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Flow-cytometric DNA content analysis of esophageal squamous cell carcinomas
GASTROENTEROLOGY
1991;101:1588-1593
Flow-Cytometric DNA Content Analysis of Esophageal Squamous Cell Carcinomas MICHEL ROBASZKIEWICZ, BRIAN J. REID, ALAIN VOLANT, JEAN MICHEL CAUVIN, PETER S. RABINOVITCH, and HERVE GOUEROU Departments Departments
of Gastroenterology and Pathology, University Hospital, Brest, France; and of Medicine and Pathology, University of Washington, Seattle, Washington
To better understand the mechanisms of esophageal carcinogenesis, abnormalities in DNA content of esophageal squamous cell carcinomas were studied. Cellular DNA content was determined by flow cytometric study of 70 endoscopic biopsy specimens obtained from 26 patients with esophageal squamous carcinoma. High-quality histograms were obtained for 23 patients. Twenty-one patients had at least one aneuploid population in their tumor. In 7 patients, multiple aneuploid peaks were detected. Specimens from 2 patients were diploid. The interpretation of the DNA histograms was difficult in 3 patients; an aneuploid population of cells was probable in 2 of them. A statistically significant relationship was found between the degree of differentiation and DNA content abnormalities in the regions of the tumors that could be evaluated by endoscopic biopsies: well-differentiated carcinomas had diploid or small aneuploid populations containing < 15% of the cells, whereas DNA histograms of moderately or poorly differentiated carcinomas were characterized by large aneuploid peaks representing 25%~90% of the cells and a higher proliferative fraction. No relationship was found between the size or the stage of the tumor and the DNA content detected in endoscopic biopsy samples. The frequency and the multiplicity of abnormal clones in esophageal squamous carcinomas indicates that this cancer, like esophageal adenocarcinoma, develops an association with an acquired genomic instability that produces abnormal clones of cells, according to the multistep model of neoplastic progression.
T
he incidence of esophageal carcinoma varies widely in different parts of the world (12). It is the fifth most common cause of cancer deaths in men in France (3). Squamous cell carcinoma of the esophagus is common in Northwest France (3), where chronic
use of alcohol and smoking have been implicated as important contributing factors as in the United States and Western Europe (4). Despite efforts at early diagnosis and advances in surgery, radiation therapy, and chemotherapy, the prognosis of esophageal cancer is poor. The etiology of the disease remains unknown, and little is known about the cellular and nuclear events that occur during carcinogenesis. DNA content flow cytometry is widely used in the study of neoplasia, because changes in DNA content have been shown to closely correlate with chromosomal changes seen in malignancy (5-7). The objective of this study was to evaluate the DNA content of esophageal squamous cell carcinomas by flow cytometry to better understand biological mechanisms of esophageal carcinogenesis.
Materials and Methods Patients The biopsy samples for the study were obtained from 26 consecutive patients who underwent esophagogastroscopy for dysphagia between February 1988 and January 1989 at Brest University Hospital. The group consisted of 23 men and 3 women whose mean age was 67.4 years [range, 41-85 years).
Endoscopy
and Biopsies
Upper endoscopy was performed with an Olympus GIF XQlO endoscope. All patients were seen at the time of diagnosis, and none had chemotherapy or radiotherapy before endoscopy. All 26 patients had a visible tumor located in the upper third of the esophagus in 3 cases, in the
Abbreviations used in this paper: CV, coefficient of variation: DI, DNA index. o 1991 by tbe American Gastroenterological Association 0016-5085/91/$3.00
December 1991
middle third in 18 cases, and in the lower third in 5 cases. The tumor was infiltrating, polypoid, or ulcerating in 3, 6, and 17 patients, respectively. In 18 cases, the esophageal lumen was obstructed, permitting only the proximal margin of the tumor to be examined. After visualizing the tumor, biopsy specimens were obtained with the Olympus FB21K biopsy forceps. Four to six biopsy samples taken from the tumor were processed for histological evaluation, and one to four biopsy specimens were taken for flow cytometry. A total of 70 specimens were analyzed by flow cytometry.
Histology The specimens for histology were fixed in Bouin’s fixative and stained with H&E saffron. Specimens were interpreted histologically by one observer (A.V.) who had no knowledge of the results of flow cytometry. Specimens were classified according to the type and differentiation of the carcinoma based on the World Health Organization histological classification of squamous cell carcinomas of the esophagus (8).
Flow Cytometry
The biopsy specimens for flow cytometry were stored at - 70°C immediately
after the endoscopy and were thawed immediately before flow cytometric analysis. The nuclei were isolated and stained with ethidium bromide and mithramycin according to a procedure derived from the method described by Barlogie et al. (9). The biopsy specimens were minced and incubated for 20 minutes in 500 FL of ethidium bromide (25 mg/L; Sigma Chemical Corp., St. Louis, MO) in 0.1 mol/L saline Tris (pH 7.4), 0.6% NaCl, 0.6% Nonidet P 40 (Sigma), 0.2% bovine serum albumin type 1 (10 mg/L; (Sigma), and 100 PL of ribonuclease Sigma). Aggregates were dissociated by forceful passage three times through a 26-gauge needle, and the nuclei were incubated for 1 hour with 500 p,L of a solution containing 50 mg/L mithramycin (Miles Pharmaceuticals, West Haven, CT), 7.5 mmol/L MgCl,, and 12.5% ethanol. Flow cytometry was performed on an ICP 22 (Ortho Diagnostic Systems, Westwood, MA). Excitation was with a 390-490-nm filter. Emission was detected above 560 nm. Data were collected on a DEC LSI II/23 computer (Digital Equipment Corp., Maynard, MA) and transfered and analyzed on a PC AT compatible computer with the Multicycle software (Phoenix Flow Systems, San Diego, CA) written by one of the authors (P.S.R.). Cell cycle parameters were analyzed by the method of Dean and Jett (10) using a zero order polymonial S phase. By this nonlinear least squares curve fitting, the diploid and aneuploid G, and G, peaks were fit using normal distributions, and the region between each G, and G, peak was fit to a distribution of cells in S phase, with special treatment of the region of overlap with G, and G,. Subnuclear-sized debris were fit by a model that accounts for the effect of slicing or cutting nuclei during sectioning; every nucleus counted is assumed to have a certain probability of being randomly sliced into portions of (usually) unequal size, and these subnuclear size portions appear in channels lower than that of the original nucleus. The probability of
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1589
being cut is determined by nonlinear least squares analysis. The model originally developed for Multicycle has been revised according to the suggestions of Bagwell et al. (11) to account for the effect of ellipsoidal shape of nuclei on the cut portion size distribution. Residual exponentially declining debris was fit by inclusion of a power function. The ratio of the aneuploid to the diploid DNA content is designated as the DNA index (DI). For tumors with a near-diploid DNA content, chicken erythrocytes were used as control cell references to determine the DNA content of the aneuploid population. A shoulder was defined as a low percentage (< 20%) of cells with an excess DNA content to the right of the G,G, peak, not showing bimodality. The coefficient of variation (CV) of the G,G, peak of the diploid or aneuploid population was expressed by the normalized standard deviation (CV = Standard Deviation/Mean Peak Position X 100). Coefficients of variation were typically 4.0%6.0% and always ~8.0%. Flow cytometric results were interpreted by two authors (M.R. and P.S.R.) without knowledge of the histological results. For statistical analysis, comparisons between groups have been tested using Fisher Exact Test and Yates’ corrected x’.
Results
Flow cytometric analysis was possible in 93% of specimens (65 of 70). High-quality histograms were obtained in 73% of biopsies (51/70). In five specimens, the tissue was necrotic, and analysis could not be performed. The histogram interpretation was difficult because of low cell number in 14 additional specimens. All of these difficult histograms came from 3 of the 26 patients; an aneuploid peak was suspected in 2 patients but could not be confirmed with certainty. Twenty-one of 26 patients had one or more aneuploid peaks. The additional peak was unique in 14 patients; 7 patients had multiple aneuploid peaks in their tumor. In 3 patients, the multiple aneuploid cell populations were present in the same biopsy specimen (Figure 1); in 4 other patients, only one aneuploid peak was present in each specimen, but specimens taken from different regions of the tumor had different DNA contents. Five patients had a combination of diploid and aneuploid biopsy specimens obtained from their tumor. In 1 patient, the aneuploidy was associated with an increased diploid G, or tetraploid fraction (20%). Two patients had no evidence of aneuploidy. One patient had normal DNA contents in two specimens, an increased G,/tetraploid fraction (10.6%) in a third biopsy, and a hyperdiploid shoulder in a fourth biopsy. The second patient had a diploid peak with a hyperdiploid shoulder.
DNA Con tent and Histology
Two kinds of DNA aneuploid histograms could be identified (Figure 2): (a) tumors with small aneu-
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ROBASZKIEWICZ ET AL.
DNA Content and Cell Proliferation Proliferative fractions (S phase and G,) of the aneuploid populations were estimated when the percentage of aneuploid cells exceeded 25% of the total cells (Figure 3). All aneuploid tumors were characterized by S phase fractions > 7%; the mean S phase fractions were 26%, with a standard deviation (SD) of 10.4% and a range of 7%48.5%. In tumors with small aneuploid peaks, the S phase could only be measured for the diploid cell population; the mean S phase fractions were 13.3% with an SD of 15.3% and a range of 4.3%20%. The two diploid tumors had lower S fractions (6.5% and 12%).
4
240
160
20
0
DNA Content and Clinical Features DNA
Content
Figure 1. Two aneuploid populations (1.6and 2.8 N) are present in this single biopsy sample from a moderately differentiated esophageal squamous cell carcinoma (channels 67 and 116). The diploid peak (2 N) (channel 84) was located by using chicken erythrocytes (channel 28) as a standard for DNA content estimation.
ploid peaks, in which the aneuploid cell population represented < 15% of examined cells; (b) tumors with larger aneuploid peaks, in which the aneuploid cells represented 25%~90% of the analyzed cells. Tumors in which the endoscopic biopsy samples showed well-differentiated squamous cell carcinoma were predominantly characterized by the first type of histograms, whereas large aneuploid peaks were seen in all but one tumor in which biopsy samples showed moderately and poorly differentiated carcinoma (Table 1; P = 0.005). In the two tumors in which biopsy specimens showed only a diploid DNA content, the corresponding histologies showed only well-differentiated carcinoma. The histological appearance of an esophageal squamous cell carcinoma may vary in different parts of the same malignancy, and in some of our tumors biopsy specimens taken for flow cytometry may not have come from the same region of the cancer as the samples for histology. Therefore, we reanalyzed the data for 15 tumors in which flow cytometric and histological biopsy samples were taken from the same region of the cancer. All eight tumors with aneuploid cell populations of < 15% came from histologically well-differentiated cancers as assessed by endoscopic biopsy specimens. However, six of seven tumors with aneuploid cell populations composed of 25%~90% of the cells came from tumors in which endoscopic biopsy specimens showed poorly differentiated carcinoma (P < 0.005).
The relationship between tumor size and the prevalence of aneuploidy and DNA content heterogeneity was studied. Patients with larger tumors had a slightly higher prevalence of aneuploidy, but it was not statistically significant. Tumor DNA content heterogeneity was not influenced by the tumor size (Table 2). No clear relation could be established between the tumor stage and the DNA pattern, in part because of the low number of cases that had surgical pathological staging to determine the true extent of the tumor. All tumors with lymph node or hepatic metastasis were aneuploid (11 of 11). Invasion of one of the diploid tumors was limited to the submucosa in the surgical specimen.
Discussion Flow cytometry is commonly used to quantitate the amount of DNA in cells by labeling the DNA stoichiometrically with a fluorescent dye. A variety of fluorescent staining techniques have been reported (12). We have chosen the two fluorochromes mithramycin and ethidium bromide, because a previous study of squamous esophageal epithelium by flow cytometry (13) showed high-debris backgrounds probably because of nonspecific dye binding. Mithramytin is a DNA-specific dye that complexes with G-C rich regions of DNA. Ethidium bromide is an intercalating agent that preferentially binds to doublestranded DNA but also to RNA. When ethidium bromide and mithramycin are used in combination, an increase in fluorescence intensity and an improvement in the DNA histogram resolution (improved CV) is noted (9), which can be explained by energy transfer between the two dyes in close proximity. Our results indicate that aneuploidy is a common finding in squamous cell carcinomas of the esophagus. The frequency with which multiple aneuploid
December
Figure 2. Relation position of diploid A.
DNA CONTENT ANALYSIS
1991
between DNA content (2 N) is indicated.
Well-differentiated
squamous
B. On the corresponding
C. Poorly D.
differentiated
The aneuploid
cell carcinoma
DNA histogram, squamous
population
and histology.
(original
the aneuploid
cell carcinoma
(3.7 N) appears
The relative
magnification cell population
(original
magnification
Ratio, cells/total examined <15%
Histological type Well-differentiated carcinomas Poorly or moderately differentiated carcinomas “x’ = 8.05; P = 0.005
0% (diploid)
is shown
as flow cytometry
channel
number,
1591
and the
x 130).
(2.7 N) is small, containing
only 5% of the cells.
x 130).
as a larger peak (38% of total cells).
Table 1. DNA Content and Histological Type aneuploid
DNA content
OF ESOPHAGEAL CARCINOMAS
small aneuploid peaks
cells
25%-90%
large aneuploid peaks
2
10
2
0
1”
8”
populations are found in association with esophageal squamous cell carcinoma is high when compared with other tumors (14). Whereas the majority of solid tumors and some dysplastic tissues have aneuploid DNA contents detectable by flow cytometry, these are usually characterized by a single population of cells. However, differences in the interpretation of flow cytometric results and in the method of sampling the tumors may contribute to variations between studies. The prevalence of aneuploidy and multiple aneuploid populations has been reported to increase with the number of samples per tumor (15). In our study, all but one case had more than two samples per tumor. Twenty-one of 26 patients had at least one aneuploid
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ET AL.
DNA
GASTROENTEROLOGY
Content
Figure 3. An aneuploid peak found in a poorly differentiated carcinoma. The aneuploid population (3.2 N), which contains 89% of the cells, is characterized by a high proliferative rate; 35% of the cells are in S phase.
population in their tumor. No aneuploid population was detected in 2 patients, but an increased G,/ tetraploid fraction was detected in 1 patient. Seven carcinomas were characterized by multiple aneuploid populations. Whereas the proportion of esophageal squamous carcinomas with multiple aneuploidies is higher than that reported for colon or gastric carcinomas (15,16), it is not as high as has been reported in adenocarcinoma arising in Barrett’s esophagus, in which 12 of 14 cancers had multiple aneuploidies (17). However, in our study we were limited to endoscopic biopsies because the standard therapy at our institution for advanced stages of squamous cell carcinoma of the esophagus is chemotherapy and radiation therapy. Our reported frequency of multiple aneuploid populations should therefore be considered a minimum value; with evaluation of the entire tumor, the prevalence of multiple aneuploid populations might increase. We have observed a relationship between the size of the aneuploid population and the histological classification of the tumor, with well-differentiated carcinomas having smaller aneuploid populations and lower S phase fractions than poorly differentiated tumors. It should be noted that this relationship was based on biopsy specimens that were taken from those regions of the tumor that were endoscopically accessible. It is possible that the genomic instability and clonal evoluTable 2. Relationship Between Tumor Size, Aneuploidy, and Tumor Heterogeneity Tumor size
< 6 cm (%)
> 6 cm (%)
Aneuploidy Tumor heterogeneity”
13 (87) 10 (67)
8 (100)
NS
2 (251
NS
“Including combination
of aneuploid
and diploid samples.
Vol. 101, No. 6
tion that produce multiple different aneuploid populations may also contribute to the well-recognized histological tumor-cell heterogeneity in squamous cell carcinomas of the esophagus (18). Thus, it is possible that other regions of these tumors that were inaccessible to endoscopic biopsies could contain additional flow cytometric and histological abnormalities. Furthermore, biopsy specimens from carcinomas may contain tumor cells, normal or precancerous epithelium, and stromal elements. Because techniques that are used to investigate the genetic basis of cancer, including flow cytometry, disrupt the architectural and cytological features required for histological diagnosis, there is as yet no perfect way to investigate the relationship between samples submitted for histology and flow cytometry or other genetic evaluations (13). In our study, samples for histology and flow cytometry that were obtained from the same region of the tumor were available for analysis in 15 cancers. In these 15 cancers, we found the same association between histological and flow cytometric abnormalities that was observed in the entire series. Although it is possible that discrepancies between the samples submitted for histological and flow cytometric evaluation might affect the results of any single tumor, it is improbable that they would affect the overall conclusions of the study. These findings are in agreement with the multistep model of neoplastic progression articulated by Nowell (19,20), which proposes that the evolution to malignancy occurs in patients who have an acquired genomic instability leading to generation of abnormal clones with progressive escape from normal growth control mechanisms and selection of a malignant clone of cells. Continued genetic instability can lead to the appearance of subclones, contributing to the heterogeneity of abnormal DNA content cell populations in the tumor. Whether these genomic abnormalities occur in dysplastic epithelia before the development of carcinoma, as is the case in Barrett’s esophagus (I 3), needs further investigation.
References 1. Day NE. The geographic pathology of cancer of the esophagus. Br Med Bull 1984;40:329-334. 2. Waterhouse J, Muir C. Cancer incidence in five continents. Volume 5. Lyon, France: IARC Sci Publ, 1988:846-847. 3. Rezvani A, Doyon F, Flamant R. Atlas de la mortalite par cancer en France. Paris:Editions INSERM, 1986. 4. Tuyns AJ, Pequignot G, Jensen OM. Le cancer de l’oesophage en Ille et Vilaine en fonction des niveaux de consommation d’alcool et de tabac. Des risques qui se multiplient. Bull Cancer 1977;64:45-60. 5. Barlogie B, Raber MN, Schumann J, Johnson TS, Drewinko B, Swartzendruber DE, Gtihde W, Andreeff M, Freireich E. Flow cytometry in clinical cancer research. Cancer Res 1983;43:39823997.
December 1991
6. Coon JS, Landay AL, Weinstein RS. Advances in flow cytometry for diagnostic pathology. Lab Invest 1987;57:453-479. 7. O’Hara MF, Bedrossian CWM, Johnson TS, Barlogie B. Flow cytometry in cancer diagnosis. Prog Clin Path01 1984;9:135153. 8. Watanabe H, Jass JR, Sobin LH. Histological typing of oesophageal and gastric tumors. World Health Organization. International Histological Classification of Turnours. 2nd ed. New York: Springer-Verlag, 1990. 9. Barlogie B, Spitzer G, Hart JS, Johnston DA, Biichner T, Schumann J, Drewinko B. DNA histogram analysis of human hemopoietic cells. Blood 1976;48:245-258. 10. Dean PN, Jett JH. Mathematical analysis of DNA distributions derived from flow microfluorometry. J Cell Biol 1974;60:523527. 11. Bagwell CB, Mayo SW, Whetstone
SD, Hitchcox SA, Baker DR, Herbert DJ, Weaver DL, Jones MA, Lovett EJ. DNA histogram debris theory and compensation. Cytometry 199O;(Suppl4):27. 12. Shapiro HM. Practical flow cytometry. 2nd ed. New York: Liss, 1988:115-198. 13. Reid BJ, Haggitt RC, Rubin CE, Rabinovitch PS. Barrett’s esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Gastroenterology 1987;93:1-11. 14. Friedlander ML, Hedley DW, Taylor IW. Clinical and biological
DNA CONTENT ANALYSIS
15.
16.
17.
18. 19. 20.
OF ESOPHAGEAL CARCINOMAS
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significance of aneuploidy in human turnours. J Clin Path01 1984;37:961-974. Sasaki K, Hashimoto T, Kawachino K, Takahashi M. Intratumoral regional differences in DNA ploidy of gastrointestinal carcinomas. Cancer 1988;62:2569-2575. de Aretxabala X, Yonemura Y, Sugiyama K, Hirose N, Kumaki T, Fushida S, Miwa K, Miyazaki I. Gastric cancer heterogeneity. Cancer 1989;63:791-798. Rabinovitch PS, Reid BJ, Haggitt RC, Norwood TN, Rubin CE. Progression to cancer in Barrett’s esophagus is associated with genomic instability. Lab Invest 1988;60:65-71. Lee RG. Esophagus. In Sternberg SS, ed. Diagnostic surgical pathology. Volume 2. New York: Raven, 1989:917-936. Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194:23-28. Nowell PC. Mechanisms of tumor progression. Cancer Res 1986;46:2203-2207.
Received October 1,199O. Accepted May 15,1991. Address requests for reprints to: Michel Robaszkiewicz, M.D., Service d’HCpatogastroent&ologie, C.H.U. Morvan, B.P. 824, 29285 Brest Cedex, France. Supported in part by grant PDT-316B from the American Cancer Society and by the Association pour la Recherche sur le Cancer and the Societe Fransaise du Cancer.
1991;101:1588-1593
Flow-Cytometric DNA Content Analysis of Esophageal Squamous Cell Carcinomas MICHEL ROBASZKIEWICZ, BRIAN J. REID, ALAIN VOLANT, JEAN MICHEL CAUVIN, PETER S. RABINOVITCH, and HERVE GOUEROU Departments Departments
of Gastroenterology and Pathology, University Hospital, Brest, France; and of Medicine and Pathology, University of Washington, Seattle, Washington
To better understand the mechanisms of esophageal carcinogenesis, abnormalities in DNA content of esophageal squamous cell carcinomas were studied. Cellular DNA content was determined by flow cytometric study of 70 endoscopic biopsy specimens obtained from 26 patients with esophageal squamous carcinoma. High-quality histograms were obtained for 23 patients. Twenty-one patients had at least one aneuploid population in their tumor. In 7 patients, multiple aneuploid peaks were detected. Specimens from 2 patients were diploid. The interpretation of the DNA histograms was difficult in 3 patients; an aneuploid population of cells was probable in 2 of them. A statistically significant relationship was found between the degree of differentiation and DNA content abnormalities in the regions of the tumors that could be evaluated by endoscopic biopsies: well-differentiated carcinomas had diploid or small aneuploid populations containing < 15% of the cells, whereas DNA histograms of moderately or poorly differentiated carcinomas were characterized by large aneuploid peaks representing 25%~90% of the cells and a higher proliferative fraction. No relationship was found between the size or the stage of the tumor and the DNA content detected in endoscopic biopsy samples. The frequency and the multiplicity of abnormal clones in esophageal squamous carcinomas indicates that this cancer, like esophageal adenocarcinoma, develops an association with an acquired genomic instability that produces abnormal clones of cells, according to the multistep model of neoplastic progression.
T
he incidence of esophageal carcinoma varies widely in different parts of the world (12). It is the fifth most common cause of cancer deaths in men in France (3). Squamous cell carcinoma of the esophagus is common in Northwest France (3), where chronic
use of alcohol and smoking have been implicated as important contributing factors as in the United States and Western Europe (4). Despite efforts at early diagnosis and advances in surgery, radiation therapy, and chemotherapy, the prognosis of esophageal cancer is poor. The etiology of the disease remains unknown, and little is known about the cellular and nuclear events that occur during carcinogenesis. DNA content flow cytometry is widely used in the study of neoplasia, because changes in DNA content have been shown to closely correlate with chromosomal changes seen in malignancy (5-7). The objective of this study was to evaluate the DNA content of esophageal squamous cell carcinomas by flow cytometry to better understand biological mechanisms of esophageal carcinogenesis.
Materials and Methods Patients The biopsy samples for the study were obtained from 26 consecutive patients who underwent esophagogastroscopy for dysphagia between February 1988 and January 1989 at Brest University Hospital. The group consisted of 23 men and 3 women whose mean age was 67.4 years [range, 41-85 years).
Endoscopy
and Biopsies
Upper endoscopy was performed with an Olympus GIF XQlO endoscope. All patients were seen at the time of diagnosis, and none had chemotherapy or radiotherapy before endoscopy. All 26 patients had a visible tumor located in the upper third of the esophagus in 3 cases, in the
Abbreviations used in this paper: CV, coefficient of variation: DI, DNA index. o 1991 by tbe American Gastroenterological Association 0016-5085/91/$3.00
December 1991
middle third in 18 cases, and in the lower third in 5 cases. The tumor was infiltrating, polypoid, or ulcerating in 3, 6, and 17 patients, respectively. In 18 cases, the esophageal lumen was obstructed, permitting only the proximal margin of the tumor to be examined. After visualizing the tumor, biopsy specimens were obtained with the Olympus FB21K biopsy forceps. Four to six biopsy samples taken from the tumor were processed for histological evaluation, and one to four biopsy specimens were taken for flow cytometry. A total of 70 specimens were analyzed by flow cytometry.
Histology The specimens for histology were fixed in Bouin’s fixative and stained with H&E saffron. Specimens were interpreted histologically by one observer (A.V.) who had no knowledge of the results of flow cytometry. Specimens were classified according to the type and differentiation of the carcinoma based on the World Health Organization histological classification of squamous cell carcinomas of the esophagus (8).
Flow Cytometry
The biopsy specimens for flow cytometry were stored at - 70°C immediately
after the endoscopy and were thawed immediately before flow cytometric analysis. The nuclei were isolated and stained with ethidium bromide and mithramycin according to a procedure derived from the method described by Barlogie et al. (9). The biopsy specimens were minced and incubated for 20 minutes in 500 FL of ethidium bromide (25 mg/L; Sigma Chemical Corp., St. Louis, MO) in 0.1 mol/L saline Tris (pH 7.4), 0.6% NaCl, 0.6% Nonidet P 40 (Sigma), 0.2% bovine serum albumin type 1 (10 mg/L; (Sigma), and 100 PL of ribonuclease Sigma). Aggregates were dissociated by forceful passage three times through a 26-gauge needle, and the nuclei were incubated for 1 hour with 500 p,L of a solution containing 50 mg/L mithramycin (Miles Pharmaceuticals, West Haven, CT), 7.5 mmol/L MgCl,, and 12.5% ethanol. Flow cytometry was performed on an ICP 22 (Ortho Diagnostic Systems, Westwood, MA). Excitation was with a 390-490-nm filter. Emission was detected above 560 nm. Data were collected on a DEC LSI II/23 computer (Digital Equipment Corp., Maynard, MA) and transfered and analyzed on a PC AT compatible computer with the Multicycle software (Phoenix Flow Systems, San Diego, CA) written by one of the authors (P.S.R.). Cell cycle parameters were analyzed by the method of Dean and Jett (10) using a zero order polymonial S phase. By this nonlinear least squares curve fitting, the diploid and aneuploid G, and G, peaks were fit using normal distributions, and the region between each G, and G, peak was fit to a distribution of cells in S phase, with special treatment of the region of overlap with G, and G,. Subnuclear-sized debris were fit by a model that accounts for the effect of slicing or cutting nuclei during sectioning; every nucleus counted is assumed to have a certain probability of being randomly sliced into portions of (usually) unequal size, and these subnuclear size portions appear in channels lower than that of the original nucleus. The probability of
DNA CONTENT ANALYSIS OF ESOPHAGEAL CARCINOMAS
1589
being cut is determined by nonlinear least squares analysis. The model originally developed for Multicycle has been revised according to the suggestions of Bagwell et al. (11) to account for the effect of ellipsoidal shape of nuclei on the cut portion size distribution. Residual exponentially declining debris was fit by inclusion of a power function. The ratio of the aneuploid to the diploid DNA content is designated as the DNA index (DI). For tumors with a near-diploid DNA content, chicken erythrocytes were used as control cell references to determine the DNA content of the aneuploid population. A shoulder was defined as a low percentage (< 20%) of cells with an excess DNA content to the right of the G,G, peak, not showing bimodality. The coefficient of variation (CV) of the G,G, peak of the diploid or aneuploid population was expressed by the normalized standard deviation (CV = Standard Deviation/Mean Peak Position X 100). Coefficients of variation were typically 4.0%6.0% and always ~8.0%. Flow cytometric results were interpreted by two authors (M.R. and P.S.R.) without knowledge of the histological results. For statistical analysis, comparisons between groups have been tested using Fisher Exact Test and Yates’ corrected x’.
Results
Flow cytometric analysis was possible in 93% of specimens (65 of 70). High-quality histograms were obtained in 73% of biopsies (51/70). In five specimens, the tissue was necrotic, and analysis could not be performed. The histogram interpretation was difficult because of low cell number in 14 additional specimens. All of these difficult histograms came from 3 of the 26 patients; an aneuploid peak was suspected in 2 patients but could not be confirmed with certainty. Twenty-one of 26 patients had one or more aneuploid peaks. The additional peak was unique in 14 patients; 7 patients had multiple aneuploid peaks in their tumor. In 3 patients, the multiple aneuploid cell populations were present in the same biopsy specimen (Figure 1); in 4 other patients, only one aneuploid peak was present in each specimen, but specimens taken from different regions of the tumor had different DNA contents. Five patients had a combination of diploid and aneuploid biopsy specimens obtained from their tumor. In 1 patient, the aneuploidy was associated with an increased diploid G, or tetraploid fraction (20%). Two patients had no evidence of aneuploidy. One patient had normal DNA contents in two specimens, an increased G,/tetraploid fraction (10.6%) in a third biopsy, and a hyperdiploid shoulder in a fourth biopsy. The second patient had a diploid peak with a hyperdiploid shoulder.
DNA Con tent and Histology
Two kinds of DNA aneuploid histograms could be identified (Figure 2): (a) tumors with small aneu-
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GASTROENTEROLOGY Vol. 101,No. 6
ROBASZKIEWICZ ET AL.
DNA Content and Cell Proliferation Proliferative fractions (S phase and G,) of the aneuploid populations were estimated when the percentage of aneuploid cells exceeded 25% of the total cells (Figure 3). All aneuploid tumors were characterized by S phase fractions > 7%; the mean S phase fractions were 26%, with a standard deviation (SD) of 10.4% and a range of 7%48.5%. In tumors with small aneuploid peaks, the S phase could only be measured for the diploid cell population; the mean S phase fractions were 13.3% with an SD of 15.3% and a range of 4.3%20%. The two diploid tumors had lower S fractions (6.5% and 12%).
4
240
160
20
0
DNA Content and Clinical Features DNA
Content
Figure 1. Two aneuploid populations (1.6and 2.8 N) are present in this single biopsy sample from a moderately differentiated esophageal squamous cell carcinoma (channels 67 and 116). The diploid peak (2 N) (channel 84) was located by using chicken erythrocytes (channel 28) as a standard for DNA content estimation.
ploid peaks, in which the aneuploid cell population represented < 15% of examined cells; (b) tumors with larger aneuploid peaks, in which the aneuploid cells represented 25%~90% of the analyzed cells. Tumors in which the endoscopic biopsy samples showed well-differentiated squamous cell carcinoma were predominantly characterized by the first type of histograms, whereas large aneuploid peaks were seen in all but one tumor in which biopsy samples showed moderately and poorly differentiated carcinoma (Table 1; P = 0.005). In the two tumors in which biopsy specimens showed only a diploid DNA content, the corresponding histologies showed only well-differentiated carcinoma. The histological appearance of an esophageal squamous cell carcinoma may vary in different parts of the same malignancy, and in some of our tumors biopsy specimens taken for flow cytometry may not have come from the same region of the cancer as the samples for histology. Therefore, we reanalyzed the data for 15 tumors in which flow cytometric and histological biopsy samples were taken from the same region of the cancer. All eight tumors with aneuploid cell populations of < 15% came from histologically well-differentiated cancers as assessed by endoscopic biopsy specimens. However, six of seven tumors with aneuploid cell populations composed of 25%~90% of the cells came from tumors in which endoscopic biopsy specimens showed poorly differentiated carcinoma (P < 0.005).
The relationship between tumor size and the prevalence of aneuploidy and DNA content heterogeneity was studied. Patients with larger tumors had a slightly higher prevalence of aneuploidy, but it was not statistically significant. Tumor DNA content heterogeneity was not influenced by the tumor size (Table 2). No clear relation could be established between the tumor stage and the DNA pattern, in part because of the low number of cases that had surgical pathological staging to determine the true extent of the tumor. All tumors with lymph node or hepatic metastasis were aneuploid (11 of 11). Invasion of one of the diploid tumors was limited to the submucosa in the surgical specimen.
Discussion Flow cytometry is commonly used to quantitate the amount of DNA in cells by labeling the DNA stoichiometrically with a fluorescent dye. A variety of fluorescent staining techniques have been reported (12). We have chosen the two fluorochromes mithramycin and ethidium bromide, because a previous study of squamous esophageal epithelium by flow cytometry (13) showed high-debris backgrounds probably because of nonspecific dye binding. Mithramytin is a DNA-specific dye that complexes with G-C rich regions of DNA. Ethidium bromide is an intercalating agent that preferentially binds to doublestranded DNA but also to RNA. When ethidium bromide and mithramycin are used in combination, an increase in fluorescence intensity and an improvement in the DNA histogram resolution (improved CV) is noted (9), which can be explained by energy transfer between the two dyes in close proximity. Our results indicate that aneuploidy is a common finding in squamous cell carcinomas of the esophagus. The frequency with which multiple aneuploid
December
Figure 2. Relation position of diploid A.
DNA CONTENT ANALYSIS
1991
between DNA content (2 N) is indicated.
Well-differentiated
squamous
B. On the corresponding
C. Poorly D.
differentiated
The aneuploid
cell carcinoma
DNA histogram, squamous
population
and histology.
(original
the aneuploid
cell carcinoma
(3.7 N) appears
The relative
magnification cell population
(original
magnification
Ratio, cells/total examined <15%
Histological type Well-differentiated carcinomas Poorly or moderately differentiated carcinomas “x’ = 8.05; P = 0.005
0% (diploid)
is shown
as flow cytometry
channel
number,
1591
and the
x 130).
(2.7 N) is small, containing
only 5% of the cells.
x 130).
as a larger peak (38% of total cells).
Table 1. DNA Content and Histological Type aneuploid
DNA content
OF ESOPHAGEAL CARCINOMAS
small aneuploid peaks
cells
25%-90%
large aneuploid peaks
2
10
2
0
1”
8”
populations are found in association with esophageal squamous cell carcinoma is high when compared with other tumors (14). Whereas the majority of solid tumors and some dysplastic tissues have aneuploid DNA contents detectable by flow cytometry, these are usually characterized by a single population of cells. However, differences in the interpretation of flow cytometric results and in the method of sampling the tumors may contribute to variations between studies. The prevalence of aneuploidy and multiple aneuploid populations has been reported to increase with the number of samples per tumor (15). In our study, all but one case had more than two samples per tumor. Twenty-one of 26 patients had at least one aneuploid
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ROBASZKIEWICZ
ET AL.
DNA
GASTROENTEROLOGY
Content
Figure 3. An aneuploid peak found in a poorly differentiated carcinoma. The aneuploid population (3.2 N), which contains 89% of the cells, is characterized by a high proliferative rate; 35% of the cells are in S phase.
population in their tumor. No aneuploid population was detected in 2 patients, but an increased G,/ tetraploid fraction was detected in 1 patient. Seven carcinomas were characterized by multiple aneuploid populations. Whereas the proportion of esophageal squamous carcinomas with multiple aneuploidies is higher than that reported for colon or gastric carcinomas (15,16), it is not as high as has been reported in adenocarcinoma arising in Barrett’s esophagus, in which 12 of 14 cancers had multiple aneuploidies (17). However, in our study we were limited to endoscopic biopsies because the standard therapy at our institution for advanced stages of squamous cell carcinoma of the esophagus is chemotherapy and radiation therapy. Our reported frequency of multiple aneuploid populations should therefore be considered a minimum value; with evaluation of the entire tumor, the prevalence of multiple aneuploid populations might increase. We have observed a relationship between the size of the aneuploid population and the histological classification of the tumor, with well-differentiated carcinomas having smaller aneuploid populations and lower S phase fractions than poorly differentiated tumors. It should be noted that this relationship was based on biopsy specimens that were taken from those regions of the tumor that were endoscopically accessible. It is possible that the genomic instability and clonal evoluTable 2. Relationship Between Tumor Size, Aneuploidy, and Tumor Heterogeneity Tumor size
< 6 cm (%)
> 6 cm (%)
Aneuploidy Tumor heterogeneity”
13 (87) 10 (67)
8 (100)
NS
2 (251
NS
“Including combination
of aneuploid
and diploid samples.
Vol. 101, No. 6
tion that produce multiple different aneuploid populations may also contribute to the well-recognized histological tumor-cell heterogeneity in squamous cell carcinomas of the esophagus (18). Thus, it is possible that other regions of these tumors that were inaccessible to endoscopic biopsies could contain additional flow cytometric and histological abnormalities. Furthermore, biopsy specimens from carcinomas may contain tumor cells, normal or precancerous epithelium, and stromal elements. Because techniques that are used to investigate the genetic basis of cancer, including flow cytometry, disrupt the architectural and cytological features required for histological diagnosis, there is as yet no perfect way to investigate the relationship between samples submitted for histology and flow cytometry or other genetic evaluations (13). In our study, samples for histology and flow cytometry that were obtained from the same region of the tumor were available for analysis in 15 cancers. In these 15 cancers, we found the same association between histological and flow cytometric abnormalities that was observed in the entire series. Although it is possible that discrepancies between the samples submitted for histological and flow cytometric evaluation might affect the results of any single tumor, it is improbable that they would affect the overall conclusions of the study. These findings are in agreement with the multistep model of neoplastic progression articulated by Nowell (19,20), which proposes that the evolution to malignancy occurs in patients who have an acquired genomic instability leading to generation of abnormal clones with progressive escape from normal growth control mechanisms and selection of a malignant clone of cells. Continued genetic instability can lead to the appearance of subclones, contributing to the heterogeneity of abnormal DNA content cell populations in the tumor. Whether these genomic abnormalities occur in dysplastic epithelia before the development of carcinoma, as is the case in Barrett’s esophagus (I 3), needs further investigation.
References 1. Day NE. The geographic pathology of cancer of the esophagus. Br Med Bull 1984;40:329-334. 2. Waterhouse J, Muir C. Cancer incidence in five continents. Volume 5. Lyon, France: IARC Sci Publ, 1988:846-847. 3. Rezvani A, Doyon F, Flamant R. Atlas de la mortalite par cancer en France. Paris:Editions INSERM, 1986. 4. Tuyns AJ, Pequignot G, Jensen OM. Le cancer de l’oesophage en Ille et Vilaine en fonction des niveaux de consommation d’alcool et de tabac. Des risques qui se multiplient. Bull Cancer 1977;64:45-60. 5. Barlogie B, Raber MN, Schumann J, Johnson TS, Drewinko B, Swartzendruber DE, Gtihde W, Andreeff M, Freireich E. Flow cytometry in clinical cancer research. Cancer Res 1983;43:39823997.
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6. Coon JS, Landay AL, Weinstein RS. Advances in flow cytometry for diagnostic pathology. Lab Invest 1987;57:453-479. 7. O’Hara MF, Bedrossian CWM, Johnson TS, Barlogie B. Flow cytometry in cancer diagnosis. Prog Clin Path01 1984;9:135153. 8. Watanabe H, Jass JR, Sobin LH. Histological typing of oesophageal and gastric tumors. World Health Organization. International Histological Classification of Turnours. 2nd ed. New York: Springer-Verlag, 1990. 9. Barlogie B, Spitzer G, Hart JS, Johnston DA, Biichner T, Schumann J, Drewinko B. DNA histogram analysis of human hemopoietic cells. Blood 1976;48:245-258. 10. Dean PN, Jett JH. Mathematical analysis of DNA distributions derived from flow microfluorometry. J Cell Biol 1974;60:523527. 11. Bagwell CB, Mayo SW, Whetstone
SD, Hitchcox SA, Baker DR, Herbert DJ, Weaver DL, Jones MA, Lovett EJ. DNA histogram debris theory and compensation. Cytometry 199O;(Suppl4):27. 12. Shapiro HM. Practical flow cytometry. 2nd ed. New York: Liss, 1988:115-198. 13. Reid BJ, Haggitt RC, Rubin CE, Rabinovitch PS. Barrett’s esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Gastroenterology 1987;93:1-11. 14. Friedlander ML, Hedley DW, Taylor IW. Clinical and biological
DNA CONTENT ANALYSIS
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18. 19. 20.
OF ESOPHAGEAL CARCINOMAS
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significance of aneuploidy in human turnours. J Clin Path01 1984;37:961-974. Sasaki K, Hashimoto T, Kawachino K, Takahashi M. Intratumoral regional differences in DNA ploidy of gastrointestinal carcinomas. Cancer 1988;62:2569-2575. de Aretxabala X, Yonemura Y, Sugiyama K, Hirose N, Kumaki T, Fushida S, Miwa K, Miyazaki I. Gastric cancer heterogeneity. Cancer 1989;63:791-798. Rabinovitch PS, Reid BJ, Haggitt RC, Norwood TN, Rubin CE. Progression to cancer in Barrett’s esophagus is associated with genomic instability. Lab Invest 1988;60:65-71. Lee RG. Esophagus. In Sternberg SS, ed. Diagnostic surgical pathology. Volume 2. New York: Raven, 1989:917-936. Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194:23-28. Nowell PC. Mechanisms of tumor progression. Cancer Res 1986;46:2203-2207.
Received October 1,199O. Accepted May 15,1991. Address requests for reprints to: Michel Robaszkiewicz, M.D., Service d’HCpatogastroent&ologie, C.H.U. Morvan, B.P. 824, 29285 Brest Cedex, France. Supported in part by grant PDT-316B from the American Cancer Society and by the Association pour la Recherche sur le Cancer and the Societe Fransaise du Cancer.