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Human DNA topoisomerase II-alpha: A new marker of cell proliferation in invasive breast cancer
Human DNA Topoisomerase ll-Alpha: A New Marker of Cell Proliferation in Invasive Breast Cancer BEVERLY J, LYNCH, MD, DONALD G, GUINEE, Jr, MD, AND JOSEPH A. HOLDEN, MD, PhD DNA topoisomerase II-alpha is the molecular target o f doxorubicin, an active drug used in the therapy o f breast cancer. From many in vitro studies, it is known that high levels o f topo H-alpha expression correlate with drug sensitivity, and low levels o f topo H-alpha correlate with drug resistance. In addition, the enzyme is known to be a marker o f cell proliferation in normal tissues. Because the number o f proliferating cells in a breast cancer has been shown to be prognostically important, and because doxoruhicin is used in the treatment o f breast cancer, we hypothesized that the measurement o f topo IIalpha in breast cancer may not only give drug sensitivity information but also may yield important data on cell proliferation. In this study, formalin-fixed, paraffin-embedded tissue from 30 specimens o f invasive breast cancer from 20 patients were immunohistochemically stained for topo II-alpha with a mouse monoclonal antibody. For each case, a topo II-alpha index was determined that represents the number o f positive-staining tumor cells divided by the total number o f tumor cells counted times 100. A similar index was determined for MIB1, a known ceil proliferation marker. Each case was also graded according to the modified Bloom-Richardson criteria and eval-
Breast cancer is a m a j o r health p r o b l e m affecting a large n u m b e r of women. T h e incidence of breast cancer in developed countries is approximately 12%. 1 T h e d e v e l o p m e n t of newer and m o r e sensitive radiological p r o c e d u r e s has helped to identify breast cancer in w o m e n when the t u m o r is still small a n d lymph n o d e metastases have not occurred. Adjuvant c h e m o t h e r a p y in w o m e n with node-negative disease has b e e n shown to improve overall disease-free survival. 25 However, it is not entirely clear that all w o m e n with node-negative breast cancer have tumors that will necessarily pursue an aggressive clinical course. Such w o m e n m i g h t be cured by local therapy alone and would not benefit f r o m systemic adjuvant chemotherapy. This has stimulated m u c h research into the possibility of using prognostic and predictive markers to d e t e r m i n e which patients would benefit f r o m chemotherapy. Two markers that have b e e n studied with respect to breast cancer include the c-erbB-2 (c-Neu) o n c o g e n e and the proliferation index. T h e c-erbB-2 gene encodes a growth factor receptor. Amplification of this gene has
From the Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT. Accepted for publication February 5, 1997. Supported by a research educational grant from Pharmacia and Upjohn, San Clemente, CA. Address correspondence and reprint requests to Joseph A. Holden, MD, PhD, Department of Pathology, University of Utah Health Sciences Center, 50 North Medical Dr, Salt Lake City, UT 84132. Copyright © 1997 by W.B. Saunders Company 0046-8177/97/2810-001055.00/0
uated for c-erbB-2 amplification, h o r m o n a l status, S-phase fraction, and mitotic index. The topo II-alpha index correlates better with the MIB1 index than with the S-phase fraction or mitotic index. The topo
II-alpha expression in breast cancer ranges from low (topo II-alpha index <1) to high (topo II-alpha index = 86). Amplification o f cerbB-2 was observed in 4 o f 28 cases (14%) but did not correlate with high topo n-alpha indices. We conclude that measurement o f topo II-alpha in invasive breast cancer can be readily p e r f o r m e d by immunohistochenfical staining, and it gives information on the number o f cycling tumor cells. In addition, b e c a m e the enzyme is the molecular target o f doxorubicin, the expression o f the enzyme may relate also to the sensitivity or resistance o f the tumor to doxorubicin-based chemotherapeutic protocols. HUM PATHOL 28:1180--1188. Copyright © 1997 by W.B. Saunders Company Key words: DNA topoisomerase H-alpha, MIB1, cell proliferation, breast cancer. Abbreviations: topo II, DNA topoisomerase II; kDNA, kinetoplast DNA; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetra-acetic acid; ATP, adenosine triphosphate.
b e e n r e p o r t e d in 28% of breast cancers. 6 Recent studies have indicated that amplification of the c-erbB-2 gene may indicate the response of breast cancer patients to chemotherapy. Patients treated with a doxorubicin-containing protocol are relatively sensitive to c h e m o t h e r apy if their tumors overexpress c-erbB-2 7 and yet are relatively resistant if the c h e m o t h e r a p y protocol does not contain doxorubicin, s Although the molecular m e c h a n i s m that serves as a basis for these results is not yet clear, these results with c-erbB-2 underscore the i m p o r t a n c e of c-erbB-2 gene amplification in predicting response to therapy. Many studies have also suggested that a high mitotic rate in a breast cancer portends a p o o r prognosis. 9-11 DNA topoisomerase II (topo II) is now known to be the molecular target of doxorubicin. 12 M a m m a 1ian cells contain two isoforms of the enzyme, t e r m e d topo II-alpha a n d topo II-beta. 13'14 Most data suggest that the alpha isoform is the primary drug target. 14-18 Interestingly, the topo II-alpha gene has b e e n shown to be coamplified along with c-erbB-2 in cell lines and in h u m a n breast cancers. 19'20 This might suggest that co-amplification of topo II-alpha with c-erbB-2 in breast cancers would sensitize the t u m o r cells to doxorubicin-based chemotherapy. T o p o II-alpha is also a m a r k e r of cell proliferation in both normal and neoplastic tissues and cells. 14'2>~5 These facts suggest that the expression of topo II-alpha in h u m a n breast cancer may show imp o r t a n t correlations with the response of the neoplasm to doxorubicin-containing protocols, c-erbB-2 amplification, and with cell proliferation data. Unfortunately, there are limited data on the ex-
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DNA TOPO II-ALPHAIN BREASTCANCER (Lynch et al) p r e s s i o n o f t o p o I I - a l p h a in h u m a n b r e a s t c a n c e r . Previous studies e m p l o y i n g p o l y c l o n a l t o p o II a n t i b o d i e s in i m m u n o h i s t o c h e m i s t r y have s h o w n t h e p r e s e n c e o f t o p o II in h u m a n b r e a s t c a n c e r . 26'27 H o w e v e r , t h e s e studies a r e p o t e n t i a l l y c o m p l i c a t e d by t h e fact t h a t s o m e p o l y c l o n a l a n t i b o d i e s , initially t h o u g h t to b e specific f o r t o p o II-alpha, a r e n o w k n o w n to r e a c t with t h e b e t a i s o f o r m as well. 28'29 T h e r o l e t h a t t o p o I I - b e t a plays in t u m o r d r u g sensitivity is n o t y e t u n d e r s t o o d . 3°-35A d d i t i o n a l w o r k u s i n g specific t o p o I I - a l p h a a n t i b o d i e s in i m m u n o h i s t o c h e m i s t r y has o n l y e v a l u a t e d a small n u m b e r o f b r e a s t c a n c e r s , a n d surprisingly, n o c o r r e l a t i o n with cell p r o l i f e r a t i o n was o b s e r v e d , s6 W e r e c e n t l y r e p o r t e d t h a t it is p o s s i b l e to d e t e c t t o p o I I - a l p h a in f o r m a l i n - f i x e d , p a r a f f i n - e m b e d d e d hum a n tissue s e c t i o n s with specific m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t t h e e n z y m e . 25 I n this r e p o r t , we i m m u n o s t a i n e d 30 s a m p l e s o f p r i m a r y a n d m e t a s t a t i c b r e a s t c a n c e r f r o m 20 p a t i e n t s f o r t o p o II-alpha. W e f o u n d t h a t t h e e x p r e s s i o n o f t h e e n z y m e c o r r e l a t e s well with MIB1, a k n o w n cell p r o l i f e r a t i o n m a r k e r . This r e s u l t suggests t h a t t o p o I I - a l p h a m a y b e a useful p r o l i f e r a t i o n m a r k e r , w h i c h can b e u s e d in s t u d y i n g b r e a s t c a n c e r . In addition, some tumors expressed high topo II-alpha levels. F r o m t h e s e data, we h y p o t h e s i z e t h a t t h e s e h i g h t o p o I I - a l p h a - e x p r e s s i n g t u m o r s m i g h t r e p r e s e n t a subclass o f n e o p l a s m s t h a t w o u l d r e s p o n d well to c h e m o therapy protocols containing doxorubicin. Doxorubicin targets t o p o II, 12 a n d m u c h d a t a a r e available t h a t s u g g e s t t h a t t h e sensitivity o f a cell to t o p o I I - t a r g e t e d d r u g s is d i r e c t l y p r o p o r t i o n a l to the a m o u n t o f c e l l u l a r t o p o II. 37-39
MATERIALS AND METHODS Sources of Tissue and Characterization Paraffin blocks were retrieved from 20 patients with primary and metastatic breast cancer from the surgical pathology files of the University of Utah Health Sciences Center. Several patients had material available from both the primary tumor as well as metastatic tumor, and so a total of 30 paraffin blocks of breast cancer were available for immunohistochemical staining. All cases were reviewed to ensure the accuracy of the diagnosis. Selection of the cases was based on the availability of sufficient material for immunohistochemical staining. The S-phase fraction of each tumor was determined by flow cytometry, as described by Holden et al. 4° Each tumor was given a modified Bloom-Richardson score by one investigator (D.G.), using established criteria. 41 The mitotic index of each tumor was determined by counting the number of mitotic figures per 500 tumor cells and was performed by one investigator (B.L.). The mitotic index is the fraction of tumor cells in mitosis.
Chemicals The sources of the chemicals were as previously described. 25 Kinetoplast DNA (kDNA) was isolated from Crithidiafasiculata as described. 4° The c-erbB-2 antibody is a monoclonal antibody that recognizes the carboxy terminus of the c-erbB-2 oncogene and was obtained from Oncogene Sciences (Cambridge, MA). The topo II-alpha antibody is a newly developed mouse monoclonal antibody similar to that described
previously.25 The antibody was purified from the hybridoma supernatant by protein G chromatography. Sodium dodecyl sulfate (SDS) gel electrophoresis of the purified fraction showed pure immunoglobulin. The concentration of the purified topo II-alpha antibody was 0.77 m g / m L . Antibodies against MIB1 were obtained from AMAC, Westbrook, ME.
Topo I[ Catalytic Activity The catalytic activity of topo II was determined by following the adenosine triphosphate (ATP)-dependent decatenation of kDNA. 4° The reaction was performed in 20-#L volumes containing 50 m m o l / L Tris-HC1 (pH 7.5), 85 m m o l / L KC1, 10 m m o l / L MgC12, 5 m m o l / L dithiothreitol, 5 m m o l / L ethylenediaminetetra-acetic acid (EDTA), 30 # g / m L bovine serum albumin, 1 m m o l / L ATP, and 250 ng kDNA. Reactions were allowed to proceed for 60 minutes at 30°C and then stopped by the addition of 2 #L 10% sodium dodecyl sulfate (SDS), 0.15 m m o l / L EDTA, 0.2% bromophenol blue, 50% glycerol. Samples were then electrophoresed on 0.8% agarose gels at 10 volts for 15 hours. The gel buffer was 40 m m o l / L Tris-HC1 (pH 7.5), 20 m m o l / L sodium acetate, 1 m m o l / L EDTA. The gels were stained in ethidium bromide (0.5 # g / m L ) for 20 minutes, rinsed with H20, and the amount of monomer circles released from the kDNA was visibly estimated by inspection of the ethidium-stained gel.
Preparation of Cellular and Nuclear Extracts Samples of breast cancer tissue were processed into nuclear or cellular extracts in procedures described in detail elsewhere. 29 Briefly, fresh tissue was obtained immediately after surgery and evaluated by frozen section for the presence of tumor. For nuclear extracts, the sample was then homogenized in a 0.3 m o l / L sucrose buffer and the nuclei isolated by centrifugation at 1,000g for 20 minutes. The nuclei were lysed by the addition of I m o l / L NaC1 and the DNA precipitated by the addition of poly(ethylene glycol) to a final concentration of 6%. After centrifugation at 20,000g for 30 minutes, topo II is recovered in the supernatant. For whole cell extracts, the tissue was first minced into 2- to 4-mm pieces with a scalpel and suspended in a 0.25 m o l / L sucrose buffer. After homogenization, the topo II was extracted by incubation of the sample at 95°C in buffer containing 1% SDS. The DNA was sheared by passing the sample through an 18-gauge needle.
Western Blotting Western blotting for topo II was performed as described. 29 Samples of whole cell extracts were electrophoresed on 8% polyacrylamide gels under denaturing conditions. After electrophoresis, the proteins were blotted onto nitrocellulose or nylon membranes with the use of a Mini-Protean II Trans Blot Cell apparatus (Bio-Rad, Hercules. CA) in 20 m m o l / L Tris-HC1 (pH 8.0), 150 m m o l / L glycine, 20% methanol at 20 volts for 16 hours at 4°C. The primary topo II antibody used to detect the protein was the monoclonal antibody used in the immunostaining procedures. The secondary antibody was an alkaline phosphatase-conjugated anti-mouse immunoglobulin (Promega, Madison, WI). Detection was with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as described. 29
lmmunohistochemical Staining Immunostaining for topo II-alpha and MIB1 was performed as described previously.25 Briefly, slides were cut in 3-
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to 5-#m sections and after deparaffinization were heated in 10 m m o l / L sodium citrate (pH 6.0) for 30 minutes on high in a Samsung microwave oven. After cooling, immunohistochemical staining was performed on a Ventana 320 automated immunohistochemical stainer in accord with the manufacturer's instructions. The topo II antibody was used at a dilution of 1:500 and the MIB1 antibody was used at a dilution of 1:40. Detection for either topo II-alpha or MIB1 was with the use of a secondary mouse immunoglobulin G linked to biotin followed by incubation with streptavidin linked to horseradish peroxidase. Color development was accomplished with diaminobenzidine. The topo II or MIB1 indices are defined as the number of positive-staining tumor cells divided by the total number of tumor cells counted times 100. At least 500 tumor cells were counted in the area of highest staining. All nuclear staining, whether strong or weak, was taken as indicative of topo IIalpha or MIB1 positivity. The indices represent the average of two i n d e p e n d e n t determinations by different observers (B.L. and J.H.). Immunostaining for c-erbB-2 (c-Neu) was performed as for topo II-alpha and MIB1 except that the heating step was not used. Evaluation was performed by one investigator (D.G.), and any distinct membrane staining was interpreted as positive] Statistics
The graphing and statistical analyses were performed with the use of the Microsoft Excel computer program. Protein Determination
The protein concentration of nuclear and crude cell extracts was estimated by the method of Bradford. 42
Lane:
Lane:
1
2
3
KDa
Origin T o p o II (~ -
-205
-97 -66
-43 BPB FIGURE 2. Whole cell extracts prepared from human breast cancer contain intact topo II-alpha immunoprotein. Whole cell extracts were prepared from a human breast cancer and subjected to Western blot as described in the Materials and Methods section. Lane 1 contains 25 #g breast cancer extract protein and Lane 2 contains 50/~g breast cancer extract protein. Lane 3 contains 4.6 #g nuclear extract protein prepared from a diffuse large cell non-Hodgkin's lymphoma known to have elevated amounts of t o p o II-alpha.25 The antibody used specifically recognizes the 170-kDa subunit of DNA topo II-alpha and is the same antibody used in the immunohistochemical staining. BPB indicates the position of the tracking dye, bromophenol blue. The molecular weight standards used are myosin heavy chain, 205 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 43 kDa.
1 2 ,3 4 5 6 RESULTS
-Network
T o p o II C a t a l y t i c A c t i v i t y a n d I m m u n o p r o t e i n A r e P r e s e n t in E x t r a c t s o f Human Breast Cancer
-Monomer
FIGURE 1. DNA topo II catalytic activity is present in a nuclear extract prepared from a human breast cancer. DNA topo II activity was assayed by the decatenation of kDNA as described in Materials and Methods. Highly c a t e n a t e d networks of kDNA remain at the origin. In the presence of functional t o p o II enzyme, monomer DNA circles are released from the kDNA and migrate into the gel. The amount of nuclear extract protein in each lane is as follows: lane 1,90 ng; lane 2, 40 ng; lane 3, 20 ng; lane 4, 10 ng; lane 5, 5 ng; lane 6, 2.5 ng.
N u c l e a r extracts p r e p a r e d f r o m a g r a d e II invasive b r e a s t c a n c e r in a 63-year-old w o m a n w e r e assayed f o r t o p o II catalytic activity. T h e p r e s e n c e o f t o p o II activity is s e e n as t h e l i b e r a t i o n o f m o n o m e r D N A circles f r o m t h e h i g h l y c a t e n a t e d kDNA, w h i c h is c o m p o s e d o f num e r o u s i n t e r l o c k e d D N A circles. Activity is o b s e r v e d in as little as 10 n g o f n u c l e a r extract, as s h o w n in F i g 1. This is in t h e s a m e r a n g e as o b s e r v e d in o t h e r h i g h l y m a l i g n a n t t u m o r s . 29 T h e lack o f o b s e r v a b l e activity with h i g h c o n c e n t r a t i o n s o f n u c l e a r e x t r a c t has b e e n obs e r v e d p r e v i o u s l y a n d p r o b a b l y reflects t h e i n h i b i t i o n o f d e c a t e n a t i o n by D N A b i n d i n g p r o t e i n s t h a t c a n b e p r e s e n t in c r u d e n u c l e a r extracts. 29 N u c l e a r extracts p r e p a r e d f r o m n o r m a l b r e a s t c o n t a i n t o p o II catalytic activity, w h i c h is at least 10 f o l d l o w e r t h a n t h a t s h o w n in F i g 1 ( H o l d e n , J, u n p u b l i s h e d d a t a ) . T h e r e f o r e , t h e activity p r e s e n t as s e e n in F i g 1 m o s t likely o r i g i n a t e s from the tumor. T h e p r e s e n c e o f t o p o I I - a l p h a p r o t e i n in t h e b r e a s t c a n c e r was c o n f i r m e d by W e s t e r n blot. As s h o w n in Fig
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DNA TOPO II-ALPHA IN BREAST CANCER (Lynch et al)
TABLE 1. Clinicopathotogic Features in 20 Patients With Invasive Breast Carcinoma Patient No.
Age (yr)
I
33
2
61
3 4 5 6 7 8 9 10
72 43 45 73 35 68 37 87
11
64
82 70 58
12 13 14
77 41
15 16
17
71
18 19
57 74
20
63
* The t The $ The § Not IENot
Site Breast Axillary Lyznph node Left breast Left axillary Lymph node Right breast Breast Breast Breast Breast Breast Breast Breast Right breast Left breast Breast Axillary Lymph node Breast Breast Breast Axillary Lymph node Breast Breast Axillary Lymph node Breast Axillary Lymph node Breast Left breast Right breast Breast Axillary Lymph node
Scarff-BloomRichardson Score
Estrogen/Progesterone Receptor Status
c-erb B-2++
0.6 1.2
6 NA II
+/+ ND
-
17.5 ND
3.6 2.8
+/-
-
NA
ND
-
23 15 18 21 26 51 18 25 20 10 1 2
5.3 4.9 1.1 9.0 2.7 8.2 3.7 3.2 3.5 ND 2.9 ND
0.4 1.0 0.2 1.4 0.2 3.8 0.6 0.4 0.2 0.6 <0.2 0.2
5 5 5 5
+/+/+ +/+ -/+
-
6 8 8 6 6 5 5 NA
+/+ +/+/+ -/+ +/+/+ +/+ ND
+ + + -
30 34 86 73
33 34 88 90
5.6 2.7 10.5 ND
1.4 1.4 2.6 4.8
6
+/+
-
9 NA
+/+ -/ND
-
12 30 29
12 36 29
3.0 2.0 ND
0.8 2.0 1.0
6
+/+
-
5
-/+
+
NA
ND
-
25 25
17 34
4.7 ND
0.8 0.8
6 NA
+/+ ND
-
22 2 1 10 5
25 2 1 11 6
4.8 1.8 ND 5.0 ND
1.0 0.2 <0.2 0.2 0.6
6 4 3 6 NA
+/+ +/+ +/+ +/+ ND
ND ND
TOPO II* Index
MIBI* Index
Flow Cytometry S-phase (%)
36 32
43 33
2.1 ND§
35 53
52 64
12 9 10 20 26 46 27 22 14 7 <1 <1
Mitotic Indext
S
5
-
-
-
Topo II and MIB1 indices are defined in the Materials and Methods section. mitotic index represents the percent of t u m o r cells in mitosis. presence of (+) or absence ( - ) of m e m b r a n o u s staining is indicated. determined. applicable.
2, a whole cell extract p r e p a r e d f r o m the t u m o r tissue a n d subjected to Western blot with the same topo IIalpha m o n o c l o n a l antibody that is used in the i m m u n o staining work described later shows the presence of an intact 170-kDa species. The subunit of topo II-alpha is 170 k O a . 43 T h e migration of this protein species is identical to the migration of the 170-kDa species present in a diffuse large cell n o n - H o d g k i n ' s lymphoma, which has previously b e e n shown to contain high levels of topo II-alpha. 25 T h e catalytic assay and Western blot indicate the presence of functional topo II activity and intact topo II-alpha protein in the t u m o r tissue and confirm the specificity of the antibody.
Immunohistochemical Staining of Breast Cancers for Topo II-Alpha and MIB1. Topo II-alpha i m m u n o s t a i n i n g of 30 samples of breast cancer f r o m 20 patients was p e r f o r m e d with an
antibody specific to the alpha isoform. For each case, a topo II index was d e t e r m i n e d that represents the n u m b e r of positive-staining t u m o r cells divided by the total n u m b e r of t u m o r cells counted times 100. T h e results are shown in Table 1 and represent the average of two i n d e p e n d e n t determinations by two different investigators (B.L. and J.H.). T h e topo II index of the tumors was highly variable and ranged from a low of < 1 to a high of 86. An example of the i m m u n o s t a i n i n g pattern of a t u m o r with a high topo II-alpha index is shown in Fig 3A, and that of a t u m o r with a low topo II-alpha index is shown in Fig 3B. A similar analysis of MIB1 in these tumors was p e r f o r m e d , and the results are also presented in Table 1. T h e MIB1 indices of the tumors ranged f r o m a low of 1 to a high of 90. T h e correlation of topo II-alpha with MIB1 in the series of breast cancers is shown in Fig 4. Clearly, topo II-alpha correlates well with MIB1 with a correlation coefficient of 0.97.
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HUMAN PATHOLOGY
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FIGURE 3. Immunohistochemical staining of human breast cancers for topo II-alpha. (A) Immunostaining of a grade Ill invasive human breast cancer for topo il-alpha shows many positive cells and the topo II-alpha index is 86. (Original magnification ×400) (B) Immunostaining of a grade II invasive human breast cancer for topo II-alpha shows only a few positive cells, and the topo ilalpha index is 12. Original (magnification ×400.) C o r r e l a t i o n o f T o p o II-Alpha With O t h e r C l i n i c o p a t h o l o g i c Features.
Because of the clear correlation of topo II-alpha with MIB1, a known marker of cell proliferation, we also correlated the presence of topo II-alpha with the S-phase fraction and mitotic index, as shown in Fig 5 and 6, respectively. T h e correlation between these parameters and topo II-alpha is not as good as it is between topo II-alpha and MIB1. The correlation coefficient between the topo II-alpha index and S-phase fraction is 0.47 and between the topo II-alpha index and mitotic index is 0.80. The grading of breast cancer has been shown to be of prognostic importance. 44 In our series, the topo II-alpha index of grade I tumors is 16.1 (range, <1 to 41), that of grade II tumors is 21.9 (range, 10 to 36), and that of grade III tumors is 48.5 (range, 27 to 86). No correlation of topo II-alpha with h o r m o n a l status was observed (Table 1). I m m u n o h i s t o c h e m i c a l Staining for c-erbB-2.
Because the topo II-alpha gene located on chromosome 17 is frequently co-amplified with c-erbB-2 in breast
cancers, 19'2°'immunostaining for this oncogene was perf o r m e d on 28 of the tumors shown in Table 1. Only four of the tumors showed the distinct m e m b r a n e staining pattern characteristics of c-erbB-2 amplification. T h e r e was no obvious correlation with the topo II-alpha index.
DISCUSSION In this study, we evaluated the expression o f topo II-alpha in 20 patients with primary and metastatic breast c a n c e r by i m m u n o h i s t o c h e m i c a l staining for enzyme protein. We f o u n d that the expression of topo II-alpha correlates well with MIB1 (correlation coefficient of 0.97). MIB1 is a molecule o f u n k n o w n f u n c t i o n that has b e e n widely used to estimate the p e r c e n t a g e of cycling cells in samples o f h u m a n tum o r tissue. 45 This suggests that topo II-alpha may yield data o n cell proliferation similar to or b e t t e r than that o b t a i n e d by MIB1 immunostaining. T o p o II-alpha has clearly b e e n shown to be a m a r k e r o f cell proliferation in n o r m a l cells and tissues, 14'2]-24 and its
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DNA TOPO II-ALPHA IN BREAST CANCER (Lynch et al) 120
100
80
Correlation of t o p o II-alpha and MIB1 indices. The topo II-alpha and MIBI index for each of the breast cancers shown in Table 1 were determined as described in the Materials and Methods section and plotted against each other, The plotfing of the points was done with Microsoft Excel. The correlation coefficient was 0.97. FIGURE 4.
60
4o
20-
000
•
10
20
O0
30
40
50
I
I
I
I
60
70
80
90
10£
3 e p o Ila I n d e x
use as a cell proliferation m a r k e r in n o n - H o d g k i n ' s l y m p h o m a s has b e e n suggested as well. 25 As expected, the topo II-alpha i n d e x also c o r r e l a t e d fairly well with mitotic i n d e x (correlation coefficient of 0.80). T h e fact that the topo II-alpha i n d e x did not correlate b e t t e r with mitotic i n d e x is probably because the n u m b e r o f cells in mitosis is only a small fraction o f the entire t u m o r cell population. T h e r e f o r e , mitotic indices between different tumors are all fairly small n u m b e r s without large variation. Thus, the mitotic i n d e x may n o t be able to accurately discriminate between tumors o f similar proliferation potential. Surprisingly, we f o u n d that the topo II-alpha index did not correlate well with t u m o r S-phase fraction measured by flow cytometry (correlation coefficient of 0.47). We believe this result points out the inherent problems of estimating the S-phase fraction of a t u m o r by flow cytometric analysis. Dilution of the sample with
normal cells as well as nuclear debris present in formalin-fixed sections can affect measurements made by flow cytometry. In agreement with our results, a recent study also shows the discordance that can be seen between measurements of cell proliferation by immunohistochemistry and flow cytometry. 46 Because of the good correlation between topo IIalpha and MIB1 as well as a fairly close correlation with mitotic index, we believe that the measurement of cell proliferation in breast cancers might be best done by immunohistochemical staining for topo II-alpha. The topo II-alpha measurement does not suffer from the problems seen with flow cytometry and, unlike mitotic index, cells staining for topo II-alpha make up a larger fraction of the overall tumor cell population. The use of the topo II-alpha index as a proliferation marker over MIB1 has several other advantages as well. T o p o II-alpha is present only during the late S and G2 phases
90"1
80'
70'
FIGURE 5. Correlation of the topo II-alpha index with percent of ceils in S-phase. The topo II-alpha index for each of the breast cancers described in Table 1 was determined as described in the Materials and Methods section. Each topo II-alpha index was then plotted against the percent of cells in S-phase as determined by flow cytometry (see Table 1). The plotting of the points was done with Microsoft Excel. The correlation coefficient was 0.47,
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Percent of Tumor Cells ill Mitoses (Mitotic Index x 100)
FIGURE 6. Correlation of the topo II-alpha index with mitotic index. The topo II alpha index and mitotic index of the breast cancers described in Table 1 was determined as described in the Materials and Methods section. The mitotic index is the fraction of tumor cells in mitosis and was plotted against the topo II-alpha index. For clarity, the mitotic index has been multiplied by 100 to give the percent of tumor cells in mitosis. The graphing of the points was done with Microsoft Excel. The correlation coefficient was 0.80.
of the cell cycle, 21 whereas MIB1 is present in all phases of the cell cycle except G0. 47 Therefore, topo II-alpha m i g h t give a better estimate of the n u m b e r of actively cycling cells than MIB1. In addition, the structure and enzymatic m e c h a n i s m of topo II is well understood, 48 and the enzyme is the molecular target of a n u m b e r of clinically useful a n t i t u m o r drugs, 49 some of which are used in the t r e a t m e n t of breast cancer. T h e function of MIB1 is not known. 45 Because m e a s u r e m e n t of cell proliferation in breast cancers has repeatedly b e e n shown to have prognostic i m p o r t a n c e ,v - l l we believe that topo II-alpha may yield similar information and, in addition, may potentially give drug targeting data. T h e r e are two isoforms of m a m m a l i a n topo II, t e r m e d alpha and beta. t3'14 In vitro, b o t h isoforms function as drug targets, 14'~a although it has b e e n suggested that the alpha f o r m is m o r e sensitive. 14 It has generally b e e n t h o u g h t that the alpha isoform is the primary drug target, a l t h o u g h the sensitivity of some breast cancer cell lines to etoposide has recently b e e n f o u n d to correlate better with topo II-beta levels than with topo IIalpha. D° We did n o t estimate topo II-beta in our work. Studies f r o m others have indicated that the beta isof o r m is readily detected in some breast cancers but not in others. ~6 T h e beta isoform does not correlate with cell proliferation.14'21 Therefore, although the alpha isof o r m clearly gives cell proliferation data, it may be the c o m b i n a t i o n of b o t h the alpha and beta isoforms that yield information on drug sensitivity. Further clinical 1186
studies employing specific topo II antibodies are n e e d e d to answer this question. Part of our interest in measuring topo II-alpha expression in breast cancer was because of its gene coamplification with c-erbB-2 that has b e e n r e p o r t e d in several in vitro systems. 19'2° Interestingly, amplification of c-erbB-2 in breast cancer was f o u n d in recent clinical studies to be associated with a relatively good response to c h e m o t h e r a p e u t i c protocols containing doxorubicin, but overexpression was associated with a relative resistance to protocols that did not include doxorubicin. 7'8 T o p o II-alpha is the molecular target of doxorubicin, 12 so it could be hypothesized that co-amplification of topo II with c-erbB-2 might explain the relative sensitivity of these overexpressing tumors to the drug. However, only 4 of 28 cases in o u r series of breast cancers were f o u n d to overexpress c-erbB-2, and these cases did not include the ones with the highest topo II-alpha index. However, co-amplification of topo II-alpha and c-erbB-2 may be a rare event in patient populations, and so our sample size m i g h t not have b e e n large enough to detect the few cases with amplification of both genes. The topo II-alpha indices in o u r series of patients ranged f r o m less than 1 to 86. Because the a m o u n t of the enzyme in a cell has b e e n shown to correlate with drug sensitivity in n u m e r o u s studies, 3739 we suggest that those tumors with high topo II-alpha indices would most likely be the ones responsive to c h e m o t h e r a p e u t i c protocols involving topo I I - t a r g e t e d drugs, and those tumors with low topo II indices would be relatively resistant. In this regard, it is worth noting that the proliferation m a r k e r Ki-S1, which has previously b e e n shown to have prognostic information in breast carcinomas, 51 has recently b e e n identified as topo I I - a l p h a ) 2 What the topo II-alpha index cut-off points for drug sensitivity and resistance are still remain to be defined, although it is possible that tumors n e e d at least a topo II-alpha index of a b o u t 50 or greater to show a good clinical response to topo I I - t a r g e t e d agents. 53 However, with the d e v e l o p m e n t of an i m m u n o h i s t o c h e m i c a l stain for the enzyme, the expression of topo II-alpha in breast cancers, as well as in other groups of neoplasms, can now be easily estimated, and the enzyme's role in determining a patient's sensitivity or resistance to chemotherapeutic drugs can be better understood.
Acknowledgment. The authors thank J. Chris Pitchford for his support.
REFERENCES 1. Harris JR, L i p p m a n ME, Veronesi U, et al: Breast cancer. N E n g l J Med 327:319-328, 1992 2. Fisher B, R e d m o n d C, Dimitrov NV, et al: A r a n d o m i z e d clinical trial evaluating sequential m e t h o t r e x a t e a n d fluorouracil in the t r e a t m e n t of patients with node-negative breast cancer who have estrogen-receptor-negative tumors. N Engl J Med 320:474-478, 1989 3. Fisher B, CostantinoJ, R e d m o n d C, et al: A randomized clinical trial evaluating tamoxifen in the t r e a t m e n t of patients with nodenegative breast cancer who have estrogen-receptor-positive tumors. N E n g l J Med 320:479-484, 1989
DNA TOPO II-ALPHA IN BREASTCANCER (Lynch et al) 4. Mansour EG, Gray R, Shatila AH, et al: Efficacy of adjuvant chemotherapy in high-risk node-negative breast cancer. N EnglJ Med 320:485-490, 1989 5. Goldhirsch A, The Ludwig Cancer Study Group: Prolonged disease-free survival after one course of perioperative adjuvant chemotherapy for node-negative breast cancer. N Engl J Med 320:491-496, 1989 6. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the HER2 / n e u proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989 7. Huss HB, Thor AD, Berry D, et al: c-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 330:1260-1266, 1994 8. Allred DC, Clark GM, Tandon AN, et al: Her2/neu in nodenegative breast cancer: Prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 10:599605, 1992 9. Meyer JS, Friedman E, McCrate M, et al: Prediction of early course of breast carcinoma by thymidine labeling. Cancer 51:18791886, 1983 10. Clark GM, Dressler LG, Owens M, et al: Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry. N EnglJ Med 320:627-633, 1989 11. Clark GM, Mathieu M, Owens MA, et al: Prognostic significance of S-phase fraction in good-risk, node-negative breast cancer patients. J Clin Oncol 10:428-432, 1992 12. Capranico G, Zunino F, Kohn KW, et al: Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: Relationship with DNA binding affinity and cytotoxicity. Biochemistry 29:562-569, 1990 13. Chung DU, Drake FH, Tan KB, et al: Characterization and immunological identification of eDNA clones encoding two human topoisomerase isozymes. Proc Natl Acad Sci U S A 86:9431-9435, 1989 14. Drake FH, Hofmann GA, Bartus HF, et al: Biochemical and pharmacological properties of p 170 and p 180 forms of topoisomerase II. Biochemistry 28:8154-8160, 1989 15. Mirski SEL, Evans CD, Almquis KC, et al: Altered topoisomerase II-alpha in a drug-resistant small cell lung cancer cell line selected in VP-16. Cancer Res 53:4866-4873, 1993 16. CampainJA, Gottesman MM, Pastan I: A novel mutant topoisomerase II-alpha present in a VP-16-resistant human melanoma cell lines has a deletion of alanine 429. Biochemistry 33:11327-11332, 1994 17. Mirski SEL, Cole SPC: Cytoplasmic localization of a mutant Mr 160,000 topoisomerase II-alpha is associated with the loss of putative bipartite nuclear localization signals in a drug-resistant human lung cancer cell line. Cancer Res 55:2129-2134, 1995 18. Hsiung Y, Jannatipour M, Rose A, et al: Functional expression of human topoisomerase II-alpha in yeast: Mutations at amino acids 450 or 803 of topoisomerase II-alpha result in enzymes that can confer resistance to anti-topoisomerase II agents. Cancer Res 56:9199, 1996 19. CouttsJ, Plumb JA, Brown R, et al: Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes. BrJ Cancer 68:793-800, 1993 20. Smith K, Houlbrook S, Greenatl M, et al: Topoisomerase IIalpha co-amplification with erbB2 in human primary breast cancer and breast cancer cell lines: Relationship to m-AMSA and mitoxantrone sensitivity. Oncogene 8:933-938, 1993 21. Woessner RD, Mattern MR, Mirabelli CK, et al: Proliferationand cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ 2:209-214, 1991 22. Heck MMS, Earnshaw WC: Topoisomerase II: A specific marker for cell proliferation. J Cell Biol 103:2569-2581, 1986 23. Capranico G, Tinelli S, Austin CA, et al: Differential patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development. Biochim Biophys Acta 132:4348, 1992 24. JuenkeJM, HoldenJA: The distribution of DNA topoisomerase II isoforms in differentiated adult mouse tissues. Biochim Biophys Acta 1216:191-196, 1993
25. HoldenJA, Perkins SL, Snow GW, et al: Immunohistochemical staining for DNA topoisomerase II in non-Hodgkin's lymphomas. A m J Clin Pathol 104:54-59, 1995 26. Efferth T, MatternJ, Volm M: Immunohistochemical detection of P glycoprotein, glutathione S transferase and DNA topoisomerase II in human tumors. Oncology 49:368-375, 1992 27. Hellemans P, van Dam PA, Geyskens M, et al: Immunohistochemical study of topoisomerase II-alpha expression in primary duetal carcinoma of the breast. J Clin Pathol 48:147-150, 1995 28. Kaufmann SH, Mclaughlin SJ, Kastan MB, et al: Topoisomerase II levels during granulocytic maturation in vitro and in vivo. Cancer Res 51:3534-3543, 1991 29. Holden JA, Rolfson DH, Wittwer CT: The distribution of immunoreactive topoisomerase II protein in human tissues and neoplasms. Oncol Res 4:157-166, 1992 30. Harker WG, Slade DL, Drake FH, et al: Mitoxantrone resistance in HL-60 leukemia cells: Reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II/3 isoform. Biochemistry 30:9952-9961, 1991 31. Khelifa T, Casabianca-Pignede M, Rene B, et al: Expression of topoisomerases II alpha and beta in chinese hamster lung cells resistant to topoisomerase II inhibitors. Mol Pharmacol 46:323-328, 1994 33. Giaccone G, van Ark-Otte J, Scagliotti G, et al: Differential expression of DNA topoisomerases in non-small cell lung cancer and normal lung. Biochim Biophys Acta 1264:337-346, 1995 34. Austin CA, Marsh KL, Wasserman RA, et al: Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II/3. J Biol Chem 270:1573915746, 1995 35. Brown GA, McPhersonJP, Lie G, et al: Relationship of DNA topoisomerase II-alpha and beta expression to cytotoxicity of antineoplastic agents in human acute lymphoblastic leukemia cell lines. Cancer Res 55:78-82, 1995 36. D'Andrea MR, Farger PA, Foglesong PD: Immunohistochemical detection of DNA topoisomerases II alpha and II beta compared with detection of Ki-67, a marker of cellular proliferation, in human tumors. Appl Immunohist 2:177-185, 1994 37. Davies SM, Robson CN, Davies SL, et al: Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins.J Biol Chem 263:17724-17729, 1988 38. Potmesil M, Hsaing Y, Liu LF, et al: Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels ofDNA topoisomerase II. Cancer Res 48:3537-3543, 1988 39. NitissJL, Ya-Xia L, Harbury P, et al: Amsacrine and etoposide hypersensitivity of yeast cell cells overexpressing DNA topoisomerase II. Cancer Res 52:4467-4472, 1992 40. HoldenJA, Rolfson DH, Wittwer CT: Human DNA topoisomerase II: Evaluation of enzyme activity in normal and neoplastic tissues. Biochemistry 29:2127-2134, 1990 41. Dalton LW, Page DL, Dupont WD: Histologie grading of breast carcinoma. Cancer 73:2765-2770, 1994 42. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248-254, 1976 43. Miller KG, Liu LF, Englund PT: A homogeneous type II DNA topoisomerase from Hela cell nuclei. J Biol Chem 256:93349339, 1981 44. Henson DE, Ries L, Freedman LS, et al: Relationship among outcome, stage of disease, and histologic grade for 22,616 cases of breast cancer: The basis for a prognostic index. Cancer 68:2142-2149, 1981 45. Duehrow M, Gerdes J, Schluter C: The proliferation-associated Ki-67 protein: Definition in molecular terms. Cell Prolif 27:235242, 1994 46. Linden MD, E1-Naggar AK, Nathanson SD, et al: Lack of correlation between flow cytometric and immunohistologic proliferation measurements of tumors. Mod Pathol 9:682-689, 1996 47. Gerdes J, Lemke H, Baisch H, et al: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715, 1984
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48. Berger JM, Gamblin SJ, Harrison SC, et al: Structure and mechanism of DNA topoisomerase II. Nature 379:225-232, 1996 49. Sinha BK: Topoisomerase inhibitors: A review of their therapeutic potential in cancer. Drugs 49:11-19, 1995 50. Houlbrook S, Addison CM, Davies SL, et al: Relationship between expression of topoisomerase II isoforms and intrinsic sensitivity to topoisomerase II inhibitors in breast cancer cell lines. Br J Cancer 72:1453-1461, 1995 51. Sampson SA, t@eipe H, Gillett CE, et al: KiS1, a novel mono-
clonal antibody that recognizes proliferating cells: Evaluation of its relationship to prognosis in mammary carcinoma. J Pathol 168:179185, 1992 52. Boege F, Andersen A, Jensen S, et al: Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II-~lpha. Am J Pathol 146:1302-1308, 1995 53. Guinee DG, Holden JA, Benfield JR, et al: Comparison of DNA topoisomerase II-alpha expression in small cell and nonsmall cell carcinoma of the lung: In search of a mechanism of chemotherapeutic response. Cancer 78:729-735, 1996
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Breast cancer is a m a j o r health p r o b l e m affecting a large n u m b e r of women. T h e incidence of breast cancer in developed countries is approximately 12%. 1 T h e d e v e l o p m e n t of newer and m o r e sensitive radiological p r o c e d u r e s has helped to identify breast cancer in w o m e n when the t u m o r is still small a n d lymph n o d e metastases have not occurred. Adjuvant c h e m o t h e r a p y in w o m e n with node-negative disease has b e e n shown to improve overall disease-free survival. 25 However, it is not entirely clear that all w o m e n with node-negative breast cancer have tumors that will necessarily pursue an aggressive clinical course. Such w o m e n m i g h t be cured by local therapy alone and would not benefit f r o m systemic adjuvant chemotherapy. This has stimulated m u c h research into the possibility of using prognostic and predictive markers to d e t e r m i n e which patients would benefit f r o m chemotherapy. Two markers that have b e e n studied with respect to breast cancer include the c-erbB-2 (c-Neu) o n c o g e n e and the proliferation index. T h e c-erbB-2 gene encodes a growth factor receptor. Amplification of this gene has
From the Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT. Accepted for publication February 5, 1997. Supported by a research educational grant from Pharmacia and Upjohn, San Clemente, CA. Address correspondence and reprint requests to Joseph A. Holden, MD, PhD, Department of Pathology, University of Utah Health Sciences Center, 50 North Medical Dr, Salt Lake City, UT 84132. Copyright © 1997 by W.B. Saunders Company 0046-8177/97/2810-001055.00/0
uated for c-erbB-2 amplification, h o r m o n a l status, S-phase fraction, and mitotic index. The topo II-alpha index correlates better with the MIB1 index than with the S-phase fraction or mitotic index. The topo
II-alpha expression in breast cancer ranges from low (topo II-alpha index <1) to high (topo II-alpha index = 86). Amplification o f cerbB-2 was observed in 4 o f 28 cases (14%) but did not correlate with high topo n-alpha indices. We conclude that measurement o f topo II-alpha in invasive breast cancer can be readily p e r f o r m e d by immunohistochenfical staining, and it gives information on the number o f cycling tumor cells. In addition, b e c a m e the enzyme is the molecular target o f doxorubicin, the expression o f the enzyme may relate also to the sensitivity or resistance o f the tumor to doxorubicin-based chemotherapeutic protocols. HUM PATHOL 28:1180--1188. Copyright © 1997 by W.B. Saunders Company Key words: DNA topoisomerase H-alpha, MIB1, cell proliferation, breast cancer. Abbreviations: topo II, DNA topoisomerase II; kDNA, kinetoplast DNA; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetra-acetic acid; ATP, adenosine triphosphate.
b e e n r e p o r t e d in 28% of breast cancers. 6 Recent studies have indicated that amplification of the c-erbB-2 gene may indicate the response of breast cancer patients to chemotherapy. Patients treated with a doxorubicin-containing protocol are relatively sensitive to c h e m o t h e r apy if their tumors overexpress c-erbB-2 7 and yet are relatively resistant if the c h e m o t h e r a p y protocol does not contain doxorubicin, s Although the molecular m e c h a n i s m that serves as a basis for these results is not yet clear, these results with c-erbB-2 underscore the i m p o r t a n c e of c-erbB-2 gene amplification in predicting response to therapy. Many studies have also suggested that a high mitotic rate in a breast cancer portends a p o o r prognosis. 9-11 DNA topoisomerase II (topo II) is now known to be the molecular target of doxorubicin. 12 M a m m a 1ian cells contain two isoforms of the enzyme, t e r m e d topo II-alpha a n d topo II-beta. 13'14 Most data suggest that the alpha isoform is the primary drug target. 14-18 Interestingly, the topo II-alpha gene has b e e n shown to be coamplified along with c-erbB-2 in cell lines and in h u m a n breast cancers. 19'20 This might suggest that co-amplification of topo II-alpha with c-erbB-2 in breast cancers would sensitize the t u m o r cells to doxorubicin-based chemotherapy. T o p o II-alpha is also a m a r k e r of cell proliferation in both normal and neoplastic tissues and cells. 14'2>~5 These facts suggest that the expression of topo II-alpha in h u m a n breast cancer may show imp o r t a n t correlations with the response of the neoplasm to doxorubicin-containing protocols, c-erbB-2 amplification, and with cell proliferation data. Unfortunately, there are limited data on the ex-
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DNA TOPO II-ALPHAIN BREASTCANCER (Lynch et al) p r e s s i o n o f t o p o I I - a l p h a in h u m a n b r e a s t c a n c e r . Previous studies e m p l o y i n g p o l y c l o n a l t o p o II a n t i b o d i e s in i m m u n o h i s t o c h e m i s t r y have s h o w n t h e p r e s e n c e o f t o p o II in h u m a n b r e a s t c a n c e r . 26'27 H o w e v e r , t h e s e studies a r e p o t e n t i a l l y c o m p l i c a t e d by t h e fact t h a t s o m e p o l y c l o n a l a n t i b o d i e s , initially t h o u g h t to b e specific f o r t o p o II-alpha, a r e n o w k n o w n to r e a c t with t h e b e t a i s o f o r m as well. 28'29 T h e r o l e t h a t t o p o I I - b e t a plays in t u m o r d r u g sensitivity is n o t y e t u n d e r s t o o d . 3°-35A d d i t i o n a l w o r k u s i n g specific t o p o I I - a l p h a a n t i b o d i e s in i m m u n o h i s t o c h e m i s t r y has o n l y e v a l u a t e d a small n u m b e r o f b r e a s t c a n c e r s , a n d surprisingly, n o c o r r e l a t i o n with cell p r o l i f e r a t i o n was o b s e r v e d , s6 W e r e c e n t l y r e p o r t e d t h a t it is p o s s i b l e to d e t e c t t o p o I I - a l p h a in f o r m a l i n - f i x e d , p a r a f f i n - e m b e d d e d hum a n tissue s e c t i o n s with specific m o n o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t t h e e n z y m e . 25 I n this r e p o r t , we i m m u n o s t a i n e d 30 s a m p l e s o f p r i m a r y a n d m e t a s t a t i c b r e a s t c a n c e r f r o m 20 p a t i e n t s f o r t o p o II-alpha. W e f o u n d t h a t t h e e x p r e s s i o n o f t h e e n z y m e c o r r e l a t e s well with MIB1, a k n o w n cell p r o l i f e r a t i o n m a r k e r . This r e s u l t suggests t h a t t o p o I I - a l p h a m a y b e a useful p r o l i f e r a t i o n m a r k e r , w h i c h can b e u s e d in s t u d y i n g b r e a s t c a n c e r . In addition, some tumors expressed high topo II-alpha levels. F r o m t h e s e data, we h y p o t h e s i z e t h a t t h e s e h i g h t o p o I I - a l p h a - e x p r e s s i n g t u m o r s m i g h t r e p r e s e n t a subclass o f n e o p l a s m s t h a t w o u l d r e s p o n d well to c h e m o therapy protocols containing doxorubicin. Doxorubicin targets t o p o II, 12 a n d m u c h d a t a a r e available t h a t s u g g e s t t h a t t h e sensitivity o f a cell to t o p o I I - t a r g e t e d d r u g s is d i r e c t l y p r o p o r t i o n a l to the a m o u n t o f c e l l u l a r t o p o II. 37-39
MATERIALS AND METHODS Sources of Tissue and Characterization Paraffin blocks were retrieved from 20 patients with primary and metastatic breast cancer from the surgical pathology files of the University of Utah Health Sciences Center. Several patients had material available from both the primary tumor as well as metastatic tumor, and so a total of 30 paraffin blocks of breast cancer were available for immunohistochemical staining. All cases were reviewed to ensure the accuracy of the diagnosis. Selection of the cases was based on the availability of sufficient material for immunohistochemical staining. The S-phase fraction of each tumor was determined by flow cytometry, as described by Holden et al. 4° Each tumor was given a modified Bloom-Richardson score by one investigator (D.G.), using established criteria. 41 The mitotic index of each tumor was determined by counting the number of mitotic figures per 500 tumor cells and was performed by one investigator (B.L.). The mitotic index is the fraction of tumor cells in mitosis.
Chemicals The sources of the chemicals were as previously described. 25 Kinetoplast DNA (kDNA) was isolated from Crithidiafasiculata as described. 4° The c-erbB-2 antibody is a monoclonal antibody that recognizes the carboxy terminus of the c-erbB-2 oncogene and was obtained from Oncogene Sciences (Cambridge, MA). The topo II-alpha antibody is a newly developed mouse monoclonal antibody similar to that described
previously.25 The antibody was purified from the hybridoma supernatant by protein G chromatography. Sodium dodecyl sulfate (SDS) gel electrophoresis of the purified fraction showed pure immunoglobulin. The concentration of the purified topo II-alpha antibody was 0.77 m g / m L . Antibodies against MIB1 were obtained from AMAC, Westbrook, ME.
Topo I[ Catalytic Activity The catalytic activity of topo II was determined by following the adenosine triphosphate (ATP)-dependent decatenation of kDNA. 4° The reaction was performed in 20-#L volumes containing 50 m m o l / L Tris-HC1 (pH 7.5), 85 m m o l / L KC1, 10 m m o l / L MgC12, 5 m m o l / L dithiothreitol, 5 m m o l / L ethylenediaminetetra-acetic acid (EDTA), 30 # g / m L bovine serum albumin, 1 m m o l / L ATP, and 250 ng kDNA. Reactions were allowed to proceed for 60 minutes at 30°C and then stopped by the addition of 2 #L 10% sodium dodecyl sulfate (SDS), 0.15 m m o l / L EDTA, 0.2% bromophenol blue, 50% glycerol. Samples were then electrophoresed on 0.8% agarose gels at 10 volts for 15 hours. The gel buffer was 40 m m o l / L Tris-HC1 (pH 7.5), 20 m m o l / L sodium acetate, 1 m m o l / L EDTA. The gels were stained in ethidium bromide (0.5 # g / m L ) for 20 minutes, rinsed with H20, and the amount of monomer circles released from the kDNA was visibly estimated by inspection of the ethidium-stained gel.
Preparation of Cellular and Nuclear Extracts Samples of breast cancer tissue were processed into nuclear or cellular extracts in procedures described in detail elsewhere. 29 Briefly, fresh tissue was obtained immediately after surgery and evaluated by frozen section for the presence of tumor. For nuclear extracts, the sample was then homogenized in a 0.3 m o l / L sucrose buffer and the nuclei isolated by centrifugation at 1,000g for 20 minutes. The nuclei were lysed by the addition of I m o l / L NaC1 and the DNA precipitated by the addition of poly(ethylene glycol) to a final concentration of 6%. After centrifugation at 20,000g for 30 minutes, topo II is recovered in the supernatant. For whole cell extracts, the tissue was first minced into 2- to 4-mm pieces with a scalpel and suspended in a 0.25 m o l / L sucrose buffer. After homogenization, the topo II was extracted by incubation of the sample at 95°C in buffer containing 1% SDS. The DNA was sheared by passing the sample through an 18-gauge needle.
Western Blotting Western blotting for topo II was performed as described. 29 Samples of whole cell extracts were electrophoresed on 8% polyacrylamide gels under denaturing conditions. After electrophoresis, the proteins were blotted onto nitrocellulose or nylon membranes with the use of a Mini-Protean II Trans Blot Cell apparatus (Bio-Rad, Hercules. CA) in 20 m m o l / L Tris-HC1 (pH 8.0), 150 m m o l / L glycine, 20% methanol at 20 volts for 16 hours at 4°C. The primary topo II antibody used to detect the protein was the monoclonal antibody used in the immunostaining procedures. The secondary antibody was an alkaline phosphatase-conjugated anti-mouse immunoglobulin (Promega, Madison, WI). Detection was with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as described. 29
lmmunohistochemical Staining Immunostaining for topo II-alpha and MIB1 was performed as described previously.25 Briefly, slides were cut in 3-
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to 5-#m sections and after deparaffinization were heated in 10 m m o l / L sodium citrate (pH 6.0) for 30 minutes on high in a Samsung microwave oven. After cooling, immunohistochemical staining was performed on a Ventana 320 automated immunohistochemical stainer in accord with the manufacturer's instructions. The topo II antibody was used at a dilution of 1:500 and the MIB1 antibody was used at a dilution of 1:40. Detection for either topo II-alpha or MIB1 was with the use of a secondary mouse immunoglobulin G linked to biotin followed by incubation with streptavidin linked to horseradish peroxidase. Color development was accomplished with diaminobenzidine. The topo II or MIB1 indices are defined as the number of positive-staining tumor cells divided by the total number of tumor cells counted times 100. At least 500 tumor cells were counted in the area of highest staining. All nuclear staining, whether strong or weak, was taken as indicative of topo IIalpha or MIB1 positivity. The indices represent the average of two i n d e p e n d e n t determinations by different observers (B.L. and J.H.). Immunostaining for c-erbB-2 (c-Neu) was performed as for topo II-alpha and MIB1 except that the heating step was not used. Evaluation was performed by one investigator (D.G.), and any distinct membrane staining was interpreted as positive] Statistics
The graphing and statistical analyses were performed with the use of the Microsoft Excel computer program. Protein Determination
The protein concentration of nuclear and crude cell extracts was estimated by the method of Bradford. 42
Lane:
Lane:
1
2
3
KDa
Origin T o p o II (~ -
-205
-97 -66
-43 BPB FIGURE 2. Whole cell extracts prepared from human breast cancer contain intact topo II-alpha immunoprotein. Whole cell extracts were prepared from a human breast cancer and subjected to Western blot as described in the Materials and Methods section. Lane 1 contains 25 #g breast cancer extract protein and Lane 2 contains 50/~g breast cancer extract protein. Lane 3 contains 4.6 #g nuclear extract protein prepared from a diffuse large cell non-Hodgkin's lymphoma known to have elevated amounts of t o p o II-alpha.25 The antibody used specifically recognizes the 170-kDa subunit of DNA topo II-alpha and is the same antibody used in the immunohistochemical staining. BPB indicates the position of the tracking dye, bromophenol blue. The molecular weight standards used are myosin heavy chain, 205 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 43 kDa.
1 2 ,3 4 5 6 RESULTS
-Network
T o p o II C a t a l y t i c A c t i v i t y a n d I m m u n o p r o t e i n A r e P r e s e n t in E x t r a c t s o f Human Breast Cancer
-Monomer
FIGURE 1. DNA topo II catalytic activity is present in a nuclear extract prepared from a human breast cancer. DNA topo II activity was assayed by the decatenation of kDNA as described in Materials and Methods. Highly c a t e n a t e d networks of kDNA remain at the origin. In the presence of functional t o p o II enzyme, monomer DNA circles are released from the kDNA and migrate into the gel. The amount of nuclear extract protein in each lane is as follows: lane 1,90 ng; lane 2, 40 ng; lane 3, 20 ng; lane 4, 10 ng; lane 5, 5 ng; lane 6, 2.5 ng.
N u c l e a r extracts p r e p a r e d f r o m a g r a d e II invasive b r e a s t c a n c e r in a 63-year-old w o m a n w e r e assayed f o r t o p o II catalytic activity. T h e p r e s e n c e o f t o p o II activity is s e e n as t h e l i b e r a t i o n o f m o n o m e r D N A circles f r o m t h e h i g h l y c a t e n a t e d kDNA, w h i c h is c o m p o s e d o f num e r o u s i n t e r l o c k e d D N A circles. Activity is o b s e r v e d in as little as 10 n g o f n u c l e a r extract, as s h o w n in F i g 1. This is in t h e s a m e r a n g e as o b s e r v e d in o t h e r h i g h l y m a l i g n a n t t u m o r s . 29 T h e lack o f o b s e r v a b l e activity with h i g h c o n c e n t r a t i o n s o f n u c l e a r e x t r a c t has b e e n obs e r v e d p r e v i o u s l y a n d p r o b a b l y reflects t h e i n h i b i t i o n o f d e c a t e n a t i o n by D N A b i n d i n g p r o t e i n s t h a t c a n b e p r e s e n t in c r u d e n u c l e a r extracts. 29 N u c l e a r extracts p r e p a r e d f r o m n o r m a l b r e a s t c o n t a i n t o p o II catalytic activity, w h i c h is at least 10 f o l d l o w e r t h a n t h a t s h o w n in F i g 1 ( H o l d e n , J, u n p u b l i s h e d d a t a ) . T h e r e f o r e , t h e activity p r e s e n t as s e e n in F i g 1 m o s t likely o r i g i n a t e s from the tumor. T h e p r e s e n c e o f t o p o I I - a l p h a p r o t e i n in t h e b r e a s t c a n c e r was c o n f i r m e d by W e s t e r n blot. As s h o w n in Fig
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DNA TOPO II-ALPHA IN BREAST CANCER (Lynch et al)
TABLE 1. Clinicopathotogic Features in 20 Patients With Invasive Breast Carcinoma Patient No.
Age (yr)
I
33
2
61
3 4 5 6 7 8 9 10
72 43 45 73 35 68 37 87
11
64
82 70 58
12 13 14
77 41
15 16
17
71
18 19
57 74
20
63
* The t The $ The § Not IENot
Site Breast Axillary Lyznph node Left breast Left axillary Lymph node Right breast Breast Breast Breast Breast Breast Breast Breast Right breast Left breast Breast Axillary Lymph node Breast Breast Breast Axillary Lymph node Breast Breast Axillary Lymph node Breast Axillary Lymph node Breast Left breast Right breast Breast Axillary Lymph node
Scarff-BloomRichardson Score
Estrogen/Progesterone Receptor Status
c-erb B-2++
0.6 1.2
6 NA II
+/+ ND
-
17.5 ND
3.6 2.8
+/-
-
NA
ND
-
23 15 18 21 26 51 18 25 20 10 1 2
5.3 4.9 1.1 9.0 2.7 8.2 3.7 3.2 3.5 ND 2.9 ND
0.4 1.0 0.2 1.4 0.2 3.8 0.6 0.4 0.2 0.6 <0.2 0.2
5 5 5 5
+/+/+ +/+ -/+
-
6 8 8 6 6 5 5 NA
+/+ +/+/+ -/+ +/+/+ +/+ ND
+ + + -
30 34 86 73
33 34 88 90
5.6 2.7 10.5 ND
1.4 1.4 2.6 4.8
6
+/+
-
9 NA
+/+ -/ND
-
12 30 29
12 36 29
3.0 2.0 ND
0.8 2.0 1.0
6
+/+
-
5
-/+
+
NA
ND
-
25 25
17 34
4.7 ND
0.8 0.8
6 NA
+/+ ND
-
22 2 1 10 5
25 2 1 11 6
4.8 1.8 ND 5.0 ND
1.0 0.2 <0.2 0.2 0.6
6 4 3 6 NA
+/+ +/+ +/+ +/+ ND
ND ND
TOPO II* Index
MIBI* Index
Flow Cytometry S-phase (%)
36 32
43 33
2.1 ND§
35 53
52 64
12 9 10 20 26 46 27 22 14 7 <1 <1
Mitotic Indext
S
5
-
-
-
Topo II and MIB1 indices are defined in the Materials and Methods section. mitotic index represents the percent of t u m o r cells in mitosis. presence of (+) or absence ( - ) of m e m b r a n o u s staining is indicated. determined. applicable.
2, a whole cell extract p r e p a r e d f r o m the t u m o r tissue a n d subjected to Western blot with the same topo IIalpha m o n o c l o n a l antibody that is used in the i m m u n o staining work described later shows the presence of an intact 170-kDa species. The subunit of topo II-alpha is 170 k O a . 43 T h e migration of this protein species is identical to the migration of the 170-kDa species present in a diffuse large cell n o n - H o d g k i n ' s lymphoma, which has previously b e e n shown to contain high levels of topo II-alpha. 25 T h e catalytic assay and Western blot indicate the presence of functional topo II activity and intact topo II-alpha protein in the t u m o r tissue and confirm the specificity of the antibody.
Immunohistochemical Staining of Breast Cancers for Topo II-Alpha and MIB1. Topo II-alpha i m m u n o s t a i n i n g of 30 samples of breast cancer f r o m 20 patients was p e r f o r m e d with an
antibody specific to the alpha isoform. For each case, a topo II index was d e t e r m i n e d that represents the n u m b e r of positive-staining t u m o r cells divided by the total n u m b e r of t u m o r cells counted times 100. T h e results are shown in Table 1 and represent the average of two i n d e p e n d e n t determinations by two different investigators (B.L. and J.H.). T h e topo II index of the tumors was highly variable and ranged from a low of < 1 to a high of 86. An example of the i m m u n o s t a i n i n g pattern of a t u m o r with a high topo II-alpha index is shown in Fig 3A, and that of a t u m o r with a low topo II-alpha index is shown in Fig 3B. A similar analysis of MIB1 in these tumors was p e r f o r m e d , and the results are also presented in Table 1. T h e MIB1 indices of the tumors ranged f r o m a low of 1 to a high of 90. T h e correlation of topo II-alpha with MIB1 in the series of breast cancers is shown in Fig 4. Clearly, topo II-alpha correlates well with MIB1 with a correlation coefficient of 0.97.
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HUMAN PATHOLOGY
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FIGURE 3. Immunohistochemical staining of human breast cancers for topo II-alpha. (A) Immunostaining of a grade Ill invasive human breast cancer for topo il-alpha shows many positive cells and the topo II-alpha index is 86. (Original magnification ×400) (B) Immunostaining of a grade II invasive human breast cancer for topo II-alpha shows only a few positive cells, and the topo ilalpha index is 12. Original (magnification ×400.) C o r r e l a t i o n o f T o p o II-Alpha With O t h e r C l i n i c o p a t h o l o g i c Features.
Because of the clear correlation of topo II-alpha with MIB1, a known marker of cell proliferation, we also correlated the presence of topo II-alpha with the S-phase fraction and mitotic index, as shown in Fig 5 and 6, respectively. T h e correlation between these parameters and topo II-alpha is not as good as it is between topo II-alpha and MIB1. The correlation coefficient between the topo II-alpha index and S-phase fraction is 0.47 and between the topo II-alpha index and mitotic index is 0.80. The grading of breast cancer has been shown to be of prognostic importance. 44 In our series, the topo II-alpha index of grade I tumors is 16.1 (range, <1 to 41), that of grade II tumors is 21.9 (range, 10 to 36), and that of grade III tumors is 48.5 (range, 27 to 86). No correlation of topo II-alpha with h o r m o n a l status was observed (Table 1). I m m u n o h i s t o c h e m i c a l Staining for c-erbB-2.
Because the topo II-alpha gene located on chromosome 17 is frequently co-amplified with c-erbB-2 in breast
cancers, 19'2°'immunostaining for this oncogene was perf o r m e d on 28 of the tumors shown in Table 1. Only four of the tumors showed the distinct m e m b r a n e staining pattern characteristics of c-erbB-2 amplification. T h e r e was no obvious correlation with the topo II-alpha index.
DISCUSSION In this study, we evaluated the expression o f topo II-alpha in 20 patients with primary and metastatic breast c a n c e r by i m m u n o h i s t o c h e m i c a l staining for enzyme protein. We f o u n d that the expression of topo II-alpha correlates well with MIB1 (correlation coefficient of 0.97). MIB1 is a molecule o f u n k n o w n f u n c t i o n that has b e e n widely used to estimate the p e r c e n t a g e of cycling cells in samples o f h u m a n tum o r tissue. 45 This suggests that topo II-alpha may yield data o n cell proliferation similar to or b e t t e r than that o b t a i n e d by MIB1 immunostaining. T o p o II-alpha has clearly b e e n shown to be a m a r k e r o f cell proliferation in n o r m a l cells and tissues, 14'2]-24 and its
1184
DNA TOPO II-ALPHA IN BREAST CANCER (Lynch et al) 120
100
80
Correlation of t o p o II-alpha and MIB1 indices. The topo II-alpha and MIBI index for each of the breast cancers shown in Table 1 were determined as described in the Materials and Methods section and plotted against each other, The plotfing of the points was done with Microsoft Excel. The correlation coefficient was 0.97. FIGURE 4.
60
4o
20-
000
•
10
20
O0
30
40
50
I
I
I
I
60
70
80
90
10£
3 e p o Ila I n d e x
use as a cell proliferation m a r k e r in n o n - H o d g k i n ' s l y m p h o m a s has b e e n suggested as well. 25 As expected, the topo II-alpha i n d e x also c o r r e l a t e d fairly well with mitotic i n d e x (correlation coefficient of 0.80). T h e fact that the topo II-alpha i n d e x did not correlate b e t t e r with mitotic i n d e x is probably because the n u m b e r o f cells in mitosis is only a small fraction o f the entire t u m o r cell population. T h e r e f o r e , mitotic indices between different tumors are all fairly small n u m b e r s without large variation. Thus, the mitotic i n d e x may n o t be able to accurately discriminate between tumors o f similar proliferation potential. Surprisingly, we f o u n d that the topo II-alpha index did not correlate well with t u m o r S-phase fraction measured by flow cytometry (correlation coefficient of 0.47). We believe this result points out the inherent problems of estimating the S-phase fraction of a t u m o r by flow cytometric analysis. Dilution of the sample with
normal cells as well as nuclear debris present in formalin-fixed sections can affect measurements made by flow cytometry. In agreement with our results, a recent study also shows the discordance that can be seen between measurements of cell proliferation by immunohistochemistry and flow cytometry. 46 Because of the good correlation between topo IIalpha and MIB1 as well as a fairly close correlation with mitotic index, we believe that the measurement of cell proliferation in breast cancers might be best done by immunohistochemical staining for topo II-alpha. The topo II-alpha measurement does not suffer from the problems seen with flow cytometry and, unlike mitotic index, cells staining for topo II-alpha make up a larger fraction of the overall tumor cell population. The use of the topo II-alpha index as a proliferation marker over MIB1 has several other advantages as well. T o p o II-alpha is present only during the late S and G2 phases
90"1
80'
70'
FIGURE 5. Correlation of the topo II-alpha index with percent of ceils in S-phase. The topo II-alpha index for each of the breast cancers described in Table 1 was determined as described in the Materials and Methods section. Each topo II-alpha index was then plotted against the percent of cells in S-phase as determined by flow cytometry (see Table 1). The plotting of the points was done with Microsoft Excel. The correlation coefficient was 0.47,
60'
50' o I
J
J
J
30' 20-
J o
10"
e,
0 0
•
2
:
I
4
6
I
I
I
I
I
I
8
10
12
14
16
18
%
1185
S-phase
20
HUMAN PATHOLOGY
Volume 28, No. 10 (October 1997)
90"
80"
70
60
_~ 50
~
40
30
4. !
Q
J
i
i
i
i
1
2
3
4
5
Percent of Tumor Cells ill Mitoses (Mitotic Index x 100)
FIGURE 6. Correlation of the topo II-alpha index with mitotic index. The topo II alpha index and mitotic index of the breast cancers described in Table 1 was determined as described in the Materials and Methods section. The mitotic index is the fraction of tumor cells in mitosis and was plotted against the topo II-alpha index. For clarity, the mitotic index has been multiplied by 100 to give the percent of tumor cells in mitosis. The graphing of the points was done with Microsoft Excel. The correlation coefficient was 0.80.
of the cell cycle, 21 whereas MIB1 is present in all phases of the cell cycle except G0. 47 Therefore, topo II-alpha m i g h t give a better estimate of the n u m b e r of actively cycling cells than MIB1. In addition, the structure and enzymatic m e c h a n i s m of topo II is well understood, 48 and the enzyme is the molecular target of a n u m b e r of clinically useful a n t i t u m o r drugs, 49 some of which are used in the t r e a t m e n t of breast cancer. T h e function of MIB1 is not known. 45 Because m e a s u r e m e n t of cell proliferation in breast cancers has repeatedly b e e n shown to have prognostic i m p o r t a n c e ,v - l l we believe that topo II-alpha may yield similar information and, in addition, may potentially give drug targeting data. T h e r e are two isoforms of m a m m a l i a n topo II, t e r m e d alpha and beta. t3'14 In vitro, b o t h isoforms function as drug targets, 14'~a although it has b e e n suggested that the alpha f o r m is m o r e sensitive. 14 It has generally b e e n t h o u g h t that the alpha isoform is the primary drug target, a l t h o u g h the sensitivity of some breast cancer cell lines to etoposide has recently b e e n f o u n d to correlate better with topo II-beta levels than with topo IIalpha. D° We did n o t estimate topo II-beta in our work. Studies f r o m others have indicated that the beta isof o r m is readily detected in some breast cancers but not in others. ~6 T h e beta isoform does not correlate with cell proliferation.14'21 Therefore, although the alpha isof o r m clearly gives cell proliferation data, it may be the c o m b i n a t i o n of b o t h the alpha and beta isoforms that yield information on drug sensitivity. Further clinical 1186
studies employing specific topo II antibodies are n e e d e d to answer this question. Part of our interest in measuring topo II-alpha expression in breast cancer was because of its gene coamplification with c-erbB-2 that has b e e n r e p o r t e d in several in vitro systems. 19'2° Interestingly, amplification of c-erbB-2 in breast cancer was f o u n d in recent clinical studies to be associated with a relatively good response to c h e m o t h e r a p e u t i c protocols containing doxorubicin, but overexpression was associated with a relative resistance to protocols that did not include doxorubicin. 7'8 T o p o II-alpha is the molecular target of doxorubicin, 12 so it could be hypothesized that co-amplification of topo II with c-erbB-2 might explain the relative sensitivity of these overexpressing tumors to the drug. However, only 4 of 28 cases in o u r series of breast cancers were f o u n d to overexpress c-erbB-2, and these cases did not include the ones with the highest topo II-alpha index. However, co-amplification of topo II-alpha and c-erbB-2 may be a rare event in patient populations, and so our sample size m i g h t not have b e e n large enough to detect the few cases with amplification of both genes. The topo II-alpha indices in o u r series of patients ranged f r o m less than 1 to 86. Because the a m o u n t of the enzyme in a cell has b e e n shown to correlate with drug sensitivity in n u m e r o u s studies, 3739 we suggest that those tumors with high topo II-alpha indices would most likely be the ones responsive to c h e m o t h e r a p e u t i c protocols involving topo I I - t a r g e t e d drugs, and those tumors with low topo II indices would be relatively resistant. In this regard, it is worth noting that the proliferation m a r k e r Ki-S1, which has previously b e e n shown to have prognostic information in breast carcinomas, 51 has recently b e e n identified as topo I I - a l p h a ) 2 What the topo II-alpha index cut-off points for drug sensitivity and resistance are still remain to be defined, although it is possible that tumors n e e d at least a topo II-alpha index of a b o u t 50 or greater to show a good clinical response to topo I I - t a r g e t e d agents. 53 However, with the d e v e l o p m e n t of an i m m u n o h i s t o c h e m i c a l stain for the enzyme, the expression of topo II-alpha in breast cancers, as well as in other groups of neoplasms, can now be easily estimated, and the enzyme's role in determining a patient's sensitivity or resistance to chemotherapeutic drugs can be better understood.
Acknowledgment. The authors thank J. Chris Pitchford for his support.
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48. Berger JM, Gamblin SJ, Harrison SC, et al: Structure and mechanism of DNA topoisomerase II. Nature 379:225-232, 1996 49. Sinha BK: Topoisomerase inhibitors: A review of their therapeutic potential in cancer. Drugs 49:11-19, 1995 50. Houlbrook S, Addison CM, Davies SL, et al: Relationship between expression of topoisomerase II isoforms and intrinsic sensitivity to topoisomerase II inhibitors in breast cancer cell lines. Br J Cancer 72:1453-1461, 1995 51. Sampson SA, t@eipe H, Gillett CE, et al: KiS1, a novel mono-
clonal antibody that recognizes proliferating cells: Evaluation of its relationship to prognosis in mammary carcinoma. J Pathol 168:179185, 1992 52. Boege F, Andersen A, Jensen S, et al: Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II-~lpha. Am J Pathol 146:1302-1308, 1995 53. Guinee DG, Holden JA, Benfield JR, et al: Comparison of DNA topoisomerase II-alpha expression in small cell and nonsmall cell carcinoma of the lung: In search of a mechanism of chemotherapeutic response. Cancer 78:729-735, 1996
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