Pol J Pathol 2003, 54, 2, PL ISSN

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1 ol J athol 3, 5,, - L ISSN 33- Bożena Lackowska, Aleksander Niezabitowski, Janusz Ryś, Jan Skołyszewski, Andrzej Stelmach 3, Andrzej Gruchała, Agnieszka Jaszcz-Gruchała S-hase Fraction and Menoausal Status as the Most Imortant rognostic Factors of Disease-Free Survival for Node Negative atients with Breast Cancer. A rosective Study Deartment of athology, Deartment of Radiation Oncology, 3 Deartment of Surgery, Oncology Center, Kraków The aim of our study was to determine a rognostic value of DNA flow cytometry measurements erformed on fresh breast cancer tissues, searately for atients grous defined by nodal status, with secial attention to histological tye of tumor. Between 3 and samles from atients were analyzed and 5 cases were included in the survival analysis. Two-hundred and nine atients from them were node negative (N). The median time of follow-u was months. In multivariate analysis of disease-free survival (DFS), S-hase fraction () and menoausal status were found to be indeendent rognostic arameters for N grou. A combination of this factors allowed us to distinguish three grous different in resect of the of recurrence. Our results showed that:. and menoausal status could be rognostically valuable factors for DFS in N breast cancer atients;. rognostic value of and loidy should be evaluated searately for each histological tye of breast cancer. Introduction Attemts for evaluation of the rognostic significance of DNA loidy and S-hase fraction () in breast cancer have been undertaken for many years, but the results remain controversial. Some of the recently ublished studies suggest that these factors are not more significant than the classically used [3, 3]. In, rognostic and redictive factors for breast cancer atients were stratified into three categories, according to their strength []. While a loidy, defined by flow cytometry, was ractically ruled out (category III), was included among factors of category II (for extensive studies). The objective of our study was to determine a rognostic value of DNA flow cytometry measurements erformed on fresh breast cancer tissues, searately for atients grous defined by nodal status, with secial attention to histological tye of tumors. Since the status of axillary lymh nodes is of secial redictive imortance among classic rognostic arameters, it should be used to verify all other otentially rognostic variables []. At resent, secial attention is aid to atients with negative nodes [, 5,, 3, ], and a majority of ublished results demonstrate a significant influence of on disease-free [5,,, 5] and overall survival [5, 5]. Commonly, the cytometric data are referred to all histological tyes of breast cancer resent in the series studied. However, some authors oint out that incidence of aneuloidy, and consequently level in articular tyes of breast carcinoma is significantly different []. Material and Methods A total of unselected breast cancer atients, consecutively oerated in the Oncology Center in Kraków between January 3 and December were analyzed for DNA content and S-hase fraction. Finally, 5 atients were included in the analysis. women were excluded because of revious treatment for malignant diseases (3 - for breast cancer), and 5 because of the chemo- or radiotheray administered before mastectomy. Among the remaining 55 atients, were lost to follow-u (time of observation for disease-free interval was shorter than months) and in 5 only simle mastectomy was erformed. Twenty-seven atients were excluded because of uninterretable DNA histogram. In all the remaining atients radical mastectomy was erformed; adjuvant chemo- and/or radiotheray was administered in all atients with ositive axillary lymh nodes and only in 3/(.%) atients without lymh node involvement. The median age of atients was 5 years (range - 5 years). At the time of diagnosis of them were re- and ost-menoausal. Median diameter of tumor measured after surgery was mm (range - mm). Metastases in axillary lymh nodes occurred in cases

2 B. Lackowska et al TABLE Univariate analysis of disease-free and overall survival for 5 atients with breast cancer Age Tumor size SBR grade Menoausal status loidy a loidy b + menoausal status Node involvement y >y mm -5mm >5mm GI GII GIII ost- re- >.% D A B W >.% ost- >.% re- none -3 N+ >3 N+ No of cases Log-rank <.. < < <. a D-diloid, A-aneuloid; b B-DI.3 or DI>. and multiloid, W-.3<DI < Log-rank < < <. TABLE Multivariate analysis of disease-free and overall survival for 5 atients with breast cancer Tumor size Menoausal status loidy a (B/W) Node involvement. <.. <. a B-DI.3 or DI>. and multiloid, W-.3<DI. ( in - 3 nodes and in >3 nodes) and the median number of metastatic lymh nodes was (range - 3). Two-hundred and nine atients had no axillary metastases. The median time of follow-u for survivors was months (range - months). Relases occurred in 5/5(3.%) atients; 35/(.%) node negative, /(3.%) - 3 nodes involved and /(.%) >3 nodes involved. Median recurrence-free survival was months (range - 5 months). Almost % of relases occurred within first 5 years of observation. At the end of the observation 3 atients were alive without evidence of disease, and 5 atients with recurrent cancer. Ninety-four atients died of breast cancer and of other causes (after 5 and months). Breast cancers were histologically classified according to the WHO recommendations []; infiltrating ductal carcinomas were graded according to Scarf-Bloom-Richardson (SBR) score []. DNA analysis was erformed on the susensions of the cell nuclei from fresh tissue secimens. After mincing with scissors, the tissue was disaggregated mechanically. Then, aliquots x cells were incubated with the staining solution (I-Calbiochem 5µg/ml, N- and RNA-se A - Sigma mg/ml). Analysis was erformed on FACScan Becton-Dickinson flow cytometer equied with DDM and CellFit software. For each DNA histogram at least, articles were analyzed. The DNA histograms were classified according to rinciles adoted at DNA Cytometry Consensus Conference []. Interretable DNA histograms were obtained in 5 cases and DI values were

3 rognostic factors in breast carcinoma TABLE 3 Univariate analysis of disease-free and overall survival for node negative atients Age Tumor size SBR grade Menoausal status loidy a loidy b + menoausal status y >y mm -5mm >5mm GI GII GIII ost- re- >.% D A B W >.% ost- >.% re- No of cases a D-diloid, A-aneuloid; b B-DI.3 or DI>. and multiloid, W-.3<DI Disease-free survival (DFS) analysis The 5-year actuarial survival rate for the whole grou of atients was %. Results of univariate analysis are resented in Table. loidy and demonstrated significant but lower influence on survival than the classically used factors. In multivariate analysis (Table ) node involvement, tumor diameter, menoausal status, and loidy in B/W categories were found to be indeendent factors, but and loidy henceforth were less significant than the clinical and histoathological arameters. After stratifying the material according to the axillary nodal status the 5-year survival rate amounted to 5%, % and % for atients with negative nodes (N), one to three nodes involved (N - 3), and more than three nodes involved (N3), resectively. Relevant data of univariate analysis are shown in Tables 3, and 5. Among node negative atients, was the most significant factor influencing recurrence estimated in all of them. could be evaluated in 3 cases. The median CV value for the whole grou amounted to 5. (range. -.). loidy of tumor cells was exressed as diloid/aneuloid (D/A). Further, the otimal borderline values of DI (DNA index) were defined through iterative analysis using the exerimentally established values; at the beginning, loidy could be ordered in seven comartments:. DI=. (diloid, cases),..<di.3 (near-diloid, 3 cases), 3..3<D. (triloid, 5 cases),..<di. (hyertriloid, cases), 5..<DI. (tetraloid, cases),. DI>. (hyertetraloid, cases),. more than one aneuloid stemline (multiloid, cases). Then, a second division was constructed, after reliminary survival analyses erformed searately for atients from each DI comartment. In result, two grous differing in rognosis were found: grou B (DI.3 or DI>. and multiloid, better rognostically) and grou W (.3<DI., worse rognostically). roliferative activity was exressed as ercentage of cells in S-hase fraction (). Otimal cut-off oint,.%, was established by iterative ste by ste analysis using the exerimental values in reference to disease-free survival. Disease-free and overall survival have been estimated by Kalan-Meier aroach. The differences between survival curves have been tested by log-rank and tests. For multile analysis roortional hazard model has been estimated. The final list of variables has been selected by stewise backward rocedure, leaving only statistically significant factors in the final model. -values not greater than.5 were considered to be statistically significant. Results 3

4 B. Lackowska et al TABLE Univariate analysis of disease-free and overall survival for atients with - 3 nodes involved Age Tumor size SBR grade Menoausal status loidy a loidy b + menoausal status y >y mm -5mm >5mm GI GII GIII ost- re- >.% D A B W >.% ost- >.% re- No of cases < a D-diloid, A-aneuloid; b B-DI.3 or DI>. and multiloid, W-.3<DI Histology and flow cytometry loidy and median values were analyzed for each histological grou of breast cancer searately. Frequency of aneuloidy differed significantly between the grous of various histology. Almost all medullary carcinomas were aneuloid, whereas in lobular and mucinous carcinomas only in one third of tumors aneuloid oulation was found. This fact was reflected in median value of, which was highest for medullary carcinoma. The relevant data are shown in Table. Distribution of histological tyes of breast cancer among defined nodal grous is shown in Table. It is worth noting that frequency of medullary carcinoma, rather low (.%) in our series of atients, standing a signi free survival. For atients with one to three nodes involved, significance was demonstrated for diameter greater than 5mm and histological grade GIII. In the grou with more than three nodes involved, only loidy attern was statistically significant. In multivariate analysis (Table ), and menoausal status were found to be indeendent rognostic arameters for N grou, diameter and histological grade for N - 3 and loidy, exressed as B/W, for N>3. Basing on the multivariate analysis erformed among node negative atients, three grous could be distinguished in resect of the of recurrence. The low grou consisted of the atients with tumor lower than.% and whatever menstrual status. The moderate grou consisted of the ost-menoausal atients with tumor greater than.%. And the high grou consisted of the re-menoausal women with tumor greater than.%. Recurrence-free survival for these three grous of atients is shown in figure. Overall survival (OS) analysis The 5-year actuarial survival rate for the whole grou of atients was 3%. Results of univariate analysis are resented in Table. Node involvement, tumor diameter and loidy in B/W categories were found to be indeendent factors in multivariate analysis (Table ). In the defined nodal grous, 5-year survival rate was 3%, % and 5% for N, N - 3, and N>3, resectively. Relevant data of univariate analysis are shown in Tables 3, and 5. Results of multivariate analysis (Table ) for node negative atients were a little different than for DFS. loidy in categories D/A was found to be the only significant factor for this grou, but lost its significance. For two nodeositive grous of atients the same factors as in DFS analysis were found to be significant.

5 rognostic factors in breast carcinoma TABLE 5 Univariate analysis of disease-free and overall survival for atients with more than three nodes involved Age Tumor size SBR grade Menoausal status loidy a loidy b + menoausal status y >y mm -5mm >5mm GI GII GIII ost- re- >.% D A B W >.% ost- >.% re- No of cases a D-diloid, A-aneuloid; b B-DI.3 or DI>. and multiloid, W-.3<DI TABLE Multivariate analysis of disease-free and overall survival according to nodal status of atients with breast cancer -3 >3-3 >3 Tumor size...3. SBR grade Menoausal status...5. loidy a (D/A) 3..3 loidy b (B/)W a D-diloid, A-aneuloid; b B-DI.3 or DI>. and multiloid, W-.3<DI. ficant art (.%) of tumors without axillary metastases (Table ) and growing u to.% in the grou of node negative, remenoausal women with highly roliferating tumours (>.%, Table ). It is clear, that this fact would be reflected in survival analysis. Then, we decided to reeat the survival analysis for node negative atients on the biggest grou of infiltrating ductal carcinoma, excluding the other histological tyes of tumors. In univariate analysis (Table ) histological grade, re-menoausal status, S-hase fraction greater than.% and DI in region.3 -. were found to be the most significant factors. None of the atients with lower than.% had any recurrence. For this reason it was imossible to erform regression analysis and to establish a relative for S-hase fraction among node negative atients. 5

6 B. Lackowska et al Fig.. Disease-free survival for atients with node negative breast cancer according to three defined grous, without consideration of histological tye of the tumor. Fig.. Disease-free survival for atients with node negative ductal infiltrating carcinoma according to three defined grous, with exclusion of other histological tumor tyes. As could be exected, a rognostic ower of combined and menoausal status was raised for grous with >.%. (Fig. ). Discussion DNA content and S-hase fraction are auxiliary rognostic arameters, therefore their clinical value should be considered along with the classical rognostic arameters. Histology Infiltrating ductal carcinoma is the most frequent tye of breast cancer, but the other histological tyes are also observed, in various number of cases. Survival analyses are usually erformed for all atients included in a study. In this aer we showed that distribution of aneuloidy, and also (usually greater in aneuloid than in diloid cases), was different in various histological tyes of breast cancer. Me-

7 rognostic factors in breast carcinoma TABLE Frequency of aneuloid cases and median according to histological tyes of breast cancer Histological tye No of cases Aneuloidy n media range Infiltrating ductal carcinoma Intraductal carcinoma Lobular carcinoma Mucinous carcinoma Medullary carcinoma Others a Total a aillary carcinoma -, carcinoma in fibroadenoma -, aget carcinoma -, signet ring carcinoma -, carcinoma with metalasia -, tubular carcinoma - TABLE Distribution of histological tyes of breast cancer according to nodal status Histology All cases -3 >3 Infiltrating ductal carcinoma Intraductal carcinoma Lobular carcinoma Mucinous carcinoma Medullary carcinoma Others a Total a aillary carcinoma -, carcinoma in fibroadenoma -, aget carcinoma -, signet ring carcinoma -, carcinoma with metalasia -, tubular carcinoma - TABLE Distribution of histological tyes of breast cancer between three grous of node negative atients defined by and menoausal status. could be determined for / cases Histology >.%, ost-menoausal >.%. re-menoausal Infiltrating ductal carcinoma Intraductal carcinoma Lobular carcinoma.. Mucinous carcinoma Medullary carcinoma.. Others a 5... Total a aillary carcinoma -, carcinoma in fibroadenoma -, aget carcinoma -, signet ring carcinoma -, carcinoma with metalasia -, tubular carcinoma - dullary carcinoma is an examle of breast cancer with almost % of aneuloid cases, extremely high, signs considered as rather unfavourable, and relatively good rognosis []. Medullary carcinoma constituted only a small ercentage of breast cancer cases in large studied grous, but its frequency becomes significant in node negative atients. The secific flow cytometric marks of this tye of breast cancer could influence final conclusions. Significant correlation between histology and loidy was found also in the other studies [, ]. Then, results of analysis trying to establish rognostic value of loidy and in mixed (from histological oint of view) grous would deend on ercentage of secific histological tyes.

8 B. Lackowska et al TABLE Univariate analysis of disease-free and overall survival for 5 node negative atients with ductal infiltrating carcinoma. could be determined for /5 cases Age Tumor size SBR grade Menoausal status loidy a loidy b + menoausal status y >y mm -5mm >5mm GI GII GIII ost- re- >.% D A B W >.% ost- >.% re- No of cases a D-diloid, A-aneuloid; b B-DI.3 or DI>. and multiloid, W-.3<DI loidy rognostic value of loidy remains controversial. Most of studies rule out loidy as an indeendent rognostic indicator [3,, 5], however some data confirm its redictive role [,, ]. The differences in atients survival were more distinct when breast cancers were evaluated according to various loidy classes [,, ]. Diloidy was ostulated to be associated with longer disease-free survival [,, 3]. Hyodiloidy and hyertetraloidy of breast cancers were regarded as less favourable [,, 3, 5]. Other studies revealed that "medium aneuloidy" (.5<DI<.5) is correlated with a shorter relase-free survival []. In this series of ductal infiltrating carcinoma, loidy considered only in categories diloid (DI=.)/aneuloid (DI.) had significant influence on overall survival only among node negative atients. However, it could be demonstrated, that there exist two sets of DI values related to rognosis:. DI.3 or DI>. and multiloid - more favourable, and..3<di. - less favourable. loidy influenced indeendently diseasefree and overall survival in atients with more than three lymh nodes involved, what is in keeing with the other studies [, 3]. DI for tumors determined as rognostically worse was in range.3 -. ("medium aneuloidy" and tetraloidy regions). We did not find that the atients with hyertetraloid tumors had worse rognosis. roliferative activity roliferative activity, when analyzed by flow cytometry, is exressed mainly as the ercentage of S-hase fraction (). Many studies demonstrated correlation of tumor diameter, grade and nodal status with [5,, ]. Indicators of roliferative activity are also good redictors of clinical outcome in breast carcinomas; recurrence-free and overall survival or both were associated with the level of roliferation exressed in different ways (as mitosis count, TLI, Ki- exression etc.) not only as [5,, 5, ]. In this study was the strongest redictor of relase for node negative atients, what is in agreement with the findings of others [5,,, 5]. However, in some studies with long term observations, a rognostic value of for atients without nodal involvement is questioned [, 3]. ossible cause of failure could be the adotion of mean or median value of as cut-off oint [3]. It seems that seeking the otimal cut-off oint is the only roer way for

9 rognostic factors in breast carcinoma establishing a suitable, rognostically valuable level of. On the other hand, in samles measured for estimation not only cancer cells are resent. Thus, gating a suboulation of cells with cytokeratin exression is suggested as more adequate []. However, some observations indicated [], that in the majority of the most undifferentiated tumors (grade GIII) only a art of cancer cells exress cytokeratin. Then, the measured only on cytokeratin-gated oulation will not be a "real " and it is not surrising that does not corresond with survival. In our study, the of relase for node negative atients with slowly roliferating tumors was minimal. It seems, that a necessity of adjuvant theray for this woman is disutable. Among atients with higher roliferating tumors, additional information about menstrual status allowed to stratify this grou into two rognostically different subgrous. Both these features taken together characterize the atients with the highest of relases (re-menoausal with high ). The resent study was erformed on unselected, consecutive breast cancer atients. The results obtained confirm the ossible clinical utility of flow cytometrically determined for node negative, but not node ositive atients with breast cancer. Breast cancer is not a homogenous disease, and the histological tye of cancer should be taken into consideration before survival analysis. References. Azzoardi JG, Cheick OF, Hartmann WM: Histological Tying of Breast Tumors. nd ed. Geneva, Switzerland, World Health Organization.. Balslev I, Christensen IJ, Rasmussen BB, Larsen JK, Lykkesfeldt AE, Thore SM, Rose C, Briand, Mouridsen HT: Flow cytometric DNA loidy defines atients with oor rognosis in node-negative breast cancer. 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10 B. Lackowska et al 5. Stäl O, Dufmats M, Hatschek T, Carstensen J, Klintenberg C, Rutqvist LE, Skoog L, Sullivan S, Wingren S, Nordenskjold B: S-hase fraction is a rognostic factor in stage I breast carcinoma. J Clin Oncol 3,, -.. Toikkanen S, Joensuu H, Klemi : The rognostic significance of nuclear DNA content in invasive breast cancer -a study with longterm follow-u. Br J Cancer,, 3-.. Visscher DW, Wykes S, Kubus J, Crissman JD: Comarison of CNA/cyclin immunohistochemistry with flow cytometric S- hase fraction in breast cancer. Breast Cancer Res Treat,, -.. Visscher DW, Wykes S, Zarbo RJ, Crissman JD: Multiarametric evaluation of flow cytometric synthesis hase fraction determination in dual-labelled breast carcinomas. Anal Quant Cytol Histol, 3, -5.. Wyss-Desserich MT, Caduff-Joos R, Wyss, Rageth C, Wight E, Unger C, Walt H, Haller U: remenoausal node-negative breast cancer: may adjuvant chemotheray be indicated by the analysis of nuclear DNA dynamics? Breast Cancer Res Treat,, Address for corresondence and rerint requests to: B. Lackowska M.D., h.d. Deartment of athology, Oncology Center Garncarska, 3-5 Kraków Fax: () () hone: () () 3 z5niezab@cyf-kr.edu.l