British Journal of Urulugy (1992), 70,628-633
01992 British Journal of Urology
Prognostic Correlation of Morphometric Values with Survival in lnvasive Transitional Cell Carcinoma of the Bladder J. A. PORTILLO, J. F. VAL-BERNAL, M. F. GARIJO, L. BUELTA and J. L. GUTIERREZ Urology Service and Department of Pathological Anatomy, University Hospital “lMarquBsde Valdecilla‘: Santander, Spain
Summary-A morphometric study was made of 95 invasive bladder tumours, differentiating 34 papillary and 61 solid carcinomas. In the invasive papillary tumours, the deeper the zone of the tumour measured, the higher the value of the mean nuclear area. A higher histological grade was also seen to correspond to a higher mean nuclear area value except for Grade IV tumours, whose nuclear area was no larger than that for Grade I l l tumours. This led u s to separate Grade IV tumours from transitional cell carcinomas and classify them as undifferentiated. A better prognosis was 0 in category T2. For T3 and T4A found for those tumours whose mean nuclear area was ~ 3 pm2 tumours no conclusive results were obtained in this respect.
Since Ooms et al. (1983) and Baak et al. (1982) first made their morphometric studies of neoplastic cells, many authors have applied this technique to many different types of tumour. It has been used to study not only tumours of the urinary tract (De Sanctis et al., 1982; Ooms et al., 1983; Montironi et al., 1984; Seldinrijk et al., 1987; Nafe and Frohreberg, 1989) but also other types of neoplasm (Baak et al., 1982; Carter et al., 1989; Payne et al., 1989; Snowden et al., 1989; Lipponen et al., 1990). Morphometry entails measuring a set of neoplastic cell parameters, particularly those related to the nucleus : area, perimeter, diameter, size and shape factor. Several studies have demonstrated that nuclear area, perimeter and size show higher mean values according to higher pre-assigned histological grades (Montironi et al., 1986; Van der Poel et al., 1988). It appears to be especially important in differentiating Grade I11 from Grades I and 11. In this retrospective study, morphometry was performed on the tumour nucleus of a series of invasive transitional cell carcinomas of the bladder Accepted for publication 3 October 1991
in an attempt to correlate the results obtained both with the survival of the patients and with histological grade.
Patients and Methods From the bladder tumours examined between January 1973 and December 1983, 95 invasive carcinomas were selected. Conditions were that evolution over a period of at least 5 years was known or that death due to the neoplasia had occurred earlier. All patients had undergone radical surgery without adjuvant chemotherapy or radiotherapy. The classification of Bergkvist et al. (1965) was used for histological grading and the series was thus divided into 34 papillary and 61 solid carcinomas. Using a semi-automatic MOP-VIDEOPLAN image analyser (Kontron, Germany), which consists of a central computer, an image processor and interactive system, together with an OLYMPUS BH2 light microscope and a camera lucida, measurements were made at a magnification of 1000 x of 100 nuclei for each tumour according to the following protocol : in the case of papillary tumours, 25 nuclei were measured from the external papillary
628
629
MORPHOMETRIC VALUES IN INVASIVE TRANSITIONAL CELL CARCINOMA OF THE BLADDER
zone of the tumour, 25 from the internal papillary zone and 50 from the invasive zone; for the solid tumours, 100 randomly selected nuclei were measured but the field was changed so that the measurements were made in different parts of the tumour. For each nucleus the following parameters were measured : nuclear area, nuclear perimeter, nuclear diameter, nuclear size (NS = 2Jarea/n) and nuclear shape factor (NSF = 4narea/perimeter). Before measurement commenced, the coefficient of variation (CV) was applied according to the method given by Fleege et al. (1988), so that the reliability of the method we were going to use could be assessed. The result was a CV of 1.7, which guaranteed that the method was reliable and, therefore, objective and reproducible. The statistical analysis was performed with the SIGMA program (Horus Hardware S.A., Madrid, Spain) in a BULL-MICRAL 30 computer.
Results Invasive papillary tumours (n = 34) Table 1 shows the results of the measurements made in the 3 previously mentioned zones. To ascertain whether the different parameters for each zone were independent or inter-related, the Pearson correlation coefficient was calculated. A high and significant correlation was found for each group between nuclear area, perimeter, diameter and size (correlation coefficient = 0.97), but not always with nuclear shape factor. Because of this high correlation, nuclear area was taken to be representative. Table 1 shows that the nuclear area is smallest in the external papillary zone and the largest nuclear area corresponds to the invasive zone of the tumour. Since the correlation between the nuclear area measurements from the external papillary, internal papillary and invasive zones was not very high, a comparison was made of the mean values for the 3
zones taken 2 at a time to establish whether it made no difference which zone was used for measurement or whether, on the other hand, there were differences depending on the zone measured. No differences were found between the external and internal papillary zones, or between the internal papillary zone and the invasive zone, but a significant difference was found between the external papillary and the invasive zones (Table 2). The overall mean nuclear area for the 3 zones was 45.1493 pm2k 13.464. Nuclear area was also compared in relation to histological grade. The 25 Grade I11 tumours were seen to have a higher mean nuclear area value than the 9 Grade I1 tumours, and the difference was significant (Table 3). Invasive solid tumours (n = 61) Table 4 gives the results of the morphometric study for these tumours. As with the invasive papillary tumours, the Pearson correlation coefficient was calculated to see if the different morphometric values were independent or inter-related. The correlation coefficient obtained was very high and also significant. In this case we again took nuclear area (mean value 42.7966 pm2k 13.8162) as the representative morphometric value. A comparative multiple-mean test was made according to histological grade and Grade I1 tumours were found to have a lower mean nuclear area value than Grade I11 and Grade IV tumours, but Grade IV tumours did not have a larger nuclear area than Grade I11 tumours (Table 5). Nuclear area of the 95 invasive tumours When the 34 papillary and the 61 solid tumours were grouped together, the mean nuclear area was found to be 43.6386 pm2 f 14.951. Figure 1 shows the different prognosis of the 95 invasive tumours in relation to their histological grade. Significant differences with regard to sur-
Table 1 Morphometric Values of the Invasive Papillary Tumour (n= 34) ~
Invasivepapillary tumour Nuclear area SD
+
External zone Internal zone
+
Nuclear size -k SD
42.1662
24.1458
8.9161
f 16.0555
& 4.5274
k 1.6523
44.1709
f 1.7988
& 0.7921
48.8101
* 25.2232 4.5497 26.8232
& 18.5216
k 4.6212
10.6391 1.8413
k 0.7842
k 15.7058 Invasive zone
Nuclear perimeter Nuclear diameter SD iSD
9.5052
+
4.0632
k 0.77124 4.1620 4.3233
Shape factor SD
+
0.87852
k 0.036623 0.88843
k 0.048639 0.82204 & 0.057241
630
BRITISH JOURNAL OF UROLOGY
Table 2 Comparison of Matched Means between External and Invasive Zones of the Invasive Papillary Tumour Invasive papillary tumour
Mean nuclear area
SD
No. of patients
External zone Invasive zone
42.1612 48.8101
16.0555 18.5216
34 34
11-111 Not significant Il-IV P
01992 British Journal of Urology
Prognostic Correlation of Morphometric Values with Survival in lnvasive Transitional Cell Carcinoma of the Bladder J. A. PORTILLO, J. F. VAL-BERNAL, M. F. GARIJO, L. BUELTA and J. L. GUTIERREZ Urology Service and Department of Pathological Anatomy, University Hospital “lMarquBsde Valdecilla‘: Santander, Spain
Summary-A morphometric study was made of 95 invasive bladder tumours, differentiating 34 papillary and 61 solid carcinomas. In the invasive papillary tumours, the deeper the zone of the tumour measured, the higher the value of the mean nuclear area. A higher histological grade was also seen to correspond to a higher mean nuclear area value except for Grade IV tumours, whose nuclear area was no larger than that for Grade I l l tumours. This led u s to separate Grade IV tumours from transitional cell carcinomas and classify them as undifferentiated. A better prognosis was 0 in category T2. For T3 and T4A found for those tumours whose mean nuclear area was ~ 3 pm2 tumours no conclusive results were obtained in this respect.
Since Ooms et al. (1983) and Baak et al. (1982) first made their morphometric studies of neoplastic cells, many authors have applied this technique to many different types of tumour. It has been used to study not only tumours of the urinary tract (De Sanctis et al., 1982; Ooms et al., 1983; Montironi et al., 1984; Seldinrijk et al., 1987; Nafe and Frohreberg, 1989) but also other types of neoplasm (Baak et al., 1982; Carter et al., 1989; Payne et al., 1989; Snowden et al., 1989; Lipponen et al., 1990). Morphometry entails measuring a set of neoplastic cell parameters, particularly those related to the nucleus : area, perimeter, diameter, size and shape factor. Several studies have demonstrated that nuclear area, perimeter and size show higher mean values according to higher pre-assigned histological grades (Montironi et al., 1986; Van der Poel et al., 1988). It appears to be especially important in differentiating Grade I11 from Grades I and 11. In this retrospective study, morphometry was performed on the tumour nucleus of a series of invasive transitional cell carcinomas of the bladder Accepted for publication 3 October 1991
in an attempt to correlate the results obtained both with the survival of the patients and with histological grade.
Patients and Methods From the bladder tumours examined between January 1973 and December 1983, 95 invasive carcinomas were selected. Conditions were that evolution over a period of at least 5 years was known or that death due to the neoplasia had occurred earlier. All patients had undergone radical surgery without adjuvant chemotherapy or radiotherapy. The classification of Bergkvist et al. (1965) was used for histological grading and the series was thus divided into 34 papillary and 61 solid carcinomas. Using a semi-automatic MOP-VIDEOPLAN image analyser (Kontron, Germany), which consists of a central computer, an image processor and interactive system, together with an OLYMPUS BH2 light microscope and a camera lucida, measurements were made at a magnification of 1000 x of 100 nuclei for each tumour according to the following protocol : in the case of papillary tumours, 25 nuclei were measured from the external papillary
628
629
MORPHOMETRIC VALUES IN INVASIVE TRANSITIONAL CELL CARCINOMA OF THE BLADDER
zone of the tumour, 25 from the internal papillary zone and 50 from the invasive zone; for the solid tumours, 100 randomly selected nuclei were measured but the field was changed so that the measurements were made in different parts of the tumour. For each nucleus the following parameters were measured : nuclear area, nuclear perimeter, nuclear diameter, nuclear size (NS = 2Jarea/n) and nuclear shape factor (NSF = 4narea/perimeter). Before measurement commenced, the coefficient of variation (CV) was applied according to the method given by Fleege et al. (1988), so that the reliability of the method we were going to use could be assessed. The result was a CV of 1.7, which guaranteed that the method was reliable and, therefore, objective and reproducible. The statistical analysis was performed with the SIGMA program (Horus Hardware S.A., Madrid, Spain) in a BULL-MICRAL 30 computer.
Results Invasive papillary tumours (n = 34) Table 1 shows the results of the measurements made in the 3 previously mentioned zones. To ascertain whether the different parameters for each zone were independent or inter-related, the Pearson correlation coefficient was calculated. A high and significant correlation was found for each group between nuclear area, perimeter, diameter and size (correlation coefficient = 0.97), but not always with nuclear shape factor. Because of this high correlation, nuclear area was taken to be representative. Table 1 shows that the nuclear area is smallest in the external papillary zone and the largest nuclear area corresponds to the invasive zone of the tumour. Since the correlation between the nuclear area measurements from the external papillary, internal papillary and invasive zones was not very high, a comparison was made of the mean values for the 3
zones taken 2 at a time to establish whether it made no difference which zone was used for measurement or whether, on the other hand, there were differences depending on the zone measured. No differences were found between the external and internal papillary zones, or between the internal papillary zone and the invasive zone, but a significant difference was found between the external papillary and the invasive zones (Table 2). The overall mean nuclear area for the 3 zones was 45.1493 pm2k 13.464. Nuclear area was also compared in relation to histological grade. The 25 Grade I11 tumours were seen to have a higher mean nuclear area value than the 9 Grade I1 tumours, and the difference was significant (Table 3). Invasive solid tumours (n = 61) Table 4 gives the results of the morphometric study for these tumours. As with the invasive papillary tumours, the Pearson correlation coefficient was calculated to see if the different morphometric values were independent or inter-related. The correlation coefficient obtained was very high and also significant. In this case we again took nuclear area (mean value 42.7966 pm2k 13.8162) as the representative morphometric value. A comparative multiple-mean test was made according to histological grade and Grade I1 tumours were found to have a lower mean nuclear area value than Grade I11 and Grade IV tumours, but Grade IV tumours did not have a larger nuclear area than Grade I11 tumours (Table 5). Nuclear area of the 95 invasive tumours When the 34 papillary and the 61 solid tumours were grouped together, the mean nuclear area was found to be 43.6386 pm2 f 14.951. Figure 1 shows the different prognosis of the 95 invasive tumours in relation to their histological grade. Significant differences with regard to sur-
Table 1 Morphometric Values of the Invasive Papillary Tumour (n= 34) ~
Invasivepapillary tumour Nuclear area SD
+
External zone Internal zone
+
Nuclear size -k SD
42.1662
24.1458
8.9161
f 16.0555
& 4.5274
k 1.6523
44.1709
f 1.7988
& 0.7921
48.8101
* 25.2232 4.5497 26.8232
& 18.5216
k 4.6212
10.6391 1.8413
k 0.7842
k 15.7058 Invasive zone
Nuclear perimeter Nuclear diameter SD iSD
9.5052
+
4.0632
k 0.77124 4.1620 4.3233
Shape factor SD
+
0.87852
k 0.036623 0.88843
k 0.048639 0.82204 & 0.057241
630
BRITISH JOURNAL OF UROLOGY
Table 2 Comparison of Matched Means between External and Invasive Zones of the Invasive Papillary Tumour Invasive papillary tumour
Mean nuclear area
SD
No. of patients
External zone Invasive zone
42.1612 48.8101
16.0555 18.5216
34 34
11-111 Not significant Il-IV P
