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Intratumoural heterogeneity of proliferation in invasive breast carcinoma evaluated with MIBI antibody
ThcBreusr(iYY7)6. 171-176 $1 1997 Pearwn Prol’essional Ltd
O R IGINAL ARTICLE
Intratumoural heterogeneity of proliferation in invasive breast carcinoma evaluated with M IBl antibody A. J. M. Connor, S. E. Pinder, C. W . Elston, J. A. Bell, P. Wencyk, J. F. R. Robertson, R. W . Blarney, R. I. Nicholson and I. 0. Ellis Department
of Histopathology,
City Hospital
NHS Trust, Nottingham,
UK
S U M MA R Y. The growth fraction has been assessed in 120 cases of primary operable breast carcinoma using the MIB 1 antibody and an index of the cycling fraction has been derived for the periphery and the centre of different histological types of breast cancer. The mean MIBl labelling index for each tumour showed a strong association with histological grade (P < 0.001) tumour type sub-group (P < O.OOl), tumour size (P = 0.029), the presence of vascular invasion (P = 0.050) and the presence of distant metastases (P = 0.043). In addition both the peripheral (P = 0.005) and mean (P = 0.048) but not the central (P = 0.069) MIB 1 labelling showed a correlation with overall survival, when those cases in the upper quartile were compared with those showing fewer cycling cells. A significantly greater MIB 1 labelling index was seen at the periphery than the centre (P < 0.001). This difference in the cycling fraction was not associated with a bias in tumour grade or size. When the data were analysed according to tumour type it was seen that in invasive ductal/no special type (NST) of grades 2 and 3, tubular mixed and lobular carcinomas the peripheral index was significantly higher than the central index (P = 0.002, P = 0.003, P = 0.024 and P = 0.004 respectively). No significant difference in peripheral and central proliferation index was seen in tubular, medullary and atypical medullary or mixed NST and lobular carcinomas (P = 0.480, P = 0.089, P = 0.069 respectively). When tumours were grouped into 3 categories according to the ratio of MIB 1 labelling (i.e. those with (i) relatively greater central, (ii) relatively greater peripheral and (iii) homogeneous cell cycling fractions), it was found that the central index was comparable (P = 0.243). Peripheral MIBl immunostaining was however significantly different (P = 0.049) in the three groups. Thus, the heterogeneity in proliferation seen in this series was not apparently caused by a relative fall in proliferation in the centre of the tumour but to a relative increase in cell cycling fraction at the periphery. INTRODUCTION
with screening practice,‘“.” with higher histological grade associated with increased tumour size.‘* It seems probable that changes in biological behaviour, such as the suggested progression of tubular carcinoma to a less differentiated form,9,‘3 may begin at the periphery. W e have therefore examined the proliferative behaviour of primary breast carcinomas by determination of the fraction of cells which are not in the GO phase of the cell cycle at the periphery and the centre using the MIBl monoclonal antibody lJ and applied this to different histological tumour types. W e aimed in this way to highlight any potential sites of disproportionate growth.
In 1957 when Bloom & Richardson published their criteria for grading carcinoma of the breast, they commented on the great heterogeneity observed within tumours.’ Since then interest in the heterogeneous nature of breast carcinoma has increased and it has been suggested that the growth rate is greater at the periphery than the centre of breast cancers.‘l However, not all authors have found this to be the case.’ Tumour progression from in situ to invasive to metastatic disease has also been reported to be associated with an increase in proliferation rate by some groups6,7but not others.8 Taylor & Norris’ first reported the relatively increased proportion of tumours of tubular type detected by breast screening. Other authors have confirmed that larger and higher grade carcinomas are seen in symptomatic compared
MATERIALS
AND METHODS
Antibody MIBl is a murine monoclonal antibody raised against recombinant parts of the Ki-67 antigen which is expressed
correspondence ro: S. E. Pinder, Department of Histopathology, City Hospital NHS Trust, Hucknall Road, Nottingham NG5 IPB, UK
Address
171
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The Breast
by cells throughout the proliferation cycle, from Gl to mitosis.‘5.‘6 The antibody can thus be used to identify the growth-fraction and can be used in formalin-fixed, paraffinembedded tissue with a microwave antigen retrieval technique making assessment of archival material possible.
Patients One hundred and twenty cases of primary invasive carcinoma of the breast were selected from the Nottingham/ Tenovus Primary Breast Carcinoma series. The only bias was to include sufficient cases of the less common types of tumour, such as pure tubular carcinoma. All patients were aged 70 or less and had primary operable invasive breast carcinoma treated under the care of one surgical team (RWB, JFRR). The patients received either mastectomy or wide local excision with intact breast irradiation, but no systemic adjuvant treatment was given to this group. All patients were followed up 3-monthly for 24 months, then 6-monthly to 5 years and annually thereafter. Patient data, including menopausal status, were known. Oestrogen receptor status was assessed in these patients by the dextran coated charcoal method and a cut off of 5 fmol/mg was used to define positivity. Tumours were incised in the fresh state, measured and a sample taken for oestrogen receptor analysis; the remainder was then fixed in phosphate buffered formalin, processed routinely and paraffin wax embedded. Routine haematoxylin and eosin stained sections were examined for determination of histological grade” and histological type.18 Lymph node stage was classified as 1,2 or 3 corresponding to none, 3 or less, or 4 or more involved lymph nodes respectively. Tumour type was also sub-classified into 4 types for further analysis as described previously.” In this classification tumours are grouped according to prognosis; lesions with an excellent prognosis (> 80% lo-year survival) include tubular, invasive cribriform, mutinous and tubulo-lobular carcinomas. Tumour with a good prognosis (6@-80% lo-year survival) include alveolar lobular, tubular mixed and mixed ductal with special type cancers. Carcinomas with an moderate prognosis (5060% lo-year survival) include classical lobular, medullary, atypical medullary and classical lobular types. The poor prognosis group (< 50% lo-year survival) includes lobular mixed, mixed ductal and lobular, ductal no special type (NST) and solid lobular carcinoma. Tumour size was defined as less than 15 mm, 15-24 mm or 25 mm or more. Vascular invasion was categorised as definite or absent.”
Immunohistochemistry MIBl
immunostaining
was performed
on 3 pm sections
mounted on slides which had previously been coated with 3-aminopropyltriethoxy-silane to increase adhesiveness which may be reduced by microwaving. The primary MIB 1 antibody was a donation from Johannes Gerdes (Borstel, Germany) and was used at 1:30 dilution. Sections were stained using a standard avidinfbiotin complex technique. After blocking endogenous peroxidase activity, sections were immersed in citrate buffer and microwaved (8OOW Panasonic) for 10 min on the high setting and then 10 min on the low setting (50% power). After incubation with the primary antibody, the sections were incubated with biotinylated goat anti-mouse/rabbit Ig G, followed by avidin/biotin complex (Dako Ltd, High Wycombe, UK) and then developed using diaminobenzidinethydrogen peroxide solution. Sections were weakly counter-stained with a solution of haematoxylin. Appropriate positive control sections of tonsil were used.
Assessment of immunostaining In order to compare the central and peripheral regions of the tumour a working definition of each was formulated: the periphery was defined as any outer microscopic field (X 40 objective lens) in which invasive tumour was seen immediately adjacent to fat or normal breast tissue. In situ carcinoma was not assessed. Any other area was considered to be ‘central’, although with large sections care was taken to examine the middle of the mass. A graticule (E 11-21 mm, 1 mm grid, from Graticules Ltd) was used with a central zone of 5 x 5 squares highlighted to reduce the area of each field (0.0255/mm2). Within this area the percentage of cells showing immunoreactivity was counted. After construction of running mean graphs, a total of 20 fields was found to be a satisfactory area of assessment (0.45 mm’), in order to counteract the effects of heterogeneity within high power fields. Within each area (peripheral or central) fields were selected randomly as for mitosis counting. Care was taken not to count either fields or individual cells more than once. Ten per cent of cases were re-assessed blind to investigate intra-observer variation.
Statistics A proliferation percentage was calculated for the central and peripheral regions from the number of cells showing any nuclear immunoreactivity and the total number of cells counted. The MIB 1 indices at the periphery and centre were compared for the entire series and in groups defined by histological type and grade. The Wilcoxon Signed Rank Test (StatView 4 programme on an Apple Macintosh) was used to compare the two regions in each group. Correlations with other pathological and clinical variables were sought
Intratumoural heterogeneity of proliferation in invasive breast carcinomas evaluted with MIB 1 antibody
173
Table 1 Comparison of central and peripheral MIB 1 labelling indices for tumour type (Wilcoxon Signed Rank) Histological type
Total number
Grade: number
2
P
NST (grade 2) NST (grade 3) Tubular mixed
18 21 23
-3.159 -2.984 -2.25 1
0.002 0.003 0.024
Lobular (all sub-types)
19
All grade 2 All grade 3 Grade 1: 15 Grade 2: 6 Grade 3: 2 Grade 1: 2 Grade 2: 14 Grade 3: 3 All grade 3
-2.857
0.004
-0.135
0.893
-0.405
0.686
-0.706 -1.069
0.480 0.285
-5.903
Medullary and atypical medullary
5
Ductal with lobular
5
Pure tubular Other ‘special types’
12 3
All histological types
112
using Kruskall-Wallis and Mann-Whitney analyses. Survival was assessed by life table analysis (Mantel-Cox).
RESULTS Eight cases were excluded from the study because there was insufficient tumour tissue or insufficient structures indicating the presence of a definite periphery. Immunostaining in all cases with the MIBI antibody was discrete and no difficulty was experienced in attributing positivity. The stain was generally confined to the nucleus, varying in intensity up to a maximum associated with mitosis. Immunostaining was scattered throughout the microscopic fields in all areas and no clear excess of nuclear positivity was seen at the stromal-tumour interface. The percentage of cells showing MIB 1 positivity ranged from 0.1 to 52.56 for the central area (median 8.68, mean 10.41) and 0.25 to 51.62 for the periphery (median 11.80, mean 12.98). On comparison the reassessment of data was found to be satisfactory with no significant difference between assessments (P = 0.824). Analysis of the series overall showed that the periphery had a greater MIBl labelling index than the centre of the breast carcinomas (P < 0.0001). The tumour types in this series were as follows: pure tubular and tubular mixed carcinoma, lobular carcinoma, medullary and atypical medullary, mixed ductal and lobular, other special types excluding pure tubular and ductal NST of histological grades 2 and 3. No grade 1 NST tumours were identified in this study. When the data were analysed according to histological type (Table l), those tumours of NST, tubular mixed and lobular types were found to demonstrate a significant difference between the central and peripheral indices. Other histological types, including mixed NST with lobular, medullary, atypical medullary and pure tubular carcinomas
Grade 2: 3 Grade 3: 2 All grade I Grade 1: 2 Grade 2: I
showed homogeneous staining throughout the surface area with no significant difference in MIBl labelling between periphery and centre of these tumour types. Correlations with other well recognized prognostic variables and survival were sought. The peripheral MIB 1 index showed a strong association with higher histological grade (P < 0.001 ), the four prognostic tumour type groups (P < 0.00 1), larger tumour size (P = 0.029), the presence of vascular invasion (P = 0.003) and of distant metastases (P = 0.010) (Table 2). The mean proliferation index for each tumour showed the same correlations with grade (P < 0.001) tumour type group (P < 0.001) tumour size (P = 0.017) vascular invasion (P = 0.018) and distant metastases (P = 0.043) and no additional associations were seen. The central MIBl labelling index showed associations with histological grade (P < 0.001) tumour type group (P < 0.001) and size (P = 0.029) only; no relationship between the central MIB 1 labelling index and the presence of either vascular invasion or distant metastatic depostis was identified. Tumours were grouped into two categories, with the carcinomas in the upper quartile for MIB 1 labelling for each area compared with those showing a lower cell cycling fraction. A significant association between the peripheral MIB 1 Table 2 MIB 1 labelling index at the periphery: correlations with other known prognostic variables Prognostic variable Histological grade (l-3) Histological type (tumour type groups - see text) Tumour size (~15. 15-24, >24 mm) Vascular invasion (absent or present) Distant metastases (absent or present) Lymph node stage (l-3) Age (<40,41-50. 51-60, >60 years) Menopausal status (pm or post menopausal) Oestrogen receptor status (5 fmol/mg protein)
P
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labelling and overall survival of the patients was seen (x2 = 8.05, P = 0.005). This association with survival was also found when the mean MIBl labelling index for the tumour overall was used (x2 = 3.92, P = 0.048). No significant difference in survival was seen between those tumours in the upper quartile of MIBl expression in the centre and those showing lower central immunostaining (x2 = 3.32, P = 0.069). However, when tumours were divided into groups around the median labelling score in each area, the peripheral (x2 = 6.05, P = 0.014), central (x’ = 5.16, P = 0.023) and mean (x2 = 7.3 1, P = 0.007) MIB 1 labelling were all significantly associated with overall survival. The ratio of peripheral to central immunoreactivity was examined and a range of values from 0.61 to 18.35 (mean 1.76) was seen. Three groups were then defined: (a) tumours with a peripheral:central index of more than 1 .l and thus with a relatively greater MIBl expression at the periphery than in the centre (71 cases); (b) those with a peripheral:central index of 0.9-l .l with essentially homogeneous immunoreactivity (24 cases); and (c) carcinomas where the ratio was less than 0.9 and which thus had greater central than peripheral MIB 1 labelling (17 cases). For these categories the pathological features which are known to show a statistical association with the proliferation index were compared; no significant difference in the size of the lesions in the three groups was found (P = 0.061) and no difference in histological grade was identified (P = 0.842). Importantly, grouped according to the ratio of cell cycling fraction and thus by the area of relatively highest proliferation, the central MIBl immunoreactivity was found to be not significantly different in the three categories (P = 0.243). The peripheral index was, however, significantly different (P = 0.049). Thus, the periphery of the tumours was the site of difference in the groups and the heterogeneity identified in MIBl labelling was apparently caused by a genuine increase in the peripheral cycling fraction rather than a fall in the central index.
DISCUSSION Many variables assessed in breast carcinomas have at some time been demonstrated to display a degree of heterogeneity. Steroid receptor heterogeneity*‘,** is well documented and positive and negative cells have been seen concurrently in tumours assayed for both oestrogen and progesterone receptors. Heterogeneous DNA ploidy,23,24 tumour suppressor gene and oncogene expression within breast tumours have also been reported.25s26Tumour heterogeneity has been noted with almost all methods of assessment of proliferation including thymidine labelling, S-phase fraction27 and bromodeoxyuridine examination.*’ Some of these, however, require examination of disaggregated
tumour and thus specific areas within an individual carcinoma cannot be assessed. Meyer & Wittlif? observed a 61% rate of heterogeneity for Thymidine Labelling Index within primary breast carcinomas. Studies of S-phase fraction and Ki-67 index in breast carcinoma have reported heterogeneity with proliferation higher near the tumour border2,29but not all series have reported similar results; in 34 breast cancer cytology samples and frozen sections Kuenen-Boumeester et al found no difference in the Ki-67 labelling index at the periphery and the centre.5 However, cryostat examination of Ki-67 immunoreactivity requires trimming of frozen blocks and the edge of the tumour may be more easily lost. By using the MIBl antibody on paraffin sections more tissue is available for block selection and the entire tumour architecture and surrounding structures can be assessed to evaluate fully any heterogeneity present. The quality and range of staining with the MIB 1 antibody reported here is comparable to data reported elsewhere3’ although the mean and median in this series are lower than those found by Pinder et al in a different series of patients.3’ This may in part be due to the different means of assessing immunostaining in the two studies; in the previous series image analysis was used. The lower mean and median values may also be related to the case bias in the present study, whereby sufficient numbers of low grade ‘special type’ carcinomas with relatively low proliferative rates were included. The associations with other known prognostic factors which have been previously reported are confirmed in this series; in particular a strong relationship between MIBl labelling and histological grade was noted at both the periphery and centre of the tumours.3’.32 Assessment of the cycling fraction also showed a significant association with overall survival for both areas and the overall mean score of the tumour when using the median value as a cut-off point. However, when the upper quartile of tumours was compared with those showing lesser labelling the central index showed no association with survival thus demonstrating the difficulties in selecting reliable cut off points for continuous variables. When tumours which had a relatively higher fraction of cycling cells at their centre than their periphery were compared with those that had either homogeneous intratumoural expression or relatively higher peripheral MIBl labelling, the central proliferation indices were found to be essentially similar overall. This was the case despite an initial selection bias which resulted in inclusion of increased numbers of those carcinomas subsequently found to show homogeneous immunoreactivity. Whilst it has been suggested that differences in proliferation within tumours may be due to central anoxia and an associated fall in central proliferation, these data do not support this argument. In this series, the turnouts overall showed an increase in
Intratumoural heteroneneitv of uroliferation in invasive breast carcinomas evaluted with MIB 1 antibodv peripheral (rather than decreased central) cycling fraction. Differences in blood supply may, nevertheless, be responsible for the heterogeneity identified, the periphery obtaining a greater blood supply through induction of angiogenesis by, for example, the acute inflammatory response (which is maximal at the periphery”). Indeed, heterogeneity in blood vessel pattern is also well recognized in breast carcinomas.3” The relationship between histological type of breast carcinoma and tumour heterogeneity has not been previously explored. In general, greater proliferative activity was seen at the periphery than the centre of the tumours in this series, as has been described by other authors, but this phenomenon was not noted in all histological types of breast carcinoma. In tubular mixed carcinomas and in NST tumours of grades 2 and 3 the MIBl labelling index was greater at the periphery than the centre (P = 0.0244, 0.0016, 0.0028 respectively). However, no difference was found between the central and peripheral indices of pure tubular carcinomas, nor was heterogeneity in the proportion of cycling cells seen in medullary or atypical medullary cancers. It has been proposed that tubular carcinomas may undergo and thus progress to a less differenti‘de-differentiation”,” ated, tubular mixed morphology” by a process better termed ‘clonal evolution’. If the NST/ductal tumour morphology which is seen at the periphery of tubular mixed carcinomas is proliferating more rapidly, as this study suggests, it seems logical to predict that with time this may become the predominant pattern of the tumour. Conversely, other tumours appear to be de novo of high histological grade and homogeneous with regard to proliferation: such a pattern is seen in medullary carcinomas. Alternative explanations for heterogeneity of MIBl expression such as variation in the length of the cell cycle or arrest of the cycle with retention of the Ki-67 antigen (without entry into mitosis) seem improbable. Indeed, prolongation of the cell cycle length, for example of Molt-4 human leukaemia cells, with progression arrest in Gl and G2 phases may result in undetectable Ki-67 antigen immunohistochemically.“’ Thus, the fraction of cells expressing Ki-67 may be smaller than the true growth fraction as estimated by 5bromodeoxyuridine labelling.3” Conversely other studies have shown that halting the cycle beyond GO but before S phase with protein kinase C inhibitors may result in a paradoxical increase in Ki-67 expression compared to thymidine uptake.3h This may be significant in that tamoxifen may function as a protein kinase C inhibitor and it has therefore been suggested that Ki-67 is not a reliable indicator of proliferation after such treatments.‘h However. in this series we have evaluated MIB 1 expression in patients who have received no adjuvant systemic treatment. It should also, however, be noted that we have not examined proliferation per se in this series of cases of invasive breast carcinoma. Although other authors have reported an
175
association between Ki-67 antigen immunoreactivity and mitotic count? we have not directly correlated mitotic count with MIBl labelling in this study. We nevertheless feel that these data support the contention that when counting mitoses as a component of histological grade the periphery of the tumour should be searched particularly thoroughly; this is most frequently the site of greatest cell cycling fraction in primary invasive breast carcinoma. References I. Bloom H J, Richardson W W. Histological grading and prognosis in breast cancer. A study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer 1957: II: 359-377. 2. Verhoeven D, Bourgeois N, Derde M P, Kaufman L. Buyssens N. Comparison of cell growth in different parts of breast cancers. Histopathology 1990; 17: 505-509. 3. Baak J P. Mitosis counting in turnours. Hum Pathol 1990: 21: 683-685. 4. Meyers J S, Wittliff J L. Regional heterogeneity in breast carcinoma: thymidine labelling index, steroid hormone receptors. DNA ploidy. Int J Cancer 1991: 47: 213-220. .5 Kuenen-Boumeester V. Van Der Kwast T H. Van Laarhoven H A, Henzen-Logmans S C. Ki-67 staining in histological subtypes of breast carcinoma and fine needle aspiration smears. J Clin Pathol 1991; 44: 208-210. 6. Pavelic Z P, Pavelic L, Lower E E, Gapany S, Barker E A, Priesler H D. c-myc. c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and non-invasive breast carcinoma. Cancer Res 1992; 2597-2602. 7. Sasa M, Komaki K, Tsuzuki H et al. Tumor progression accompanied by increase in proliferation rate in breast cancer: a preliminary report. J Surg Oncol 1993; 54: 255-259. 8. Pence J C, Kilibash A L, Kerns B-J 0, Marks J R. Iglehart J D. Proliferation index in various stages of breast cancer determined by Ki-67 immunostaining. J Surg Oncol I99 I; 48: I I-20. 9. Taylor H B, Norris H J. Well-differentiated carcinoma of the breast. Cancer 1970; 6877692. IO. Pinder S E, Ellis I 0. Breast screening pathology. Surgery 1994: 12: 277-278. Il. Ellis I 0, Galea M H, Locker A et al. Early experience in breast cancer screening: emphasis on development of protocols for triple assessment. The Breast 1993; 2: 1488153. 12. Duffy S W. Tabar L. Fagerberg G et al. Breast screening. prognostic factors and survival-results from the Swedish two county study. Br J Cancer 1991; 64: 1133-l 138. 13. Cooper H S, Patchefsky A S, Krall R A. Tubular carcinoma of the breast. Cancer 1978: 42: 2334-2342. 14. Cattoretti Cl, Becker M H, Key G et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB I and MIB3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992; 168: 357-363. 15. Gerdes J, Schwab U. Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983; 3 I : 13-20. 16. Gerdes J, Lemke J, Baisch H. Walker H H. Schwab U, Stein H. Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984: 133: 1710-1715. I7 Elston C W, Ellis 1 0. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow up. Histopathology 1991; 19: 403410. IX Ellis I 0, Galea M, Broughton N, Locker A. Blarney R W, Elston C W. Pathological prognostic factors in breast cancer. II. Histological type. Relationship with survival in a large study with long-term follow-up. Histopathology 1992: 20: 479489.
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19. Pereira H, Pinder S E, Sibbering D M et al. Pathological prognostic factors in breast cancer IV. Should you be a typer or grader? A comparative study of two histological prognostic features in operable breast cancer. Histopathology 1995; 27: 219-226. 20. Pinder S E, Ellis I 0, Galea M, O’Rourke S, Blarney R W, Elston C W. Pathological prognostic factors in breast cancer. III. Vascular invasion: relationship with recurrence and survival in a large study with long-term follow-up. Histopathology 1994; 24: 4147. 21. Davis B W, Zava D T, Lecher Cl W, Goldhirsch A, Hartman W H. Receptor heterogeneity of human breast cancer as measured by multiple intratumoral assays of estrogen and progesterone receptor. Br J Cancer Clin Oncol 1984; 20: 375-382. 22. Poulson H S, Jensen J, Hermansen C. Human breast cancer: heterogeneity of estrogen binding sites. Cancer 1981; 43: 1791. 23. Visscher D W, Shaheen C, Drozdowicz S, Crissman J D. Image cytophotometric DNA histogram heterogeneity in adenocarcinoma of the breast. Anal Quant Cytol Histol 1993; 15: 206-212. 24. Beerman H, Smit V T, Kluin P M, Bonsing B A, Hermans .I, Cornelisse C J. Flow cytometric analysis of DNA stemline heterogeneity in primary and metastatic breast cancer. Cytometry 1991; 12: 147-154. 25. Pavelic Z P, Pavelic K, Carter C P, Pavelic L. Heterogeneity of c-myc expression in histologically similar infiltrating ductal carcinomas of the breast. J Cancer Res Clin Oncol 1992; 118: 16-22. 26. Trudel M, Mulligan L, Cavenee W, Margolese R, Cote J, Gariepy G. Retinoblastoma and ~53 gene product expression in breast carcinoma: immunohistochemical analysis and clinicopathologic correlation. Hum Pathol 1992; 23: 1388-1394. 27. Siitonen S M, Isola J J, Rantala I S, Helin H J. Intratumor variation in cell proliferation in breast carcinoma as determined by antiproliferating cell nuclear antigen monoclonal antibody and
automated image analysis. Am J Clin Path01 1993; 99: 22623 1. 28. Going J J, Stanton P D, Cooke T G. Influences on measurement of cellular proliferation by histology and flow cytometry in mammary carcinomas labelled in vivo with bromodeoxyuridine. Am J Clin Path01 1993; 100: 218-222. 29. Lee A K, Wiley B, Loda M et al. DNA ploidy, proliferation, and neu-oncogene protein overexpression in breast carcinoma. Mod Path01 1992; 5: 61-67. 30. Barbareschi M, Girlando S, Mauri F M et al. Quantitative growth fraction evaluation with MIBl and Ki67 antibodies in breast carcinomas. Am J Clin Path01 1994; 102: 171-175. 3 1. Pinder S E, Wencyk P, Sibbering D M et al. Assessment of the new proliferation marker MIB 1 in breast carcinoma using image analysis: associations with other prognostic factors and survival. Br J Cancer 1995; 71: 146149. 32. Weidner N, Moore D, Vartanian R. Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel ‘paraffin’-reactive MIB 1 antibody. Hum Path01 1994; 25: 337-342. 33. Weidner N, Semple J P, Welch W R, Folkman J. Tumor angiogenesis and metastatis - correlation in invasive breast carcinoma. N Engl J Med 1991; 324: 1-8. 34. Verhoeven D, Van Marck E. Proliferation, basement membrane changes, metastasis and vascularization patterns in human breast cancer. Path01 Res Pratt 1993; 189: 851-861. 35. Tsurusawa M, Fujimoto T. Cell cycle progression and phenotypic modification of Ki67 antigen-negative Gl- and G2-phase cells in phorbol ester-treated Molt-4 human leukaemia cell. Cytometry 1995; 20: 146-153. 36. Couldwell W T, Weiss M H, Law R E, Hinton D R. Paradoxical elevation of Ki-67 labelling with protein kinase inhibition in malignant gliomas. 3 Neurosurg 1995; 82: 461-468.
O R IGINAL ARTICLE
Intratumoural heterogeneity of proliferation in invasive breast carcinoma evaluated with M IBl antibody A. J. M. Connor, S. E. Pinder, C. W . Elston, J. A. Bell, P. Wencyk, J. F. R. Robertson, R. W . Blarney, R. I. Nicholson and I. 0. Ellis Department
of Histopathology,
City Hospital
NHS Trust, Nottingham,
UK
S U M MA R Y. The growth fraction has been assessed in 120 cases of primary operable breast carcinoma using the MIB 1 antibody and an index of the cycling fraction has been derived for the periphery and the centre of different histological types of breast cancer. The mean MIBl labelling index for each tumour showed a strong association with histological grade (P < 0.001) tumour type sub-group (P < O.OOl), tumour size (P = 0.029), the presence of vascular invasion (P = 0.050) and the presence of distant metastases (P = 0.043). In addition both the peripheral (P = 0.005) and mean (P = 0.048) but not the central (P = 0.069) MIB 1 labelling showed a correlation with overall survival, when those cases in the upper quartile were compared with those showing fewer cycling cells. A significantly greater MIB 1 labelling index was seen at the periphery than the centre (P < 0.001). This difference in the cycling fraction was not associated with a bias in tumour grade or size. When the data were analysed according to tumour type it was seen that in invasive ductal/no special type (NST) of grades 2 and 3, tubular mixed and lobular carcinomas the peripheral index was significantly higher than the central index (P = 0.002, P = 0.003, P = 0.024 and P = 0.004 respectively). No significant difference in peripheral and central proliferation index was seen in tubular, medullary and atypical medullary or mixed NST and lobular carcinomas (P = 0.480, P = 0.089, P = 0.069 respectively). When tumours were grouped into 3 categories according to the ratio of MIB 1 labelling (i.e. those with (i) relatively greater central, (ii) relatively greater peripheral and (iii) homogeneous cell cycling fractions), it was found that the central index was comparable (P = 0.243). Peripheral MIBl immunostaining was however significantly different (P = 0.049) in the three groups. Thus, the heterogeneity in proliferation seen in this series was not apparently caused by a relative fall in proliferation in the centre of the tumour but to a relative increase in cell cycling fraction at the periphery. INTRODUCTION
with screening practice,‘“.” with higher histological grade associated with increased tumour size.‘* It seems probable that changes in biological behaviour, such as the suggested progression of tubular carcinoma to a less differentiated form,9,‘3 may begin at the periphery. W e have therefore examined the proliferative behaviour of primary breast carcinomas by determination of the fraction of cells which are not in the GO phase of the cell cycle at the periphery and the centre using the MIBl monoclonal antibody lJ and applied this to different histological tumour types. W e aimed in this way to highlight any potential sites of disproportionate growth.
In 1957 when Bloom & Richardson published their criteria for grading carcinoma of the breast, they commented on the great heterogeneity observed within tumours.’ Since then interest in the heterogeneous nature of breast carcinoma has increased and it has been suggested that the growth rate is greater at the periphery than the centre of breast cancers.‘l However, not all authors have found this to be the case.’ Tumour progression from in situ to invasive to metastatic disease has also been reported to be associated with an increase in proliferation rate by some groups6,7but not others.8 Taylor & Norris’ first reported the relatively increased proportion of tumours of tubular type detected by breast screening. Other authors have confirmed that larger and higher grade carcinomas are seen in symptomatic compared
MATERIALS
AND METHODS
Antibody MIBl is a murine monoclonal antibody raised against recombinant parts of the Ki-67 antigen which is expressed
correspondence ro: S. E. Pinder, Department of Histopathology, City Hospital NHS Trust, Hucknall Road, Nottingham NG5 IPB, UK
Address
171
172
The Breast
by cells throughout the proliferation cycle, from Gl to mitosis.‘5.‘6 The antibody can thus be used to identify the growth-fraction and can be used in formalin-fixed, paraffinembedded tissue with a microwave antigen retrieval technique making assessment of archival material possible.
Patients One hundred and twenty cases of primary invasive carcinoma of the breast were selected from the Nottingham/ Tenovus Primary Breast Carcinoma series. The only bias was to include sufficient cases of the less common types of tumour, such as pure tubular carcinoma. All patients were aged 70 or less and had primary operable invasive breast carcinoma treated under the care of one surgical team (RWB, JFRR). The patients received either mastectomy or wide local excision with intact breast irradiation, but no systemic adjuvant treatment was given to this group. All patients were followed up 3-monthly for 24 months, then 6-monthly to 5 years and annually thereafter. Patient data, including menopausal status, were known. Oestrogen receptor status was assessed in these patients by the dextran coated charcoal method and a cut off of 5 fmol/mg was used to define positivity. Tumours were incised in the fresh state, measured and a sample taken for oestrogen receptor analysis; the remainder was then fixed in phosphate buffered formalin, processed routinely and paraffin wax embedded. Routine haematoxylin and eosin stained sections were examined for determination of histological grade” and histological type.18 Lymph node stage was classified as 1,2 or 3 corresponding to none, 3 or less, or 4 or more involved lymph nodes respectively. Tumour type was also sub-classified into 4 types for further analysis as described previously.” In this classification tumours are grouped according to prognosis; lesions with an excellent prognosis (> 80% lo-year survival) include tubular, invasive cribriform, mutinous and tubulo-lobular carcinomas. Tumour with a good prognosis (6@-80% lo-year survival) include alveolar lobular, tubular mixed and mixed ductal with special type cancers. Carcinomas with an moderate prognosis (5060% lo-year survival) include classical lobular, medullary, atypical medullary and classical lobular types. The poor prognosis group (< 50% lo-year survival) includes lobular mixed, mixed ductal and lobular, ductal no special type (NST) and solid lobular carcinoma. Tumour size was defined as less than 15 mm, 15-24 mm or 25 mm or more. Vascular invasion was categorised as definite or absent.”
Immunohistochemistry MIBl
immunostaining
was performed
on 3 pm sections
mounted on slides which had previously been coated with 3-aminopropyltriethoxy-silane to increase adhesiveness which may be reduced by microwaving. The primary MIB 1 antibody was a donation from Johannes Gerdes (Borstel, Germany) and was used at 1:30 dilution. Sections were stained using a standard avidinfbiotin complex technique. After blocking endogenous peroxidase activity, sections were immersed in citrate buffer and microwaved (8OOW Panasonic) for 10 min on the high setting and then 10 min on the low setting (50% power). After incubation with the primary antibody, the sections were incubated with biotinylated goat anti-mouse/rabbit Ig G, followed by avidin/biotin complex (Dako Ltd, High Wycombe, UK) and then developed using diaminobenzidinethydrogen peroxide solution. Sections were weakly counter-stained with a solution of haematoxylin. Appropriate positive control sections of tonsil were used.
Assessment of immunostaining In order to compare the central and peripheral regions of the tumour a working definition of each was formulated: the periphery was defined as any outer microscopic field (X 40 objective lens) in which invasive tumour was seen immediately adjacent to fat or normal breast tissue. In situ carcinoma was not assessed. Any other area was considered to be ‘central’, although with large sections care was taken to examine the middle of the mass. A graticule (E 11-21 mm, 1 mm grid, from Graticules Ltd) was used with a central zone of 5 x 5 squares highlighted to reduce the area of each field (0.0255/mm2). Within this area the percentage of cells showing immunoreactivity was counted. After construction of running mean graphs, a total of 20 fields was found to be a satisfactory area of assessment (0.45 mm’), in order to counteract the effects of heterogeneity within high power fields. Within each area (peripheral or central) fields were selected randomly as for mitosis counting. Care was taken not to count either fields or individual cells more than once. Ten per cent of cases were re-assessed blind to investigate intra-observer variation.
Statistics A proliferation percentage was calculated for the central and peripheral regions from the number of cells showing any nuclear immunoreactivity and the total number of cells counted. The MIB 1 indices at the periphery and centre were compared for the entire series and in groups defined by histological type and grade. The Wilcoxon Signed Rank Test (StatView 4 programme on an Apple Macintosh) was used to compare the two regions in each group. Correlations with other pathological and clinical variables were sought
Intratumoural heterogeneity of proliferation in invasive breast carcinomas evaluted with MIB 1 antibody
173
Table 1 Comparison of central and peripheral MIB 1 labelling indices for tumour type (Wilcoxon Signed Rank) Histological type
Total number
Grade: number
2
P
NST (grade 2) NST (grade 3) Tubular mixed
18 21 23
-3.159 -2.984 -2.25 1
0.002 0.003 0.024
Lobular (all sub-types)
19
All grade 2 All grade 3 Grade 1: 15 Grade 2: 6 Grade 3: 2 Grade 1: 2 Grade 2: 14 Grade 3: 3 All grade 3
-2.857
0.004
-0.135
0.893
-0.405
0.686
-0.706 -1.069
0.480 0.285
-5.903
Medullary and atypical medullary
5
Ductal with lobular
5
Pure tubular Other ‘special types’
12 3
All histological types
112
using Kruskall-Wallis and Mann-Whitney analyses. Survival was assessed by life table analysis (Mantel-Cox).
RESULTS Eight cases were excluded from the study because there was insufficient tumour tissue or insufficient structures indicating the presence of a definite periphery. Immunostaining in all cases with the MIBI antibody was discrete and no difficulty was experienced in attributing positivity. The stain was generally confined to the nucleus, varying in intensity up to a maximum associated with mitosis. Immunostaining was scattered throughout the microscopic fields in all areas and no clear excess of nuclear positivity was seen at the stromal-tumour interface. The percentage of cells showing MIB 1 positivity ranged from 0.1 to 52.56 for the central area (median 8.68, mean 10.41) and 0.25 to 51.62 for the periphery (median 11.80, mean 12.98). On comparison the reassessment of data was found to be satisfactory with no significant difference between assessments (P = 0.824). Analysis of the series overall showed that the periphery had a greater MIBl labelling index than the centre of the breast carcinomas (P < 0.0001). The tumour types in this series were as follows: pure tubular and tubular mixed carcinoma, lobular carcinoma, medullary and atypical medullary, mixed ductal and lobular, other special types excluding pure tubular and ductal NST of histological grades 2 and 3. No grade 1 NST tumours were identified in this study. When the data were analysed according to histological type (Table l), those tumours of NST, tubular mixed and lobular types were found to demonstrate a significant difference between the central and peripheral indices. Other histological types, including mixed NST with lobular, medullary, atypical medullary and pure tubular carcinomas
Grade 2: 3 Grade 3: 2 All grade I Grade 1: 2 Grade 2: I
showed homogeneous staining throughout the surface area with no significant difference in MIBl labelling between periphery and centre of these tumour types. Correlations with other well recognized prognostic variables and survival were sought. The peripheral MIB 1 index showed a strong association with higher histological grade (P < 0.001 ), the four prognostic tumour type groups (P < 0.00 1), larger tumour size (P = 0.029), the presence of vascular invasion (P = 0.003) and of distant metastases (P = 0.010) (Table 2). The mean proliferation index for each tumour showed the same correlations with grade (P < 0.001) tumour type group (P < 0.001) tumour size (P = 0.017) vascular invasion (P = 0.018) and distant metastases (P = 0.043) and no additional associations were seen. The central MIBl labelling index showed associations with histological grade (P < 0.001) tumour type group (P < 0.001) and size (P = 0.029) only; no relationship between the central MIB 1 labelling index and the presence of either vascular invasion or distant metastatic depostis was identified. Tumours were grouped into two categories, with the carcinomas in the upper quartile for MIB 1 labelling for each area compared with those showing a lower cell cycling fraction. A significant association between the peripheral MIB 1 Table 2 MIB 1 labelling index at the periphery: correlations with other known prognostic variables Prognostic variable Histological grade (l-3) Histological type (tumour type groups - see text) Tumour size (~15. 15-24, >24 mm) Vascular invasion (absent or present) Distant metastases (absent or present) Lymph node stage (l-3) Age (<40,41-50. 51-60, >60 years) Menopausal status (pm or post menopausal) Oestrogen receptor status (5 fmol/mg protein)
P
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labelling and overall survival of the patients was seen (x2 = 8.05, P = 0.005). This association with survival was also found when the mean MIBl labelling index for the tumour overall was used (x2 = 3.92, P = 0.048). No significant difference in survival was seen between those tumours in the upper quartile of MIBl expression in the centre and those showing lower central immunostaining (x2 = 3.32, P = 0.069). However, when tumours were divided into groups around the median labelling score in each area, the peripheral (x2 = 6.05, P = 0.014), central (x’ = 5.16, P = 0.023) and mean (x2 = 7.3 1, P = 0.007) MIB 1 labelling were all significantly associated with overall survival. The ratio of peripheral to central immunoreactivity was examined and a range of values from 0.61 to 18.35 (mean 1.76) was seen. Three groups were then defined: (a) tumours with a peripheral:central index of more than 1 .l and thus with a relatively greater MIBl expression at the periphery than in the centre (71 cases); (b) those with a peripheral:central index of 0.9-l .l with essentially homogeneous immunoreactivity (24 cases); and (c) carcinomas where the ratio was less than 0.9 and which thus had greater central than peripheral MIB 1 labelling (17 cases). For these categories the pathological features which are known to show a statistical association with the proliferation index were compared; no significant difference in the size of the lesions in the three groups was found (P = 0.061) and no difference in histological grade was identified (P = 0.842). Importantly, grouped according to the ratio of cell cycling fraction and thus by the area of relatively highest proliferation, the central MIBl immunoreactivity was found to be not significantly different in the three categories (P = 0.243). The peripheral index was, however, significantly different (P = 0.049). Thus, the periphery of the tumours was the site of difference in the groups and the heterogeneity identified in MIBl labelling was apparently caused by a genuine increase in the peripheral cycling fraction rather than a fall in the central index.
DISCUSSION Many variables assessed in breast carcinomas have at some time been demonstrated to display a degree of heterogeneity. Steroid receptor heterogeneity*‘,** is well documented and positive and negative cells have been seen concurrently in tumours assayed for both oestrogen and progesterone receptors. Heterogeneous DNA ploidy,23,24 tumour suppressor gene and oncogene expression within breast tumours have also been reported.25s26Tumour heterogeneity has been noted with almost all methods of assessment of proliferation including thymidine labelling, S-phase fraction27 and bromodeoxyuridine examination.*’ Some of these, however, require examination of disaggregated
tumour and thus specific areas within an individual carcinoma cannot be assessed. Meyer & Wittlif? observed a 61% rate of heterogeneity for Thymidine Labelling Index within primary breast carcinomas. Studies of S-phase fraction and Ki-67 index in breast carcinoma have reported heterogeneity with proliferation higher near the tumour border2,29but not all series have reported similar results; in 34 breast cancer cytology samples and frozen sections Kuenen-Boumeester et al found no difference in the Ki-67 labelling index at the periphery and the centre.5 However, cryostat examination of Ki-67 immunoreactivity requires trimming of frozen blocks and the edge of the tumour may be more easily lost. By using the MIBl antibody on paraffin sections more tissue is available for block selection and the entire tumour architecture and surrounding structures can be assessed to evaluate fully any heterogeneity present. The quality and range of staining with the MIB 1 antibody reported here is comparable to data reported elsewhere3’ although the mean and median in this series are lower than those found by Pinder et al in a different series of patients.3’ This may in part be due to the different means of assessing immunostaining in the two studies; in the previous series image analysis was used. The lower mean and median values may also be related to the case bias in the present study, whereby sufficient numbers of low grade ‘special type’ carcinomas with relatively low proliferative rates were included. The associations with other known prognostic factors which have been previously reported are confirmed in this series; in particular a strong relationship between MIBl labelling and histological grade was noted at both the periphery and centre of the tumours.3’.32 Assessment of the cycling fraction also showed a significant association with overall survival for both areas and the overall mean score of the tumour when using the median value as a cut-off point. However, when the upper quartile of tumours was compared with those showing lesser labelling the central index showed no association with survival thus demonstrating the difficulties in selecting reliable cut off points for continuous variables. When tumours which had a relatively higher fraction of cycling cells at their centre than their periphery were compared with those that had either homogeneous intratumoural expression or relatively higher peripheral MIBl labelling, the central proliferation indices were found to be essentially similar overall. This was the case despite an initial selection bias which resulted in inclusion of increased numbers of those carcinomas subsequently found to show homogeneous immunoreactivity. Whilst it has been suggested that differences in proliferation within tumours may be due to central anoxia and an associated fall in central proliferation, these data do not support this argument. In this series, the turnouts overall showed an increase in
Intratumoural heteroneneitv of uroliferation in invasive breast carcinomas evaluted with MIB 1 antibodv peripheral (rather than decreased central) cycling fraction. Differences in blood supply may, nevertheless, be responsible for the heterogeneity identified, the periphery obtaining a greater blood supply through induction of angiogenesis by, for example, the acute inflammatory response (which is maximal at the periphery”). Indeed, heterogeneity in blood vessel pattern is also well recognized in breast carcinomas.3” The relationship between histological type of breast carcinoma and tumour heterogeneity has not been previously explored. In general, greater proliferative activity was seen at the periphery than the centre of the tumours in this series, as has been described by other authors, but this phenomenon was not noted in all histological types of breast carcinoma. In tubular mixed carcinomas and in NST tumours of grades 2 and 3 the MIBl labelling index was greater at the periphery than the centre (P = 0.0244, 0.0016, 0.0028 respectively). However, no difference was found between the central and peripheral indices of pure tubular carcinomas, nor was heterogeneity in the proportion of cycling cells seen in medullary or atypical medullary cancers. It has been proposed that tubular carcinomas may undergo and thus progress to a less differenti‘de-differentiation”,” ated, tubular mixed morphology” by a process better termed ‘clonal evolution’. If the NST/ductal tumour morphology which is seen at the periphery of tubular mixed carcinomas is proliferating more rapidly, as this study suggests, it seems logical to predict that with time this may become the predominant pattern of the tumour. Conversely, other tumours appear to be de novo of high histological grade and homogeneous with regard to proliferation: such a pattern is seen in medullary carcinomas. Alternative explanations for heterogeneity of MIBl expression such as variation in the length of the cell cycle or arrest of the cycle with retention of the Ki-67 antigen (without entry into mitosis) seem improbable. Indeed, prolongation of the cell cycle length, for example of Molt-4 human leukaemia cells, with progression arrest in Gl and G2 phases may result in undetectable Ki-67 antigen immunohistochemically.“’ Thus, the fraction of cells expressing Ki-67 may be smaller than the true growth fraction as estimated by 5bromodeoxyuridine labelling.3” Conversely other studies have shown that halting the cycle beyond GO but before S phase with protein kinase C inhibitors may result in a paradoxical increase in Ki-67 expression compared to thymidine uptake.3h This may be significant in that tamoxifen may function as a protein kinase C inhibitor and it has therefore been suggested that Ki-67 is not a reliable indicator of proliferation after such treatments.‘h However. in this series we have evaluated MIB 1 expression in patients who have received no adjuvant systemic treatment. It should also, however, be noted that we have not examined proliferation per se in this series of cases of invasive breast carcinoma. Although other authors have reported an
175
association between Ki-67 antigen immunoreactivity and mitotic count? we have not directly correlated mitotic count with MIBl labelling in this study. We nevertheless feel that these data support the contention that when counting mitoses as a component of histological grade the periphery of the tumour should be searched particularly thoroughly; this is most frequently the site of greatest cell cycling fraction in primary invasive breast carcinoma. References I. Bloom H J, Richardson W W. Histological grading and prognosis in breast cancer. A study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer 1957: II: 359-377. 2. Verhoeven D, Bourgeois N, Derde M P, Kaufman L. Buyssens N. Comparison of cell growth in different parts of breast cancers. Histopathology 1990; 17: 505-509. 3. Baak J P. Mitosis counting in turnours. Hum Pathol 1990: 21: 683-685. 4. Meyers J S, Wittliff J L. Regional heterogeneity in breast carcinoma: thymidine labelling index, steroid hormone receptors. DNA ploidy. Int J Cancer 1991: 47: 213-220. .5 Kuenen-Boumeester V. Van Der Kwast T H. Van Laarhoven H A, Henzen-Logmans S C. Ki-67 staining in histological subtypes of breast carcinoma and fine needle aspiration smears. J Clin Pathol 1991; 44: 208-210. 6. Pavelic Z P, Pavelic L, Lower E E, Gapany S, Barker E A, Priesler H D. c-myc. c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and non-invasive breast carcinoma. Cancer Res 1992; 2597-2602. 7. Sasa M, Komaki K, Tsuzuki H et al. Tumor progression accompanied by increase in proliferation rate in breast cancer: a preliminary report. J Surg Oncol 1993; 54: 255-259. 8. Pence J C, Kilibash A L, Kerns B-J 0, Marks J R. Iglehart J D. Proliferation index in various stages of breast cancer determined by Ki-67 immunostaining. J Surg Oncol I99 I; 48: I I-20. 9. Taylor H B, Norris H J. Well-differentiated carcinoma of the breast. Cancer 1970; 6877692. IO. Pinder S E, Ellis I 0. Breast screening pathology. Surgery 1994: 12: 277-278. Il. Ellis I 0, Galea M H, Locker A et al. Early experience in breast cancer screening: emphasis on development of protocols for triple assessment. The Breast 1993; 2: 1488153. 12. Duffy S W. Tabar L. Fagerberg G et al. Breast screening. prognostic factors and survival-results from the Swedish two county study. Br J Cancer 1991; 64: 1133-l 138. 13. Cooper H S, Patchefsky A S, Krall R A. Tubular carcinoma of the breast. Cancer 1978: 42: 2334-2342. 14. Cattoretti Cl, Becker M H, Key G et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB I and MIB3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992; 168: 357-363. 15. Gerdes J, Schwab U. Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983; 3 I : 13-20. 16. Gerdes J, Lemke J, Baisch H. Walker H H. Schwab U, Stein H. Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984: 133: 1710-1715. I7 Elston C W, Ellis 1 0. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow up. Histopathology 1991; 19: 403410. IX Ellis I 0, Galea M, Broughton N, Locker A. Blarney R W, Elston C W. Pathological prognostic factors in breast cancer. II. Histological type. Relationship with survival in a large study with long-term follow-up. Histopathology 1992: 20: 479489.
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automated image analysis. Am J Clin Path01 1993; 99: 22623 1. 28. Going J J, Stanton P D, Cooke T G. Influences on measurement of cellular proliferation by histology and flow cytometry in mammary carcinomas labelled in vivo with bromodeoxyuridine. Am J Clin Path01 1993; 100: 218-222. 29. Lee A K, Wiley B, Loda M et al. DNA ploidy, proliferation, and neu-oncogene protein overexpression in breast carcinoma. Mod Path01 1992; 5: 61-67. 30. Barbareschi M, Girlando S, Mauri F M et al. Quantitative growth fraction evaluation with MIBl and Ki67 antibodies in breast carcinomas. Am J Clin Path01 1994; 102: 171-175. 3 1. Pinder S E, Wencyk P, Sibbering D M et al. Assessment of the new proliferation marker MIB 1 in breast carcinoma using image analysis: associations with other prognostic factors and survival. Br J Cancer 1995; 71: 146149. 32. Weidner N, Moore D, Vartanian R. Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel ‘paraffin’-reactive MIB 1 antibody. Hum Path01 1994; 25: 337-342. 33. Weidner N, Semple J P, Welch W R, Folkman J. Tumor angiogenesis and metastatis - correlation in invasive breast carcinoma. N Engl J Med 1991; 324: 1-8. 34. Verhoeven D, Van Marck E. Proliferation, basement membrane changes, metastasis and vascularization patterns in human breast cancer. Path01 Res Pratt 1993; 189: 851-861. 35. Tsurusawa M, Fujimoto T. Cell cycle progression and phenotypic modification of Ki67 antigen-negative Gl- and G2-phase cells in phorbol ester-treated Molt-4 human leukaemia cell. Cytometry 1995; 20: 146-153. 36. Couldwell W T, Weiss M H, Law R E, Hinton D R. Paradoxical elevation of Ki-67 labelling with protein kinase inhibition in malignant gliomas. 3 Neurosurg 1995; 82: 461-468.