Breast Cancer Research and Treatment 15: 149-160, 1990. © 1990KluwerAcademic Publishers. Printedin the Netherlands. Report
Cytometric and histopathologic features of tumors detected in a randomized mammography screening program: correlation and relative prognostic influence T. Hatschek, 10. Grrntoft, 2 G. Fagerberg, 30. St~l, 1 S. Sullivan, 1J. Carstensen, 1 and B. Nordenskjrld 1 1Departments o f Oncology, 2 Pathology, and 3Diagnostic Radiology, University Hospital, S-581 85 Linkdping, Sweden
Key words: breast cancer, cytometry, histopathology, mammography, prognosis, screening Summary
Cytometric determination of S-phase fraction and ploidy type was performed on 430 tumors detected within a randomized trial of mammographic screening. The results were compared to several histopathologic features. A high S-phase fraction was estimated in tumors with a high grade of malignancy and other histopathologic findings related to rapid tumor progression, including lack of tubule formation, a high mitotic index, marked nuclear pleomorphism, multifocal cancer growth, tumor emboli in lymphatic and blood vessels, tumor necrosis, and inflammatory reaction. DNA aneuploidy was correlated with a high malignancy grade, frequent mitoses, a high degree of nuclear pleomorphism, vascular invasion, necrosis, and the presence of noninvasive ductal carcinoma. Both cytometric variables were inversely related to the degree of elastosis. Positive nodes, large tumor size, DNA aneuploidy, a high S-phase fraction, high grade of malignancy, lack of tubule formation, as well as high mitotic index and pleomorphism, presence of multifocal cancer, and vascular invasion, predicted a significantly shorter distant recurrence-free interval after a median follow-up time of 46.6 months. Elastosis and the presence of estrogen and progesterone receptors indicated favorable prognosis. In the multivariate analysis, only lymph node status, tumor size, S-phase fraction, and multifocal growth pattern had independent prognostic value.
Introduction
Histologic malignancy grading according to the WHO [1] and estimation of the nuclear grade [2] are widely used to characterize breast cancer growth, but a large number of other pathologic findings, including elastosis, vessel invasion, and sinus histiocytosis have been investigated, along with the established prognostic factors: axillary node status and tumor size. In recent years, several authors have found that tumor cell ploidy and Sphase level either as separate variables [3-7] or in
combination [8-9] can be used to predict the clinical outcome in breast carcinomas. Furthermore, several studies have shown that both ploidy and S-phase level correlate with other prognostic factors, i.e. node involvement, tumor size, estrogen receptor content, and histologic grade [3, 4, 10]. Little is known about the correlation between cytometric and histologic variables other than those used in malignancy grading. Some studies comparing morphologic findings and proliferation using thymidine labeling inde [11] or flow cytometry [12] have been published recently.
Address for offprints: T. Hatschek,Dept. of Oncology,UniversityHospital, S-58185 Linkrping, Sweden
150
T Hatschek et al.
In the present study on the relationship between histologic and cytometric variables and their prognostic value, we used tumors from a randomized population-based mammographic screening program which was started in 1978 as part of the Swedish Two-County study. Tumors detected during the second and third screening rounds, or diagnosed in the intervals between the screenings or in the control group during the same time period, were investigated. Recently, we have reported on the results of cytometry [13]. A more extensive work on malignancy grading based on the same mammographic screening trial has been published previously [14].
Material and methods
The screening program
In 1978, a controlled trial evaluating mammographic screening was started in the county of Osterg6tland, Sweden, as one part of the Swedish Two-County study. Including all women aged 40 years and older, the trial enrolled 92,934 women [15]. After randomization, 47,001 were allocated to the study group and 45,933 to the control group. Women in the study group were offered repeated mammographic screening, while those in the control group received no such invitation. The first screening was started in May 1978; second and third screenings were performed between April
Table 1. Distribution of tumor types according to WHO (all
1981 and July 1985, with screening intervals being approximately two years. As a result of the first screening, the cancer prevalence rate among women 40 years or older rose to 8.1 per 1000 in the study group but was 6.6 per 1000 when limited to women 40 to 74 years [15], compared to a yearly incidence rate of 1.9 per 1000 women in the control group. The proportion of tumors at early stages and with a low grade of malignancy was noticeably higher in the study group compared to the control group [14]. In the following screening rounds, the yearly breast cancer incidence in the study group, which included interval cases and non-participants, decreased to approximately the same level as the incidence rate in the control group [15]. A similar redistribution was found for the malignancy grade, while the difference in stage distribution was still in favor of the study group [14].
Patients
During the second and third screening rounds, 212 breast carcinomas were detected by mammography. Ninety-eight cancers were diagnosed in the screening intervals due to clinical symptoms, 54 of these between the first and second screenings and 44 between the second and third screening rounds. Among women in the study group who had been offered screening but had not participated, 88 breast cancers were diagnosed. In women allocated to the control group, 317 breast tumors were found in routine medical care during the same period. Carcinomas diagnosed among women allocated
stages) Tumor type
Number
Percent
Ductal Lobular Medullary Mucinous Tubular Apocrine Paget's disease Other types 1
306 72 14 14 11 4 2 7
71.2 16.7 3.3 3.3 2.6 0.9 0.5 1.6
1Argyrophile cancer (1), undifferentiated cancer (6).
Table 2. Stage distribution among study groups and controls (percentage in parentheses)
Stage
I II III IV
All patients
229(53) 177 (41) 15 (4) 9 (2)
Study group
Control group
Screened cancers
Interval cancers
120 (75) 37 (23) 3 (2) 1 (1)
21 (42) 27 (54) 2 (4) 0
88 113 10 8
(40) (52) (5) (4)
Cytometric and histopathologic features of tumors to the study group who did not follow the invitation to screening (non-participants) were not investigated in our study. F u r t h e r m o r e , patients with breast cancer diagnosed previous to entry into the study or with bilateral tumors, and those treated with preoperative radiotherapy, were excluded. Of 627 tumors, 430 were available for cytometric investigation. The m e a n age at diagnosis was 61.7 years, with no significant differences between the study and control groups.
Table3. Distribution of patients according to histologic characteristics of their tumors. All stages included Histologic variable
Tubule formation
Category
No. of patients
absent 174 moderate 113 marked 10 Mitotic activity low 83 moderate 113 high 101 Nuclear pleomorphism none 32 moderate 119 marked 147 Tumor border circumscribed100 stellate 227 absent edge 70 Multifocal cancer none 224 moderate 142 marked 23 Vascular invasion absent 333 present 30 Necrosis absent 317 present 47 Lymphoid reaction absent 185 moderate 156 marked 53 Elastosis slight 124 moderate 149 marked 141 Lobular cancerization absent 290 moderate 48 marked 11 Ductal cancer in situ absent 173 moderate 138 marked 91 Lobular cancer in situ absent 325 moderate 43 marked 16
151
Primary treatment Stage I cancers were removed by either modified radical mastectomy or quadrantectomy. Both methods included an axillary lymph node dissection. All w o m e n with tumors treated with breastconserving surgery and those with stage II cancers removed by mastectomy and axillary dissection received postoperative radiotherapy. Two hundred and three (46%) of the patients were included in adjuvant trials with either tamoxifen or combined chemotherapy.
Histopathology and staging
Percent
59 38 3 28 38 34 11 40 49 25 57 18 58 37 6 92 8 87 13 47 40 13 30 36 34 83 14 3 43 34 23 85 11 4
Breast cancer diagnosis was confirmed histologically in all cases. After fixation in formalin or, for approximately 10% of all tumors, Carnoy's solution, the specimens were routinely stained with hematoxylin & eosin and elastin. All histologic slides were reviewed by one pathologist ( O G ) . Malignancy grading and histologic classification followed the recommendations of the World Health Organization [1, 16]. The distribution among histologic types is shown in Table 1. T u m o r size (largest diameter) was obtained both macro- and microscopically, and in most cases whole organ sections were used [17]. The n u m b e r of axillary nodes involved could be determined in 417 of the 430 cases including all stages. Distant metastases were excluded by chest X-ray and bone scan. Stage I tumors dominated among screening detected cancers, while the stage distribution among interval cases was approximately the same as in the control group (Table 2). The histopathologic features investigated are shown in Table 3. Tubular differentiation; hyperchromatism and mitosis; and irregularity of size, shape and staining of the nuclei were used both as separate variables and as components of malignancy grading [1]. In the present material, these variables and malignancy grading were applied only in carcinomas of ductal type, of which 64 (21%) had a low, 97 (32%) an intermediate, and 144 (47%) a high grade of malignancy when all stages were included. Further, we investigated the
152
T Hatschek et al.
mode of infiltrative growth (tumor border), the presence of satellites in surrounding breast tissue (multifocal cancer), tumor emboli in lymphatic and blood vessels, tumor necrosis, the degree of inflammatory reaction, presence of elastosis, and simultaneous presence of noninvasive tumor independent of the invasive carcinoma formed either as lobular cancerization [18], or ductal or lobular carcinoma in situ.
D N A cytometry
The method of cell preparation has been previously described [19]. From the embedded specimens, 50/,m tissue sections were deparaffinized with xylene followed by stepwise hydration in ethanol (99.5, 95, 70, and 40%) and distilled water. The tissue was disaggregated with 0.5% pepsin (Sigma) in 0.9% NaC1, pH 2.0, for 30 minutes in a shaking water-bath at 37 ° C. After filtration through a nylon mesh, pore size 41/xm, the cell suspension was centrifuged onto microscope slides and then fixed
in a mixture of methanol, formaldehyde (35% w/ v), and glacial acetic acid (85/10/5 by volume) for one hour. The preparations were stained in a solution containing 1/xg Hoechst 33258 per ml in 400 mM NaC1 and 100 mM sodium phosphate buffer, pH 7.0. Specimens were analyzed in a phase contrast microscope which allowed selection of intact nuclei under visual control. We used a Leitz MPV 3 cytophotometer (Leitz GmbH, Wetzlar, FRG) in connection with a Luxor ABC 800 microcomputer. The FLOWRA computer program [20] enabled continuous fast measurements while passing selected nuclei through the excitation light beam. Between 300 and 450 tumor ceils from each specimen were analyzed. In addition, lymphocytes equal to 10% of the number of measured tumor cells from each specimen served as an internal standard. The FLOWRA program allowed computation of histograms for estimation of D N A index related to the internal standard and for calculation of S-phase levels by use of a rectangular model. Additional
Table 4. C o r r e l a t i o n (r) b e t w e e n the h i s t o l o g i c variables. A l l s t a g e s are i n c l u d e d Noninvasive lobular cancer Tubule formation 1 Mitotic index I Nuclear pleomorphism 1 Tumor border Multi focal cancer Vascular invasion Necrosis Lymphoid reaction Elastosis Lobular cancerization Ductal cancer insitu
Ductal cancer in situ
Elastosis
Lymphoid reaction
Necrosis
Vessel invasion
Multifocal cancer
Tumor border
PleoMitotic morphism 1 index I
- 0.12
0.08
0.15"
- 0.28***
0.15"
0.25***
0.17"*
0.25***
0.02
0.22***
- 0.05
0.09
0.04
- 0.38***
0.20***
0.33***
0.23***
0.29***
0.26***
0.70***
- 0.16"**
0.06
0.03
- 0.34***
0.17"**
0.27***
0.20***
0.30***
0.27***
0.20***
- 0.07
0.01
0.20***
- 0.18"**
0.08
0.31"**
0.00
0.04
0.09
- 0.11"
- 0.06
0.00
- 0.14" *
- 0.01
0.03 0.07
- 0.21"**
0.03
- 0 . 2 3 * **
0.05
- 0.17"**
0 . 2 0 " ** - 0.04
0.34***
- 0.15"*
i O n l y ductal c a r c i n o m a s , *p----- 0 . 0 5 ,
Lobular cancerization
**p-
2.05 ' a n e u p l o i d ' , a n d b e t w e e n 1.96 a n d 2.05 'tetraploid'. W h e n all stages were i n c l u d e d , 272 ( 6 3 . 3 % ) t u m o r s were a n e u p l o i d . I n the analysis r e g a r d i n g the predictive v a l u e of the variables in l i m i t e d disease, diploid a n d t e t r a p l o i d t u m o r s were t r e a t e d as o n e g r o u p (euploid) since the relative risk for r e c u r r e n c e was similar in b o t h [13]. T h e m e a n S-phase level was 6 . 9 % , m e d i a n was 6 . 0 % , r a n g i n g b e t w e e n 1% a n d 27%. I n the present study, S-phase was divided into t h r e e subgroups with a p p r o x i m a t e l y e q u a l size: < 4 % , 4 - 7 % , a n d -> 8 % . Classification of the D N A hist o g r a m s was d o n e i n d e p e n d e n t of i n f o r m a t i o n o n the clinical o u t c o m e .
assays were p e r f o r m e d as described by W r a n g e et al. [21, 22]. Cytosol was i n c u b a t e d with 5 n M 3H-estradiol ( E R ) or 1 0 0 n M R5020 (PgR), respectively, a n d the receptors were isolated by isoelectric focusing in p o l y a c r y l a m i d e gel. T h e a m o u n t of r e c e p t o r was r e l a t e d to the t u m o r D N A c o n t e n t . C a r c i n o m a s with h o r m o n e r e c e p t o r content - 0 . 1 fmol//zg D N A were j u d g e d as positive. O f 306 t u m o r s e x a m i n e d , 207 (68%) were E R positive a n d 120 (39%) P g R positive.
Follow-up T h e p a t i e n t s were r e - e x a m i n e d every t h r e e m o n t h s d u r i n g the first t h r e e years after surgery a n d every six m o n t h s thereafter. Chest X-ray, b o n e scan, a n d m a m m o g r a p h y were p e r f o r m e d once a y e a r in a s y m p t o m a t i c patients. T h e m e d i a n follow-up pe-
Table5. Correlation coefficients (r) between traditional and cytometric prognostic factors and the histopathologic1variables. All stages are included
Malignancy grading4 Tubule formation4 Mitotic index4 Pleomorphism4 Tumor border Multifocal growth Vascular invasion Necrosis Lymphoid reaction Elastosis Lobular cancerization Ductal ca in situ Lobular ca in situ
Axillary nodes
Tumor size
Estrogen receptor
S-phase2
DNA-index3
0.22"** 0.10 0.25*** 0.17** 0.02 0.28*** 0.35*** 0.20*** 0.07 - 0.04 0.02 0.09 - 0.02
0.33"** 0.14" 0.37*** 0.32* ** 0.10" 0.34*** 0.24*** 0.21"** 0.08 - 0.04 0.01 0.00 - 0.03
-
0.43"** 0.29'** 0.40*** 0.33 *** 0.01 0.18*** 0.18"** 0.38*** 0.31'** - 0.21"** 0.01 0.01 - 0.09
0.29"** 0.05 0.26*** 0.27*** 0.05 0.06 0.14"* 0.11" 0.06 - 0.11 0.04 0.15"* - 0.14"*
-
0.21"* 0.17" 0.24*** 0.13* 0.04 0.13* 0.18"* 0.31"** 0.30"** 0.16"* 0.09 0.02 0.02
aThe categories of the histopathologic variables were scored according to Table 3. 2S-phase was scored '1' if < 4, '2' if 4-7, and '3' if ->8%. 3DNA ploidy was scored '1' if euploid (diploid + tetraploid) and '2' if aneuploid. 4Only ductal carcinomas. * P ~ 0.05, ** P --
Cytometric and histopathologic features of tumors detected in a randomized mammography screening program: correlation and relative prognostic influence T. Hatschek, 10. Grrntoft, 2 G. Fagerberg, 30. St~l, 1 S. Sullivan, 1J. Carstensen, 1 and B. Nordenskjrld 1 1Departments o f Oncology, 2 Pathology, and 3Diagnostic Radiology, University Hospital, S-581 85 Linkdping, Sweden
Key words: breast cancer, cytometry, histopathology, mammography, prognosis, screening Summary
Cytometric determination of S-phase fraction and ploidy type was performed on 430 tumors detected within a randomized trial of mammographic screening. The results were compared to several histopathologic features. A high S-phase fraction was estimated in tumors with a high grade of malignancy and other histopathologic findings related to rapid tumor progression, including lack of tubule formation, a high mitotic index, marked nuclear pleomorphism, multifocal cancer growth, tumor emboli in lymphatic and blood vessels, tumor necrosis, and inflammatory reaction. DNA aneuploidy was correlated with a high malignancy grade, frequent mitoses, a high degree of nuclear pleomorphism, vascular invasion, necrosis, and the presence of noninvasive ductal carcinoma. Both cytometric variables were inversely related to the degree of elastosis. Positive nodes, large tumor size, DNA aneuploidy, a high S-phase fraction, high grade of malignancy, lack of tubule formation, as well as high mitotic index and pleomorphism, presence of multifocal cancer, and vascular invasion, predicted a significantly shorter distant recurrence-free interval after a median follow-up time of 46.6 months. Elastosis and the presence of estrogen and progesterone receptors indicated favorable prognosis. In the multivariate analysis, only lymph node status, tumor size, S-phase fraction, and multifocal growth pattern had independent prognostic value.
Introduction
Histologic malignancy grading according to the WHO [1] and estimation of the nuclear grade [2] are widely used to characterize breast cancer growth, but a large number of other pathologic findings, including elastosis, vessel invasion, and sinus histiocytosis have been investigated, along with the established prognostic factors: axillary node status and tumor size. In recent years, several authors have found that tumor cell ploidy and Sphase level either as separate variables [3-7] or in
combination [8-9] can be used to predict the clinical outcome in breast carcinomas. Furthermore, several studies have shown that both ploidy and S-phase level correlate with other prognostic factors, i.e. node involvement, tumor size, estrogen receptor content, and histologic grade [3, 4, 10]. Little is known about the correlation between cytometric and histologic variables other than those used in malignancy grading. Some studies comparing morphologic findings and proliferation using thymidine labeling inde [11] or flow cytometry [12] have been published recently.
Address for offprints: T. Hatschek,Dept. of Oncology,UniversityHospital, S-58185 Linkrping, Sweden
150
T Hatschek et al.
In the present study on the relationship between histologic and cytometric variables and their prognostic value, we used tumors from a randomized population-based mammographic screening program which was started in 1978 as part of the Swedish Two-County study. Tumors detected during the second and third screening rounds, or diagnosed in the intervals between the screenings or in the control group during the same time period, were investigated. Recently, we have reported on the results of cytometry [13]. A more extensive work on malignancy grading based on the same mammographic screening trial has been published previously [14].
Material and methods
The screening program
In 1978, a controlled trial evaluating mammographic screening was started in the county of Osterg6tland, Sweden, as one part of the Swedish Two-County study. Including all women aged 40 years and older, the trial enrolled 92,934 women [15]. After randomization, 47,001 were allocated to the study group and 45,933 to the control group. Women in the study group were offered repeated mammographic screening, while those in the control group received no such invitation. The first screening was started in May 1978; second and third screenings were performed between April
Table 1. Distribution of tumor types according to WHO (all
1981 and July 1985, with screening intervals being approximately two years. As a result of the first screening, the cancer prevalence rate among women 40 years or older rose to 8.1 per 1000 in the study group but was 6.6 per 1000 when limited to women 40 to 74 years [15], compared to a yearly incidence rate of 1.9 per 1000 women in the control group. The proportion of tumors at early stages and with a low grade of malignancy was noticeably higher in the study group compared to the control group [14]. In the following screening rounds, the yearly breast cancer incidence in the study group, which included interval cases and non-participants, decreased to approximately the same level as the incidence rate in the control group [15]. A similar redistribution was found for the malignancy grade, while the difference in stage distribution was still in favor of the study group [14].
Patients
During the second and third screening rounds, 212 breast carcinomas were detected by mammography. Ninety-eight cancers were diagnosed in the screening intervals due to clinical symptoms, 54 of these between the first and second screenings and 44 between the second and third screening rounds. Among women in the study group who had been offered screening but had not participated, 88 breast cancers were diagnosed. In women allocated to the control group, 317 breast tumors were found in routine medical care during the same period. Carcinomas diagnosed among women allocated
stages) Tumor type
Number
Percent
Ductal Lobular Medullary Mucinous Tubular Apocrine Paget's disease Other types 1
306 72 14 14 11 4 2 7
71.2 16.7 3.3 3.3 2.6 0.9 0.5 1.6
1Argyrophile cancer (1), undifferentiated cancer (6).
Table 2. Stage distribution among study groups and controls (percentage in parentheses)
Stage
I II III IV
All patients
229(53) 177 (41) 15 (4) 9 (2)
Study group
Control group
Screened cancers
Interval cancers
120 (75) 37 (23) 3 (2) 1 (1)
21 (42) 27 (54) 2 (4) 0
88 113 10 8
(40) (52) (5) (4)
Cytometric and histopathologic features of tumors to the study group who did not follow the invitation to screening (non-participants) were not investigated in our study. F u r t h e r m o r e , patients with breast cancer diagnosed previous to entry into the study or with bilateral tumors, and those treated with preoperative radiotherapy, were excluded. Of 627 tumors, 430 were available for cytometric investigation. The m e a n age at diagnosis was 61.7 years, with no significant differences between the study and control groups.
Table3. Distribution of patients according to histologic characteristics of their tumors. All stages included Histologic variable
Tubule formation
Category
No. of patients
absent 174 moderate 113 marked 10 Mitotic activity low 83 moderate 113 high 101 Nuclear pleomorphism none 32 moderate 119 marked 147 Tumor border circumscribed100 stellate 227 absent edge 70 Multifocal cancer none 224 moderate 142 marked 23 Vascular invasion absent 333 present 30 Necrosis absent 317 present 47 Lymphoid reaction absent 185 moderate 156 marked 53 Elastosis slight 124 moderate 149 marked 141 Lobular cancerization absent 290 moderate 48 marked 11 Ductal cancer in situ absent 173 moderate 138 marked 91 Lobular cancer in situ absent 325 moderate 43 marked 16
151
Primary treatment Stage I cancers were removed by either modified radical mastectomy or quadrantectomy. Both methods included an axillary lymph node dissection. All w o m e n with tumors treated with breastconserving surgery and those with stage II cancers removed by mastectomy and axillary dissection received postoperative radiotherapy. Two hundred and three (46%) of the patients were included in adjuvant trials with either tamoxifen or combined chemotherapy.
Histopathology and staging
Percent
59 38 3 28 38 34 11 40 49 25 57 18 58 37 6 92 8 87 13 47 40 13 30 36 34 83 14 3 43 34 23 85 11 4
Breast cancer diagnosis was confirmed histologically in all cases. After fixation in formalin or, for approximately 10% of all tumors, Carnoy's solution, the specimens were routinely stained with hematoxylin & eosin and elastin. All histologic slides were reviewed by one pathologist ( O G ) . Malignancy grading and histologic classification followed the recommendations of the World Health Organization [1, 16]. The distribution among histologic types is shown in Table 1. T u m o r size (largest diameter) was obtained both macro- and microscopically, and in most cases whole organ sections were used [17]. The n u m b e r of axillary nodes involved could be determined in 417 of the 430 cases including all stages. Distant metastases were excluded by chest X-ray and bone scan. Stage I tumors dominated among screening detected cancers, while the stage distribution among interval cases was approximately the same as in the control group (Table 2). The histopathologic features investigated are shown in Table 3. Tubular differentiation; hyperchromatism and mitosis; and irregularity of size, shape and staining of the nuclei were used both as separate variables and as components of malignancy grading [1]. In the present material, these variables and malignancy grading were applied only in carcinomas of ductal type, of which 64 (21%) had a low, 97 (32%) an intermediate, and 144 (47%) a high grade of malignancy when all stages were included. Further, we investigated the
152
T Hatschek et al.
mode of infiltrative growth (tumor border), the presence of satellites in surrounding breast tissue (multifocal cancer), tumor emboli in lymphatic and blood vessels, tumor necrosis, the degree of inflammatory reaction, presence of elastosis, and simultaneous presence of noninvasive tumor independent of the invasive carcinoma formed either as lobular cancerization [18], or ductal or lobular carcinoma in situ.
D N A cytometry
The method of cell preparation has been previously described [19]. From the embedded specimens, 50/,m tissue sections were deparaffinized with xylene followed by stepwise hydration in ethanol (99.5, 95, 70, and 40%) and distilled water. The tissue was disaggregated with 0.5% pepsin (Sigma) in 0.9% NaC1, pH 2.0, for 30 minutes in a shaking water-bath at 37 ° C. After filtration through a nylon mesh, pore size 41/xm, the cell suspension was centrifuged onto microscope slides and then fixed
in a mixture of methanol, formaldehyde (35% w/ v), and glacial acetic acid (85/10/5 by volume) for one hour. The preparations were stained in a solution containing 1/xg Hoechst 33258 per ml in 400 mM NaC1 and 100 mM sodium phosphate buffer, pH 7.0. Specimens were analyzed in a phase contrast microscope which allowed selection of intact nuclei under visual control. We used a Leitz MPV 3 cytophotometer (Leitz GmbH, Wetzlar, FRG) in connection with a Luxor ABC 800 microcomputer. The FLOWRA computer program [20] enabled continuous fast measurements while passing selected nuclei through the excitation light beam. Between 300 and 450 tumor ceils from each specimen were analyzed. In addition, lymphocytes equal to 10% of the number of measured tumor cells from each specimen served as an internal standard. The FLOWRA program allowed computation of histograms for estimation of D N A index related to the internal standard and for calculation of S-phase levels by use of a rectangular model. Additional
Table 4. C o r r e l a t i o n (r) b e t w e e n the h i s t o l o g i c variables. A l l s t a g e s are i n c l u d e d Noninvasive lobular cancer Tubule formation 1 Mitotic index I Nuclear pleomorphism 1 Tumor border Multi focal cancer Vascular invasion Necrosis Lymphoid reaction Elastosis Lobular cancerization Ductal cancer insitu
Ductal cancer in situ
Elastosis
Lymphoid reaction
Necrosis
Vessel invasion
Multifocal cancer
Tumor border
PleoMitotic morphism 1 index I
- 0.12
0.08
0.15"
- 0.28***
0.15"
0.25***
0.17"*
0.25***
0.02
0.22***
- 0.05
0.09
0.04
- 0.38***
0.20***
0.33***
0.23***
0.29***
0.26***
0.70***
- 0.16"**
0.06
0.03
- 0.34***
0.17"**
0.27***
0.20***
0.30***
0.27***
0.20***
- 0.07
0.01
0.20***
- 0.18"**
0.08
0.31"**
0.00
0.04
0.09
- 0.11"
- 0.06
0.00
- 0.14" *
- 0.01
0.03 0.07
- 0.21"**
0.03
- 0 . 2 3 * **
0.05
- 0.17"**
0 . 2 0 " ** - 0.04
0.34***
- 0.15"*
i O n l y ductal c a r c i n o m a s , *p----- 0 . 0 5 ,
Lobular cancerization
**p-
2.05 ' a n e u p l o i d ' , a n d b e t w e e n 1.96 a n d 2.05 'tetraploid'. W h e n all stages were i n c l u d e d , 272 ( 6 3 . 3 % ) t u m o r s were a n e u p l o i d . I n the analysis r e g a r d i n g the predictive v a l u e of the variables in l i m i t e d disease, diploid a n d t e t r a p l o i d t u m o r s were t r e a t e d as o n e g r o u p (euploid) since the relative risk for r e c u r r e n c e was similar in b o t h [13]. T h e m e a n S-phase level was 6 . 9 % , m e d i a n was 6 . 0 % , r a n g i n g b e t w e e n 1% a n d 27%. I n the present study, S-phase was divided into t h r e e subgroups with a p p r o x i m a t e l y e q u a l size: < 4 % , 4 - 7 % , a n d -> 8 % . Classification of the D N A hist o g r a m s was d o n e i n d e p e n d e n t of i n f o r m a t i o n o n the clinical o u t c o m e .
assays were p e r f o r m e d as described by W r a n g e et al. [21, 22]. Cytosol was i n c u b a t e d with 5 n M 3H-estradiol ( E R ) or 1 0 0 n M R5020 (PgR), respectively, a n d the receptors were isolated by isoelectric focusing in p o l y a c r y l a m i d e gel. T h e a m o u n t of r e c e p t o r was r e l a t e d to the t u m o r D N A c o n t e n t . C a r c i n o m a s with h o r m o n e r e c e p t o r content - 0 . 1 fmol//zg D N A were j u d g e d as positive. O f 306 t u m o r s e x a m i n e d , 207 (68%) were E R positive a n d 120 (39%) P g R positive.
Follow-up T h e p a t i e n t s were r e - e x a m i n e d every t h r e e m o n t h s d u r i n g the first t h r e e years after surgery a n d every six m o n t h s thereafter. Chest X-ray, b o n e scan, a n d m a m m o g r a p h y were p e r f o r m e d once a y e a r in a s y m p t o m a t i c patients. T h e m e d i a n follow-up pe-
Table5. Correlation coefficients (r) between traditional and cytometric prognostic factors and the histopathologic1variables. All stages are included
Malignancy grading4 Tubule formation4 Mitotic index4 Pleomorphism4 Tumor border Multifocal growth Vascular invasion Necrosis Lymphoid reaction Elastosis Lobular cancerization Ductal ca in situ Lobular ca in situ
Axillary nodes
Tumor size
Estrogen receptor
S-phase2
DNA-index3
0.22"** 0.10 0.25*** 0.17** 0.02 0.28*** 0.35*** 0.20*** 0.07 - 0.04 0.02 0.09 - 0.02
0.33"** 0.14" 0.37*** 0.32* ** 0.10" 0.34*** 0.24*** 0.21"** 0.08 - 0.04 0.01 0.00 - 0.03
-
0.43"** 0.29'** 0.40*** 0.33 *** 0.01 0.18*** 0.18"** 0.38*** 0.31'** - 0.21"** 0.01 0.01 - 0.09
0.29"** 0.05 0.26*** 0.27*** 0.05 0.06 0.14"* 0.11" 0.06 - 0.11 0.04 0.15"* - 0.14"*
-
0.21"* 0.17" 0.24*** 0.13* 0.04 0.13* 0.18"* 0.31"** 0.30"** 0.16"* 0.09 0.02 0.02
aThe categories of the histopathologic variables were scored according to Table 3. 2S-phase was scored '1' if < 4, '2' if 4-7, and '3' if ->8%. 3DNA ploidy was scored '1' if euploid (diploid + tetraploid) and '2' if aneuploid. 4Only ductal carcinomas. * P ~ 0.05, ** P --
