Europ. J. CancerVol. 13, pp. 1155-1162. Pergamon Press 1977. Printed in Great Britain
The Clinical and Histopathological Significance of Feulgen DNA-Values in Transitional Cell Carcinoma of the Human Urinary Bladder* S. D. FOSS~,~++ O. KAALHUS§ and O. SCOTT-KNUDSEN[[ ~General Department and [IDepartment of Pathology, The Norwegian Radium Hospital, Oslo, Norway, and Departments of +Tissue Culture and §Biophysics, Norsk Hydro's Institutefor CancerResearch, The Norwegian Radium Hospital, Oslo, Norway Abstract--Scanning absorption cytophotometric measurements were performed in Feulgen-stained cell nuclei in imprintsfrom 123 biopsy specimensfrom human transitional cell carcinoma ( TCC). The results were correlatedwith the histopathological grade and the clinical stage of the tumour. Nearly all histologically well differentiated bladder carcinomas and tumours of clinical stage I had diploid DNA-stemlines, while non-diploid DNAstemlines werefound in most of the dedifferentiated carcinomas and in most of the advanced cases. Increasing clinical stage and decreasing histological differentiation were correlatedwith low numbers of DNA-stemline values. Within the group of locally advancedcarcinomas,patients with non-diploid turnouts had a worse prognosis than those with diploid TCC. Distant metastases in 3 patients showed non-diploid DNA-stemlines. In some turnouts the DNA-histogram showed regional differences.
INTRODUCTION IN 1948 Boivin et al. [1] found that the nuclear DNA-content was usually constant in cells from different tissues in the same species. A few years later it became evident that the variations in the nuclear DNA-content which can be observed in certain tissues may be explained as a result of nuclear polyploidy and by nuclear DNAsynthesis [2]. The DNA-content in carcinoma cell nuclei may differ from that o f n o r m a l cells [3]. M a n y studies have dealt with the relationship between the nuclear DNA-content and the histopathology in human tumours. In addition, some authors have considered nuclear DNA-content in relation to clinical aspects suggesting that measurement of the nuclear DNA-content might
Accepted 31 March 1977. *This work was supported by grants from Grosserer N. A. Stang's Legat til KreftsykdommersBekjempelse, Knut Knutsen's O.A.S. fond for Kreftforskning,Nansenfondet and The Norwegian Cancer Society. 1155
be of prognostic value in patients With a malignant disease [4]. In this study we correlated hist0pathologica! and clinical aspects of transitional cell carcinoma (TCC) of the h u m a n urinary bladder with the results of cytophotometric DNAmeasurements of individual Feulgen-stained cell nuclei in imprints from bladder tumour biopsies.
MATERIAL AND METHODS Imprints were made from biopsies of histologically proven transitional cell carcinoma from 123 patients admitted to the Norwegian Radium Hospital during the year 1974 and from 1st October 1975 to 31st March 1976. The biopsy material ("actual biopsy") was taken from areas thought to be representative of the whole tumour, either by transurethral resection or by cystotomy. A single biopsy was taken in 118 patients, whereas both a central and a peripheral tumour area were biopsied in the remaining 5 patients (Fig. 2): In the latter the results from the
1156
S. D. Fossd, O. Kaalhus and O. Scott-Knudsen b)
c)
- iii\
i~ I00 L -qP*'*\l~-. a)
?-"80 /I \L
°~°-O~ 0
~0~0~0~0~
60
c~
--..
'"°-°-o\o. o
20
O0
I
i
6
12
1
18
I
24
I
6
12
I
I
18
I
24
6
1
12
1
IB
1
2/,
Time (months after actual biopsy)
Fig. 1. Actuarial survival in patients with bladder carcinoma calculateclfrom the actual biopsy. Tumour~ with diploid D.,~24-stemline,t (e • •). Tumoar,~ with non-diploid DNA-stemlines (© © ©). (a) All turnouts (63 diploid~60 non-diploid) (b) Stage 1 turnouts (43 diploid/20 non-diploid) (c) Stage Ii I I I turnouts (8 diploid/18 non-diploid) and Stage IV tumours (12 diplcfid/22 nondiploid).
°°',1
Central region
t
Central regbon
d)
". 20 0
x
o
x
II o
"6
• 40 20
I
0
• te [AU]I Perip/t~ral regiorl I
•
te
Per¢W'~ral re(jlon
fAU] 40
60
C e r ~ nK~on
b)k --
te [AU]
o 20 0
l
•
x
o
•
W [AU.]
•
~[AU]
Per~
60
e)
Central recjion
I °I
Cenlral rt'gul
"6 o
• .
1
J_m__
I Per il~r~,ral region
0
Fig 2
I, x
Peript'~ml r~jon
o
•
te [ A U ]
O
~
le [AU]
DNA-histograms from 'biopsy @eeimensjion~ a central and a peripheral region in 5 bladder carcinoma~L x diploid ] @ tetraploid ~ D,Ni~t-value. • octoploid J
peripheral biopsy were used in the sampled material of 123 patients (Tables 2-5 and Fig. 1 ). In 3 patients biopsies were also taken from distant metastases (Fig. 3). A control group consisted of 32 specimens of macroscopically normal bladder mucosa. These were collected ti'om patients with benign disor-
ders of the lower urinary tract (mostly benign prostatic hyperplasia) or were taken immediately after death from patients who died without urological disorders. The age distribution in the T C C group and the control group was similar. In 20 patients the bladder carcinoma was
The Clinical and Histopathological Significance of Feulgen D NA- Values
when the actual biopsy was taken, regardless of previous surgical intervention. As it was often impossible to distinguish between stage II and stage III in previously operated patients these stages were grouped together. This stage I I - I I I was defined by the presence of a firm mobile induration of any size in the bladder wall, or of a tumour with histologically proven infiltration of the muscular layer of the bladder wall. Chest X-rays were performed in all patients. Pedal lymphography was done in 29 patients. Only patients with unquestionable evidence of disseminated disease were considered to have metastases. Table 1 shows the treatment of the patients, following the actual biopsy. Preparation, fixation, staining and mounting of the imprints were done as described previously [6]. The total extinction (le) of a Feulgen-stained cell nucleus, here defined as Feulgen DNAvalue, was measured by means of a scanning microscope photometer SMP05 (Zeiss, Oberkochen, W. Germany) [6]. A total of 100 round or elipsoid cell nuclei with reticular chromatin pattern was measured in randomly selected regions of the imprints. The mean DNA-value of 30 polymorphonuclear leukocytes in each imprint was used as a
40
~o
Bladde r t u m o u r
o
oo
40
Lyrn~ node metastasis
20
te [AU.I
Bladder tumour
_~ 20 >o 0 o 40: Subcutar~ous metastasis
20
0~uJ
,g
o
w
Z
Fig. 3. DNA-histograms .from biopsy specimens from patients with bladdercarcinomaand distant metastases. x diploid ~ etraploid~ DNA-value. octoploid .}
Table 1.
Treatmentin 123 patients with bladder carcinoma Treatment
Clinical stage
Stage I StageII III Stage IV
Radical surgery
47 2
1157
Nonradical surgery
n u m b e r of patients
Radical surgery + local chemotherapy
Nonradical surgery + external irradiation
14 5 1
17 27
General chemotherapy
Total 63 26 34
Local chemotherapy: 30 mg T h i o t e p a intravesically for 30 min after transurethral resection. External irradiation: 60 Gy/6 weeks to the pelvis by a betatron 33 mV.
General chemotherapy: 5-fluorouracil intravenously 12 mg/kg/week. verified histologically by the actual biopsy, while 103 patients were admitted with tumour recurrence after surgery in other hospitals. In these patients information was not available to determine the clinical stage of the bladder carcinoma when the initial diagnosis was made. The TNM-system, used for staging of the bladder tumour [5] in all patients, was therefore slightly adjusted in the present study: The terms, stages I - I V , were here used to describe the clinical extent of the bladder tumour at the time
reference to the actual specimen's diploid DNAvalue (internal standard) [3]. T h e frequency distribution of 100 Feulgen DNA-values in each specimen (DNAhistogram) was drawn using the following definitions: The diploid region was defined as the diploid DNA-value+_25(}/o. The tetraploid and octoploid regions were 2-fold and 4-fold the diploid DNA-value respectively,+25c~Jo. The diploid, tetraploid and octoploid regions were each
1158
S. D. Fossd, O. Kaalhus and O. ScoU-Knudsen
<
Z~
<
c~
<
r
~r.,I
q %
Z
i
7c
~
The Clinical and Histopathologkal Significance of Feulgen D N A - Values subdivided into 5 intervals, each comprising 10% of the diploid, tetraploid and octoploid figure respectively. The definition of a DNAstemline has been described elsewhere [6]. DNA-values within the DNA-stemline region were called DNA-stemline values. The mean of the DNA-stemline values yielded the modal DNA-value [4]. Following imprint preparation the tissue fragments were fixed in formalin and embedded in paraffin. Grading of the histological haematoxylineosin-stained sections was done according to Mostofi [7] denominating all epithelial bladder tumours with cellular anaplasia as carcinomas, regardless whether infiltrative growth was demonstrated or not. Statistical evaluation The actuarial survival of patients was calculated from the date of the actual biopsy using the life table method [8]. Differences between survival rates at a definite point of time were tested by the Student's t test. Differences between survival curves for different groups of patients were also tested by the logrank test [9]. The original test is not directly applicable for data grouped in time. The scheme was therefore modified making all possible rankings within each group equally probable when evaluating the estimator (e) for the logarithm of the survival curve. The numbers for death expectancy Ej obtained by cumulating the e-values for the j'th group of patients, were compared to the number of deaths 0j by the Z2 test. RESULTS
In each of the control specimens a diploid DNA-stemline was found. The control group comprised, on the average, 2.4°/'o tetraploid and 0.1°/J.o octoploid DNA-values. In 6 individual control specimens a frequency of 8 - 1 0 % polyploid DNA-values was found. Table 2 shows the distribution of DNAstemlines in relation to histological grade and Table 4.
1159
clinical stage. Sixty-three specimens showed a diploid DNA-stemline, while in 60 the stemline was non-diploid. O f the latter, 35 were tetraploid and 6 showed bimodality (diploid and tetraploid DNA-stemline). The relative frequency of non-diploid tumours increased with decreasing histological differentiation and increasing clinical stage. In Table 3 the mean percentage of DNAstemline values is shown for diploid and nondiploid tumours. Within the same histological grade the number of DNA-stemline values tended to decrease with increasing clinical stage, most clearly demonstrated when comparing stage I with more advanced tumours. Especially in diploid tumours increasing clinical stage and decreasing histological differentiation were correlated with low numbers of DNA-stemline values. Non-diploid tumours showed no such clear correlation, but the DNA-stemlines in nondiploid tumours generally comprised less DNAstemline values than observed in diploid tumours. Table 4 shows the relationship between the DNA-stemline ploidy and the number of patients with obvious metastases found either at the time of the actual biopsy or during follow-up. Metastases were twice as often associated with non-diploid tumours as with diploid carcinomas. A particularly high frequency of metastases was noted in non-diploid stage IV tumours. The actuarial survival of patients with diploid T C C was compared to that of patients with nondiploid tumours (Fig. l a). After 24 months more patients with diploid tumours than with non-diploid tumours were alive (P<0.05). The logrank test showed a statistically significant difference between the survival curves. One obvious reason for this is that 43 of 63 diploid carcinomas were at stage I. Therefore, in Fig. l(b) and l(c) the survival curves were given separately for patients with stage I disease and with advanced stages (stages I I - I I I and stage IV). Patients with advanced diploid tumours had a higher cumulative survival rate at 18 months than those with non-diploid tumours
Correlation between clinical stage and tumour's DNA-stemline ploidy in 27 patients with metastasizing bladder carcinoma
Clinical stage
Stage I
Stage II-III
Stage IV
Total
Diploid tumours
Number of patients
3 (43)
2(8)
4(12)
9 (63)
Non-diploid tumours
Number of patients
3 (20)
2 (18)
13(22)
18(60)
Figures in brackets refer to the total number of patients within each subgroup.
1160
S. D. Fossd, O. Kaalhus and O. Scott-Knud~en
Table 5. Number oJ patients with poorly dijfcrentiated and undifferentiated locally advanced bladder carcinoma surviving 18 months related to the lumour's D,,~'3t-stemline ploidy
Number of patients DNA-stcmline ploidy Diploid Non-diploid Total
Total
Alive
Dead
10 30 40
6 I0 16
4 2(I 24
(P-~ 0.10). After 24 months the survival rate was the same for patients with diploid and nondiploid advanced bladder tumours. No significant difference was observed in the survival regarding patients with stage I disease. Table 5 shows the number of patients with poorly differentiated and undifferentiated locally advanced bladder carcinoma (grade II! and grade 1V, stages I I - I I I and stage IV) surviving 18 months. Patients with diploid turnouts seem to do better than those with nondiploid tumours. In 3 of the 5 patients with two biopsy specimens from the same tumour, different modal DNA-values could be demonstrated in the central and peripheral tumour area, de, monstrated both in stage I tumours (Figs. 2c and 2d) and in a stages II I l l turnout (Fig. 2e). Comparison of the DNA-histogram from a bladder tumour with that of a distant metastasis was made in each of 3 patients (Fig. 3). The metastases in these patients had non-diploid DNA-stemlines, even in the patient with a diploid bladder tumour (Fig. 3a). DISCUSSION We have based our control group on specimens taken from patients with benign urological disease or from deceased individuals without urological disease. As the bladder mucosa often shows inflammatory reactions in patients with urological disease it was necessary to accept the presence of a slight degree of inflammation in some of the first-mentioned controls. We found a low mean percentage (2.4~)'o) of tetraploid DNA-values in our control group while some individual control specimens showed between 8 and 10(~ polyploid cell nuclei. Levi et al. [ l 0] found about 25 o~ polyploid cell nuclei in human urothelium. Tribukait et al.I11 ] a n d F r e n i et al. [12] examined bladder washings and urine from patients with benign urothelial disorders by tlow-cytometry. They found an almost pure diploid DNA-stemline with only few
polyploid DNA-values. Freni et al. [12] observed about 1000 non-diploid DNA-values in urothelial cells obtained by scraping normal bladder mucosa. Some of the discrepancies found in the frequency of polyploid cells in the bladder mucosa in different studies may be explained by different techniques in obtaining the specimens and by different methods in selection of cell nuclei to be measured. Based on our own studies and Freni's et al. [ 12] observations we assume that normal urothelium does not contain more than 15'~o non-diploid DNA-values, most of" them in the tetraploid range. Sixty-three bladder carcinomas were characterized as diploid and 60 as non-diploid. These results were based on DNA-measurements in a single tumour biopsy. However, intratumoural variations of the DNA-stemline ploidy may occur (Fig. 2). This limitation of the method, used in this study, should be regarded when evaluating the present results. In this study the cytophotometric results were correlated with clinical stage and histological grade of the bladder tumour, though the clinical stage according to the TNM-system [5] does not always describe the real extent of the disease [13[. However, no other clinical classification system is available which can easily be used in all patients with bladder carcinoma. The preponderance of non-diploid tumours found in clinically advanced and histologically dedifl'erentiated carcinomas is in agreement with the results of others [10, 14-171. We tbund, however, that 5 out of 10 undifferentiated bladder turnouts had diploid DNA-stemlines. Contrary to what is observed in well dittErentiated tumours, these undifferentiated diploid bladder carcinomas had "weak" DNA-stemlines with low"numbers of DNA-stemline values. The number of DNA-values outside the DNAstemline increased with decreasing histological differentiation. In deditt'erentiated bladder tumours an increased uptake of 3H-thymidine has been observed as compared to well ditt~rentiated carcinomas [18, 19]. The raised numbers of non-DNA-stemline values may therefore indicate an increased growth fi'action in dedifferentiated tumours as compared to well differentiated ones. In addition, the increased frequency of non-DNA-stemline values in dedifferentiated bladder carcinomas may also reflect that such tumours have lost the relative homogeneity of the normal urothelium and that aneuploid and polyploid cell clones are developing as a result of chromosomal changes. In the 3 patients with metastasizing bladder
The Clinical and Histopathological Significance of Feulgen D NA- Values carcinoma a weak DNA-stemline and numerous non-diploid cells were observed in the primary tumour. Non-diploid tumour cells may have selective advantages leading to the predominance of these cells in metastases, even in those from diploid bladder tumours. The observation that DNA-stemlines in metastases may show a higher degree of ploidization than those in the correspondin~ primary tumours, has also been made by Avtandilov et al. [20] and Rabotti {21]. From the literature, only limited information is available about the prognostic significance of cytophotometric DNA-measurements in human bladder carcinoma [22]. In the present study the survival of patients was calculated from the date when the actual biopsy was taken, as only at this time comparable results of the clinical, histological and cytophotometric examination could be obtained, In our series of 123 patients those with diploid carcinomas had a higher survival rate than those with non-diploid tumours (Fig. la). However, the prognostic significance of cytophotometric DNA-studies can only be proven when comparing patients with the same clinical stage and with the same histological grade. In this study the prognostic value of DNA-measurements was indicated by the observation that patients with diploid locally advanced dedifferentiated tumours had a better prognosis than those with nondiploid carcinomas. Clinically, non-diploid bladder carcinomas seemed to be more aggressive than diploid
tumours, by more extensive local growth, by metastasizing more frequently and by leading to the patient's death within a shorter period of time. Another possible explanation of the observed differences between the survival curves might be that diploid bladder carcinomas corresponded differently to treatment than non-diploid ones. O f 16 patients with advanced diploid bladder tumours treated by non-radical surgery and radiotherapy, 6 were without tumour after 18 months whereas only 4 of 26 likewisely treated patients with non-diploid carcinomas were tumour-free. Whatever the explanation may be for the better survival of patients with diploid bladder carcinomas the present study indicates that cytophotometric DNA-measurements have clinical and histopathological significance which may be valuable in determining the prognosis in patients with TCC. Cytophotometric DNA-studies in imprints from solid tumours are time-consuming. Only a limited number of cell nuclei can be evaluated in each specimen. More rapid DNA-determination by pulse-cytophotometry may therefore offer some advantages, but this method's role in studies of human bladder carcinoma needs further investigation [ 11, 12].
Acknowledgements--The valuable assistanceof Ashton Miller, M.D., F.R.C.S., during this work is gratefully acknowledged. The authors thank Per F. Marton, M.D., for help with evaluation of the histopathologicalsections and for valuable criticismof the manuscript.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9.
1161
A. BOXVIN, R. VENDRELY and C. VENDRELY, L'acide d6soxiribonucldique du noyau cellulaire, ddpositaire des caract+res h6rdditaires; arguments d'ordre analytique. C.R. Acad. Sci. (Paris) 226, 1061 (1948). C. LEUCHTENBERGER,R. LEUCHTENBERGERand A. M. DAVIS,A microspectrophotometric study of the desoxyribose nucleic acid (DNA) content in cells of normal and malignant human tissues. Amer. J. Path. 30, 65 (1954). N. B6H~ and W. SANDRITTER,DNA in human tumors: a cytophotometric study. Curr. Top. Path. 60, 151 (1975). N.B. ATKIN,B. M. RICHARDSand A.J. Ross, The deoxyribonucleic acid content of carcinoma of the uterus: An assessment of its possible significance in relation to histopathology and clinical course, based on data from 165 cases. Brit. J. Cancer13, 773 (1959). UIIC (International Union Against Cancer), T N M Classification of Malignant Tumours. 2nd edn., p. 79. International Union Against Cancer, Geneva (1974). S.D. Foss2x, Feulgen DNA-values in transitional cell carcinoma of the human urinary b]~dder. Beitr. Path. 155, 44 (1975). F.K. MOSTOFI,Pathology of cancer of bladder. Acta Un. int. Cancr. 18, 611 (1962). S.J. CUTLERand F. EDERER, Maximum utilization of the life table method in analyzing survival. J. chron. Dis. 8, 699 (1958). R. PETO and J. PETO, Asymptotically efficient rank invariant test procedures. J. roy. statist. Soc. A 135 (Part 2), 185 (1972).
1162
S. D. Fossd, O. Kaalhus and O. Scott-Knu&en 10.
l l.
12.
13. 14. 15. 16. 17.
l g. 19.
90.
21. 22.
P . E . LEVI, E. H. COOPER, C. K. ANDERSON,M. C. PATH and R. E. WILLIAMS, Analyses of DNA content, nuclear size and cell proliferation of transitional cell carcinoma in man. Cancer (Philad.)23, 1074 (1969). B. TRIBUKAIT,G. MOBERGER and A. ZETTERBERG, Methodological aspects of rapid-flow cytofluorometry for DNA analysis of human urinary" bladder cells. In 1st Int. Syrup. Pulse-Cytophotometry (Part I) (Edited by" C. A. M. HAANEN,H. F. P. HILLEN andJ. M. C. WESSELS)p. 50. European Press Medicon, Ghent (1975). S.C. FRENI,O. REIJNDERS-WARNERand H . J . DE VOOGT, M. E. BEYER-BooN and J. A. M. BRUSSEE, Flow fluorophotometry on urinary cells compared with conventional cytology. I n 1st Int. Syrup. Pulse-Cytopholometry (Part I I I ) (Edited by" C. A. M. HAANEN,H. F. P. HILLEN andJ. M. C. WESSELS)p. 194. European Press Medicon, Ghent (1975 ). G . M . KENNY, G . J . HARDNER and G. P. MURPHY, Clinical staging of bladder tumors. J. Urol. 104, 720 (1970). D. LAMB, Correlation of chromosome counts with histological appearances and prognosis in transitional-cell carcinoma of bladder. Brit. reed. J. 1, 273 (1967). M . E . SPOONER and E. H. COOPER, Chromosome constituents of transitional cell carcinoma of the urinary bladder. Cancer (Philad.) 29, 1401 (1972). S. SHIOEMATSU,Significance of the chromosome in vesical cancer. In Int. Soc. Urol. 13th Congr., London 1964, Vol. 2, p. 111. E. & E. Livingstone, London (1965). B. LEDERER,G. MiKuz, W. Gi~'TTERand G. ZUR NEDDEN, Zytophotometrische Untersuchungen yon Tumoren des l~bergangsepithels der Harnblase. Vergleich zytophotometrischer Untersuchungsergebnisse mit dem histologischen Grading. Beitr. Path. 147, 370 (1972). H . H . SKY-PECK, Effects of chemotherapy on the incorporation of 3H-thymidine into DNA of human neoplastic tissue. Nat. CancerInst. Monogr. 34, 197 (1971). B. HAINAU and P. DOMBERNOWSKY,Histology and cell proliferation in human bladder tumors. An autoradiographic study. Cancer (Philad.) :33, 115 (1974). G.G. AVTANDILOVand L. V. CHERVONNAYA,Comparative microspectrophotometric investigation of the DNA content in cells of primary foci and metastases of malignant melanoma. Bull. exp. Biol. Med. 75, 432 (1973). G. RABOTTI, Ploidy of primary and metastatic human tumours. Nature (Lond.) 183, t276 (1959). E . H . COOPER, Biology of human bladder cancer. In The Biology and Clinical .14anagemenl qfBladder Cancer. (Edited by' E. H. COOPER and R. E. W~LHAMS)p. 50. Blackwell Scientific Publications, Oxford-London--Edinburgh-Melbourne (1975).