with diploid tumor (P = .004; Fig. 5). No such difference was observed with high (>10%) and low (<10%) S-phase fraction (P = .3043; Fig. 6), presence and absence of venous invasion (P = .9273;Fig. 7), and 5/10HPF and >5/10HPF mitotic rate (P = .159; Fig. 8). Of 21 cases where information for metastasis was available, the incidence of aneuploidy was higher in patients who developed metastasis as compared with patients who did not develop metastasis (P = .007;Table 4). In overall analysis, among all markers, DNA ploidy was the only statistically significant independent prognosticator (P = .007; Table 5).
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FIG. 6. Kaplan-Meier survival curve of patients with high (>10%) S-phase fraction and low (<10%) S-phase fraction tumor.
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FIG. 7. Kaplan-Meier survival curve of patients with presence and absence of venous invasion in tumors.
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FIG. 8. Kaplan-Meier survival curve of patients with low (<5HPF) and high (>5HPF) mitotic index tumors.
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DISCUSSION
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It is thought that approximately 10% of pheochromocytomas or paragangliomas that arise from the adrenal medulla or paraganglia have malignant potential.19,20 Adrenal cortical tumors that are very large and/or with metastasis may be easy to diagnose as malignant. However, for most such tumors in adults, malignant potential is difficult to predict even with detailed endocrinologic and imaging studies and we have to depend on pathologic examinations. But it has been difficult for pathologists to discriminate benign from malignant pheochromocytoma/paraganglioma from microscopic examination. There is a multifactorial analysis of nine criteria to differentiate metastasizing tumors from nonmetastasizing tumors.7,21
In the recent years it has become apparent that nuclear DNA analysis of various human neoplasms can provide important information that cannot be obtained by conventional histopathologic study. Only a few studies on patients with adrenal pheochromocytoma or paraganglioma with DNA analysis and correlation with malignant potential outcome have been published.13,14,16,22–24
In the present study we tried to correlate various histological features of paraganglioma/pheochromocytoma with their potential malignant behavior and also with the results obtained from flow cytometric analysis of these tumors (Table 3). Although individual histopathological parameters like cell size, cell size variation in different tumor areas, nuclear pleomorphism, golden yellow to brown pigment in the tumor, mitotic rate, venous invasion, and necrosis failed to correlate directly with the malignant potential, two parameters showed good correlation with aneuploidy found on flow cytometric analysis. In the present study, there was a significant correlation between mitotic rate and incidence of aneuploidy (P < .05; Table 3). However, this parameter and venous invasion do not add additional information to metastatic potential of the tumor. This is similar to another study, which reported that DNA ploidy was correlated with mitotic rate and nuclear grade but did not correlate well with clinical outcome.7 Although this criterion is a valuable method for pathologic analysis, it does not always predict definite metastatic potential.
DNA analysis of all 34 patients showed 50% tumors with aneuploidy. Of these 34 patients, follow-up data were available only in 21 patients. Of these 21 patients, 11 developed or had metastasis at the time of diagnosis (Table 4). Of these 11 patients, 64% (07/11) showed aneuploidy on flow cytometric analysis. Of the 10 patients who did not develop the metastasis in the follow-up period of approximately 24 months, only 2 had (20%) aneuploidy. Table 4 also shows results of S-phase fraction in these 21 patients. It shows that S-phase fraction did not correlate well with metastasis. The P value for aneuploid tumors developing metastasis was significant (.007). Our results are thus comparable with other studies that showed that aneuploid (hyperdiploid and tetraploid) tumors have greater metastatic potential than diploid tumors.14,15 Further aneuploidy also correlated with poor patient survival (Fig. 5).
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CONCLUSION
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From the results obtained from this study of 34 patients with paraganglioma/pheochromocytoma and follow-up study of 21 patients of approximately 24 months, we conclude that aneuploidy in tumors correlated well with the metastatic potential of these tumors and poor patient outcome. Histological features were not much help in prognostication of these tumors. However, mitotic rate and presence or absence of golden brown pigment correlated with aneuploidy.
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Acknowledgments
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This study was supported by a grant from The Gujarat Cancer Society, Ahmedabad, India. The authors thank the cooperation of Dr. D. D. Patel, Director, and Dr. P. M. Shah, Additional Director, The Gujarat Cancer & Research Institute, Ahmedabad, India for their support in this research work and Ms. Priti O. Pandey, statistician, for statistical help.
The acknowledgments are available online at www.annalssurgicaloncology.org.
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Footnotes
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A retrospective study of pheochromocytomas and paragangliomas (n = 34) was performed using histological features and DNA flow cytometric analysis. Patients with aneuploid tumors on DNA analysis were shown to have greater metastatic potential and poor overall survival.
Received for publication April 8, 2002.
Accepted for publication November 7, 2002.
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REFERENCES
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- Schlumberger M, Gicquel C, Lumbroso J, et al. Malignant pheochromocytoma: clinical, biological, histologic and therapeutic data in a series of 20 patients with distant metastases. J Endocrinol Invest 1992; 15: 631–42.[Medline]
- Proye C, Vix M, Goropoulos A, Kerlo P, Lecomte-Houcke M. High incidence of malignant pheochromocytoma in a surgical unit. 26 cases out of 100 patients operated from 1971 to 1991. J Endocrinol Invest 1992; 15: 651–63.[Medline]
- Heinbecker P, O’Neal LW, Ackerman LV. Functioning and nonfunctioning adrenal cortical tumors. Surg Gynecol Obstract 1957; 105: 21–33.
- Lack EE. Afip Atlas of Tumor Pathology: Tumors of the Adrenal Gland and Extra Adrenal Paraganglia. Washington, DC: American Registry of Pathology, 1997.
- Olson JL, Salyer WR. Mediastinal paragangliomas (aortic body tumor): a report of four cases and a review of the literature. Cancer 1978; 41: 2405–12.[CrossRef][Medline]
- Lack EE, Cubilla AL, Woodruff JM, Lieberman PH. Extra-adrenal paragangliomas of the retroperitoneum: a clinicopathologic study of 12 tumors. Am J Surg Pathol 1980; 4: 109–20.[Medline]
- Linnoila RI, Keiser HR, Steinberg SM, Lack EE. Histopathology of benign versus malignant sympathoadrenal paragangliomas: clinicopathologic study of 120 cases including unusual histologic features. Hum Pathol 1990; 21: 1168–80.[CrossRef][Medline]
- Moyer TP, Jiang NS, Tyce GM, Sheps SG. Analysis for urinary catecholamines by liquid chromatography with amperometric detection: methodology and clinical interpretation of results. Clin Chem 1979; 25: 256–63.[Abstract/Free Full Text]
- Cheung PS, Thompson NW, Dmuchowski CF, Sisson JC. Spectrum of pheochromocytoma in the 131I-MIBG era. World J Surg 1988; 12: 546–51.[CrossRef][Medline]
- Nakada T, Kubota Y, Sasagawa I, Yagisawa T, Watanabe M, Ishigooka M. Remarkably suppressed manganese superoxide dismutase activity in malignant pheochromocytoma. J Urol 1995; 153: 1787–90.[CrossRef][Medline]
- Kubota Y, Nakada T, Sasagawa I, Yanai H, Itoh K. Elevated levels of telomerase activity in malignant pheochromocytoma. Cancer 1998; 82: 176–9.[CrossRef][Medline]
- Hirano Y, Fujita K, Suzuki K, Ushiyama T, Ohtawara Y, Tsuda F. Telomerase activity as an indicator of potentially malignant adrenal tumors. Cancer 1998; 83: 772–6.[CrossRef][Medline]
- Hosaka Y, Rainwater LM, Grant CS, Farrow GM, van Heerden JA, Lieber MM. Pheochromocytoma: nuclear deoxyribonucleic acid patterns studied by flow cytometry. Surgery 1986; 100: 1003–10.[Medline]
- Gonzalez-Campora R, Diaz Cano S, Lerma-Puertas E, et al. Paragangliomas. Static cytometric studies of nuclear DNA patterns. Cancer 1993; 71: 820–4.[CrossRef][Medline]
- Nativ O, Grant CS, Sheps SG, et al. The clinical significance of nuclear DNA ploidy pattern in 184 patients with pheochromocytoma. Cancer 1992; 69: 2683–7.[CrossRef][Medline]
- Hedley DW, Friedlander ML, Taylor IW, Rugg CA, Musgrove EA. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem 1983; 31: 1333–5.[Abstract]
- Norusis MJ. SPSS for Windows, Advanced Statistics, Release 6.0. Chicago, IL: SPSS Inc., 1993.
- Kaplan EL, Meier P. Non-parametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457–81.[CrossRef]
- Modlin IM, Farndon JR, Shepherd A, et al. Phaeochromocytomas in 72 patients: clinical and diagnostic features, treatment and long term results. Br J Surg 1979; 66: 456–65.[Medline]
- Remine WH, Chong GC, Van Heerden JA, Sheps SG, Harrison EG Jr. Current management of pheochromocytoma. Ann Surg 1974; 179: 740–8.[Medline]
- Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg 1984; 8: 163–9.
- Klein FA, Kay S, Ratliff JE, White FK, Newsome HH. Flow cytometric determinations of ploidy and proliferation patterns of adrenal neoplasms: an adjunct to histological classification. J Urol 1985; 134: 862–6.[Medline]
- Lewis PD. A cytophotometric study of benign and malignant phaeochromocytomas. Virchows Arch B Cell Pathol 1971; 9: 371–6.
- Amberson JB, Vaughan ED Jr, Gray GF, Naus GJ. Flow cytometric analysis of nuclear DNA from adrenocortical neoplasms. A retrospective study using paraffin-embedded tissue. Cancer 1987; 59: 2091–5.[CrossRef][Medline]
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ABSTRACT
INTRODUCTION
