Flow cytometry in node‐positive breast cancer: Cancer and leukemia group B protocol 8869

Abstract

This report describes a companion flow cytometry study (Cancer and Leukemia Group B (CALGB)-8869) using tumors derived from patients enrolled in a large randomized clinical trial (CALG6-8541) performed on 1,572 patients with early stage, node-positive breast cancer. The CALGB initiated an adjuvant breast cancer trial in 1985 to determine if dose intensification (dose of drug per unit time) of chemotherapy was related to relapse-free and overall survival. Patients were randomized by pretreatment clinical variables to one of three different dosage regimens of chemotherapy. Using a tumor enrichment procedure, 442 paraffinembedded blocks were analyzed by flow cytometry, and S-phase fraction (SPF) was analyzed by three different methods. Ploidy analysis was performed using standard procedures. Tissue from 90% of the patients was suitable for ploidy analysis, whereas only 68% could be assessed for SPF. With a median follow-up time of 80 months, our results show that ploidy status had no clinical utility, whereas high SPF predicted poorer overall survival. The rectangular fit model for SPF was more predictive of outcome than both the area fit model and a computer fit model (modfit) for SPF. In univariate analysis, patients with a low SPF (< 10%) had a better prognosis than those patients with a high SPF (> lo%), but they responded equally well to the different treatment regimens. Patients with high SPF (> 10%) had longer relapse-free and overall survival to high dose chemotherapy compared to low or standard dose chemotherapy. Multivariate analysis indicated that treatment intensity as well as the number of positive nodes, tumor size, steroid receptor status, and c-erb 8-2 expression were significant in predicting overall and disease-free survival. The multivariate analysis, however, revealed that SPF was significant in predicting overall but not diseasefree survival, but there was no longer any relationship among SPF, dose intensity, and outcome. Key terms: Flow cytometry, breast cancer, standardization, S-phase, prognosisThe use of flow cytometry analysis for determining prognosis in patients with early breast cancer depends on several different factors, including tissue preparation procedure, quality of the DNA histogram, methods of histogram analysis, patient characteristics, and treatment. Some reports indicate that DNA content (ploidy) and cell cycle kinetic (S-phase fraction, %S) data are useful in predicting outcome ( 1-3), whereas other reports have shown no predictive value for this information (4-6). A recent NIH consensus conference on breast cancer concluded that ploidy is of limited prognostic use, whereas S-phase fraction (SPF) has prognostic value if performed correctly (7). At the present time, and in spite of continuing controversy, many laboratories, both academic and commercial, are performing both ploidy and SPF on breast cancer tissues for clinical use.The CALGB initiated an adjuvant breast cancer protocol for node-positive patients in 1985 to determine if ~~~~~ ~

Combined flow cytometry determination of S-phase fraction and DNA ploidy is an independent prognostic factor in node-negative invasive breast carcinoma: analysis of a series of 271 patients with stage I and II breast cancer

“…In node-positive patients, we did not find any correlation between SPF and prognosis, confirming the conclusions of two large-scale studies with randomized adjuvant therapy [2,34]. In subgroups defined by SBR grade, we did not find any prognostic impact for SPF in contrast to that reported by Chassevent et al [28], probably because of the small number of patients in the subgroups.…”

“…SPF values and/or DNA ploidy have already been demonstrated to be prognostic factors for the risk of breast cancer recurrence [2,9,10,16,18,19,[21][22][23][24][25]. However, three reports have emphasized the limitations of the extensive use of FCM DNA measurement in clinical practice mainly due to the lack of standardization of procedures to prepare and analyze samples and interpret histograms.…”

“…However, despite extensive literature on flow cytometry analysis of breast carcinomas, the results obtained by this method are still not widely used in everyday practice. However, a high ''tumour proliferation'' index has been associated with a higher risk of metastatic recurrence in surgically treated breast cancer particularly in node-negative patients [1][2][3][4][5][6][7][8]. Moreover, it has been suggested that a high tumour proliferation also influences the response to chemotherapy in neoadjuvant, adjuvant, and metastatic settings [9][10][11].…”

“…''Tumour proliferation'' in invasive breast cancer has been evaluated by several techniques, including immunohistochemical detection of proliferation-associated antigens [5,8,12], thymidine kinase assay [13,14], labelling index [7,15] as well as flow cytometry determination of S-phase-fraction (SPF) associated with simultaneous analysis of DNA ploidy [2,[9][10][11][16][17][18][19][20][21][22][23][24][25]. In 1992, the DNA Cytometry Consensus Conference drew up practical guidelines confirming the relationships between the observed SPF and disease-free survival (DFS) [24], subsequently confirmed by the American Society of Clinical Oncology Tumor Marker Panel [17,26].…”

The glutathione-related detoxification system is increased in human breast cancer in correlation with clinical and histopathological features

“…Such tumours undoubtedly bene®t from these changes in elevating resistance capacities against oxidative stress that is known to induce apoptosis and thereby to signi®cantly inhibit cell proliferation (Hall 1999). This could also explain why patients with elevated S-phase fraction require higher dose chemotherapy (Kute et al 1995). This would emphasise the poor prognosis value attributed to such tumours.…”