ANNALS O F C O N ICA L AND LABORATORY SCIENCE, Vol. 28, No. 6Copyright © 1998, Institute for Clinical Science, Inc.

Comparison of Proliferation Activity in Breast Carcinoma by Flow Cytometry Analysis of S-phase and Quantitative Analysis of MIB-1*

SHAHLA MASOOD, MD, FCAP, MIAC MARILYN M. BUI, MD, PhD

and LEO LU MD

Department of Pathology, University of Florida Health Science Center,Jacksonville, FL 32209

ABSTRACT

The S-phase which assesses tumor proliferation has been considered to be an independent prognostic factor for breast carcinoma. Quantitative analysis of MIB-1 immunoreactivity is a newly recognized method of determining cellular proliferation that offers some advantages over flow cytometry when limited tumor tissue is avail­able. However, it has been controversial whether there is a significant correlation between MIB-1 immunostaining and S-phase in defining proliferation activity in breast cancer. In order to explore the usefulness of MIB-1 as an additional prolif­eration parameter and a potential prognostic factor for breast cancer, we analyzed 94 cases of invasive ductal carcinoma of the breast by both flow cytometry (for S-phase and DNA ploidy) and quantitative MIB-1 immunohistochemical analysis using for­malin-fixed paraffin-embedded tissue. MIB-1 staining was quantitatively analyzed by image analysis and by visual scoring. Forty-six cases were diploid by flow, while the remaining 48 cases were aneuploid tumors. T-test results indicated that S-phase means were significantly greater (p = 0.0001) in aneuploid cases (mean = 18) com­pared to diploid cases (mean = 7). MIB-1 means were also greater in aneuploid patients, but these differences were only marginally significant (p = 0.05). S-phase was positively correlated with MIB-1 (r = 0.36, p = 0.003 for image analysis and r = 0.34, p = 0.001 for visual scoring). ROC curve analysis indicated that MIB-1 quan­titation is a good predictor of high S-phase (ie, >10 percent) in aneuploid cases. A MIB-1 cutoff value of 25 percent for image analysis achieved 82 percent specificity and 80 percent sensitivity for aneuploid high S-phase, while a MIB-1 cutoff value of 40 percent for visual scoring was 73 percent specific and 85 percent sensitive. However, in diploid cases, no comparable MIB-1 cutoffs could be achieved for detecting high S-phase. In summary, our study demonstrated that aneuploid breast carcinomas proliferate more aggressively than diploid tumors. Although linear correlation between MIB-1 and S-phase was weak, MIB-1 was considered to be a

* Shahla Masood, M D, FCAP, MIAC, Professor and Associate Chair o f Department o f Pathology, Assistant Dean for Research, University o f Florida Health Science Center, Jacksonville; Chief o f Pathology, University Medical Center, 655 West Eighth Street, University Medical Center, Jacksonville, FL 32209, (904) 549-4219 (Phone), (904) 549-4290 (Fax), sm@umc5.umc.ufl.edu (E-mail).

3150091-7370/98/1100-0315 $02.25 © Institute for Clinical Science, Inc.

3 1 6 MASOOD, BUI, AND LU

good predictor of high S-phase in aneuploid breast cancer patients, possibly due to a threshold effect. Image analysis and visual scoring of MIB-1 immunoreac- tivity appeared to be comparable in analyzing proliferative activity in breast cancer. Thus MIB-1 assessed by visual scoring may be a less expensive alternative to image analysis.

Introduction

In 1988, the National Cancer Institute sug­gested a benefit from adjuvant therapy in women with node-negative breast cancer patients.1 This was based on two trials with median follow-ups of 3.5 years and the sugges­tion of an improved disease free survival. Given the natural history of breast cancer, however, this was a short follow-up period and the impact on overall survival could not be m easured. N evertheless, this suggestion brought a debate on the optimal treatment of patients with node-negative breast cancer. While the majority of oncologists considered treating their premenopausal node-negative wom en with estrogen recep tor negative tumors with adjuvant chemotherapy, there were others who preferred to undergo the challenge of identifying those 30 percent of node-negative breast cancer patients who were typically at a higher risk of relapse and subse­quent death from breast cancer.2’3’4’5,6,7’8’9

This method is of remarkable interest in the study of prognostic indicators in breast cancer. At the present time, tumor size, histologic type, estrogen and progesterone receptors and nuclear grade represent the established prog­nostic factors for use in the management of women with node-negative breast cancer.10’11

Reports on the solidity of proliferation rate as the predictor of biologic behavior of patients with breast cancer has also been consis­tent.12 There are also several newly recog­nized prognostic markers that are still under intense investigation.

Our previous study on 100 node-negative breast cancer patients identified proliferation rate as determined by the S-phase as an inde­pendent prognostic indicator. We demon­strated that S-phase fraction of more than

10 percent particularly in aneuploid tumors was associated with a poor outcome in the studied patients.12

S-phase is measured by flow cytometry, which is a technically dependent procedure, and the result may occasionally not represent the proliferation rate of a given tumor. This is due to features intrinsic to the tissue sample, such as contamination by non-tumoral ele­ments (benign epithelial cells, stromal cells, infiltrating cells and presence of cellular debris). Similar assessment of S-phase in tumors with multiple stem lines is difficult. In addition, as the sample size of breast cancer at the time of diagnosis is decreasing, the small sample size may also preclude flow cyto­metric analysis.

Recent development of antibodies against proliferation-associated nuclear antigens such, as MIB-I, now has provided another alterna­tive for assessment of proliferation rate in breast cancer specimens. MIB-I is applicable to formalin-fixed paraffin-embedded tissue and highlights the nuclei of proliferating cells in all cell cycles phases except those in resting phases (Go/GJ.13-14-15

In the current study, we compared the pro­liferation activity of breast carcinomas as determined by flow cytometric analysis versus quantitative MIB-I immunostaining on tissue sections obtained from formalin-fixed paraffin- embedded tissue blocks of breast cancer.

Materials and Methods

S p e c i m e n s

Formalin-fixed paraffin-embedded tissue blocks of 94 cases of invasive ductal carcinoma of the breast were selected from the surgical pathology archives of the D epartm ent of

FLOW CYTOMETRY ANALYSIS 3 1 7

Pathology at the University of Florida Health Science Center/Jacksonville. These cases were diagnosed and treated at our institute from 1983 to 1986. The information of S-phase and DNA ploidy status of these cases was retrieved from the Tumor Analysis Laboratory at Uni­versity Medical Center, Jacksonville, FL.

DNA F l o w C y t o m e t r y

All cases were fixed in 10 percent neutral buffered formalin and embedded in paraffin blocks. Five 50 |xm serial sections were cut from each block and placed into a 16 x 100 mm glass tube. Each sample was deparaf- finized by two changes of 10 ml of xylene treat­ment. Each treatment was about 15 minutes. Then the samples were dehydrated by two changes of 100 percent ethanol for 10 minutes, followed by rehydration with 10 ml of 95 per­cent, 70 percent, and 50 percent ethanol for 10 minutes respectively. Complete dehydration was achieved by adding distilled water to the specimen and allowing it to sit in a refrigerator overnight. The next day, single cell suspen­sions were obtained from the serial 50 |xrn sec­tions according to the established method.16,17 Briefly, specimens were minced and incubated with 3 ml pepsin in a 37°C shaker water bath for 30 minutes. Then the specimen was fil­tered and washed with buffer twice. A cell count was performed and adjusted to 1 million cells per milliliter. After treating the cells with trypsin and RNase, the cell suspensions were then stained with propidium iodide. For each run of flow cytometry analysis, embedded ton­sil cells were used as a control sample. The samples were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using a doublet discrimination module. Most of the DNA histograms were generated using the CellFIT Cell-cycle Analysis Program Version 2.01.2 (Becton Dickinson). Few cases collected in 1986 were also analyzed using ModFit Software Program Version 1.0 from the same company. The results analyzed by different software programs appeared to cor­relate well. The cell proliferation status index generated by these programs was S-phase per­

centage. S-phase fraction (SPF) was the sum of S-phase value and the percent of cells in G2-M.

MIB-1 I m m u n o h i s t o c h e m is t r y

From the same paraffin blocks used for flow cytometry analysis, sections were cut at 4 |xrn and placed onto silanized slides. After being dried for one hour at 65°C, they were depar- affinized with three changes of xylene, rehy­drated with graded ethanol and distilled water. A standard high temperature antigen recovery procedure in 1 mM citrate buffer at pH 6.0 was applied. We used a 900 w. Panasonic microwave oven set for two minutes on high to boil, followed by 15 minutes on medium-low. After the slides were cooled for 30 minutes, they were washed with phosphate-buffered saline (PBS). Endogenous peroxidase was blocked using 3 percent hydrogen peroxide for 15 minutes. Slides were rinsed with PBS, fol­lowed by removing excess PBS. Then mono­clonal antibody against MIB-1 (ImmunoTech Cat. No. 5050, Westbrook, ME) was diluted 1:200. The slides were incubated with primary MIB-1 antibody dilution at 4°C overnight. The next day we allowed the slides to reach room temperature for one hour. Sections were well rinsed in PBS, followed by a standard labeled streptavidin-biotin detection method (DAKO LSAB 2 Detection Kit, Carpintería, CA) using diaminobenzidine (DAB) as a substrate. The slides were counterstained using 0.4 percent aqueous ethyl green in pH 4.0 acetic acid buffer for 10 minutes, followed by successive rinsing in acetone, alcohol:xylene (1 :1 ), and xylene, and mounted in cytoseal. Each case was run in parallel with a negative control slide by using PBS to replace the primary antibody against MIB-1. Positive controls were slides of normal tonsils.

Q u a n t it a t iv e A n a l y s is o f MIB-1

Quantitation of MIB-1 was conducted by both image analysis and visual scoring to obtain the percentage of positively stained nuclei. Image analysis was performed by using

3 1 8 MASOOD, BUI, AND LU

a CAS 200 Image Analysis System (Becton Dickinson, Mountain View, CA) and its Quan­titative Proliferation Index (QPI) program (version 2.0). The proliferation index is the count of the number of MIB-l-tagged nuclei divided by the total number of cell nuclei. A uniform level of counting was set at 400-600 positive nuclei. Total area scanned per slide was between 20,000-25,000 |xm2. The average fields counted were four per slide. Visual scor­ing was done by three independent viewers simultaneously using a multihead microscope (Nikon Optiphot-2, Japan). By scanning the entire tumor areas on the slides at low power and scoring at 400x magnification, the per­centage of positively stained nuclei was calcu­lated. The average of the results from the three viewers was obtained. The inter-rater and intra-rater reliability were excellent.

St a t is t ic a l M e t h o d s

The Pearson correlation coefficient was used to assess pairwise linear correlation among S-phase, S-phase fraction (SPF), MIB-1 quantitative analysis by image analysis and visual scoring. Linear regression was used to estimate intercept and slope for quantitative analysis of MIB-1 as predictors of S-phase or SPF in order to test for significant difference among these estimates from zero. A dummy variable for ploidy was used to determine if regression lines of quantitative MIB-1 analysis for aneuploid and diploid patients were non­parallel or parallel but non-coincident. The independent sample t-test was used to com­pare mean S-phase, SPF and quantitative analysis of MIB-1 levels between aneuploid and diploid groups. Receiver-operator charac­teristic (ROC) curve analysis was used to assess the diagnostic performance of quantita­tive MIB-1 analysis (image analysis and visual scoring) as predictors of high S-phase (>10 percent). Each unique image analysis or visual scoring of MIB-1 value was used as cutoff to classify patients as highly likely or highly unlikely to have a high S-phase. Sensitivity and specificity for high S-phase, and percentage of

patients correctly classified as high S-phase, were computed to generate cutpoint for image analysis or visual scoring of MIB-1. Sensitivity/ specificity pairs were sorted by increasing specificity and sensitivity plotted against speci­ficity. The area under the ROC curve, which will vary between zero and one, was computed for aneuploid and diploid patient groups both separately and combined.

Results

P l o id y , S - p h a s e , SPF, a n d Q u a n t it a t iv e

A n a l y s is D a t a

By flow cytometry, 46 cases were diploid tumors, while 48 cases were aneuploid. The representative histogram of both a diploid and an aneuploid case as well as their S-phase value and SPF are demonstrated in figure 1. The result of quantitative analyses of MIB-1 determined by CAS 200 image analysis is not shown here. The representative visual scoring of MIB-1 immunoreactivity is demonstrated in figure 2 .

C o r r e l a t io n a m o n g S - p h a s e , SPF, a n d

V a l u e s o f MIB-1 b y Q u a n t it a t iv e A n a l y s is

For aneuploid group, S-phase was positively correlated with SPF (r = 0.70, p = 0.001), MIB-1 values (r = 0.34, p = 0.02 and r = 0.48, p = 0.008) by image analysis and by visual scor­ing respectively. SPF was positively correlated with MIB-1 by visual scoring (r = 0.37, p = 0.012). MIB-1 by image analysis was positively correlated with MIB-1 by visual scoring (r = 0.84, p = 0.0001). S-phase was positively cor­related with MIB-1 (r = 0.36, p = 0.003 for image analysis and r = 0.34, p = 0.001 for visual scoring) in both diploid and aneuploid cases.

For diploid group, S-phase was positively correlated with SPF (r = 0.74, p = 0.0001), MIB-1 by image analysis (r = 0.32, p = 0.026), and MIB-1 by visual scoring (r = 0.36, p =0.012). MIB-1 by image analysis was positively correlated with MIB-1 by visual scoring (r =0.77, p = 0.0001).

FLOW CYTOMETRY ANALYSIS 3 1 9

Content

DIPLOID:100.00%Dip G0-G1: 96.97% at 49.11 Dip G2-M: 1.28% at 98.21 Dip S: 1.75% G2/G 1:2.00 Dip %CV: 3.61

Total S-Phase: 1.75%

Diploid B.A.D.: 4.17%

DIPLOID: 61.64%Dip G0-G1: 89.93% at 50.26 Dip G2-M: 2.39% at 100.52 Dip S: 7.69% G2/G1: 2.00 Dip %CV: 6.01

ANEUPLOID 1: 38.36%An1 G0-G1: 65.28% at 81.81 An1 G2-M:0.00% at 172.47 An1 S: 34.72% G2/G1: 2.11 An1 %CV: 3.68 Dl: 1.63

Total S-Phase: 18.06%

Diploid B.A.D.: 9.77%

Aneuploid B.A.D.: 18.22%

F i g u r e 1. DNA histogram (gen­erated by ModFit Software Program Version 1.0). (A) a diploid tumor showing one CJ,/CJ, peak at channel 49.11 with S-phase o f 1.75% and SPF o f 3.01 percent. (B) an aneuploid tumor showing two distinct Gq/G, peaks. The diploid G,/G] peak is at 50 .26 . T he aneuploid peak is at 81 .81 w ith S-phase and SPF o f 34.72 percent.

100 150DNA ContentT T f f l 1 1 |

200 250

For aneuploid and diploid combined group, S-phase was positively correlated with SPF (r = 0.80, p = 0.0001), M1B-1 by image analysis (r = 0.36, p = 0.0003), and MIB-1 by visual scor­ing (r = 0.45, p = 0.0001). SPF was positively correlated with MIB-1 by image analysis (r =0.31, p = 0.002) and by visual scoring (r = 0.37, p = 0.0002). MIB-1 by image analysis was posi­tively correlated with MIB-1 by visual scoring (r = 0.81, p = 0.0001).

C o m p a r is o n o f R e g r e s s io n L i n e s a n d

M e a n s b e t w e e n A n e u p l o i d a n d

D ip l o id G r o u p s

Use of a dummy variable for ploidy group indicated parallel regression lines and a signifi­cant difference between aneuploid and diploid intercepts for MIB-1 by image analysis as a predictor of S-phase (p = 0.0001) and SPF (p = 0.0001), and MIB-1 by visual scoring

320 MASOOD, BUI, AND LU

A B

C

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m * 'w&

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F ig u r e 2 . Visual scoring o f MIB-1 immunohistochemi- cal staining (A) high positive, (B) intermediate positive, and (C) low positive.

as a predictor of SPF (p = 0.0001). Aneuploid and diploid regression lines for MIB-1 by visual scoring as a predictor of S-phase were found to be nonparallel (p = 0.032) with simi­lar intercepts.

T-test results indicated that S-phase mean (figure 3) were significantly greater in aneu­ploid cases (p = 0.0001, mean = 18) compared to diploid cases (mean = 7). SPF mean was also significantly greater in aneuploid cases (p = 0.0001) compared to diploid cases. MIB-1 means were also greater in aneuploid patients, but these differences were only marginally sig­nificant (p = 0.053 and 0.050 respectively).

ROC C u r v e A n a l y s is o f MIB-1 Q u a n t it a t io n a s a P r e d ic t o r o f

H ig h S - p h a s e

ROC curve analysis indicated MIB-1 values by image analysis to be good predictors of high S-phase in aneuploid patients (area under the ROC curve was 84.5 percent), with the best sensitivity/specificity pair noted at MIB-1 value of 25 percent (80.0 percent/81.8 per­cent) (figure 4). MIB-1 by image analysis was a

poor to moderate predictor of high S-phase in diploid patients (area under the ROC curve was 69.5 percent), with the best sensitivity/ specificity pair noted at MIB-1 value of 33 per­cent (60.0 percent/73.7 percent). MIB-1 by image analysis was also a poor to moderate predictor of high S-phase in aneuploid and diploid cases combined (area under the ROC curve was 74.7 percent), with the best sensi­tivity/specificity pair noted at MIB-1 value of 28 percent (71.1 percent/65.3 percent).

Similarly, ROC curve analysis indicated MIB-1 by visual scoring to be a good predictor of high S-phase in aneuploid cases (area under the ROC curve was 85.1 percent), with the best sensitivity/specificity pair noted at MIB-1 value of 40 percent (85.7 percent/72.7 per­cent). MIB-1 by visual scoring was a poor to moderate predictor of high S-phase in diploid patients (area under the ROC curve was 69.1 percent), with the best sensitivity/specificity pair noted at MIB-1 value of 60 percent (60.0 percent/65.8 percent). MIB-1 by visual scoring was also a poor to moderate predictor of high S-phase in aneuploid and diploid cases com­bined (area under the ROC curve was 76.3

FLOW CYTOMETRY ANALYSIS 321

8-PHASE BY PLOIDY

9

8

i

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oA D

PICMDY

F igure 3. Box plot o f S-phase determination grouped by ploidy. Aneuploid group (A) has a mean of 18.0 per­cent. Diploid group (D) has a mean o f 7.0 percent.

percent), with the best sensitivity/specificity pair noted at MIB-1 value of 45 percent (75.6 percent/63.3 percent).

Discussions

S-phase measured by flow cytometry is a gold standard to assess tumor proliferation that is well accepted as an independent prognostic factor for breast cancer. However, flow cytom­etry requires a flow cytometer as well as a large amount of fresh tissue specimen. Thus its application is compromised when fixed tissue is the sole material or when there is limited availability of specimen obtained by fine- needle aspiration biopsy of the breast. For practical purposes, an alternative method of assessing proliferation activity of breast carci­nomas that can be used routinely in an average pathology laboratory is on a demand basis.

Such a method should be highly specific, highly sensitive and technically easy to per­form. Ki67 immunohistochemical staining allows the assessment of cellular proliferation in fresh tissue. Newly recognized MIB-1 anti­body has the advantage of detecting Ki67 anti­gen in formalin-fixed paraffin-embedded tis­sue section. Our study investigated the validity of quantitation of MIB-1 by both image analy­sis and visual scoring by comparing the tumor cell proliferation rate with the results obtained by flow cytometric analysis. Our study indi­cated that S-phase and SPF was positively cor­related with MIB-1 quantitated by both image analysis and visual scoring. This finding is con­sistent with the results of other investigators.18 It seems that the existence of positive correla­tion is currently well accepted by the research­ers in this field. However, the degree of cor­relation may vary in different studies.

The results of our study also once again demonstrated the proliferation differences betw een aneuploid and diploid tum ors. Although the value of the MIB-1 mean was not useful to distinguish these two groups of

HOC CURVE ( M J C - M . S X )

«recm ctT Y n m h w h s -p h m e (%>

F igure 4. ROC curve for MIB-1 (determined by image analysis) as a predictor o f high S-phase (i.e. >10 percent) in aneuploid patients. Area under the ROC curve was 84.5 percent with the best sensitivity/specificity pair (80.0 percent/81.8 percent) at MIB-1 value o f 25 percent.

3 2 2 MASOOD, BUI, AND LU

tumors, MIB-1 is a good predictor of high S-phase in aneuploid breast cancer patients. When high S-phase is defined as greater than 10 percent, MIB-1 cutoff values (25 percent for image analysis and 40 percent for visual scoring) are both specific and sensi­tive for predicting aneuploid high S-phase. Unfortunately, no comparable MIB-1 cutoffs could be achieved for detecting high S-phase in diploid tumors. The explanation for this observation is currently unknown to us. This finding suggests that MIB-1 quantitation by immunocytochemistry offers an attractive alternative to assess the status of tumor cell proliferation in breast cancer cases. This is par­ticularly important for breast cancer cases that are either too small for flow cytometric analysis or for which this technique is not readily avail­able. With growing interest in the use of screening mammography and detection of small-sized breast cancer cases, as well as the use of breast fine-needle aspiration biopsy, less tissue is available for traditional flow cytometry study. In these scenarios, the immunostaining for MIB-1 becomes valid. Recently, the reli­ability of MIB-1 immunostaining of breast cancer cells by FNAB cytology has been proved by another researcher.19 MIB-1 quan­titation will become an important technique in assessing tumor cell proliferation to compli­ment FNAB in the diagnosis and prognosis of breast cancer.

In addition, we have demonstrated that the visual assessment of the tumor prolif­eration by MIB-1 immunoreactivity is of equal value compared to cell image analy­sis. Due to the expensive cost of instruments and personnel when flow cytom etry and image analysis are used, these techniques may actually be prohibitive for daily routine use in a pathology practice. It appears that M IB-1 im m u n o h isto ch em istry and the visual assessment of proliferating cells in breast cancer cases is an accurate and less expensive alternative.

Finally, since MIB-1 quantitation is a unique way of assessing tumor cell prolifera­tion, further studies are warranted to investi­

gate the feasibility of MIB-1 as an independent prognostic factor for breast cancer, especially for the aneuploid cancer group.

Acknowledgement

The authors are sincerely grateful to Mr. Mike Carwar- dine, who is a former developmental technologist at the Tumor Analysis Laboratory o f University Medical Center/ Jacksonville, FL. Without his dedication in collecting data for this project, this manuscript would not be possible. The authors would also like to thank Mr. Paul S. Kubilis, who was our consultant of statistical analysis when he was work­ing for the Department o f Biostatistics at the University o f Florida Health Science Center/Gainesville, FL.

References

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