The prognostic significance of selective nuclear morphometry in urinary bladder carcinoma

The Prognostic Significance of Selective Nuclear Morphometry in Urinary Bladder Carcinoma C. E. M. BLOMJOUS, MD, PHD, W. VOS, MD, N. W. SCHIPPER, MSc, PHD, A. M, UYTERLINDE, MD, PHD, J, P. A. B&X, MD, H. J. DEVOOGT, MD, PtiD, AND C. J. L. M. MEIJER, MD, PHD Transurethral primary

tumor

specimens

of 61 patients with a

and1untreated bladder

resected

carcinoma

were studied by selec-

tive nuclear morphometry, significant

enlargement

a method recently described

by us. A

of the mean nuclear

area was found with the advance of tumor grade and stage (Wilcoxon, P < .OOOl and P < .OOOl). The heterogeneity of the grade 2 patient group and the additional

value of morphomerry

were demonstrated

by ob-

serving the 5-year survival rates. Patients with grade 2 carcinoma could be separated

into one subgroup

with small nuclei

nuclear area s 95 pm*) having a favorable vival rate: ~lOO’%),and into another (mean nuclear area :> 95 pm’) was not significantly

1 (5-year survival

subgroup

with large nuclei

showing a worse prognosis

(5-year

P = .Ol). The outcome of these

survival rate: 63.2’S! (Mantel-Cox, subgroups

(mean

outcome (5-year sur-

different

from that of the grade

rate: 93.8%) and grade 3 (5-year survival

50%) patients, respectively

rate:

P = .45 and P = .57).

(Mantel-Cox,

The value of selective nuclear morphometry,

in addition to tumor

staging, wa!s indicated by rhe association of nuclear enlargement (mean nuclear area > 95 pm’)

with progressive

of

the patients

15 patients;

33%,) among

treated superficial demonstrate

carcinoma

tsotumor grading

ogeneous

group

ficial

tumors

(five

conservatively

value of selective nuclear

and staging, especially

of grade 2 carcinomas

(stages Ta and Tl).

1990 by W.B. Saunders

recurrence

(stages Ta and Tl). The findings

the supplementary

phomerry

with

HUM

and the group

PATHOL

mor-

in the heterof super-

21:409-413.

CC

Company.

~l‘umor g,rade and stage are the most important parameters for the therapeutic management and prognosis of transitional cell carcinoma of. the urinar) bladder.‘,’ The value of histologic grading is. however, impaired by the inconsistency of pathologists’ observations. ’ Moreover. grade 2 carcinomas (World Health Organization [WHO] classification) represent a large and hetero,geneous group in which both noninvasive and invasive tumors occur and in which parient sur~r.ival rate varies widely.“.” ‘I‘umor staging is a considerable proalso prone to error.’ .4dditionally, portion r)f the patients with superficial carcinoma (stages Ta and ‘1‘1)will develop recurrent disease after the usua’l conservative treatment by transurethral resection. and 15% to 2.54 will ultimatelv progress to

MATERIALS AND METHODS

Patientsand Materials Fu)m21series of 121 consecutive patients who had IIIIdergone transurethral resection of a primal-~ and untreated transitional cell carcinoma of the urinary bladder between September 1974 and August 1979 in our hospital, 61 were included in this study. Conditions were that: cauterization artifacts of the tumor specimens were not abundant, the tissue had been fixed in 3% neutral buffered formalin, and archival material was still available. ‘There were no differences in age, sex, and tumor stage or grade between the original and selected group. .I‘he quantity of transurethrallv resrctrd material was

the Departments of Pathology and Prolog\, Free Uni\ersit! Hwpital. Amsterdam, The Netherlands; and the DepxtInent of Pathology. Stichting Samenwerking Delftse Ziekenhuizen. Delft. The iKetherlands. Accepted f’or publication August 24. 1989. Kry rtrr,ni.~: IIU&YII- n~orphometry. urinar\ bladtle~- tarcintrtna. From

pr~~gn&s. S-year survival. Addrrs\ ~orresponclenre

requests to C. E. M. Frw L’nivtrsity Hospi1a1,De Botrlelaarl I 1 17, 1007 MB .4mstcrtlam. The Netherlands. ‘C IWI bv W.13. Saunders (Iompany. Blon~~jous. hll).

Department

high-grade carcinoma (grade 3) or muscle-invasive tumor.“.’ Most of these recurrences actually represent new tumors derived from field changes of the mucosa adjacent to the primary tumor. In order to find new objective criteria to predict the clinical outcome, efforts have been made to identify supplementary features such as cytogenetic pattel’ns, flow-cytometric DNA ploidy status, immunohistochemical characteristics (expression of ABO( H) isoantigens, ‘T antigen or carcinoembryonic antigen), and ultrastructural features of the cell membrane.x-‘3 ‘To date, only DNA flow cytometry has proven to be both a practicable method to predict tumor behavior, and suitable for routine clinical use. In recent years, several studies have demonstrated that morphometry provides a valuable and easy method of investigation of urinary bladder carcinoma.“*-‘5 Although these studies differed in the morphometric methods used, they unequivocally emphasized the usefulness of morphometry as an objective alternative for histologic grading. However, no further attempts have been made to evaluate the im1ydct on tumor recurrence and patient survival rates. In view of the methodological differences in the literature, t-1-17 we have recentlv undertaken a comparative study of a variety of ‘morphometric methods.‘# The measurement of the nuclear areas of selectively sampled nuclei emerged from that study as the most suitable method for tumor grading. In this study, the prognostic importance of selective nuclear morphometry is demonstrated. In particular. attention is paid to the correlation with survival among patients with grade 2 carcinoma and the development of progressive recurrence in superficial carcinomas (stages Ta and ‘1‘1).

and reprint

of Pathology.

11046.81 Tii!IOi:! 10~-000ti$5.~~~1/0

409

HUMANPATHOLOGY

Volume 21, No. 4 (April 1990)

dependent on the tumor size and varied from 0.5 to 10 cm’. Accordingly, the number of tissue blocks of each specimen ranged from one (42 cases; 69%) to five. For histologic and morphometric investigation 4-pm thick sections were routinely stained with hematoxylin and eosin. The slides were examined independently by two pathologists (C.E.M.B. and W.V.) and the histologic grade was assessed according to the WHO classification. ” Thus, together with the tumor grade arrived at by routine diagnostic procedures, three observations of each specimen resulted. Complete agreement between these observations was obtained in 75% of the cases. In the remaining 25%, no differences of more than one grade occurred, and in these cases the prevailing grade was assessed. Tumors also had been staged by the TNM classification (Table l).“’

erators, including pathologists of. different laboratories, were compared. In all these cases the correlation coefficients of the mean nuclear area and the standard deviation exceeded 0.90, and the regression coefficients were between 0.75 and 1.25. Table 2 demonstrates the reproducibility of selective morphometry by the same pathologist (A 1 and A2), as well as in the hands of various operators from different laboratories (A and B from Free University Hospital, Amsterdam, The Netherlands: C from Stichting Samenwerking Delftse Ziekenhuizen, Delft. The Netherlands).

Statistical Analysis The significance of the mean and standard deviations of the nuclear area was determined by Wilcoxon’s test. Survival (Kaplan-Meier) curves were analyzed, using the Mantel-Cox statistics. This analysis takes into account the length of time at risk from transurethral resection of the primary tumor to either death by tumor or date of last follow-up. The x2 test was used to evaluate the relation between the nuclear area and progressive recurrence. As a level of signiflcance, P = .05 was taken.

Clinical Features Clinical data were available from 58 patients; three patients were unavailable for follow-up. Forty-four patients were male and 14 were female. Age ranged from 46 to 88 years, mean 68.9 years. All patients with superficial carcinoma (stages Ta and Tl) had been initially treated by transurethral resection. Patients with advanced disease (stages T2 to T4; n = 21) received different methods of therapy, depending on general health condition and likelihood of cure. They were either treated by local radium implantation (three), radiation pretreatment followed by radical cystectomy (seven), or conservative radiotherapy (nine), or they remained untreated (two).

1

RESULTS

increased significantly with the advance of tumor grade (see Table 3). The prognostic significance of nuclear morphometry, in addition to histologic grading, is demonstrated in Fig 2 (n = 58; three patients were unavailable for follow-up). The 5-year survival rates of the patient groups with grades 1, 2. and 3 carcinoma were 93.8%,, 78% and 50%, respectively (Mantel-Cox, P = .Ol; Fig 2A). Subdivision of’ the grade 2 patient group based on the measurement of the mean nuclear area revealed a subgroup with small nuclei (mean nuclear area s 95 km-) that showed a favorable outcome (5-year survival rate: lOO%), approaching the grade 1 patient group, and another subgroup (mean nuclear area > 95 km’) with a poorer prognosis (5-year survival rate: 63.2%) (Mantel-Cox, P = .Ol; Fig 2B). There were no significant differences between the 5-year survival rates of both these subgroups and the grades 1 and 3 patient groups, respectively (Mantel-Cox, P = .45 and P = .57). Table 4 shows the mean and standard deviations of the nuclear area in various tumor stages. Among the superficial tumors (stages Ta and Tl) an increased presence of stromal invasion (stage Tl) corresponded with a significant increase of both values (Wilcoxon, P

Morphometric analysis was done with a projection microscope equipped with a 100X oil immersion objective, projecting at a graphic tablet (MOP-Videoplan, Kontron, Munich, FRG) with an optical magnification of 2000X. If more than one section was available (19 cases; Yl%), one representative slide of the tumor was chosen for morphometry by the two pathologists. The nuclear areas of selectively sampled nuclei were measured by a previously described morphometric method. I* Briefly, according to this method, the most atypical histologic area (size: approximately 5 lowpower fields, 10x) of the slide is selected by the operator. using criteria of histologic and cytologic abnormality as described in detail by Koss.” From this area 10 distinctly abnormal nuclei are selected because of their large size. Their nuclear areas are subsequently measured by tracing the nuclei with a graphic pencil. Our previous methodological study has shown that this selective procedure was most useful to discriminate between different tumor gradesIx The method resulted in a considerable score of 93.2% correctly classified cases from a learning set of bladder carcinomas. The reproducibility of this method was evidenced by comparing the results of repeated selection and measurement of nuclei by the same pathologist (test set: 12 bladder carcinomas of various grades). Also, the results of various op-

TABLE 2. Correlation and Regression Coefficients Between the Morphometrically Assessed Mean Nuclear Areas BY Three Different Operators (A B and C)

TABLE 1. Distribution of Tumour Grade and Stage Stage T1

T2

T3

Grade 1 Grade 2 Grade 3

14 7 0

2

0

12 2

7 5

Total

21

16

12

distribution of the mean nuclear area and grade is shown in Fig 1. The mean values

The histologic

Morphometric Analysis

Ta

v

T4

Total

0

0

I 1

.5 2

16 32

2

7

(:C and R(:

10

CC and

Al

vs A2:

0.99

0.w

Al

vs C:

0.97

0.90

A2

vs 8:

0.94

I .lJ7

A:! \‘s B:

(I.94

I.14

B YS C::

Note:

Al

and

Abbreviation,:

410

R(:

Al

by pathologist

58

CC: and

A2 represent

the first

and

sccvnd

vs <::

series

0.97

0.97

0.94

0.77

of na~surements

A. (I:.

WITelatton

cuefticient.

RC:

KC:. regression

coefficient.

MORPHOMmlY IN URINARY BLADDER CARCINOMA [Blomjous et al)

275

mar grade in bladder carcinoma. 14-’’ The results of the present investigation demonstrate that morphometry also provides prognostic information in addition to that obtained by grading and staging, a finding which, to our knowledge, has not been reported before. ‘The morphometric method used in the current study necessarily carries the risk of subjectivity. as selection of atypical histologic areas and of large nuclei are essential steps in the procedure. It has however been shown in a previous methodological study done by our group that selection combined with quantitative analysis results in satisfactory reproducibility.lX Similar results have also been reported. in morphometric studies done by pathologists frotn different laboratories. Selective morphometric analysis is apparently easily reproducible if the operator is welltrained and the guidelines are carefully followed. Increased nuclear areas in advanced tumor grade is in keeping with various classification systems bv which nuclear enlargement is designated as a ma,jt;r criterion in the grading of’ transitional cell carcitlotna.L).~.‘!’ The nuclear enlargement in advanced malignancy corresponds with abnormal nuclear DNA content found by cytogenetic and flowcytometric analyses. y’ The correlation between nuclear size and DNA content has, moreover, also been evidenced by combined karyometric and DNA photocytometric studies of bladder carcinonla.‘4~“2 The values of the mean nuclear area found in this study exceeded those of previous reports.‘“-” This can be explained by the different sampling technique, as the measurement of selected large and abnormal nuclei inevitably results in larger values than the measurement of random nuclei practiced in many studies. Although our values constituted a continuous spectrum, there was little overlap between tumors of different histologic grade. This contrasts with other findings in the literature. 14-17 Morphometric discrimination is apparently improved by the selective manner of sampling of the present study. IH The importance of morphometry in addition to histologic grading was shown by the varying prognosis of patients with grade 2 carcinoma. These patients could be separated into a subgroup with relatively small nuclei (mean nuclear area s 95 pm’), having a 5-year survival rate (100%) similar to that of the patients with grade 1 carcinoma (5-year survival rate: 93.8’%), and into a subgroup (mean nuclear area > 95 pm’) in which the 5-year survival rate (63.2%) approached that of the grade 3 patients (5-year survival rate: 50%). Although perhaps influenced by varying therapy regimens, this finding still reflects the fact

a

T

/ 725 I

!

175

Mean Vurlear

i

T

I

/

i

2

*

FIGURE 1. Mribution of the mean nuclear area and histologic grade. Some overlap isshown between tumors of various grades. (Grade 1, n = 16, grade 2, n = 34; grade 3, n = 11.) = .005). Similarly, the mean values of the advanced lesions (stages -1‘2 to T3) were significantly larger than those of the superficial tumors (stages Ta and ‘1‘1; Wilcoxon, P < ,000 1). The differences between the advanced stages (stages T2, T3. and T4) were not significanlt. The additional value of nuclear morphometry to tumor staging was demonstrated, especially in patients with superficial carcinoma (stages Ta and Tl). Of the 37 patients with superficial tumors, seven developed progressive recurrent disease within an intcrval of 6 to 120 months (median: 33 months) after transurethral resection of the primary lesion. ‘Two of them had progression from low-grade (grade 1 or 2) to high-grade (grade 3) carcinoma without obvious muscle invasion, but in one case accompanied by flat carcinoma in situ. One patient underwent cystectomy because of‘ multiple recurrences and, although no deep invasion had been demonstrated. subsequently developed abdominal metastases. Four patients had muscle-invasive recurrent tumor. Large nuclei appeared to be predictive for progressive recurrence. Of 22 paltients with relatively small nuclei (mean nuclear area 5 95 km”) only two (9%) had progression, Lqhereas of’ the other 15 patients who had large nuclei (mean nuclear area > 95 pm’) five (33%) had pro-

gressive

wcllrrent

disease

(x2, P = .06).

DlSCUSSllON Prior- studies ( lear morphomrtry TABLE

have shown the usefulness in the objective assessment

of nuof tu-

TABLE

3. Mean and Standard Deviation of the Nuclear Area in Grade 1, 2 and 3 Carcinomas (km’,

411

4.

Mean and Standard Area in Various Stages

Deviation

of the Nuclear

of Disease [pm’,

Volume 21, No. 4 [April 1990)

HUMAN PATHOLOGY

(2a)

Ii)

(2bl

(31

0

10

20

3a

40

SO

60

0

10

20

30

40

50

I 60

months

months

FIGURE 2. Kaplan-Meier survival curves for patients with various tumour grades. (Left) (1) Grade 1, n = 16; (2) grade 2, n = 32; and (3) grade 3, n = IO, (Right) The grade 2 patient group is separated into different prognostic subgroups based on the measurement of the mean nuclear area (MNA). [I) Grade 1 (n = 16); (2a) grade 2 (MNA s 95 prn2) (n = 9); (2b) grade 2 (MNA 2 95 Frn2] (n = 23); and (3) grade 3 [n = 10).

analysis of tumor nuclei in urinary bladder carcinoma provides additional prognostic information to histologic grading, especially in the heterogeneous group of grade 2 carcinomas. Moreover, the finding of large nuclei (mean nuclear area > 95 km2) in the initial superficial tumor (stages Ta and Tl) suggests that a more aggressive treatment is warranted, in view of the increased risk of progressive recurrent disease.

that grade 2 carcinomas comprise a broad spectrum of tumors, some resembling grade 1 and some grade 3. It may be argued moreover that the discrimination of only two, instead of three, grading classes using morphometry would be more profitable (see Fig 2, right). From a clinical point of view, such a simplified classification would possibly facilitate a more effective therapeutic approach. Nuclear enlargement as a hallmark of aggressive tumor behavior is also apparent from its association with advanced tumor stage. The advanced tumors (stages T2 to T4) showed larger nuclei than the superficial lesions (stages Ta and Tl). In addition, the superficial tumors with superficial invasion (stage Tl) had significantly larger nuclei than those without (stage Ta). Although this might suggest a more aggressive potential for the stage Tl lesions, local treatment by transurethral resection is commonly sufficient for both stage Ta and Tl carcinoma. None of our 37 patients with superficial carcinoma had residual tumor. Despite complete resection of the primary tumor, up to 25% of the patients with superficial carcinoma nevertheless will suffer progressive recurrence (actually new occurrence).“.7 This occurred in seven of our 37 patients. Progression was presaged by the finding of large nuclei (mean nuclear area > 95 pm2) in the initial tumor, although this tendency was not significant (P = .06), possibly due to the small number of cases. Morphometry of the primary tumor apparently predicts the propensity of the adjacent mucosa to give rise to progressive recurrence. A similar finding has been reported by flow-cytometric studies that showed the prediction of progressive recurrence by DNA aneuploidy of the primary tumor.23 Future studies on larger numbers of patients are required to clarify the prognostic specificity of morphometry, and especially its value in comparison with DNA flow cytometry. It is our conclusion that selective morphometric

REFERENCES 1. Kern WH: The grade and pathological stage of bladder cancer. Cancer 53:1184-l 189, 1984 2. Koss LG: Tumors of the urinary bladder. In Atlas of Tumor Pathology, series 2, fascicle 11, Washington DC, Armed Forces Institute of Pathology, 1975, pp 16.I7 3. Ooms ECM, Anderson WAD, Alons CL, et al: Analysis of the performance of pathologists in the grading of bladder tumors. HUM PATHOL 14:140-143, 1983 4. Bergkvist A, Ljungkvist A. Moberger G: Classification of bladder tumours based on the cellular pattern: Preliminary report of a clinical-pathological study of 300 cases with a minimum followup of 8 years. Acta Chir &and 130:371-378, 1965 5. Schmidt JD, Weinstein SH: Pitfalls in clinical staging of bladder tumors. Urol Clin North Am 3: 107-127, 1976 6. Melicow MM: Tumors of the urinary bladder: A clinicopathological analysis of over 2,500 specimens and biopsies. J Ural 74:498-503, 1955 7. Barnes R, Hadley H, Dick A, et al: Changes in grade and stage of recurrent bladder turnours. J Ural 118:177-178, 1977 8. Spooner ME, Cooper EH: Chromosome constitution of transitional cell carcinoma of the urinary bladder. Cancer 29: 14011412, 1972 9. Gustafson H, Tribukait cinoma by flow DNA analysis.

B: Characterisation of bladder Eur Urol 11:410-417, 1985

car-

10. Decenzo JM, Howard P, Irish CE: Antigenetic deletion and prognosis of patients with stage A transitional cell bladder carcinoma. J Urol 114:874-878, 1975 11. Coon JS, Weinstein RS, Summers JL: Blood group precursor T-antigen expression in human urinary bladder carcinoma. Am J Clin Pathol 77:692-699, 1982 12. Wiley EL, Mendelsohn G, Droller M. et al: Immunoperoxidase detection of carcinoembryonic antigen and blood group

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MORPHOMETRY IN URINARY BLADDER CARCINOMA (Blomjous et al)

substances in papillary transitional cell carcinoma of the bladder. J Urol 128:?76-280. 1982 13. Pauli BU. Cohen SM, Alroy 1, et al: Desmosome ultrastructure an’d the biological behavior of chemical carcinogen induced urinal-v bladder carcinoma. Cancer Res 38:3276-3285. 1978 14. De Sanctis PN, Tannenbaum M, Tannenbaum S,‘et al: Morphologic quantitation of nuclear size in various grades of transitional cell carcinoma of urinary bladder. Urology 20: 196-199. 1982 15. Bjelkenkrantz K, Herder A, Grontoft 0, et al: Cytophotometric characterization of the WHO grades of transitional cell neoplasms. Pathol Res Pratt 174:68-77. i982 16. Ooms ECM. Kurver PHI. Veldhuizen RW. et al: Moruhometric grading of. bladder tumors in comparison with histologic grading by pathologists. HUM PATHOL 14:144-150, 1983 17. Helander K. Hofer PA, Holmberg G: Karyometric investigations on urinary bladder carcinoma, correlated to histopathological grading. Virchow Arch [A] 403:117-125, 1984 18. Blomjous CEM. Smeulders AWM, Vos W, et al: A comparative study in morphometric grading of transitional cell carci_I

noma of the urinary bladder. Anal Quant Cytol Histol, 1990 (in press) 19. Mostofi FK, Sobin LH, Torloni H: Histologic typing of urinarv bladder tumours. In: International Histological Classification of Tumours, no. 10. Geneva, Switzerland, Woyld Health Organization, 1973, pp 16-17 20. Harmer MH: TNM classification of malignant tumours (ed 3). Geneva, Switzerland. International Union Against Cancer, 1978 21. Wijkstroem H, Granberg-Oehman I. Tribukait B: Chromosomal and DNA patterns in transitional cell bladder carcinoma. A comparative cytogenetic and flow cytofluorometric DNA study. Cancer 53:1718-1723, 1984 22. Helander KG, Kirkhu B, Iversen OH, et al: Studies on urinary bladder carcinoma by morphometry. flow- cytometry and light microscopic grading with special reference to grade II tumours. Virchow Arch [A] 408: 117, 1985 23. Gustafson H, Tribukait B. Esposti PL: DNA profile and tumour progression in patients with superficial bladder tumours. Urol Res 10:13-18. 1982

413