Histopathologg 1992,21,257-262
Intra-patient variation between breast cancer axillary lymph node metastases using quantifiable features P. J .VAN DIEST, J. C .FLEEGE, E.MATZE-COK, J. P. A.BAAK lnstitwte for Pathology, Free University Hospital, Amsterdam, The Netherlands Date of submission 17 September 1991 Accepted for publication 13 March 1992
VAN DIEST P.J., PLEEGE J.C.,
MATZE-COK E. & B A A K J.P.A.
(1992) Histopathology 21, 257-262
Intra-patient variation between breast cancer axillary lymph node metastases using quantifiable features The intra-patient variations of some clinically relevant quantifiablefeatures, between axillary lymph node metastases were evaluated in 44 breast cancer patients. In all lymph node metastases detected (range 2-33 per patient), the mitotic figures were counted, the volume percentage epithelium was assessed and the mean nuclear area was measured. The intra-patient variation for each quantifiable feature was expressed by the coefficient of variation (CV). Since the measurement techniques used introduce a certain, well known variation themselves because of sampling and measurement errors, the CVs found had to be greater than methodological tolerance limits (established in previous studies) to be interpreted as indicating biological variation. The CVs exceeded the methodological tolerance l i t s in 86%of the cases for the mitotic count, in 48%of the cases for the volume percentage epithelium, and in 47%of the cases for the mean nuclear area. This indicated that in these cases, the variation found in the quantifiable features could not be explained by sampling or measurement errors and should be regarded as real biological variation. Furthermore, the variation in the quantifiable features studied showed a significant positive correlation with the number of lymph node metastases. Thus. there may be considerable intra-patient variation in quantifiable features between axillary lymph node metastases in breast cancer. This may indicate that these lymph node metastases originate independently from different clones within the primary tumour, that they are independently formed in different stages of tumour development,or that they, as an expression of intrinsic tumour heterogeneity, may develop in different directionsfrom the start. Keywords: breast cancer, proliferation, morphometry, stereology, lymph node metastases
Introduction Considering the relatively poor prognosis of breast cancer patients in spite of radical surgery, there is a strong need for prognostic factors that can identify patients at high risk of relapse to guide adjuvant therapy strategies. Lymph node status is a widely used prognostic factor', but is clearly not accurate enough in view of the fact that 5 years after the diagnosis, about 40% of patients are still alive despite the presence of lymph node metastases and 35% of patients without lymph node metastases have already diedz. Recently, additional Address for correspondence: Professor J.P.A.Baak.Institute for Pathology, Free University Hospital, PO Box 7057. 1007 IMB. Amsterdam, The Netherlands.
prognostic factors have been identified for both lymph node p ~ s i t i v e and ~ - ~lymph node negative breast cancer patients3e9*10.In lymph node positive patients, besides primary tumour factor^'^^*^*"-'^, quantitative data derived from the lymph node metastases such as proliferative activity and nuclear and tissue morphometric features may also be ~ s e f u l ~ - However, ~ , * ~ . despite the heterogeneity in tumour tissue that is generally observed, only one lymph node metastasis was evaluated per case in most of these studies, apparently assuming that the variation between lymph node metastases within the same patient was negligible. However, this assumption has not been shown to be correct. The aim of this study was therefore to evaluate the intrapatient variation of some clinically relevant quantifiable 257
2 58 P.1. van Diest et al.
features betweeq breast cancer axillary lymph node metastases.
Materials and methods PATIENTS' DETAILS A N D SPECIMEN PREPARATION
A total of 52 patients with invasive breast cancer metastatic to the axillary lymph nodes, diagnosed between 1971 and 1981, were considered for this study. All patients were treated with radical or modified radical mastectomy. Lymph nodes were identified in the fresh axillary dissection preparation, fixed in neutral buffered formalin and embedded in paraffin. Sections 4 pm thick were cut and stained with haematoxylin and eosin. All identified lymph nodes were reviewed and all lymph nodes containing metastatic breast cancer cells were included for all patients. Seven patients with only one lymph node metastasis and one patient having two lymph node metastases, one of them being unevaluable because of poor morphology, were excluded. The remaining 44 patients had, on average, 6.9 positive lymph nodes (range 2-33). A total of 305 lymph node metastases were evaluated. QUANTITATIVE METHODS
The most cellular area containing the highest density of mitotic figures was chosen subjectively by the same observer in all lymph node metastases of each case, rejecting areas with necrosis or many non-tumour cells. Although theoretically this may introduce bias, this procedure has been shown in several large studies, to be repr~ducible"*'~*'~~~~. In the selected area, a measurement field of approximately 0.5 x 0.5 cm2 (or the entire field containing metastatic cells in the case of metastases smaller than that size) was marked for counting mitotic figures, and the assessmentof volume percentage epithelium and nuclear area. These features were chosen because of their merent pathological significance (mitotic rate indicating proliferation, and epithelial percentage and nuclear area pointing at tissue and cellular differentiation, respectively) and their previously established prognostic ~ a l u e " ~ * ' ~ , Mitotic figures were counted in 10 consecutive high power fields (HPFs) at a x 4 0 0 magnification using a x 40 objective (numeric aperture of 0.75, field diameter 450 pm), starting at the region within the measurement area with the highest density of mitotic figures and from there on scanning in a random way. In order to cope with micro-metastases having less than 10 HPFs (15% of the lymph node metastases), the number of mitoses counted was expressed as the average number of mitoses
per field (the mitotic count). The mitotic count (MC) was assessed in all positive lymph nodes of all patients. The volume percentage of epithelium (VPE)versus stroma within the metastasis was assessed by point counting on a projection microscope at a magnification of approximately x 200 using a 42-points Weibel grid (test line length 1 5 mm). The whole measurement field was considered in each case, regarding only tumour stroma as stroma. The percentage of points falling on each compartment were taken as the volume percentages. In very small metastases, point counting was performed at x 4 0 0 magnification in order to obtain adequate sample sizes. Approximately 500 points were counted per lymph node metastasis. Nuclear morphometry was performed in 36 cases (174 lymph node metastases in total) using an interactive digitizing video overlay system (PRODIT, MSI Europe, Tilburg, The Netherlands) at a final magnification of approximately x 3000. Nuclei were systematically sampled16from the entire measurement field up to a sample size of 100 (or the maximum number of malignant nuclei), and the mean nuclear area (MNA) was calculated. Data evaluation For each patient and each feature (MC, VPE and MNA), the mean, SD, coefficient of variation (CV, 100% (SD/ mean)) and range (maximum -minimum) were calculated from the individual lymph node metastases data. Since the measurement techniques used introduce a certain variation themselves because of sampling and measurement errors, the CVs found had to be greater than the methodological tolerance limits (established in previous studies) to be interpreted as indicating real biological variation. These tolerance limits, established by long-term experience with quantitative techniques in many thousands of patients including large scale intraand inter-observer reproducibility studies3.11*12*1p16, were set to 20% for the MC. 10% for the VPE, and 10% for the MNA. Intra-patient CVs above these thresholds were therefore taken to indicate biological variation, but C V s lower than these thresholds could be explained by sampling and measurement errors. In order to evaluate whether the variation between lymph node metastases depended on their number, linear regression analysis was performed between the number of lymph node metastases on the one hand and range and CV on the other, taking a Pvalue of 0.05 as the level of significance.
Results Table 1 shows the CVs and ranges for the MC, VPE and MNA for the patients studied. In 38 of the 44 cases (86%)
Morphometrical variations in breast cancer metastases
259
Table 1. Intra-patient coefficient of variation and range between lymph node metastases of 45 breast cancer patients
Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
No. of lymph nodes 6 9 2 3 8 3 6 7 9 22 24 12 13 10 8 9 2 2 33 3 4 3 4 3 4 2 12 4 5 2 2 5 2
4 2 3 4
4 10 13 4 3 9 6
Mitotic count
Volume percentage epithelium
Mean nuclear area
cv
Range
cv
Range
cv
Range
53 65 24 15 42 22 29 48 24 49 54 54 61 27 89 24 6 0 56 19 28 13 40 62 45 113 26 40 25 76 87 20 42 36 76 134 53 67 41 63 83 29 77 69
0.1-0.8 0.4-3.1 0.5-0.7 0.4-0.7 0.1-0.5 6.5-10.2 2.2-5.5 0.9-3.4 1.2-3.7 0.6-8.1 0.2-1.9 0.25-3.0 0.0-2.5 1.5-3.7 0.0-5.8 0.54.4 1.1-1.2 2.5-2.5 0.5-7.4 0.8-1.1 0.3-0.6 2.4-3.1 1.o-2.2 0.3-1.4 0.8-2.6 0.3-2.7 1.2-4.8 0.3-0.8 1.l-1.8 0.1-0.3 0.7-3.0 0.4-0.6 0.7-1.3 1.1-2.9 0.1-0.3 0.0-4.3 1.5-5.0 0.1-0.5 0.6-3.2 0.5-9.9 0.0-1.4 0.6-1.1 0.0-1.1 0.9-6.9
3 3 28 17 1 4 3 13 9 5 38 11 21 2 23 9 8 0 22 5 4 9 22 7 2 5 8 8 19 3 12 12 34 5 6 36 13 41 13 14 19 46 2 21
92-98 89-97 25-38 66-90 90-99 91-99 88-95 72-99 73-99 82-99 21-97 6 7-9 9 36-96 93-98 50-9 3 73-98 86-96 75-75 46-9 7 88-98 67-73 74-88 46-71 81-92 95-98 89-96 75-98 79-9 5 62-92 92-96 82-9 7 73-97 44-72 85-98 86-9 3 47-99 67-91 52-95 63-90 63-97 66-9 5 38-89 92-97 56-92
14 17 6 6 10 6 6 16 14 NA NA NA NA NA NA NA 9 10 NA 11 4 21 15
5 5-76 50-77 59-65 52-58 37-50 53-60 67-76 49-76 56-88 NA NA NA NA NA NA NA 54-62 67-77 NA 43-52 54-60 55-83 83-1 1 5 59-62 49-84 44-8 3 64-8 5 53-64 69-82 46-48 61-67 71-84 55-77 69-75 46-50 62-78 47-57 47-56 70-99 61-75 75-78 53-66 46-60 58-71
NA=not available: CV =coefficient of variation.
3 28 43 8 8 7 3 6 10 23 4 6 16 8 13 12 8 2 11 9 7
260 P.].vun Diest et ul.
Figure 1. Illustration of variation in proliferation between lymph node metastases in case 40. a Lymph node metastasis showing low proliferation (mitotic count=0.5). b Lymph node metastasis with high proliferation (mitotic count= 9.9).
the CV for the MC was above the 20% tolerance limit, indicating that in most patients, there was real biological variation in proliferation between lymph node metastases. In case 40, the lowest and highest MCs found in the lymph node metastases were 0.5 (Figure l a ) and 9.9 (Figure lb), respectively, which denotes a difference as high as 9 4 mitoses per 10HPF. For the VPE, 2 1of the 44 cases (48%) showed CVs above the tolerance limit of lo%,with the highest range observed (case 11)of 21% to 9 7%. For the MNA, 1 7 of the 36 cases evaluated (47%) showed CVs above the 10%threshold. Case 26 showed the highest variation in MNA between lymph nodes ranging from 44.0 to 82.9 pm2. Linear regression showed significant positive linear correlations between the number of lymph node metastases and the MC range (P
Intra-patient variation between breast cancer axillary lymph node metastases using quantifiable features P. J .VAN DIEST, J. C .FLEEGE, E.MATZE-COK, J. P. A.BAAK lnstitwte for Pathology, Free University Hospital, Amsterdam, The Netherlands Date of submission 17 September 1991 Accepted for publication 13 March 1992
VAN DIEST P.J., PLEEGE J.C.,
MATZE-COK E. & B A A K J.P.A.
(1992) Histopathology 21, 257-262
Intra-patient variation between breast cancer axillary lymph node metastases using quantifiable features The intra-patient variations of some clinically relevant quantifiablefeatures, between axillary lymph node metastases were evaluated in 44 breast cancer patients. In all lymph node metastases detected (range 2-33 per patient), the mitotic figures were counted, the volume percentage epithelium was assessed and the mean nuclear area was measured. The intra-patient variation for each quantifiable feature was expressed by the coefficient of variation (CV). Since the measurement techniques used introduce a certain, well known variation themselves because of sampling and measurement errors, the CVs found had to be greater than methodological tolerance limits (established in previous studies) to be interpreted as indicating biological variation. The CVs exceeded the methodological tolerance l i t s in 86%of the cases for the mitotic count, in 48%of the cases for the volume percentage epithelium, and in 47%of the cases for the mean nuclear area. This indicated that in these cases, the variation found in the quantifiable features could not be explained by sampling or measurement errors and should be regarded as real biological variation. Furthermore, the variation in the quantifiable features studied showed a significant positive correlation with the number of lymph node metastases. Thus. there may be considerable intra-patient variation in quantifiable features between axillary lymph node metastases in breast cancer. This may indicate that these lymph node metastases originate independently from different clones within the primary tumour, that they are independently formed in different stages of tumour development,or that they, as an expression of intrinsic tumour heterogeneity, may develop in different directionsfrom the start. Keywords: breast cancer, proliferation, morphometry, stereology, lymph node metastases
Introduction Considering the relatively poor prognosis of breast cancer patients in spite of radical surgery, there is a strong need for prognostic factors that can identify patients at high risk of relapse to guide adjuvant therapy strategies. Lymph node status is a widely used prognostic factor', but is clearly not accurate enough in view of the fact that 5 years after the diagnosis, about 40% of patients are still alive despite the presence of lymph node metastases and 35% of patients without lymph node metastases have already diedz. Recently, additional Address for correspondence: Professor J.P.A.Baak.Institute for Pathology, Free University Hospital, PO Box 7057. 1007 IMB. Amsterdam, The Netherlands.
prognostic factors have been identified for both lymph node p ~ s i t i v e and ~ - ~lymph node negative breast cancer patients3e9*10.In lymph node positive patients, besides primary tumour factor^'^^*^*"-'^, quantitative data derived from the lymph node metastases such as proliferative activity and nuclear and tissue morphometric features may also be ~ s e f u l ~ - However, ~ , * ~ . despite the heterogeneity in tumour tissue that is generally observed, only one lymph node metastasis was evaluated per case in most of these studies, apparently assuming that the variation between lymph node metastases within the same patient was negligible. However, this assumption has not been shown to be correct. The aim of this study was therefore to evaluate the intrapatient variation of some clinically relevant quantifiable 257
2 58 P.1. van Diest et al.
features betweeq breast cancer axillary lymph node metastases.
Materials and methods PATIENTS' DETAILS A N D SPECIMEN PREPARATION
A total of 52 patients with invasive breast cancer metastatic to the axillary lymph nodes, diagnosed between 1971 and 1981, were considered for this study. All patients were treated with radical or modified radical mastectomy. Lymph nodes were identified in the fresh axillary dissection preparation, fixed in neutral buffered formalin and embedded in paraffin. Sections 4 pm thick were cut and stained with haematoxylin and eosin. All identified lymph nodes were reviewed and all lymph nodes containing metastatic breast cancer cells were included for all patients. Seven patients with only one lymph node metastasis and one patient having two lymph node metastases, one of them being unevaluable because of poor morphology, were excluded. The remaining 44 patients had, on average, 6.9 positive lymph nodes (range 2-33). A total of 305 lymph node metastases were evaluated. QUANTITATIVE METHODS
The most cellular area containing the highest density of mitotic figures was chosen subjectively by the same observer in all lymph node metastases of each case, rejecting areas with necrosis or many non-tumour cells. Although theoretically this may introduce bias, this procedure has been shown in several large studies, to be repr~ducible"*'~*'~~~~. In the selected area, a measurement field of approximately 0.5 x 0.5 cm2 (or the entire field containing metastatic cells in the case of metastases smaller than that size) was marked for counting mitotic figures, and the assessmentof volume percentage epithelium and nuclear area. These features were chosen because of their merent pathological significance (mitotic rate indicating proliferation, and epithelial percentage and nuclear area pointing at tissue and cellular differentiation, respectively) and their previously established prognostic ~ a l u e " ~ * ' ~ , Mitotic figures were counted in 10 consecutive high power fields (HPFs) at a x 4 0 0 magnification using a x 40 objective (numeric aperture of 0.75, field diameter 450 pm), starting at the region within the measurement area with the highest density of mitotic figures and from there on scanning in a random way. In order to cope with micro-metastases having less than 10 HPFs (15% of the lymph node metastases), the number of mitoses counted was expressed as the average number of mitoses
per field (the mitotic count). The mitotic count (MC) was assessed in all positive lymph nodes of all patients. The volume percentage of epithelium (VPE)versus stroma within the metastasis was assessed by point counting on a projection microscope at a magnification of approximately x 200 using a 42-points Weibel grid (test line length 1 5 mm). The whole measurement field was considered in each case, regarding only tumour stroma as stroma. The percentage of points falling on each compartment were taken as the volume percentages. In very small metastases, point counting was performed at x 4 0 0 magnification in order to obtain adequate sample sizes. Approximately 500 points were counted per lymph node metastasis. Nuclear morphometry was performed in 36 cases (174 lymph node metastases in total) using an interactive digitizing video overlay system (PRODIT, MSI Europe, Tilburg, The Netherlands) at a final magnification of approximately x 3000. Nuclei were systematically sampled16from the entire measurement field up to a sample size of 100 (or the maximum number of malignant nuclei), and the mean nuclear area (MNA) was calculated. Data evaluation For each patient and each feature (MC, VPE and MNA), the mean, SD, coefficient of variation (CV, 100% (SD/ mean)) and range (maximum -minimum) were calculated from the individual lymph node metastases data. Since the measurement techniques used introduce a certain variation themselves because of sampling and measurement errors, the CVs found had to be greater than the methodological tolerance limits (established in previous studies) to be interpreted as indicating real biological variation. These tolerance limits, established by long-term experience with quantitative techniques in many thousands of patients including large scale intraand inter-observer reproducibility studies3.11*12*1p16, were set to 20% for the MC. 10% for the VPE, and 10% for the MNA. Intra-patient CVs above these thresholds were therefore taken to indicate biological variation, but C V s lower than these thresholds could be explained by sampling and measurement errors. In order to evaluate whether the variation between lymph node metastases depended on their number, linear regression analysis was performed between the number of lymph node metastases on the one hand and range and CV on the other, taking a Pvalue of 0.05 as the level of significance.
Results Table 1 shows the CVs and ranges for the MC, VPE and MNA for the patients studied. In 38 of the 44 cases (86%)
Morphometrical variations in breast cancer metastases
259
Table 1. Intra-patient coefficient of variation and range between lymph node metastases of 45 breast cancer patients
Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
No. of lymph nodes 6 9 2 3 8 3 6 7 9 22 24 12 13 10 8 9 2 2 33 3 4 3 4 3 4 2 12 4 5 2 2 5 2
4 2 3 4
4 10 13 4 3 9 6
Mitotic count
Volume percentage epithelium
Mean nuclear area
cv
Range
cv
Range
cv
Range
53 65 24 15 42 22 29 48 24 49 54 54 61 27 89 24 6 0 56 19 28 13 40 62 45 113 26 40 25 76 87 20 42 36 76 134 53 67 41 63 83 29 77 69
0.1-0.8 0.4-3.1 0.5-0.7 0.4-0.7 0.1-0.5 6.5-10.2 2.2-5.5 0.9-3.4 1.2-3.7 0.6-8.1 0.2-1.9 0.25-3.0 0.0-2.5 1.5-3.7 0.0-5.8 0.54.4 1.1-1.2 2.5-2.5 0.5-7.4 0.8-1.1 0.3-0.6 2.4-3.1 1.o-2.2 0.3-1.4 0.8-2.6 0.3-2.7 1.2-4.8 0.3-0.8 1.l-1.8 0.1-0.3 0.7-3.0 0.4-0.6 0.7-1.3 1.1-2.9 0.1-0.3 0.0-4.3 1.5-5.0 0.1-0.5 0.6-3.2 0.5-9.9 0.0-1.4 0.6-1.1 0.0-1.1 0.9-6.9
3 3 28 17 1 4 3 13 9 5 38 11 21 2 23 9 8 0 22 5 4 9 22 7 2 5 8 8 19 3 12 12 34 5 6 36 13 41 13 14 19 46 2 21
92-98 89-97 25-38 66-90 90-99 91-99 88-95 72-99 73-99 82-99 21-97 6 7-9 9 36-96 93-98 50-9 3 73-98 86-96 75-75 46-9 7 88-98 67-73 74-88 46-71 81-92 95-98 89-96 75-98 79-9 5 62-92 92-96 82-9 7 73-97 44-72 85-98 86-9 3 47-99 67-91 52-95 63-90 63-97 66-9 5 38-89 92-97 56-92
14 17 6 6 10 6 6 16 14 NA NA NA NA NA NA NA 9 10 NA 11 4 21 15
5 5-76 50-77 59-65 52-58 37-50 53-60 67-76 49-76 56-88 NA NA NA NA NA NA NA 54-62 67-77 NA 43-52 54-60 55-83 83-1 1 5 59-62 49-84 44-8 3 64-8 5 53-64 69-82 46-48 61-67 71-84 55-77 69-75 46-50 62-78 47-57 47-56 70-99 61-75 75-78 53-66 46-60 58-71
NA=not available: CV =coefficient of variation.
3 28 43 8 8 7 3 6 10 23 4 6 16 8 13 12 8 2 11 9 7
260 P.].vun Diest et ul.
Figure 1. Illustration of variation in proliferation between lymph node metastases in case 40. a Lymph node metastasis showing low proliferation (mitotic count=0.5). b Lymph node metastasis with high proliferation (mitotic count= 9.9).
the CV for the MC was above the 20% tolerance limit, indicating that in most patients, there was real biological variation in proliferation between lymph node metastases. In case 40, the lowest and highest MCs found in the lymph node metastases were 0.5 (Figure l a ) and 9.9 (Figure lb), respectively, which denotes a difference as high as 9 4 mitoses per 10HPF. For the VPE, 2 1of the 44 cases (48%) showed CVs above the tolerance limit of lo%,with the highest range observed (case 11)of 21% to 9 7%. For the MNA, 1 7 of the 36 cases evaluated (47%) showed CVs above the 10%threshold. Case 26 showed the highest variation in MNA between lymph nodes ranging from 44.0 to 82.9 pm2. Linear regression showed significant positive linear correlations between the number of lymph node metastases and the MC range (P
