yielded comparable results in terms of DNA content distribution. Staining and Flow Cytometry. Twenty microliters of Nonidet P40 (Fluka, Buchs,. Switzerland) ...
0 1984 Alan R. Liss, Inc.
Cytometry 5:63-70 (1984)
Cvtometricallv Determined Relative DNA Content i s an Indicator of Neoplasia in Gastric Lesions’7’ Laura Teodori, Lucio Capurso, Eugenia Cordelli, Raffaele De Vita, Maurizio Koch, Maurizio Tarquini, Francesco Pallone, and Francesco Mauro Laboratorio di Dosimetria e Biofisica, ENEA-Casaccia,00060 Roma, Italy (L.T., E.C., R.D.V., F.M.), Cattedra di Scienze dell’Alimentazione,Universita di Camerino, 62032 Camerino (Macerata),Italy (L.C.),Servizio di Gastroenterologia, Ospedale San Filippo Neri, 00135 Roma, Italy (L.C., M.K., M.T.), Cattedra di Gastroenterologia,Universita di Roma, 00185 Roma, Italy (F.P.), Istituto Medico e di Ricerca Scientifica,00184 Roma, Italy (L.T.) Received for publication February 18, 1983; accepted August 24,1983
Cellular samples from human gastric endoscopic biopsies were analysed in order to detect possible DNA content alterations as markers of cancerous and precancerous lesions of the digestive tract. Samples were derived from the stomach of normal donors (17 cases), and from patients clinically classified as affected by stomach adenocarcinoma (18 cases), chronic atrophic gastritis (20 cases), or other nonneoplastic lesions (17 cases). Sample processing was performed by mechanical and enzymatic treatment to obtain monodispersed cells. Staining for flow cytometric analysis was achieved with ethidium bromide and mithramycin. Samples from normal donors constantly exhibited a single cell population with diploid DNA content. All but three neoplastic specimens exhibited both a diploid and an aneuploid cell subpopulation, with the DNA index of the aneuploid peak ranging
Cytogenetic investigations indicate that karyotypic abnormalities are present in both hematologic and solid human malignant tumors (9, 11, 20). These aberrations may correspond to alterations of relative cellular DNA content. Flow cytometry is a reliable tool for measuring relative DNA content in cellular samples from normal and pathological tissue (1, 7, 8). In other words, cytometrically determined aneuploidy can be used as a tumor marker (2). In particular, the present state of the art seems t o indicate that aneuploidy is a condition exclusive to some (or the majority of, malignant tumors. So far, no clear-cut evidence has been reported of aneuploidy as a feature of benign tumors. Still, aneuploidy could be present in the precancerous state either as a marker of some kind of transition from the benign to malignant condition or as an indicator of the presence of a small fraction of already malignant cells. The use of gastric endoscopies as a source of cells for cytometric determinations has been reported by other authors (4, 19).In some studies, chronic atrophic gastri-
from 1.10 to 1.85 (except a single instance with a value of 3.13). The presence of a recognizable aneuploid subpopulation was also observed in 9 out of 20 chronic atrophic gastritis specimens. Such aneuploidy is similar to that observed for the adenocarcinoma, even if the fraction of aneuploid cells appears to be generally higher in the tumor than in the gastritis cases. All other cases of gastritis and of nonneoplastic disease exhibited diploid cells only. The meaning of aneuploidy in some gastritis specimens is a phenomenon not yet fully explained. Still, aneuploidy appears to be a useful marker for recognizing the presence of suspect malignant cells in gastric lesions. Key terms: Gastric adenocarcinoma, chronic atrophic gastritis, endoscopic biopsy, DNA content determination, flow cytometry
tis has been also discussed (22, 23). However, in the latter reports ploidy level and “shape” of the DNA content pattern have been jointly used as cytometric indicators. In the present work, the attempt to use only a strictly defined criterion of aneuploidy as a tumor marker will be followed.
MATERIALS AND METHODS Sample Preparations Gastric biopsies, obtained by endoscopy and in some instances by surgery, were collected from patients and
‘Partially supported by Progetto Finalizzato CNR “Controllo della Crescita Neoplastica,” contract no. 80.01586.06. ‘Presented a t the Combined International Conference on Analytical Cytology and Cytometry IX and VI International Symposium on Flow Cytometry, Schloss Elmau, Mittenwald, Bavaria, West Germany, October 18-23, 1982. Address reprint requests to Laura Teodori, Laboratorio di Dosimetria e Biofisica, ENEA-Casaccia, Via Anguillarese, 00060 Roma, Italy.
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TEODORI ET AL
stored a t 4” C in RPMI medium (Gibco, New York, NY), supplemented with 5% fetal calf serum. Biopsy processing was performed on the day of sampling; single cell suspensions were obtained by mechanical and enzymatic treatments. The specimens were minced with scissors, washed in calcium- and magnesium-free buffer, and treated with 0.05% pepsin (Serva, Heidelberg, FR Germany) for 10 min a t room temperature. Tris-buffer was then added, the samples were centrifuged at 200 g for 5 min, and the pellet resuspended in the same buffer. Pepsin treatment was selected (and used in all cases reported in the present work) as the least time consuming; preliminary tests indicated that either pepsin or collagenase and hyaluronidase (0.05% and 0.01%, respectively, for 15 min a t 37” C) or collagenase followed by papain (300 Anson unitdm1 in 0.1 N Na-citrate/ NaOH, pH = 6.4, for 60 seconds a t room temperature) yielded comparable results in terms of DNA content distribution.
Staining and Flow Cytometry Twenty microliters of Nonidet P40 (Fluka, Buchs, Switzerland), 1 ml of 25 pg/ml ethidium bromide (Serva: Heidelberg, FR Germany), and 1 ml of 25 pg/ml mithra. mycin (courtesy of Pfizer, Rome, Italy) were added to 0.2 ml of cell suspension (3, 14). The samples were then measured with a n ICP 22 arc-lamp pulse cytophotometer (Ortho, Westwood, MA), equipped with a glass flow chamber kindly supplied hy Dr. W. G h d e . The number of cells measured was generally about 10,000 per histogram.
Data Analysis
Table 1 List of Patients Under Investigation Diagnosis “Normal” Gastric adenocarcinomaa Chronic atrophic gastritisb Superficial gastritis Benign gastric ulcersd Hyperplastic polyps
No. of Patients
Age Range (Years)
17 18 20‘ 5 10 2
33-55 42-83 31-79 35-72 21-65 60-89
Sex M
F
10 12 7 3 9 2
7 6 13 2 1 0
aAccording to Lauren’s histological classification (13): 18 were of the intestinal type, and 2 (CAP G 54 and CAP G 73) of the diffuse type. bWith intestinal metaplasia. ‘2 of these cases refer to patients affected by both adenocarcinoma and gastritis, i n which distinct endoscopic samples were collected. all cases, endoscopic and histological healing was observed by 30 days after the beginning of therapy.
patients for whom corticosteroid or antiinflammatory therapy was planned). These patients have been used, with their consent, as “normal” donors in the present study. The list of the patients, classified in terms of diagnosis, age, and sex is reported in Table 1.
Clinical and Histopathological Evaluation The type and site of disease were initially diagnosed on the basis of objective and endoscopic criteria and then confirmed by a professional pathologist (F.P.). Microscopic and cytometric samples were prepared by dividing the same biopsy specimen. When possible, more than one endoscopic biopsy from the same area were collected and analyzed both separately and as a mixture. Histopathological and cytometric analyses were performed independently as a blind test. In the case of discrepancies, further evaluation was achieved by a second endoscopic sampling when possible or by analysis of the surgical biopsy if the patient underwent a n operation a t a later time. Slides for histopathology were stained with hematoxylin-eosin.
DNA content distributions were accumulated in a multichannel analyzer, and data analysis performed on a Nuclear Data ND620 computer (Schaumburg, IL). In all cases, the diploid peak in the distribution was localized by adding to the sample cells from a normal area of the stomach from the same patient andor from a “donor” patient (see below). DNA index (DI) was calculated as the ratio of the Gl,o aneuploid peak modal channel to the G1,0 diploid peak modal channel. No corrections for RESULTS background and cell aggregations were applied to the Normal Samples histograms in the Figures which show the actual data. Figure 1 illustrates the typical cytometrically deterHowever, background correction was applied to obtain the data of Table 4. Correction for cell aggregates was mined DNA content distribution obtained from a specimen of “normal ” stomach. All “normal” specimens applied only in the specific cases discussed in the text. constantly exhibited a normal diploid content, that is, DJ = 1.00. No relevant differences were noted among Patients various sites of the stomach including antrum, cardia, Flow cytometric measurements were performed on corpus, and fundus. samples from 70 patients. All of them had been referred Neoplastic Samples for a gastrointestinal checkup, were clinically examined by one of us (L.C.), and underwent the first endoscopy In Table 2, 18 cases of adenocarcinoma of the stomach on the same occasion. Seventeen of them were patients are listed. Fifteen out of eighteen revealed the presence under study for pathological conditions in organs other of both diploid and aneuploid cell subpopulations, the DI than those in the gastrointestinal tract, and for whom of the latter ranging from 1.10 to 1.85, with an extreme a n endoscopic control of the stomach was requested by value in a specific case (CAP G70) of 3.13. A representathe referring physician for precautionary reasons (eg, tive histogram is shown in Figure 2. The histogram
65
DNA FLOW CYTOMETRY OF GASTRIC LESIONS
exhibits a relatively large “debris” area, evidenced by some neoplastic samples (as well as by some gastritis samples and, more rarely, by “normal” samples). Ancillary experiments have indicated that this area can be reduced if, instead of pepsin, papain is used (as indicated in “Materials and Methods”). It is likely that, as is suggested by microscopic observations, this area also includes both pycnotic cells and nuclear debris. We preferred to avoid any correction of the histograms so that we could show the actual experimental observations. In a particular case (CAP G62), two aneuploid subpopulations, with DIs of 1.50 and 1.81, were present together with the diploid subpopulation. The presence of two aneuploid subpopulations was confirmed after flow cytometry of a surgical biopsy from the same patient. In two other cases (CAP G73 and GAS 03) of carcinoma, diploid cells only were observed.
v , -A 2
U W
Chronic Atrophic Gastritis Samples DNA content measurements of samples from 20 patients with a diagnosis of chronic atrophic gastritis are presented in Table 3. Nine out of twenty samples revealed the presence of a n aneuploid cell clone similar to that which is typical of carcinoma; the remaining eleven out of twenty samples exhibited a diploid cell population only.
I
I
CHANKL NUMBER FIG1. Flow cytometrically determined DNA content distribution of normal stomach tissue. Such a distribution is also typical of superficial gastritis, benign ulcers, and hyperplastic polyps. The histogram refers to a total of 7,460 cells (3,914 cells in the modal channel) and exhibits a coefficient of variation (CV) that equals 3.1.
D
Table 2 Gastric adenocarcinomaa Patient CapG Cap G CapG CapG CapG CapG CapG Cap G CapG CapG CapG CapG CapG CapG CapG Gas Gas Gas
Site
01 07 32 48 53 54 58 62 64 65 67 67B 70 73 84 01 03 19
Prepylorus Antrum Cardia Fundus Corpus Corpus Cardia Corpus Fundus Corpus Corpus Corpus Fundus Angulus Fundus Corpus Fundus Fundus
D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
A1
A2
Al/D
1.18 1.40 1.23 1.68 1.10 1.13 1.35 1.50 1.81 1.27 1.85 1.75 1.54 3.13
0.50 0.60 0.30 1.45 0.10 0.10 0.35 0.25 0.85 0.21 0.02 0.55 0.10
1.37 1.76
0.07 0.05
1.60
0.16
A2/D
v,
1.35
=The DI’s of all subpopulations are reported. D, diploid peak; Al, first aneuploid peak; A2, second aneuploid peak AID, ratio of the number of cells in the aneuploid peak to the number of cells in the diploid peak.
U
8
9 I
I
I
------I
CHANNEL N W R FIG 2. Representative histogram of a gastric adenocarcinoma. D indicates the GI,* peak of the diploid cells, and A the aneuploid cells. The histogram refers to a total of 13,601 cells (1,700 cells in the modal channel) and exhibits a CV that equals 5.1.
66
TEODORI ET AL
Table 3 Atrophic Chronic Gastritis” Patient
Site
CapG 01 Fundus CapG 07 Fundus CapG 08 Fundus CapG 27 Antrum CapG 30 Antrum CapG 31 Antrum CapG 34 Antrum CapG 35 Antrum CapG 38 Antrum CapG 39 Antrum CapG 43 Antrum Antrum CapG 50 CapG 51 Fundus CapG 52 Fundus CapG 56 Antrum CapG 61 Fundus CapG 66 Fundus CapG 94 Fundus CapG 97 Fundus CapG 107 Corpus aAbbreviations as in Table 2.
0
D
A
AID
1.00
1.08 1.40
0.09
1.14 1.31 1.30
0.03 0.33
1.21
0.26
1.10
0.18
1.16
0.03
1.44
0.21 __
1.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.00
0.10
0.05
1.00
1.00 1.00 1.00
1.00 1.00 1.00 1.00 1.00
l i CHANNEL N W R
FIG4. Representative histogram of a chronic atrophic gastritis exhibiting diploid cells only. The histogram refers to 4,820 cells (1,962 cells in the modal channel) and exhibits a CV that equals 3.4.
QwfawREn FIG3. Representative histogram of a chronic atrophic gastritis exhibiting an aneuploid cell subpopulation. The histogram refers to 37,081 cells (2,930 in the modal channel) and exhibits a CV that equals 8.5.
In Tables 2 and 3, the AJD peak height ratios for the aneuploid and normal peaks of the carcinoma and chronic atrophic gastritis cases are also reported. Representative histograms of gastritis with and without aneuploid peak are shown in Figures 3 and 4 respectively. It must be noted that two particular cases (CAP GO1 and CAP G07) refer to patients affected by gastric adenocarcinoma in a specific area of the stomach (see Table 2) and by chronic atrophic gastritis in other areas (Table 3). In both cases, the samples from supposedly nonneoplastic areas also revealed an aneuploid cell subpopulation. In the specific case of CAP G07, the same DNA index characterized the aneuploid peaks of the tumor and the gastritis samples. A histogram, in which an appreciable peak of cells around the hexaploid value and a very small aneuploid peak are present, is shown in Figure 5. Peaks around the hexaploid value can sometimes be observed in stomach specimens independently of the type of disease. As far as these cases are concerned, the use of longer exposures to pepsin and microscopic observations has demonstrated that such peaks are essentially due to cell triplets. Of course, a s is evident in the case of Figure 5, the magnitude of the G ~ + peak M is affected by the presence of doublets. We considered the “small-clone” aneuploidy of Figure 5 a s a bona fide positive case because
67
DNA FLOW CYTOMETRY OF GASTRIC LESIONS 0
b
h
I
A
Ik _ I
f
I
--
CHAVWNEL N W R
FIG 5. Histogram of a chronic atrophic gastritis characterized by “small clone” aneuploidy [see text). The histogram refers to a total of 19,156 cells (7,962 cells in the modal channel) and exhibits a CV that equals 2.4. The “small clone” represents 4.7% of the total cell population.
(a) such a peak occurred in repeated readings of the same sample; (b) the peak remained visible even when histogram corrections for background and cell aggregates were applied; and (c) aneuploidy was still evident in samples taken a t a later date during follow-up. “Small-clone” aneuploidy was present in three (CAP G30, CAP G34, CAP G52) of the nine cytometrically positive gastritis cases; in the other five aneuploid gastritis cases with a n aneuploid peak, the histograms were relatively similar to those reported in Figures 2 and 7. In three cases of gastritis with an aneuploid peak (CAP G31, CAP G43, CAP G52), aneuploidy was still present in the specimens from a second endoscopy performed some 6 months later. In the last case (CAP G52), the follow-up a t 6 months revealed the presence of a cytometrically positive (Fig. 6) endoscopically ‘kuspect” area not confirmed by histology, in spite of the fact that some ten endoscopic biopsies were collected and microscopically analyzed. At a later time the patient underwent surgery because of a pyloric stenosis. After surgery, the microscopic analysis of tissue sections indicated the presence of a small neoplastic focus, classified as early gastric cancer. In fact, a new cytometric analysis con-
FIG 6. Histogram of case CAP G52, initially classified as severe chronic atrophic gastritis. In this histogram, the presence of’ a relatively small (3% of total; D1 = 1.16)cell subpopulation is evident. The histogram refers to a total of 15,913 cells (1,535 cells in the modal channel) and exhibits a CV that equals 3.4.
firmed the occurrence of a relatively large aneuploid cell subpopulation (Fig. 7).
Superficial Gastritis, Benign Gastric Ulcers, and Hyperplastic Polyps In all instances, no aneuploid cell subpopulation was observed. The histograms were similar to those reported in Figures 1and 4. Cell Cycle Phase Analysis The percentage of cells in the various phases of the cell cycle was estimated on the basis of the DNA content histograms, for the majority of the diploid cases. In the instance of normal tissue, diploid adenocarcinoma, diploid chronic atrophic gastritis, superficial gastritis, and benign ulcers, the percentages evaluated (5, 61, and reported in Table 4, clearly indicate that no statistically significant difference between samples can be detected.
DISCUSSION The present study confirms the possibility of detecting cellular DNA content alterations in tissue samples from stomach endoscopic biopsies. Ploidy abnormalities in
68
TEODORI ET AL.
specimens of adenocarcinoma of different areas of the stomach were present in all but two of the cases examined. In the majority of cases, aneuploidy fell within a narrow range of DI (mean value = 1.44).Only four cases exhibited a different situation. In the first one, two aneuploid peaks were observed. In another case, the aneuploid cell subpopulation was characterized by a DI of 3.13. Perhaps such peculiarities could be related to
0
C H M L NlMBER FIG7. Histogram of case CAP G52 after surgery, classified as early gastric cancer. The histogram refers to a total of 18,900 cells (7,207 cells in the modal channel) and exhibits a CV = 6.4. The aneuploid population is 23.1% of total; DI = 1.64.
some specific histological feature of the tumor. In fact, interestingly enough, these two cases refer to advanced states with deeper involvement of the underlying tissue layer. In the remaining two instances, no aneuploid peak was revealed. The observation that aneuploidy is present in 89% (16/ 18)adenocarcinoma cases is in agreement with the initial report (100%) of Barlogie et a1 (2) and with a later confirmation by the same group (4) on a larger number of cases (88%, 21/24). In contrast, other authors (18, 22) have reported a frequency of aneuploidy of around of 50-60%. However, it must be noted that these authors (18)obtained a different percentage when they broke the data down by institution (13% and 69% for two hospitals). Moreover, they reported aneuploidy as more typical of differentiated (57% and 79% for the same two hospitals) than undifferentiated carcinoma. Unfortunately, the histological classification used in the present work (13)is not exactly comparable with the classification followed by these authors. Still, it is interesting to note that one of the diploid cases observed by us (CAP G73) refers to a n adenocarcinoma of the diffuse (scattered pattern) type and so is likely to correspond to the diploid “undifferentiated dissociated” carcinoma reported by Petrova et al (18).At the same time, it cannot be excluded that the diploid cases are due to technical problems in the endoscopy as well as to the occurrence of a cytometrically undetectable quasi-diploidy. Furthermore, other considerations can he made on the reports of the above-mentioned authors (18, 22, 23). In particular, they classify some histograms according to definitions such a s “irregular S-phase . . . with extra peak” and “split G~+M.’’ In our opinion, there is no reason to consider “extra peaks” as different from aneuploid peaks. If the observations of these authors are classified in terms of aneuploidy only (ie, a t least some of the “extra peaks” are considered as aneuploid peaks), then the percent of cytometrically positive gastric cancer they observe would approach the values reported by Barlogie’s and our groups. The data obtained in the present study reveal less cellular heterogeneity in gas-
Table 4 Cell Cycle Phases Analysisa
Gz
+M
Diagnosis
No. of Cases
GlIO
S
“Normal”
17
84.7 & 5.7 (74.4 - 88.5) 87.1 (84.6 - 89.6) 87.9 k 4.2 (82.4 - 95.8)
7.9 10.3 (1.3 - 24.6) 8.0 (7.9 - 8.0) 9.7 3.5 (3.8 - 15.8)
5.0 i 5.3 (0.5 - 12.9) 4.9 (7.4 - 2.4) 2.5 i 2.0 (0.1 - 6.1)
86.1 & 2.4 (84.1 - 90.1) 82.8 & 9.9 (71.6 - 90.7)
8.6 i 1.7 (8.1 - 10.5) 12.8 i 10.4 (6.7 - 24.8)
5.3 (4.0 4.4 (2.4
Adenocarcinoma diploid Chronic atrophic gastritis (diploid) Superficial gastritis Benign ulcers
2 11
5
10
*
*
aMean values f SD. The values in parentheses are the ranges.
* * -
-
1.2 0.9) 2.5 7.3)
DNA FLOW CYTOMETRY OF GASTRIC LESIONS
tric tumors than in other solid tumors. In fact, multiclonality has been reported by other authors for colorectal carcinoma (16, 17). In our previous investigations on lung, head, and neck carcinoma (14, 15,21), up to six clones per single tumor were observed with a very broad range of DI (1.2-8). By contrast, with the exception of case CAP G62, the presence of one aneuploid subpopulation only seems to be the rule for gastric cancer. In the specific case of the stomach, aneuploidy is sometimes apparently associated (22) with a disease such as chronic atrophic gastritis (10) that is clinically considered to be precancerous. In fact, while normal tissue, superficial gastritis, ulcers, and benign polyps, as examined in the present work or reported by other authors (22, 231, constantly exhibited single diploid cell populations, 9 out of 20 chronic atrophic gastritis studied by us were cytometrically characterized by the concomitant presence of a diploid and a n aneuploid cell subpopulation. The level of aneuploidy in the nine cytometrically positive gastritis cases is comparable to that observed in gastric tumors, with DI ranging from 1.08 to 1.44.As already reported in our “Results,” the fraction of aneuploid cells (as measured in terms of A/D peak height ratio) is relatively lower in aneuploid gastritis than in adenocarcinoma. In our experience, the majority of instances of apparent polyploidy could be explained in terms of triplets and multiplets. Clearly, the problem of the actual existence of aneuploid chronic atrophic gastritis is open. Weiss et a1 (22) reported 3% aneuploid gastritis and even a 90% gastritis with a n “S phase irregular pattern.” Of course the cases observed by us could be interpreted as false-negative clinical and histological diagnoses of actual malignant lesions. Case CAP G52 may well represent the typical false-negative in which malignant neoplasia is later recognized. Furthermore, cases CAP GO1 and CAP GO7 can also be interpreted as reflecting the infiltration of tumor cells outside of the endoscopically determined tumor area. However, the alternative hypothesis that aneuploidy is typical of the precancerous condition of the stomach mucosa (where precancerous refers to a n epithelial atypia preceeding the stage of early gastric cancer), should also be considered, even if such a hypothesis is in contrast with the assumption that aneuploidy is indeed a tumor marker. In fact, these cytometrically positive gastritis cases are cytometrically indistinguishable, apart from the relative fraction of aneuploid cells, from gastric cancer. Perhaps the correct hypothesis should be that the occurrence of aneuploidy in a gastritis case is indicative not of epithelial atypia but of a n already existing early gastric cancer or carcinoma in situ. The possibility of detecting, by DNA cytophotometry, gastric carcinomas of a high grade of malignancy, even in early stages of development, has been recently reported (12). Obviously, the collection of a larger number of cases, studied cytometrically at various stages of the history of the disease, is necessary to solve these problems. How-
69
ever, it can already be said that aneuploidy may represent a marker to be used in addition to traditional procedures to classify gastric lesions. The same cannot be said, a t least in our experience, for markers such as the percentage of cells in various cycle phases. This does not mean that the phase distribution pattern should not be different in the various pathological states of the gastric mucosa. The absence of a significant difference is likely to be due simply to the technical limits of the endoscopic sampling. Therefore, only large differences could be revealed by this approach. In fact, we have been able to observe (unpublished data) a significant decrease of the G ~ + M fraction in ulcer patients treated with cimetidine, and a significant increase of the same fraction in patients after gastric resection.
LITERATURE CITED 1. Barlogie B, Drewinko B, Raber MN, Swarzendruber DE: Cell
kinetics in clinical oncology. In: Cell Growth, Nicolini C (ed), Vol. 2: Plenum Press, New York, 1982, pp 773-798. 2. Barlogie B, Drewinko B, Schumann J, Gijhde W, Dosik G, Latreille J, Johnston DA, and Freireich E J Cellular DNA content as a marker of neoplasia in man. Am J Med 69:195-203, 1980. 3. Barlogie B, Spitzer G, Hart JS, Johnston DA, Buchner T, Schumann J, Drewinko B: DNA histogram analysis of human hemopoietic cells. Blood 48:245-258, 1976. 4. Bennets R, Stroehlein J, Barlogie B: Ploidy determination by DNA flow cytometry of malignant and benign gastric tissue obtained by endoscopic biopsy. Gastroenterology 76:1099, 1976. 5. Bruni C, Curzi L, Koch G, Rossi C: Biological aspects and mathematical analysis of the inversc problcm in flow cytometry. Rapport0 IASI no. R16: Consiglio Nazionale delle Ricerche, Rome, 1981. 6. Bruni C, Koch G, Lucidi S: Analysis of DNA distribution from flow cytometry: Validation of automatic procedure against simulated data. Rapport0 IASI R58: Consiglio Nazionale delle Ricerche, Rome, 1983. 7. Christensen I, Vindelav LL: A method for the analysis of a mixture of DNA distributions measured by flow cytometry. VI International Symposium on Flow Cytometry, Schloss Elmau, 1982, p. 41. 8. Gijhde W, Schumann J, Buchner T, Otto F, Barlogie B: Pulse cytophotometry: Application in tumor cell biology and clinical oncology. In: Flow Cytometry and Sorting, Melamed M, Mullaney PF, and Mendelson ML (eds), John Wiley & Sons, New York, 1979, pp 599-620. 9. Gripenberg U, Ahlqvist J, Stenstrom R, Gripenberg L: Two chromosomally different cell populations in a human neoplasm. Hereditas 875-56, 1977. 10. Heilman K, Burghardt HU, K: Der intestinale und diffuse Typ des Megenkarzinoms. Z Gastroent 16422-430, 1978. 11. Hossfeld DK, Schmidt CG: Chromosome findings in effusions from patients with Hodgkin’s disease. Int J Cancer 21:147-156, 1978. 12. Inokuchi K, Kodama Y, Sasaki 0, Kamegawa T, Okamura T Differentiation of growth patterns of early gastric carcinoma determined by cytophotometric DNA analysis. Cancer 51:11381141, 1983. 13. Morson BC, Dawson I M P Malignant epithelial tumours: The Lauren classification. In: Gastrointestinal Pathology, Morson BC, Dawson IMP (eds), Blackwell, Oxford, 1979, pp 157-159. 14. Nervi C, Badaracco G, Maisto A, Mauro F, Tirindelli-Danesi D, Starace G: Cytometric evidence of cytogenetic and proliferative heterogeneity of human solid tumors. Cytometry 2:303-307, 1982. 15. Nervi C, Badaracco G, Morelli M, Starace G: Cytokinetic evaluation in human head and neck cancer by autoradiography and DNA cytofluorimetry. Cancer 45452-459, 1980. 16. Petersen SE: The progression of colo-rectal tumours investigated with flow cytometric analysis of DNA ploidy. VI International Symposium on Flow Cytometry, Schloss Elmau, 1982, p 159.
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ier, New York, 1981,pp 468485. 17. Petersen SE, Bichel P, Lorentzen M Flow-cytometric demonstration of tumour cell subpopulations with different DNA content in 21. Teodori L, Tirindelli-Danesi D, Mauro F, De Vita R, Uccelli R, Botti C, Modini C, Nervi C, and Stipa S Nonsmall-cell lung carcihuman colo-rectal carcinoma. Eur J Cancer 15:383-386,1978. noma: Tumor characterization on the basis of flow cytometrically 18. Petrova AS, Subrichina GN, Techistjakova OV, Lukina TA, Weiss determined cellular heterogeneity.Cytometry, 4:174-183,1983. H, Wildner G Flow cytofluorometry, cytomorphology and histol22. Weiss H, Gutz HJ, Wildner GP, Ebeling K, Schmidt W: Characterogy in gastric carcinoma. Oncology 37:318-324,1980. ization of chronic atrophic gastritis by means of pulse cytophoto19. Petrova AS, Subrichina GN, Tschistjakova OV, Rottenberg WI, metry. In: Flow Cytometry IV,Laerum OD, Lindmo T, Thorud E. Weiss H, Giitz HJ, Steenbeck L, Wildner G P Flow cytometry, (eds), Universitetsforlaget, Bergen, 1980,pp 473-477. cytomorphology, histology and autoradiography in human gastric hyperplastic polyps and the surrounding mucosa. Oncology 39:308- 23. Weiss H, Wildner GP, Gutz HJ, Ebeling K, Steinhoff G, Tanneberger S DNA distribution patterns of preneoplastic cells and their 313,1982. interpretation. Oncology 38:210-218,1981. 20. Sandberg AA: Tumors of the alimentary tract. In: The Chromo8omes in Human Cancer and Leukemia, Sandberg AA (ed), Elsev-
