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DNA ploidy of breast cancer analyzed in association with classical morphologic factors and hormonal receptors
TheBreosf(l994)3,221-229 Q Longman
Group Ltd 1994
loidy of breast cancer analyzed in association with c morphologic factors and hormonal receptors %. Veneti, L. Ioannidou-Mouzaka, D. Papaioamrou, J. Viglas and G. Papadimitriou Breast Center and Cytology Department, ‘LETO’ Maternity Hospital, Athens, Greece $ U M M A HiY. The DNA content of 114 breast caaPcinomas was measured by Wow cytometry estimating the DNA index in each case. Our purpose was to evaluate the significance of this parameter in relation to morpbological factors (size and grade of the tumour, lymph node and blood vessel infiltration) and hormone receptor status (oestrogen and progesterone). Statistical analysis demonstrated tbat the amount of NA in the nuclei of the cells expressed by ploidy and measured by flow cytometry was not significantly correlated with the other parameters tested. These results suggest that DNA ploidy expressing the aggressiveness of the tumour may be an independent prognostic variable of the disease, but this needs to be verified by the fohow-up of our patients and the evaluation of disease free interval and overall survival rate,
clearer perspective on the potential value of DNA ploidy in breast cancer. Carcinoma of the breast is the most common malignant neoplasm afflicting women and despite well-documented successes in diagnosis and treatment, the survival rates have not'improved.'.2 It is evident that the peculiarities of breast ‘carcinoma such as the slow rate of proliferation combined with early dissemination force us to rethink the general philosophy of treatment and apply more individualized therapeutic schemes. For this purpose, we need precise identification of patients at high risk of recurrence. Glassical morphologic factors are insufficient in individual cases and they are subjective and susceptible to inter-observer variation.’ Other prognostic parameters have been added such as hormonal receptors, turnour markers, oncogenes, DNA index and proliferation inclex3-g in an attempt to individualize prognosis to help select for example, those node negative patients who have a relatively high risk of relapse and who may benefit from systemic therapy. We are entering ~JSera of highly selective therapy based on sophisticated analysis of the primary cancer.‘O Which parameters are importan for predicting patient outcome have not been cjearly defined. In this study we have investigated DNA index of breast carcinomas and correlated this with the size and grade of the tumour, lymph node and blood vessel infiltration, oestrogen and progesterone receptor status in an effort to obtain information that might provide a
IALS AND MET
The DNA content of breast cancers from 114 patients was measured by flow cytometry. The ages of the patients varied from 30 t0 85 years, mean age 53.7 * 14.2. In Figure 1, the material smdied and the number of cases tested with each parameter are outlined. Flow cytometric measurements were performed on paraffin embedded tumour specimens. The sites selected were from areas with abundant cancer cells. 5Q-micron slices of blocks were deparaffinized, rehydrated and the nuclei of the cells were disaggregated by mechanical and enzymatic means and stained with ethidium bromide, the substance responsible for the red fluorescence emitted
114
Address cqrrespondence to: L. Ioannidou-Mouzaka, 6 Kifissios Aver.&, 115 26 Athens, Greece
Fig. I-Number parameters. 221
Ca/DNA
t
of cases studied for DNA index and other
228 The Breast Table 1
Classification of breast tumours according to their DNA
index DNA index (DI) =
Aneuploid
Table 2 Values of significance (p) correlating DI with other prognostic factors. First univariate statistical analysis
GnGI (path.)
DI correlation with
‘X2
P
G”G1 (normaI)
Size Grade Lymph nodes inf. Vascular infiltration ER PgR
0.28 3.80 0.09 0.01 3.07 0.19
0.60 0.15 0.77 0.87 0.22 0.67
Diploid: Hypotetraploid: Tetraploid: Hypertetraploid:
DI < 1.20 DI 1.21-1.80 DI 1.81-2.20 DI>2.21
and counted by the flow cytometer (ACR/1250 Bruker, France). A histogram from each case was further analyzed, so that the DNA index was calculated (Table 1) representing the ratio of the G,G, pathologic peak to the GoG, normal peak in the histogram. The cases studied were thus classified according to their DNA index (Table 1). For practical purposes, in our analysis all tumours with DI < 1.20 have been considered diploid (euploid) and all tumours with DI > 1.21 have been considered aneuploid. There were 70 diploid and 44 aneuploid carcinomas.
RESULTS Figure 2 outlines the percentage of malignant factors and number of cases that correspond with aneuploidy, while Figure 3 presents what percentage and number of cases of benign factors shows euploidy. It is evident from the above diagrams that euploidy corresponds
Table 3 New classification of breast tumours in euploid and aneuploid categories
+ANEUI’LOID L
Diploid: Hypotetraploid: Tetraploid: Hypertetraploid:
DI<1.20+ DI 1.21-1.80 DI 1.81-2.202 DI > 2.21
EUI’LOID
better with the classical benign prognostic factors and the presence of hormonal receptors than aneuploidy which does not correspond as well with classical malignant prognostic factors and the absence of hormonal receptors. In fact a high percentage of the ‘good’ turnouts are diploid, but a high percentage of the ‘bad’ tumours are not aneuploid. Further statistical analysis was performed using the SPSS/PC+VAO statistical software package.” Correlating separately each factor with ploidy in a univariate analysis there were no significant values. (Table 2). After these unpromising aneuploidy correlations we performed a second umvariate statistical analysis and included as euploid both diploid, and tetraploid turnours, as outlined in Table 3 of the 114 breast cancers now 92 were classified as euploid and 22 as aneuploid. Reanalysis did not demonstrate correlation between ploidy and any other factor. A multivariate analysis was also performed looking at the relationship of all other factors and ploidy using a backward stepwise logistic regression analysis” and demonstrated no significant correlations with DNA index.
DISCUSSION Fig. 2-Aneuploid
Fig. &Diploid
turnouts (44).
turnouts (70).
Management of patients with breast cancer requires an individualized approach that is based on known prognostic factors. A review of the literature relating DNA ploidy obtained by flow cytometry with other parameters shows a diversity of results’2-24and definitive conclusions regarding the value of these markers are not possible. Several factors may explain the diversity of these observations such as prior systemic or local treatment, heterogeneity within turnours, variations in methodology and equipment, the use of paraffin embedded material and different definitions of aneuploidy. The need for quality control to ensure comparability of findings obtained is clearly necessary.25 Our results demonstrate that the amount of DNA in the nuclei of the cells expressed by ploidy and measured by flow cytometry is not significantly correlated with
DNA ploidy of breast cancer
other known prognostic parameters. However, euploidy has some association with benign morphologic factors and the presence of hormonal receptors. These findings agree with the better prognosis of diploid mammary carcinomas reported by other authors. 26Aneuploidy, in contrast was not associated with the other parameters of poor prognosis. The independence of the DNA index may mean that if it is shown to be of value in prognosis that it adds extra information not provided by other commonliy used factors.
Our work has been financially Hospital, Athens, Greece.
supported
by
‘LETO’
Maternity
Visscher D W, Zarbo R J, Greenawalk K A, G&man J D. Prognostic significance of morphological parameters and flow cytometric DNA analysis in carcinoma of the breast. Pathology Annual 1990; Part I. Haagensen C D. Diseases of the breast. 3rd edn. Philadelphia: Saunders, 1988; ch. 21. Kallioniemi 0 P, Blanc0 6, Alavaikko M. Tumor DNA ploidy as an independent prognostic factor in breast cancer. Br J Cancer 1987; 56: 637-642. Dowie C S, Owainati A, Robins A. Prognostic significance of DNA content of human breast cancer. Br J Surg 1987; 14: 133-136. Domagala W, Lubinski J, Weber K, Osborn M. Intermediate filarrent typing of tumor cells by means of monoclonal antibodies. Acta Cytol 1986; 30: 214. McCarty K S, Barton T K, Fetter B P. Correlation of estrogen and progesterone receptors with histologic differentiation in mammary carcinoma. Cancer 1980; 46: 2851. Dierendonck J H, Keijzer R, Velde C J, Comelisse C J. Nuclear distribution of Ki-67 antigen during the cell cycle: comparison with growth fraction in hunan breast cancer cells. Cancer Res 1989; 49: 2999-3006. O’Reiliy S M, Barnes D M, Campejohn R S et al. The relationship between c-erbB-2 expression, S-phase fraction and prognosis in breast cancer. Br J Cancer 1991; 63: 444446. Bask J P, Chin D, Van Diest P J et al. Comparative long-term prognostic value of quantitative HER-2/neu protein expression, DNA p!oidy, and morphametric and clinical features in paraffin embedded invasive breast cancer. Lab Invest 1991; 64(2): 215-223.
229
10. Cady B. New diagnostic, staging and therapeutic aspects of early breast cancer. Cancer 1990; 65(Suppl 3): 634-647. 11. Norusis M J: SPSS/pC+ for the IBM PC/XT/AT. Ed. SPSS Inc. 1990; Chicago. 12. Beerman H, Kluin P M, Hermans J. Van de Velde C J, Comelisse C J. Prognostic significance of DNA pioidy in a series of 690 primary breast cancer patients. Int J Cancer 1990; 45(l): 34-39. 13. Merkel D E, Osborne C K. Prognostic factors in breast cancer. Hematol Oncol Clin North Am 1989; 3(4): 641-652. 14. Toikkanen ST Joenssuu H, Klemi P. Tine prognostic significance of nuclear DNA content in invasive breast cancer. Br J Cancer 1989; 60(5): 693-700. 15. Muss H B, Kute T E, Case L D et al. The relation of flow cytometry to clinical and biologic characteristics in women with node negative primary breast cancer. Cancer 1989; 64(9): 18941900. 16. Von Rosen A. Aneuploidy as a prognostic factor in breast cancer. Med Oncol Tumor Pharmacother 1989; 6(2): 117-120. 17. Saez R A, McGuire W L, Clark G M. Prognostic factors in breast cancer. Semin Surg Oncoi 1989; S(2): 102-l 10. 18. S&IO, Wingren S, Carstensen J et al. Prognostic value of DNA ploidy and S-phase fraction in relation to estrogen receptor content and clinicopatbological variables in primary breast cancer. Eur .I Cancer Clin Qncol 1989; 25: 301-309. 19. Van der Linden J C, Lindeman J, Baak J P, Meijer C J, Herman C J. The multivariate prognostic index and nuclei DNA content are Tndependent prognostic factors in primary breast cancer patients. Cytometry 1989; 10: 56-61. 20. Del Bino G, Silvestrini R. Zucconi M R et al. DNA ploidy of human breast cancer. Anal Cell Path01 1989; l(4): 215-223. 21. Winchester D J: Duda R B, August C Z et al. The importance of DNA flow cytometry in node negative breast cancer. Arch Surg 1990; 125(7): 886-889. 22 Lewis W E. Prognostic significance of flow cytometric DNA analysis in node negative breast cancer. Cancer 1990; 65(10): 2315-2322. 23 O’Reilly S M, Camplejohn R S, Barnes D M et al. DNA index, S-phase fractin, histologic grade and prognosis in breast cancer. Br J Cancer 1990; 61: 671-674. 24 Sigurdsson H, Baldetorp B, Borg A et al. Indicators of prognosis in node negative breast cancer. N Engl J Med 1990; 322(15): 1045-1053. 25 Fisher B, Gtmduz N, Constantino 3 et al. DNA flow cytome+&c analysis of primary operable breast cancer. Cancer 1991; 68: 1465-1475. 26. Feichter G E, Kaufmann M, Mtiller A et al. DNA index and cell cycle analysis of primary breast cancer and synchronous axillary lymph node metastases. Breast Cancer Res Treat 1989; 13(l): 17-22.
Date received 10 November 1993 Date accepted 24 March 1994
Group Ltd 1994
loidy of breast cancer analyzed in association with c morphologic factors and hormonal receptors %. Veneti, L. Ioannidou-Mouzaka, D. Papaioamrou, J. Viglas and G. Papadimitriou Breast Center and Cytology Department, ‘LETO’ Maternity Hospital, Athens, Greece $ U M M A HiY. The DNA content of 114 breast caaPcinomas was measured by Wow cytometry estimating the DNA index in each case. Our purpose was to evaluate the significance of this parameter in relation to morpbological factors (size and grade of the tumour, lymph node and blood vessel infiltration) and hormone receptor status (oestrogen and progesterone). Statistical analysis demonstrated tbat the amount of NA in the nuclei of the cells expressed by ploidy and measured by flow cytometry was not significantly correlated with the other parameters tested. These results suggest that DNA ploidy expressing the aggressiveness of the tumour may be an independent prognostic variable of the disease, but this needs to be verified by the fohow-up of our patients and the evaluation of disease free interval and overall survival rate,
clearer perspective on the potential value of DNA ploidy in breast cancer. Carcinoma of the breast is the most common malignant neoplasm afflicting women and despite well-documented successes in diagnosis and treatment, the survival rates have not'improved.'.2 It is evident that the peculiarities of breast ‘carcinoma such as the slow rate of proliferation combined with early dissemination force us to rethink the general philosophy of treatment and apply more individualized therapeutic schemes. For this purpose, we need precise identification of patients at high risk of recurrence. Glassical morphologic factors are insufficient in individual cases and they are subjective and susceptible to inter-observer variation.’ Other prognostic parameters have been added such as hormonal receptors, turnour markers, oncogenes, DNA index and proliferation inclex3-g in an attempt to individualize prognosis to help select for example, those node negative patients who have a relatively high risk of relapse and who may benefit from systemic therapy. We are entering ~JSera of highly selective therapy based on sophisticated analysis of the primary cancer.‘O Which parameters are importan for predicting patient outcome have not been cjearly defined. In this study we have investigated DNA index of breast carcinomas and correlated this with the size and grade of the tumour, lymph node and blood vessel infiltration, oestrogen and progesterone receptor status in an effort to obtain information that might provide a
IALS AND MET
The DNA content of breast cancers from 114 patients was measured by flow cytometry. The ages of the patients varied from 30 t0 85 years, mean age 53.7 * 14.2. In Figure 1, the material smdied and the number of cases tested with each parameter are outlined. Flow cytometric measurements were performed on paraffin embedded tumour specimens. The sites selected were from areas with abundant cancer cells. 5Q-micron slices of blocks were deparaffinized, rehydrated and the nuclei of the cells were disaggregated by mechanical and enzymatic means and stained with ethidium bromide, the substance responsible for the red fluorescence emitted
114
Address cqrrespondence to: L. Ioannidou-Mouzaka, 6 Kifissios Aver.&, 115 26 Athens, Greece
Fig. I-Number parameters. 221
Ca/DNA
t
of cases studied for DNA index and other
228 The Breast Table 1
Classification of breast tumours according to their DNA
index DNA index (DI) =
Aneuploid
Table 2 Values of significance (p) correlating DI with other prognostic factors. First univariate statistical analysis
GnGI (path.)
DI correlation with
‘X2
P
G”G1 (normaI)
Size Grade Lymph nodes inf. Vascular infiltration ER PgR
0.28 3.80 0.09 0.01 3.07 0.19
0.60 0.15 0.77 0.87 0.22 0.67
Diploid: Hypotetraploid: Tetraploid: Hypertetraploid:
DI < 1.20 DI 1.21-1.80 DI 1.81-2.20 DI>2.21
and counted by the flow cytometer (ACR/1250 Bruker, France). A histogram from each case was further analyzed, so that the DNA index was calculated (Table 1) representing the ratio of the G,G, pathologic peak to the GoG, normal peak in the histogram. The cases studied were thus classified according to their DNA index (Table 1). For practical purposes, in our analysis all tumours with DI < 1.20 have been considered diploid (euploid) and all tumours with DI > 1.21 have been considered aneuploid. There were 70 diploid and 44 aneuploid carcinomas.
RESULTS Figure 2 outlines the percentage of malignant factors and number of cases that correspond with aneuploidy, while Figure 3 presents what percentage and number of cases of benign factors shows euploidy. It is evident from the above diagrams that euploidy corresponds
Table 3 New classification of breast tumours in euploid and aneuploid categories
+ANEUI’LOID L
Diploid: Hypotetraploid: Tetraploid: Hypertetraploid:
DI<1.20+ DI 1.21-1.80 DI 1.81-2.202 DI > 2.21
EUI’LOID
better with the classical benign prognostic factors and the presence of hormonal receptors than aneuploidy which does not correspond as well with classical malignant prognostic factors and the absence of hormonal receptors. In fact a high percentage of the ‘good’ turnouts are diploid, but a high percentage of the ‘bad’ tumours are not aneuploid. Further statistical analysis was performed using the SPSS/PC+VAO statistical software package.” Correlating separately each factor with ploidy in a univariate analysis there were no significant values. (Table 2). After these unpromising aneuploidy correlations we performed a second umvariate statistical analysis and included as euploid both diploid, and tetraploid turnours, as outlined in Table 3 of the 114 breast cancers now 92 were classified as euploid and 22 as aneuploid. Reanalysis did not demonstrate correlation between ploidy and any other factor. A multivariate analysis was also performed looking at the relationship of all other factors and ploidy using a backward stepwise logistic regression analysis” and demonstrated no significant correlations with DNA index.
DISCUSSION Fig. 2-Aneuploid
Fig. &Diploid
turnouts (44).
turnouts (70).
Management of patients with breast cancer requires an individualized approach that is based on known prognostic factors. A review of the literature relating DNA ploidy obtained by flow cytometry with other parameters shows a diversity of results’2-24and definitive conclusions regarding the value of these markers are not possible. Several factors may explain the diversity of these observations such as prior systemic or local treatment, heterogeneity within turnours, variations in methodology and equipment, the use of paraffin embedded material and different definitions of aneuploidy. The need for quality control to ensure comparability of findings obtained is clearly necessary.25 Our results demonstrate that the amount of DNA in the nuclei of the cells expressed by ploidy and measured by flow cytometry is not significantly correlated with
DNA ploidy of breast cancer
other known prognostic parameters. However, euploidy has some association with benign morphologic factors and the presence of hormonal receptors. These findings agree with the better prognosis of diploid mammary carcinomas reported by other authors. 26Aneuploidy, in contrast was not associated with the other parameters of poor prognosis. The independence of the DNA index may mean that if it is shown to be of value in prognosis that it adds extra information not provided by other commonliy used factors.
Our work has been financially Hospital, Athens, Greece.
supported
by
‘LETO’
Maternity
Visscher D W, Zarbo R J, Greenawalk K A, G&man J D. Prognostic significance of morphological parameters and flow cytometric DNA analysis in carcinoma of the breast. Pathology Annual 1990; Part I. Haagensen C D. Diseases of the breast. 3rd edn. Philadelphia: Saunders, 1988; ch. 21. Kallioniemi 0 P, Blanc0 6, Alavaikko M. Tumor DNA ploidy as an independent prognostic factor in breast cancer. Br J Cancer 1987; 56: 637-642. Dowie C S, Owainati A, Robins A. Prognostic significance of DNA content of human breast cancer. Br J Surg 1987; 14: 133-136. Domagala W, Lubinski J, Weber K, Osborn M. Intermediate filarrent typing of tumor cells by means of monoclonal antibodies. Acta Cytol 1986; 30: 214. McCarty K S, Barton T K, Fetter B P. Correlation of estrogen and progesterone receptors with histologic differentiation in mammary carcinoma. Cancer 1980; 46: 2851. Dierendonck J H, Keijzer R, Velde C J, Comelisse C J. Nuclear distribution of Ki-67 antigen during the cell cycle: comparison with growth fraction in hunan breast cancer cells. Cancer Res 1989; 49: 2999-3006. O’Reiliy S M, Barnes D M, Campejohn R S et al. The relationship between c-erbB-2 expression, S-phase fraction and prognosis in breast cancer. Br J Cancer 1991; 63: 444446. Bask J P, Chin D, Van Diest P J et al. Comparative long-term prognostic value of quantitative HER-2/neu protein expression, DNA p!oidy, and morphametric and clinical features in paraffin embedded invasive breast cancer. Lab Invest 1991; 64(2): 215-223.
229
10. Cady B. New diagnostic, staging and therapeutic aspects of early breast cancer. Cancer 1990; 65(Suppl 3): 634-647. 11. Norusis M J: SPSS/pC+ for the IBM PC/XT/AT. Ed. SPSS Inc. 1990; Chicago. 12. Beerman H, Kluin P M, Hermans J. Van de Velde C J, Comelisse C J. Prognostic significance of DNA pioidy in a series of 690 primary breast cancer patients. Int J Cancer 1990; 45(l): 34-39. 13. Merkel D E, Osborne C K. Prognostic factors in breast cancer. Hematol Oncol Clin North Am 1989; 3(4): 641-652. 14. Toikkanen ST Joenssuu H, Klemi P. Tine prognostic significance of nuclear DNA content in invasive breast cancer. Br J Cancer 1989; 60(5): 693-700. 15. Muss H B, Kute T E, Case L D et al. The relation of flow cytometry to clinical and biologic characteristics in women with node negative primary breast cancer. Cancer 1989; 64(9): 18941900. 16. Von Rosen A. Aneuploidy as a prognostic factor in breast cancer. Med Oncol Tumor Pharmacother 1989; 6(2): 117-120. 17. Saez R A, McGuire W L, Clark G M. Prognostic factors in breast cancer. Semin Surg Oncoi 1989; S(2): 102-l 10. 18. S&IO, Wingren S, Carstensen J et al. Prognostic value of DNA ploidy and S-phase fraction in relation to estrogen receptor content and clinicopatbological variables in primary breast cancer. Eur .I Cancer Clin Qncol 1989; 25: 301-309. 19. Van der Linden J C, Lindeman J, Baak J P, Meijer C J, Herman C J. The multivariate prognostic index and nuclei DNA content are Tndependent prognostic factors in primary breast cancer patients. Cytometry 1989; 10: 56-61. 20. Del Bino G, Silvestrini R. Zucconi M R et al. DNA ploidy of human breast cancer. Anal Cell Path01 1989; l(4): 215-223. 21. Winchester D J: Duda R B, August C Z et al. The importance of DNA flow cytometry in node negative breast cancer. Arch Surg 1990; 125(7): 886-889. 22 Lewis W E. Prognostic significance of flow cytometric DNA analysis in node negative breast cancer. Cancer 1990; 65(10): 2315-2322. 23 O’Reilly S M, Camplejohn R S, Barnes D M et al. DNA index, S-phase fractin, histologic grade and prognosis in breast cancer. Br J Cancer 1990; 61: 671-674. 24 Sigurdsson H, Baldetorp B, Borg A et al. Indicators of prognosis in node negative breast cancer. N Engl J Med 1990; 322(15): 1045-1053. 25 Fisher B, Gtmduz N, Constantino 3 et al. DNA flow cytome+&c analysis of primary operable breast cancer. Cancer 1991; 68: 1465-1475. 26. Feichter G E, Kaufmann M, Mtiller A et al. DNA index and cell cycle analysis of primary breast cancer and synchronous axillary lymph node metastases. Breast Cancer Res Treat 1989; 13(l): 17-22.
Date received 10 November 1993 Date accepted 24 March 1994