carcinoma of the breast.

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1 J Clin Pathol 1997;5: Hedley Atkins Pathology Laboratory, Imperial Cancer Research Fnd Clinical Oncology Unit, Gy's Hospital, London SE1 9RT, UK Correspondence to: A H S Lee, University Department of Pathology, Mailpoint 813, Level E, Soth Block, Sothampton General Hospital, Sothampton S16 6YD, UK. Accepted for pblication 12 Jne 1997 Angiogenesis and inflammation in invasive carcinoma of the breast A H S Lee, L C Happerfield, L G Bobrow, R R Millis Abstract Aim-To investigate the relation between angiogenesis and inflammation in invasive carcinoma of the breast. Methods-Sections from 75 invasive carcinomas of the breast were stained sing immnohistochemistry for von Willebrand factor, CD3, CD8, CD45RO, CD45RA, CD2, CD68, and c-erbb-2. Tmor vasclarity was assessed by conting vessels in the three most vasclar areas, and calclating the average (x4 magnification, field.168 mm2). Each pattern of inflammation was scored semiqantitatively. Reslts-The main pattern of inflammation was a diffse infiltrate of macrophages, and to a lesser extent T cells. Perivasclar and periloblar clsters of B and T cells were noted at the edge of the carcinomas, bt were less prominent than the diffse inflammation. Diffse inflammation, particlarly macrophages, was associated with high tmor grade, tmor necrosis, large tmor size, and c-erbb-2 expression. Perivasclar and periloblar inflammation also increased with tmor grade. Tmor vasclarity increased slightly with intensity of diffse inflammation (Spearman's rank correlation coefficient r. =.17, p =.8), and was inversely related to periloblar inflammation (r, = -.23, p =.3). Conclsions-The correlations between inflammation and vasclarity were weak in this stdy (r2 abot.4) and ths there was no evidence of an important relation. Discrepancies between this and other stdies may be resolved by stdying expression of angiogenic cytokines and proteolytic enzymes by tmor infiltrating inflammatory cells, and their relation to tmor vasclarity. (J Clin Pathol 1997;5: ) Keywords: breast; carcinoma; inflammation; neovasclarisation Angiogenesis is essential for tmor growth. Several stdies have assessed vasclarity in invasive carcinomas of the breast sing sections stained for endothelial markers. Highly vasclar tmors are associated with an increased risk of metastasis in abot half the stdies, and the majority have shown that highly vasclar carcinomas have a less favorable prognosis than tmors with low vasclarity.' Different patterns of inflammation may be seen in 669 invasive carcinoma of the breast. The predominant pattern is a diffse infiltrate in the stroma between tmor cells composed of macrophages and T lymphocytes.6 Less prominent are perivasclar and periloblar clsters of B and T lymphocytes.7 An association of inflammation with expression of c-erbb-2 by the carcinoma is described.8 The significance of the inflammatory infiltrate in mammary carcinoma is controversial. Different stdies, even with mltivariate analysis, have shown that intense inflammation in the tmor is associated with good prognosis,8 poor prognosis,9 or no effect.' "1 It has been thoght that the inflammation is a reflection of an immne response to the tmor. There is recent evidence that if this is the case the response may be ineffective,'2 perhaps becase of inhibition by tmor cells. 3 On the other hand, inflammatory cells are a potentially- important sorce of angiogenic cytokines. They can also release enzymes that digest the extracelllar matrix and ths either facilitate vessel formation or release angiogenic factors which are present in the stroma." '5 A recent stdy'" sggested that more highly vasclar carcinomas had more intense macrophage "hotspots" within the tmor. The aim of the present stdy was to assess the relations between the different patterns of inflammation and tmor vasclarity in invasive carcinoma of the breast. Methods Seventy five consective invasive carcinomas of the breast were stdied (the majority were also inclded in a previos stdy7). All patients were operated on at the Imperial Cancer Research Fnd clinical oncology breast nit at Gy's Hospital. Patients with a previos carcinoma of the breast, or mltiple mammary carcinomas at presentation, were exclded. The tmors were typed sing Azzopardi's criteria.'7 Tmors were classified as mixed if they contained more than 1% of at least two tmor types. The following were recorded for each tmor: necrosis in the invasive tmor; invasion of lymphatics or blood vessels; the nmber of axillary lymph nodes examined, and the nmber of these involved by tmor; and tmor size (measred microscopically in tmors p to 2 mm across, and macroscopically in larger tmors). All carcinomas were graded sing the modified Bloom and Richardson method.'8 The tmor vasclarity and inflammation were assessed in the first srgical specimen in J Clin Pathol: first pblished as /jcp on 1 Agst Downloaded from on 14 Agst 218 by gest. Protected by copyright.

2 67 Lee, Happerfield, Bobrow, Millis Table 1 Panel of antibodies Pretreatment Antibody CD Specificity (mintes) Sorce von Willebrand factor - Endothelim Trypsin (1) Dako CD3 CD3 T cells Microwave (3) Dako CD8/144B CD8 T sppressor cells Microwave (3) Dr D Mason, Oxford UCHL1 CD45RO Primed T helper cells, macrophages, - Prof PCL Beverley, ICRF B cells London SN13 CD45RA Naive T helper cells, macrophages, - Dr G Janossy, Royal Free B cells Hospital, London L26 CD2 B cells Microwave (15) Dako PGM1 CD68 Macrophages Trypsin (1) Dako c-erbb-2 c-erbb-2 Dako patients ndergoing more than one procedre, to avoid changes associated with previos srgery. Immnohistochemistry sing a panel of antibodies with appropriate pretreatment (table 1), and a streptavidin-biotin techniqe7 was performed on sections from one selected phenol formalin fixed block from each case. We chose von Willebrand factor as the endothelial marker becase in a preliminary stdy this gave better reslts than with CD3 1, CD34, and Ulex eropaes. The inflammatory cell markers were chosen to stain T and B lymphocytes and macrophages. Before application of the primary antibodies to von Willebrand factor, c-erbb-2, and CD3, normal swine serm dilted 1/5 was applied for 15 mintes. In two biopsies there was only methacarn fixed tisse available, so von Willebrand factor cold not be assessed, as the antibody does not work with this fixative. EVALUATION OF INFLAMMATION The inflammation was assessed on haematoxylin and eosin sections, and each of the sections was stained for inflammatory cell markers. Three patterns of inflammation were recognised: (1) diffse in the stroma arond and between tmor cells, (2) perivasclar clsters, and (3) periloblar clsters. The intensity of each pattern of inflammatory infiltrate, (a) within and (b) at the edge of the invasive tmor, was graded as absent or minimal (), mild (1), moderate (2), or marked (3). Definite membrane staining for c-erbb-2 was interpreted as positive. Very weak and focal staining were scored as negative. EVALUATION OF VASCULARITY Vessels were assessed within the invasive carcinoma in sections stained for von Willebrand factor. The most vasclar areas, or hotspots, Figre 1 Correlations of the intensity of the diffse infiltrate of macrophages with other histological featres. For rs =. 28, p =. 9; rs =. 32, p =. 3; r, =. 39, p =. 3; rs =.58,p < 1i-. were identified by examination at low power. The vessel conts from the three most vasclar fields (.168 mm', x4 magnification) were recorded, and the average calclated. No dcts or lobles, either normal or involved by carcinoma in sit, were allowed in the field. All discrete clsters or single cells stained for von Willebrand factor were conted as a vessel. A lmen was not reqired to cont as a vessel. The findings of a recent stdy noted that more vasclar carcinomas had more intense macrophage hotspots within the tmor. 16 This prompted s to look at the distribtion of macrophages (CD68) and their relation to vessels in 42 tmors. Seven tmors, selected from those showing heterogeneos vasclarity, were frther stdied sing doble immnohistochemistry. Antibody to von Willebrand factor was applied first and visalised with a streptavadin biotin techniqe, followed by antibody to CD68 demonstrated with an alkaline phosphatase anti-alkaline phosphatase techniqe.'7 Comparisons between grops were made with the Mann-Whitney U method, and correlation was assessed sing Spearman's rank method. The Wilcoxon signed rank test was sed to analyse paired data. Reslts The 75 carcinomas were classified as 5 dctal, five tblar or cribriform, nine loblar, five mixed dctal and loblar, and one mcinos carcinoma. There were 23 grade I, 33 grade II, and 19 grade III. Axillary lymph node stats was known in 71 patients, and metastatic carcinoma was present in 38. The median tmor size was 15 mm (range 2 to 1 mm). Vasclar invasion was present in 18 and necrosis in 14 tmors. Clear membrane staining for c-erbb-2 was seen in 1 tmors (14%). PATTERNS OF INFLAMMATION Diffse inflammation was seen in 36 tmors and was the most prominent pattern of inflammation noted in this stdy. It was sally evenly distribted within the tmor, with accentation at the tmor edge in some tmors. The main cell types were macrophages and T cells, with the former predominant-the intensity of the diffse infiltrate of macrophages exceeded or was similar to the intensity of T cells in almost all the tmors (Wilcoxon statistic 992, p <.1). Periloblar and perivasclar inflammation was seen in 2 and 41 tmors respectively. Both patterns of inflammation were more prominent at the tmor edge, and J Clin Pathol: first pblished as /jcp on 1 Agst Downloaded from on 14 Agst 218 by gest. Protected by copyright.

3 Angiogenesis and inflammation in breast cancer 671 both were mainly composed of B and T cells, with few macrophages. Intense diffse inflammation, especially macrophages, was associated with high tmor grade of dctal carcinomas, larger tmor size, tmor necrosis, and c-erbb-2 expression (fig 1). Intense periloblar inflammation, composed of B and T cells, was associated with high tmor grade of dctal carcinomas (Spearman's coefficient rs =.28 to.35, p =.16 to.3), tmor necrosis (r, =.41 to.44, p <.1) and larger tmor size (rs =.22 to.25, p =.3 to.1). Intense perivasclar inflammation, composed of B and T cells, was seen particlarly in high grade dctal carcinomas (rs =.32 to.39, p =.6 to.1). TUMOUR VASCULARITY The tmor blood vessels were either distribted evenly throgh the tmor (abot half the tmors), or the vasclarity was highest towards the periphery of the invasive tmor. Marked variation of vasclarity, appreciable at low power examination, was noted in only one fifth of tmors. The median tmor vasclar density was 143/mm2 (range 54 to 333/mm2). Vasclar density was not correlated with nodal stats (r, = -.2), histological grade (rs = -.4), tmor size (rs = -.2), vasclar invasion (r, =.8), or c-erbb-2 expression (rs = -.5). RELATIVE DISTRIBUTION OF MACROPHAGES AND VESSELS Macrophages were diffsely distribted throgh the tmor. In a qarter there was increased density of macrophages at the tmor edge, particlarly in the normal tisse adjacent to the carcinoma. Macrophage E 'a U) cn Q 4 Co I: 4 r o Hotspot vasclar densitylmm2 (divided into tertiles) Intensity of diffse inflammation within tmor Intensity of diffse inflammation within tmor Figre 2 The hotspot vasclar density and intensity of the diffse inflammation within the tmor; r =. 17,p = E UE m c Un Co. U, Ir [ I El HEJ Fo o Li IlI o 1 2 Intensity of periloblar inflammation at tmor edge Figre 3 The hotspot vasclar density and intensity of the periloblar inflammation at the tmor edge; r = -. 23, p =.3. hotspots were noted in a qarter of tmors, often both at the tmor edge and within the tmor. Those within the tmor were often in areas of fibros tisse and only occasionally related to necrosis. As noted above, the blood vessels were sally either distribted evenly throgh the tmor or were more marked towards the tmor edge. Marked variation of vasclarity was seen in only one fifth. There was no consistent pattern to the relative distribtions of the vessels and macrophages. Seven tmors, selected from those showing heterogeneos vasclarity, were frther stdied sing doble immnohistochemistry for von Willebrand factor and CD68. The macrophages were diffsely distribted in all bt two tmors. In one the macrophages were associated with an area of necrosis, and in one were in a central area of fibrosis. The macrophage hotspots in these two tmors were at least 7 mm from the vasclar hotspots. VASCULARITY AND INFLAMMATION Increasing vasclar density correlated weakly with increasing intensity of diffse inflammation within the tmor (fig 2). This small effect was seen with T cells, bt not with macrophages (CD3 rs =.16, CD8 rs =. 19, Table 2 Intensity of diffse inflammation within tmor and hotspot vasclar density (divided into tertiles) 3 J Clin Pathol: first pblished as /jcp on 1 Agst Downloaded from on 14 Agst 218 by gest. Protected by copyright.

4 672 Lee, Happerfield, Bobrow, Millis CD45RO r, =.14, CD68 r, = -.1). We also analysed or data sing Leek's method to allow comparison between the two stdies. The tmors were divided by vasclar density into tertiles, and the diffse inflammation in the three grops of tmors compared. Using the overall measre obtained from haematoxylin and eosin sections, we fond that the inflammation in the highest tertile was higher than that in the lower two tertiles (p =.4, Mann- Whitney U test). However, tmors in the lowest tertile for vasclarity had more diffse inflammation than tmors in the middle tertile (table 2). Similar reslts were obtained for CD3 cells, with more CD3 cells in the highest tertile (p =.55). By contrast, the scores for CD68 cells were similar in the three tertiles. Vasclar density decreased slightly with increasing intensity of periloblar inflammation at the tmor edge (fig 3). This pattern was seen with all cell types except macrophages (rs = -.17 to -. 18). Vasclar density was not correlated with perivasclar inflammation at the tmor edge (rs = -.4). Discssion We fond three main patterns of inflammation, confirming a previos smaller stdy.7 The predominant pattern was a diffse infiltrate of macrophages and T cells. Periloblar and perivasclar inflammation was composed of B and T cells and was seen particlarly at the tmor edge. Most previos stdies have not attempted to sbdivide the inflammation into different patterns. Nevertheless, reported associations of inflammation with histological grade,2 21 necrosis,2 and c-erbb-2 expression8 are consistent with or findings for diffse inflammation. One previos stdy has looked at the relation between inflammation and vasclarity in invasive carcinoma of the breast. Leek et al16 assessed the density of macrophages in hotspots in a manner similar to the assessment of vasclarity in this stdy. They fond that tmors with vasclarity in the pper tertile had higher macrophage hotspot conts (average of abot 2), compared with tmors in the two lower tertiles for vasclarity (both average of abot 15). This difference was significant (p =.3), bt small. The macrophage and vasclar hotspots did not coincide. They sggested that the macrophage hotspots in the tmor centre were stimlated by hypoxia to prodce angiogenic factors casing angiogenesis at the tmor edge. We fond a weak correlation between the intensity of diffse inflammation and vasclarity (r. =.17, p =.8). This relation was de to T cells and not macrophages. There was also a weak inverse relation between periloblar inflammation at the tmor edge and vasclarity (rs = -.23, p =.3). These correlation coefficients are small (r2 abot.4) sggesting if there is a relation it is of little importance. Using a method of analysis like that of Leek et al,'6 we fond more intense diffse inflammation in tmors in the highest tertile for vasclarity. As with the correlation analysis, this relation was de to T cells and not macrophages. Ths, despite different methods of assessing the inflammation, both stdies have shown weak associations between inflammation and angiogenesis, althogh of different cell types. Where we differ is in the interpretation. Leek et al thoght the reslt important. We feel the magnitde of the relation is small and therefore not of major importance. We looked for macrophage hotspots bt fond the macrophages were sally diffsely distribted throgh a tmor, and if anything the inflammation was more marked at the tmor edge. Macrophage hotspots within the tmor were present in a minority of cases. Separate hotspots of vessels and macrophages, as described by Leek et al,'6 were shown by doble immnohistochemistry in two of seven tmors selected becase they contained vasclar hotspots. Leek at al sed a more sophisticated conting method, so it is possible that we have missed a sbtle relation between the distribtions of macrophages and vessels. The macrophage and vasclar hotspots were often far apart, and ths an important relation between the two wold appear nlikely. The evenness of the distribtion of macrophages in most tmors in this stdy, and in another stdy sing macrophage conts,2" sggests that an overall score of macrophage density (rather than hotspot conting) is a reasonable measre of the intensity of macrophages. By contrast with the present stdy, in dctal carcinoma in sit we previosly fond a moderately strong correlation between perivasclar clsters of B and T cells and vasclarity in the interdctal stroma.2" Similar methods were sed in both stdies, sggesting that this comparison is valid. One potential explanation of the discrepancy with the present stdy is that the main pattern of inflammation associated with dctal carcinoma in sit is perivasclar, whereas it is a mch smaller component of the inflammation fond in invasive carcinomas. Also, the increased vasclarity seen in dctal carcinoma in sit is otside rather than within the carcinoma. The absence of a strong association between the intensity of inflammation and vasclarity does not exclde a possible role for inflammation in angiogenesis. It may be possible to demonstrate sbtle changes with cell conts that are not apparent sing inflammation scores. Also, in this stdy simple markers of cell type were sed. For example CD68 is a panmacrophage marker that stains macrophages in different sitations, and probably of different fnction.24 We were ths not able to distingish qiescent, antigen presenting, phagocytic, or angiogenic macrophages. There is evidence that macrophages need to be activated before they can indce angiogenesis,'5 and ths only a proportion of macrophages have an angiogenic phenotype. Assessment of inflammatory cell fnction, rather than type, may be more meaningfl. Stdies of expression of digestive enzymes, angiogenic factors, and inhibitors of angiogenesis by inflammatory and carcinoma cells, and their relation to tmor vasclarity, may J Clin Pathol: first pblished as /jcp on 1 Agst Downloaded from on 14 Agst 218 by gest. Protected by copyright.

5 Angiogenesis and inflammation in breast cancer explain the absence of a relation between inflammation and vasclarity in invasive carcinoma, compared with the associations fond in dctal carcinoma in sit.2" Urokinase type plasminogen activator, one enzyme that has been stdied in carcinoma of the breast, may be important in digesting extracelllar matrix and ths promoting angiogenesis. Urokinase plasminogen activator levels (measred by enzyme linked immnosorbent assay (ELISA) on tmor extracts) correlate with tmor vasclarity2" 26; and rokinase plasminogen activator (assessed by both immnohistochemistry and ELISA) correlates with macrophage density Unfortnately these stdies did not assess the relation between intensity of inflammation and vasclarity. Many angiogenic factors and inhibitors of angiogenesis can be secreted by inflammatory cells,'4 fibroblasts, and endothelial cells, as well as by the carcinoma cells. Most stdies of angiogenic cytokines have concentrated on expression in carcinoma cells,283 rather than stromal cells." Investigation of expression of digestive enzymes and angiogenic factors, particlarly by inflammatory cells, and their relation to tmor vasclarity may explain the differences in invasive carcinoma and dctal carcinoma in sit. 1 Weidner N, Semple JP, Welch WR, Folkmnan J. Tmor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl Jf Med 199 1;324: Weidner N, Folkman J, Pozza F, Bevilacqa P, Alldred EN, Moore DH, et al. Tmor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. 7 Natl Cancer Inst 1992;84: Horak ER, Leek R, Klenk N, Lejene S, Smith K, Start N, et al. Angiogenesis, assessed by platelet/endothelial cell adhesion molecle antibodies, as indicator of node metastases and srvival in breast cancer. Lancet 1992;34: Bhan AK, DesMarais CL. 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