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Clinical Cancer Research Vol. 7, 909-917, April 2001
© 2001 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

S-Phase Fraction and DNA Ploidy in 633 T1T2 Breast Cancers

A Standardized Flow Cytometric Study

Agnès Chassevent1, Marie-Lise Jourdan, Sylvie Romain, Françoise Descotes, Marc Colonna, Pierre-Marie Martin, Michel Bolla2, Frédérique Spyratos and for Multicenter Study Group PHRC953

Centre Paul Papin, Angers, France [A. C.]; Centre Hospitalier Universitaire, Tours, France [M-L. J.]; AP-HM, Marseille, France [S. R., P-M. M.]; CHU-Lyon Sud, France [F. D.]; CHU, Grenoble, France [M. C., M. B.]; and Centre René Huguenin, St-Cloud, France [F. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The lack of a standardized methodology for quantifying DNA ploidy and S-phase fraction (SPF) by flow cytometry is hindering routine use of these markers in breast cancer management. In a retrospective clinical multicenter study, we validated a standardized flow cytometry protocol. We tested 633 frozen T1T2, N0N1, M0 breast tumors obtained in four institutions. Cell preparation was standardized, and precise rules for data interpretation were followed. Three SPF classes were defined on the basis of tertiles after adjustment for ploidy. DNA aneuploidy was observed in 61.0% of cases. No significant difference was observed among centers. Aneuploidy and high SPF were associated with large tumor size, node involvement, high histological grade, and hormone receptor negativity. In the overall population (median follow-up, 69 months), patients with medium and high SPF values had shorter disease-free survival (DFS) than those with low SPF values (P < 0.0001). Ploidy had no significant influence. By Cox analysis, SPF, pN, and estrogen receptor status were independent predictors of DFS (P = 0.0002, P = 0.001, and P = 0.05). In node-negative patients, SPF was the only predictor of DFS (P = 0.01), whereas in node-positive patients, the risk of relapse increased with both high SPF (P = 0.003) and estrogen receptor negativity (P = 0.004). Low SPF values distinguished grade II tumors with a particularly good outcome. Our results strongly support the use of SPF in multicenter studies and clinical trials and suggest that node-negative patients with slowly proliferating tumors do not require systemic adjuvant therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High tumor proliferation is an adverse prognostic factor in surgically treated breast cancers (1, 2, 3, 4, 5) , particularly in node-negative patients, in whom it can be used to distinguish those with the highest risk of metastasis (6, 7, 8, 9, 10, 11, 12, 13, 14) . Some studies have suggested that high proliferation also influences the response to chemotherapy in neoadjuvant, adjuvant, and metastatic settings (15, 16, 17, 18, 19, 20) .

Several approaches have been developed to study tumor proliferation, including labeling index (6 , 11 , 21 , 22) , immunohistochemical detection of proliferation-associated antigens (10 , 12 , 14 , 23) , and thymidine kinase assay (24 , 25) . However, the most widely studied technique is SPF4 measurement by FCM, which simultaneously provides DNA ploidy (26) . Abundant data have been published on SPF and DNA ploidy in breast cancer (5 , 15, 16, 17, 18, 19, 20 , 26, 27, 28, 29, 30, 31, 32, 33, 34) .

In 1992, the DNA Cytometry Consensus Conference, supported by the National Cancer Institute, concluded that the literature clearly showed a link between high SPF values and an increased risk of breast cancer recurrence (1) and proposed practical guidelines (31) . These conclusions were confirmed in 1996 and 1998 by the American Society of Clinical Oncology Tumor Marker Panel (35 , 36) . All three reports underlined the limitations of the extensive use of FCM DNA measurement in clinical practice, due mainly to the lack of standardized procedures to prepare and analyze samples and interpret histograms. However, many advances in technology have been performed with more efficient instruments and software programs. It has been shown that agreement among laboratories can be improved by following recommendations on software use (37 , 38) . However, full standardization of FCM DNA analysis is still lacking.

Three experienced French laboratories (15 , 37 , 39 , 40) subsequently established a standardized procedure for cell preparation and precise rules for software use and data interpretation (40) . The retrospective multicenter study described here was designed to clinically validate these procedures. Given the increasingly frequent diagnosis of small tumors, the study focused on T1T2, N0N1, M0 breast cancers. Six hundred and thirty-three frozen samples of primary breast cancers obtained in four French institutions were analyzed. Correlations with classical prognostic factors and DFS were investigated in the entire population and in patient subgroups defined by lymph node status, histological grade, and histological type.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Characteristics of the Patients.
The study involved 633 patients diagnosed and treated in four French cancer centers (Angers, 150 patients; Marseille, 190 patients; St-Cloud, 131 patients; Tours, 162 patients) between early 1987 and late 1992. Patients were included if they fulfilled the following criteria: (a) female primary unilateral invasive breast carcinoma without previous or concomitant malignancies; (b) T1T2, N0N1, M0 staging according to UICC criteria (41) ; (c) <75 years of age; and (d) surgical first-line treatment. The following information was recorded for each patient (Table 1)Citation : (a) age at diagnosis; (b) clinical stage; (c) pathological tumor size; (d) histological type and axillary lymph node involvement; (e) histological SBR grade (42) ; (f) steroid receptor status; (g) type of surgery (tumorectomy or mastectomy); and (h) adjuvant treatment (chemotherapy and/or hormone therapy).


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  		Table 1 Characteristics of the overall population (n = 633) and of the patients with calculated SPF values (n = 462)

 
The median age was 55.3 years (range, 24–75 years). The first-line treatment was tumorectomy with axillary dissection in 80.3% of patients or modified radical mastectomy in 19.7% of patients. In the overall population, 93.8% of patients had received radiotherapy after surgery. Adjuvant treatment consisted of chemotherapy in 22.0% of patients, hormone therapy in 25.8% of patients, and both in 13.2% of patients. Adjuvant chemotherapy was given to 20.1% of node-negative patients and to 52.6% of node-positive patients.

Hormone Receptor Assays.
The hormone receptor status of the tumor was recorded at the time of surgery. The tumor was considered to be steroid receptor positive if estrogen and PR values exceeded 10 fmol/mg-1 protein in radioligand assay and 15 fmol/mg-1 protein in enzyme immunoassay (Abbott Laboratories, Abbott Park, IL). Quality control was based on regular testing of both European Organization for Research and Treatment of Cancer and internal controls (39) .

Flow Cytometric DNA Analysis and SPF Measurement.
Three of the four participating centers possessed flow cytometers, consisting of two FACScan (Becton Dickinson, San Jose, CA) devices (Angers and St-Cloud) and one Coulter (Hialeah, FL) Epics Profile II apparatus (Tours), respectively, equipped with Modfit 5.2 (Verity Software House, Topsham, ME) and Multicycle AV (Phoenix Flow Systems, San Diego, CA) software. The three centers analyzed their own tumors, and Angers also analyzed samples originating from Marseille (samples were transported on dry ice).

Cell preparation was standardized as follows: (a) quick specimen thawing was performed; (b) tumor imprints were done systematically, stained with May Grunwald Giemsa, and observed by a pathologist to control the presence of malignant cells; (c) fine-needle aspiration and mechanical dissociation were performed; and (d) DNA staining was performed according to Vindelov’s method (43) . FCM was performed as follows: the DNA diploid peak was located on DNA histograms according to an external standardization procedure using normal human lymphocytes positioned at the fifth part of the red fluorescence scale; when a slight DNA abnormality was suspected, thawed normal human lymphocytes were added to the cell suspensions before DNA staining.

Rules established during a previous interlaboratory control procedure (40) were applied for use of the different cell cycle software models and for objective interpretation of DNA histograms. They included graphic aggregate subtraction.

The seven ploidy subclasses described in Table 2Citation were defined according to tumor DI values, the number of G0-G1 peaks, and the proportion of cells with an abnormal DNA content (aneuploid fraction). For example, a tumor was classified as DNA tetraploid when 1.90–2.05 DI cells exceeded 10% of total cells. Multiploid tumors were characterized by DNA histograms showing two or more aneuploid G0-G1 peaks. Automatic procedures were not used. SPF was calculated when the CV of the G0-G1 peaks was lower than 5%, background was lower than 20% of total acquired events, and a minimal aneuploid fraction was present (expressed in Table 2Citation according to DI subclasses). When a unimodal histogram was obtained, the diploid option of the software programs was used. In case of abnormal DNA content, only the aneuploid SPF was taken into account (the diploid SPF was not calculated). In every case, the rectangular option was chosen for SPF calculation. Whatever the software, the background subtraction option was always used.


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  		Table 2 Guidelines for DNA ploidy validation and SPF calculation

 
Statistical Methods.
Comparisons between DNA ploidy or SPF and patient characteristics were made using Pearson’s {chi}2 test. Two-sided Ps < 0.05 were considered significant. DFS was the interval between first treatment and primary failure, defined as a locoregional and/or distant recurrence. Actuarial survival rates were computed using the Kaplan-Meier method and compared using the log-rank test. The influence of DNA ploidy and SPF on outcome, adjusted for the other prognostic factors, was assessed by multivariate analysis using the Cox proportional hazards regression model in a forward stepwise procedure. The ascending method was used for a block by block construction (clinical variables, and then biological variables). Candidate variables were categorized as follows: (a) age at first treatment (<=45 years; 46–55 years; >=56 years); (b) pathological tumor size (<=2 cm; >2 cm); (c) lymph node involvement (none; 1–3 N+; >=4 N+); (d) histological grade (SBR grade I; SBR grade II; SBR grade III); (e) ER status (positive; negative); (f) PR status (positive; negative); (g) DNA ploidy (DIP; ANEUP); and (h) SPF adjusted for ploidy (<=33rd; 33rd–66th; >66th percentile). To take differences among centers into account, multivariate analysis was stratified according to the center. For patient subgroup analyses, results of multivariate analysis are only reported when the proportion of patients with local or distant recurrences exceeded 15%.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distributions
DNA Ploidy.
DNA ploidy was determined for all of the tumors. The mean CV of the G0-G1 peak was 2.5% for diploid samples and 2.9% for aneuploid samples.

A unimodal histogram with a normal DI (DI = 1) was obtained in 31.1% of cases (Table 2)Citation . The near-diploid subclass (DI, 0.95–1.10) represented 7.9% of tumors. The population also included 7.1% hypodiploid tumors, 28.6% aneuploid tumors with DI = 1.10–1.90 [including 60 near-triploid tumors (9.5%) with DI between 1.35 and 1.65], 6.8% DNA tetraploid tumors, 3.8% hypertetraploid tumors (DI > 2.05), and 14.7% multiploid tumors.

Because the clinical, histological, and biological features of the near-diploid tumors were not significantly different from those of the DNA diploid tumors (data not shown), these two subclasses were pooled in a "diploid" group (DIP; n = 247). The subclasses with an abnormal DNA content were pooled in an "aneuploid" group (ANEUP; n = 386).

No significant difference was found with regard to the DI subclass distribution among the four centers (P = 0.21). An abnormal DNA content was observed in 62.0%, 65.8%, 54.2%, and 59.9% of cases.

SPF.
According to the restrictive conditions listed in Table 2Citation , 462 specimens (73.0%) could be evaluated for SPF (89.1.% of the DIP group and 62.7.% of the ANEUP group). The main reason for missing SPF data in the ANEUP group was an insufficient aneuploid fraction. As shown in Table 1Citation , the characteristics of the patients with calculated SPF values were not significantly different from those of the overall population. Tumors from 71.9% of node-negative patients and 73.9% of node-positive patients were assessed for SPF.

SPF values were higher in the ANEUP group than in the DIP group (P < 0.001). Mean percentages were 6.4% and 2.2%, respectively (median values, 5.1% and 1.8%). The highest mean SPF values were observed in the near-triploid (8.0%), hypertetraploid (7.8%), and hypodiploid subgroups (6.6%), whereas the lowest values occurred in the multiploid (5.1%), tetraploid (5.3%), and 1.10–1.35 DI subgroups (5.1%).

SPF values were similarly distributed in the four participating centers (P = 0.43). The SPF tertile thresholds (33rd and 66th percentiles) of each DNA ploidy group are shown in Table 3Citation by center. In all subsequent analyses, a "low-SPF" group was formed by pooling the samples included in the first SPF tertile of the DIP and ANEUP groups. The "medium-SPF" and "high-SPF" groups were respectively composed of the second and third tertiles of the DIP and ANEUP groups.


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  		Table 3 SPF 33rd and 66th percentiles for DNA ploidy groups according to the center

 
Correlations
The frequency of abnormal DNA content increased significantly with pathological tumor size (P = 0.03), histological lymph node involvement (P = .0001), SBR grade (P < 0.00001), and ER (P = 0.009) and PR (P = 0.00007) negativity. IDCs were more often aneuploid than were invasive lobular tumors and other histological types (P < 0.00001).

As shown in Table 4Citation , high SPF was also more frequent in tumors with large pathological tumor size (P = 0.003), high SBR grade (P < 0.00001), and hormone receptor negativity (P < 0.00001). A weaker association was observed with clinical node status (P = 0.009) and histological node involvement (P = 0.02). Young patients (<=45 years) tended to have tumors with higher SPF values than older patients (P = 0.006).


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  		Table 4 Correlation between SPF and patient characteristics (n = 462)

 
Patient Outcome
The median follow-up was 69 months (maximum, 121 months). At the cutoff date for this analysis, there had been 51 local recurrences (8.1%) and 113 distant recurrences (17.9%), and 74 patients had died of cancer (11.7%). The 5-year DFS rate was 81.3% in the overall population, 89.4% among node-negative patients, and 76.7% among node-positive patients. The corresponding values among the SBR grade I, II, and III subgroups were 87.4%, 82.6%, and 73.4%, respectively. The 5-year DFS rate associated with ILC was 82.8%.

Univariate Survival Analysis
DNA Ploidy.
In the overall population, the log-rank test showed no prognostic impact of DNA ploidy (Table 5)Citation . The 5-year DFS rate was 84.6% in the DIP group and 79.7% in the ANEUP group. According to DI subclasses, the lowest 5-year DFS rates were associated with near-triploid (72.9%) and hypodiploid tumors (79.1%).


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  		Table 5 Five-year DFS rates and log-rank test for the overall population and node status subgroups

 
When DFS was studied in the subgroups defined by node status and histological grade, no significant difference was observed between patients with DIP and ANEUP tumors. However, a trend toward longer survival was observed for patients with DIP tumors in the node-positive (Table 5)Citation or SBR grade II (Table 6)Citation subgroups.


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  		Table 6 Five-year DFS rates and log-rank test for subgroups defined by histological grade

 
The 5-year DFS rate among patients with ILC was 90.0% in the DIP group and 67.7% in the ANEUP group, but the difference did not quite reach statistical significance (P = 0.06).

SPF.
In the overall population, the highest SPF values were strongly predictive of shorter DFS (P < 0.0001). Survival rates are shown in Table 5Citation . The DFS curve corresponding to the intermediate tertile showed a long-term prognosis similar to that in the third tertile (Fig. 1A)Citation . The first tertile was associated with the best outcome. The 5-year DFS rate of the 171 patients (83.5%) without calculated SPF values was intermediate between the low and medium SPF groups.



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  		Fig. 1. DFS according to SPF tertiles in the overall population and subgroups. A, overall population (P < 0.0001); B, node-negative patients (P = 0.008); C, node-positive patients (P = 0.003); D, SBR grade II patients (P = 0.0003).

 
In node-negative patients with calculated SPF values (n = 243), high and medium SPF values were associated with the shortest DFS (P = 0.008; Fig. 1BCitation ; Table 5Citation ), as also seen in the overall population. Similar results (P = 0.003) were obtained for the node-positive patients (n = 215; Fig. 1CCitation ; Table 5Citation ). The node-negative subgroup with high SPF values had almost the same 5-year DFS rate (77.1%) as the entire node-positive population (76.7%). Similarly, the node-positive subset with low SPF values had almost the same rate (89.7%) as the entire node-negative population (89.4%).

SPF was not significantly predictive of DFS in the SBR grade I (n = 75) and SBR grade III subgroups (n = 112; Table 6Citation ). In contrast, in the SBR grade II subgroup (n = 258), patients with low SPF values had particularly good survival compared to those with medium and high SPF values (P = 0.0003; Table 6Citation ). This good prognosis was observed even among patients with lymph node involvement (5-year DFS, 94.9%).

In patients with ILC (n = 62), the 5-year DFS rates were 90.6%, 81.8%, and 77.4% for low, medium, and high SPF values, respectively, but these results did not reach statistical significance.

Multivariate Cox Analysis
Complete clinical and biological data were available for 415 patients (Table 7)Citation . SPF, pathological node involvement, and ER status were independent predictors of DFS in the overall population (P = 0.0002, P = 0.001, and P = 0.05, respectively).


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  		Table 7 Cox analysis of overall population and subgroups for DFS

 
In node-negative patients (n = 219), SPF was the only variable selected by the Cox model (P = 0.01), with a relative risk of 3.66 (95% CI, 1.33–10.0) when SPF was high.

In node-positive patients (n = 196), SPF and ER were independent predictors of DFS (P = 0.003 and P = 0.004, respectively). A relative risk of 3.27 (95% CI, 1.45–7.40) was associated with high SPF.

Multivariate analysis was also performed in the SBR grade II subgroup (n = 241), where SPF was the only variable retained, with a relative risk of 4.92 when SPF was high (P = 0.0001).

In patients with ILC (n = 60), pathological tumor size was the only independent factor (P = 0.03; data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We investigated the prognostic value of DNA ploidy and the SPF in a large retrospective series of T1T2, N0N1, M0 primary breast tumors originating from four French institutions.

The recommendations of the 1992 DNA Cytometry Consensus Conference (1 , 31) have appeared insufficient to ensure interlaboratory reproducibility (37 , 38 , 44 , 45) . We thus established a standardized procedure for cell preparation and precise objective rules for using software programs and interpreting DNA histograms (40) . This protocol has helped us to minimize fluctuations in SPF measurements when using different cytometers and softwares. The use of frozen samples generated high-quality DNA histograms. To our knowledge, this is the first time that the clinical value of FCM DNA analysis parameters has been tested in a retrospective multicenter study after preliminary interlaboratory standardization.

Validating this standardization step, we observed no significant difference in the DI subclass and SPF distribution according to the center. The frequency of aneuploidy (61.0%) and the percentage of cases evaluated for SPF (73.0%) were within the range of values obtained in studies published since guidelines were first published (1 , 31 , 33) . The characteristics of the patients with calculated SPF values were similar to those of the overall population. In this study, SPF values were lower than most published values. This can be explained by several factors: (a) background is reduced by using frozen samples; (b) aggregates (which are rare with Vindelov’s method) were always subtracted; and (c) CVs were quite good. The importance of using strict guidelines to obtain reliable SPF values has recently been emphasized (34) . Studies are under way to estimate the prognosis of patients whose tumor SPF cannot be calculated on the basis of a prognostic model derived from FCM DNA histograms (46) .

The frequency of aneuploidy and high SPF increased significantly with pathological tumor size, histological node involvement, SBR grade, and hormone receptor negativity, as described previously (1 , 2) . We also confirmed previous reports of higher proliferation rates in younger patients (47) .

Our study confirms that ploidy status had no prognostic impact in the overall population or in subgroups defined by lymph node status, histological grade, and histological type (1 , 5 , 13 , 27 , 48) . However, among the aneuploid subclasses, near triploidy and, to a lesser degree, hypodiploidy tended to be associated with a poorer 5-year prognosis, as observed previously (47 , 49, 50, 51) .

As recommended by the DNA Cytometry Consensus Conference, SPF classes were defined according to tertile thresholds after adjustment for ploidy (DIP or ANEUP). In the overall population, patients with medium and high SPF values had a clearly lower 5-year DFS rate than patients with low SPF values. In multivariate analysis, SPF was an independent predictor, in agreement with studies performed in single laboratories (2 , 13 , 28 , 30 , 49) . Histological grade was not selected when SPF was introduced in the Cox model, probably because of the strong correlation between the two parameters.

In the node-negative subgroup, high SPF values distinguished patients with a higher risk of recurrence, in agreement with the findings of others (7 , 8 , 12, 13, 14) . In the Cox model, SPF was the only significant variable; SBR grade was not selected, as also observed in the NSABP B14 trial (13) and in a retrospective multivariate study (12) . In our series, node-negative patients with high SPF values had DFS rates similar to those of node-positive patients. In contrast, a particularly good prognosis was observed among node-negative patients with low SPF values, suggesting that the need for additional therapy is questionable in this subgroup. This was also suggested by the San Antonio Specialized Programs of Research Excellence breast cancer database (33) and is probably insufficiently respected in clinical practice.

In node-positive patients, a high SPF remained a detrimental factor in the multivariate analysis, whereas in two large studies with randomized adjuvant therapy, SPF was not a significant predictor of DFS (5 , 52) . This discrepancy may be explained by the difference in FCM DNA analyses that were performed on paraffin-embedded samples in these studies. Our results do not exclude the possibility that some patients with high SPF values may have benefited from chemotherapy (5 , 17 , 19) , but the treatment protocols in this retrospective multicenter study were too heterogeneous to draw firm conclusions. Again, SBR grade was not selected by the Cox model, contrary to the results of the Eastern Cooperative Oncology Group Companion study (52) , in which mitotic counts and grade were more predictive of DFS than SPF. Node-positive patients with low SPF values (79.2% of whom received hormone therapy) had survival rates comparable to those observed in the node-negative subgroup.

The predictive value of SPF was also studied in subgroups defined by SBR grade, which is one of the most widely used histological grading systems (42) and is often used as a decision tool in therapeutic protocols (4 , 53) . Despite criticisms over reproducibility (54) , SBR grade III is strongly associated with a higher risk of recurrence, and SBR grade I is associated with a lower risk of recurrence when determined by experienced pathologists (7 , 55 , 56) . SBR grade II tumors, including about half of resectable breast carcinomas, are known to be associated with varying prognosis (55) . No difference was observed among our SBR grade III patients according to the SPF tertiles. Adjuvant chemotherapy was received by a number of these patients and, as described previously, this treatment might have benefited some of those with high-SPF tumors. Among patients with SBR grade I tumors in our study, the small subset with high SPF values tended to have a worse DFS. However, among patients with SBR grade II tumors, a low SPF was strongly associated with a particularly good outcome. This should be taken into account in therapeutic protocols, particularly for node-negative patients.

FCM DNA analysis parameters were analyzed in our 83 patients with ILCs. This histological subtype is infrequent in breast cancer, and previous FCM DNA prognostic studies performed on frozen tumors were restricted to fewer than 50 samples. We confirmed the low frequency of aneuploidy associated with these tumors relative to IDC (48) . Diploid lobular tumors were associated with a longer DFS than aneuploid ones, but the difference was not significant. Contrary to the findings of Toikkanen et al. (48) , we did not find that high SPF values were associated with a bad prognosis in such patients.

In conclusion, this standardized multicenter FCM DNA protocol allowed us to demonstrate, in patients with T1T2, N0N1, M0 breast cancers, a clear link between the risk of recurrence and high or medium SPF. SPF gave better prognostic information than SBR histological grade in both node-negative and node-positive patients and furthermore separated low-risk patients from high-risk patients among those with SBR grade II tumors. These results strongly support the use of SPF in multicenter studies and clinical trials. They also suggest that node-negative patients with slowly proliferating tumors do not require systemic adjuvant treatment. Randomized, well-controlled studies of homogeneous chemotherapy protocols are required to settle this issue.


   ACKNOWLEDGMENTS
 
Clinical teams that participated in this study are indicated by city: (a) Angers, F. Bertrand, G. Bertrand, S. Cadeau, P. Cellier, I. Dalifard, N. Dauver, A. Daver, R. Delva, E. Fondrinier, E. Gamelin, J. Geslin, O. Guérin, V. Guérin-Meyer, E. Jadaud, F. Larra, G. Lorimier, A. Lortholary, P. Maillart, F. Mancel, C. Mayras, M. Mège, J. P. Muratet, P. Pabot du Chatelard, G. de Rauglaudre, C. Tuchais, and V. Verrièle; (b) Marseille, P. Bonnier, J. M. Brandone, C. Bressac, J. M. Blanc, L. Cals, C. Charpin, J. Del Grande, J. Guidon, A. Lachard, P. Nouyrigat, L. Piana, and M. Pizzi-Anselme; (c) St-Cloud, E. Allot, V. Becette, J. Berlie, F. Bertrand, A. Boudinet, E. Brain, M. Briffod, P. Cherel, C. Cohen Solal, C. Corone, F. Cvitkovic, B. de la Lande, C. de Maulmont, J. L. Floiras, A. Gentile, A. Goupil, K. Hacene, C. Hagay, J. M. Hannoun-Levy, M. Janvier, S. Lasry, V. Le Doussal, P. Moisson, C. Nogues, O. Ouhioun, E. Pain, C. Pallud, A. Pecking, P. Petot, M. M. Plantet, J. Rouësse, M. Trassard, M. Tubiana-Hulin, F. Turpin, and S. Yacoub; and (d) Tours, G. Body, P. Bougnoux, G. Calais, S. Chapet, F. Fetissof, A. Fignon, J. Lansac, O. Le Floch, T. Lefrancq, A. Reynaud-Bougnoux, and A. Veret.

We thank Denise Joyaux, Cécile Henry, Brigitte Travaillard, and Magali Ferrero-Poüs for performing FCM DNA analyses.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Centre Paul Papin, CRLCC, 2 rue Moll, 49036 Angers Cedex 01, France. Phone: 33-2-41-35-27-00; Fax: 33-2-41-48-31-90; E-mail: a.chassevent{at}unimedia.fr Back

2 M. B. was the coordinator of the Projet Hospitalier de Recherche Clinique (PHRC95) sponsored by the Ministère Français de la Santé. Back

3 Supported by a grant from the Ministère Français de la Santé (1995). Back

4 The abbreviations used are: SPF, S-phase fraction; FCM, flow cytometry; DI, DNA index; DIP, DNA diploid; ANEUP, DNA aneuploid; CV, coefficient of variation; SBR, Scarff, Bloom, and Richardson histological grade; ER, estrogen receptor; PR, progesterone receptor; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; DFS, disease-free survival; CI, confidence interval; UICC, Union Internationale Contre le Cancer. Back

Received 10/ 2/00; revised 12/26/00; accepted 1/ 2/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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