Node-negative breast cancer: prognostic subgroups defined by tumor size and flow cytometry

Adjuvant systemic therapy for women with node-negative breast cancer is most easily justified for those patients at highest risk of relapse. We have examined the impact of tumor size, histologic grade, estrogen receptor (ER) status, tumor ploidy, and S-phase fraction (SPF) on relapse-free survival (RFS) for 169 patients with node-negative breast cancer in order to identify groups of patients at high and low risk of relapse. Patients with small tumors (less than or equal to 1.0 cm) had a significantly better RFS than those with larger tumors (P = .005), with 96% remaining relapse-free at 5 years. Patients with tumors less than or equal to 1.0 cm were thus excluded from analysis when attempting to define a group with a poor prognosis. Within the group of patients with tumors greater than 1.0 cm, tumor ploidy (P = .63), ER status (P = .3), or progesterone receptor (PgR) status (P = .24) did not predict for RFS. Patients with grade 1 or 2 infiltrating ductal tumors had a significantly better prognosis than those with grade 3 tumors (P = .04). The prognostic factor that gave the widest separation between subgroups, however, was SPF. Patients whose tumors were greater than 1.0 cm with an SPF less than or equal to 10% had a 5-year RFS of 78% compared with a 5-year RFS of 52% for those with an SPF greater than 10% (P = .006). We have combined tumor size and SPF to identify three prognostic groups: (1) tumor less than or equal to 1.0 cm, 5-year RFS 96%; (2) tumor greater than 1.0 cm plus SPF less than or equal to 10%, 5-year RFS 78%; 3) tumor greater than 1.0 cm plus SPF greater than 10%, 5-year RFS 52%. These prognostic groupings may help identify patients most suitable for adjuvant therapy.     

DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.     

Prognostic significance of S-phase fraction in good-risk, node-negative breast cancer patients.

PURPOSE: Formalin-fixed, paraffin-embedded tissues from axillary node-negative breast cancer patients were analyzed by flow cytometry to determine the prognostic significance of DNA ploidy and S-phase fraction (SPF). PATIENTS AND METHODS: All patients were registered on a good-risk control arm of an intergroup clinical trial. They had small- to intermediate-sized (less than 3 cm), estrogen receptor (ER)-positive tumors and received no adjuvant therapy after modified radical mastectomy or total mastectomy with low axillary-node sampling. The median follow-up was 4.8 years. RESULTS: Assessable ploidy results were obtained from 92% of the 298 specimens studied (51% diploid, 49% aneuploid), and SPFs were assessable for 83% of the tumors. SPFs for diploid tumors ranged from 0.7% to 11.9% (median, 3.6%), compared with a range of 1.2% to 26.7% (median, 7.6%) for aneuploid tumors (P less than .0001). No significant differences in disease-free or overall survival were observed between patients with diploid and aneuploid tumors. Using different SPF cutoffs by ploidy status (4.4% for diploid, 7.0% for aneuploid), patients with low SPFs had significantly longer disease-free survival rates than patients with high SPFs (P = .0008). The actuarial 5-year relapse rates were 15% and 32% for patients with low (n = 142) and high SPFs (n = 105), respectively. Similar relationships between SPF and clinical outcome were observed for patients with diploid tumors (P = .053) and for patients with aneuploid tumors (P = .0012). CONCLUSION: S-phase fraction provides additional prognostic information for predicting disease-free survival for axillary node-negative breast cancer patients with small, ER-positive tumors.     

Risk-group discrimination in node-negative breast cancer using invasion and proliferation markers: 6-year median follow-up.

Factors reflecting two major aspects of tumour biology, invasion (urokinase-type plasminogen activator (uPA), plasminogen activator inhibiter (PAI-1), cathepsin D) and proliferation (S-phase fraction (SPF), Ki-67, p53, HER-2/neu), were assessed in 125 node-negative breast cancer patients without adjuvant systemic therapy. Median follow-up time was 76 months. Antigen levels of uPA, PAI-1 and cathepsin D were immunoenzymatically determined in tumour tissue extracts. SPF and ploidy were determined flow-cytometrically, Ki"'-67, p53, and HER-2/neu immunohistochemically in adjacent paraffin sections. Their prognostic impact on disease-free (DFS) and overall survival (OS) was compared to that of traditional factors (tumour size, grading, hormone receptor status). Univariate analysis determined PAI-1 (P < 0.001), uPA (P = 0.008), cathepsin D (P = 0.004) and SPF (P = 0.023) as significant for DFS. All other factors failed to be of significant prognostic value. In a Cox model, only PAI-1 was significant for DFS (P < 0.001, relative risk (RR) 6.2). In CART analysis for DFS, the combination of PAI-1 and uPA gave the best risk group discrimination. For OS, PAI-1, cathepsin D, tumour size and ploidy were statistically significant in univariate, but PAI-1 was the only independently significant factor in Cox analysis (P < 0.001, RR 8.9). In particular, this analysis shows that PAI-1 is still a strong and independent prognostic factor in node-negative breast cancer after extended 6-year median follow-up.     

c-erbB-2 oncoprotein overexpression identifies a subgroup of estrogen receptor positive (ER+) breast cancer patients with poor prognosis

Purpose:To investigate the predictive value of c-erbB-2 oncoprotein expression as compared with established histopathological and cytometric indicators of disease evolution in breast carcinoma. Patients and methods:A short-term retrospective study was conducted on a series of 306 breast cancer patients. Classic prognostic factors included tumour size, nodal involvement, histological grading, and hormone receptor status. Flow cytometric DNA ploidy and S-phase fraction (SPF) were also assessed. A Cox proportional hazards regression model was used for multivariate statistical analysis. Results:c-erbB-2 overexpression was present in 43 out of 295 (14.6%) tumours, and showed a statistically significant correlation with high histological grade, DNA aneuploidy, high SPF and lack of estrogen receptors (ER). Univariate analysis revealed its association with worse disease-free survival (DFS) and overall survival (OS). The combined evaluation of c-erbB-2 with ploidy and SPF defines a variable (P + S + c) that showed a significant correlation with disease outcome. By multivariate analysis, only nodal status (P < 0.001) and P + S + c subgrouping (group 2: P = 0.002; group 3: P = 0.001) in relation to DFS, and nodal status (P = 0.001) and DNA ploidy (P = 0.006) in relation to OS, retained independent prognostic significance. Subset analyses showed that cytometric parameters, P + S + c subgrouping and hormone receptors were significantly correlated with disease outcome in node-positive patients, whereas in node-negative subgroup no prognostic indicators were found. c-erbB-2 overexpression exhibited a trend in node-positive breast cancer (DFS: P = 0.068; OS: P = 0.086), and significant correlation with poor clinical evolution in ER positive patients (DFS: P = 0.015; OS: P = 0.004), mostly receiving tamoxifen. Conclusions:c-erbB-2 is an independent prognostic indicator of DFS when evaluated in conjunction with ploidy and SPF. It also seems to predict response to tamoxifen therapy, by identifying a subgroup of ER positive (ER+) breast cancer patients with poor prognosis.     

Significance of S-phase fraction and hormone receptor content in the management of young breast cancer patients.

Tumours from 336 breast cancer patients under the age of 50 were analysed for hormone receptor content and by DNA flow cytometry. Sixty-six percent of the tumours were positive for estrogen receptors (ER), 60% were progesterone receptor (PR) positive and 42% showed DNA diploid profiles. DNA hypodiploid tumours were relatively frequent (7%), especially in patients aged 40 years or less (11%). S-phase fraction (SPF), with a mean of 10%, correlated significantly with receptor status, DNA ploidy, lymph node status, tumour size and age. With a median follow-up period of 34 months, the distant recurrence-free interval was independently predicted by lymph node status, tumour size, SPF and PR content. Amongst the 212 patients who had not received adjuvant systemic treatment, receptor status was, in addition to lymph node status and SPF, independently related to distant recurrence rate. A high SPF identified a subgroup with high recurrence rate, comprising approximately one third of the node-negative patients. Similarly, the one third of node-positive patients who had PR-positive tumours with a low S-phase fraction formed a subgroup with low recurrence rate. We conclude that hormone receptor assays and DNA flow cytometry should be useful tools in the management of breast cancer patients less than 50 years of age.     

Measures of cell turnover (proliferation and apoptosis) and their association with survival in breast cancer.

Our objective was to investigate the prognostic significance of cell turnover (apoptosis and proliferation) in breast cancer patients. Apoptosis was microscopically quantitated on histological sections from 791 breast cancer patients with long-term follow-up (median, 16.3 years). Apoptotic counts were also compared with proliferation data (mitotic counts and MIB-1 labeling); apoptosis data derived from terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay; and pathobiological variables, including p53, erbB-2, and estrogen receptor (ER). High apoptotic counts were associated with increased cellular proliferation, ER negativity, immunopositivity of erbB-2 and p53 (P < 0.0001), and shortened disease-specific survival (DSS; P = 0.0009) and disease-free survival (DFS; P = 0.0006). Other factors associated with shortened DFS and DSS by univariate analysis were high tumor grade, nodal metastases, and large tumor size (P < 0.0001 for each). Multivariate analysis of data for all of the patients demonstrated that tumor size, nodal status, ER, histological grade, and erbB-2 showed independent prognostic value. In node-negative patients, tumor size and mitotic rate per 1000 cells independently predicted DFS (P = 0.0055). Tumor grade was the only independent predictor of DSS. For node-positive patients, tumor size, nodal status, ER, and erbB-2 were independent prognostic factors. The number of mitoses per 1000 was independently associated with DFS (P = 0.043) but not with DSS. Apoptosis data did not provide independent prognostic value in any, node-positive or node-negative, breast cancer patients.     

Prognostic significance of flow cytometric DNA analysis and estrogen receptor content in breast carcinomas — a 10 year survival study

Summary The prospective prognostic significance of flow cytometry derived DNA-ploidy status, the level of the S-phase fraction (SPF), estrogen receptor (ER) content, and combinations of these factors, was evaluated with respect to overall survival (OS) in a series of 516 breast cancer patients who were without signs of residual or distant disease after primary completed treatment. The median duration of survival follow-up time was ten years (range, 95–148 months) for surviving patients.Of the single factors, ER was the only significant predictor among node-negative patients; the ten-year OS rate was 71% in cases with ER-rich tumors vs. 62% for ER-poor tumors (p=0.03). Where tumors were both non-diploid and ER-poor, the ten-year OS rate was 58%, as compared to 75% for the remaining node-negative patients (p=0.003), who constituted a low-risk group whose survival was comparable with that in the age-matched normal population.Among patients with 1–3 positive nodes, the ten-year OS rate was 65% in patients whose tumors had an SPF <7.3% vs. 50% if the SPF was 7.3% (p=0.01), and 58% in cases with ER-rich tumors vs. 45% where the tumors were ER-poor (p=0.02).In a multivariate analysis, apart from age and menopausal status the combination of ploidy status and ER content was the significant (p=0.002) predictor of OS in node-negative patients. Thus, combining ploidy and ER status, both of which are variables easily determined, enabled the selection of a subgroup of patients at high risk of relapse and reduced survival whose prognosis should be improved by effective adjuvant systemic treatment, whereas the remaining low risk N0 patients can not be expected to derive any survival benefit from adjuvant therapy since their predicted survival is already on a par with that of the general population.     

Prognosis of a series of 763 consecutive node-negative invasive breast cancer patients without adjuvant therapy: analysis of clinicopathological prognostic factor.

BACKGROUND AND OBJECTIVES: The objectives of this study were to confirm the favorable outcome of Japanese invasive breast cancer patients without lymph node metastasis, after treatment with surgery alone, and to evaluate clinicopathological prognostic factors in this population. METHODS: The subjects were 763 consecutive node-negative invasive breast cancer patients who underwent surgery without adjuvant therapies between 1988 and 1993 at our hospital. Disease-free survival (DFS) and overall survival (OS) rates were analyzed by clinicopathological factors. RESULTS: The median age of the patients at surgery was 52 years and the median follow-up period of patients was 74 months. At 5 years, the respective DFS and OS rates of all patients were 90.8% and 93.9%. Patients with a pathological tumor size of invasive component of more than 2 cm (319 patients) had a significantly lower DFS than those with tumors measuring 2 cm or less (361 patients) (P = 0.045). Patients with positive hormone receptor status (280 patients) (estrogen and/or progesterone receptor positive) tended to have a better OS than those negative for both hormone receptors (92 patients) (P = 0.078). Meanwhile, patients with tumors of histological grade 3 (328 patients) had a much poorer prognosis than those with tumors of histological grade 1 or 2 (413 patients) (P = 0.008 for OS and P = 0.042 for DFS). The respective 5-year DFS and OS rates of patients with histological grade 3 tumors larger than 2 cm in pathological tumor size of invasive component (195 patients) were 85.5% and 87.6%, indicating that these node-negative patients form a high risk group. CONCLUSIONS: Japanese invasive breast cancer patients without lymph node metastasis tended to show a survival advantage compared with their Caucasian counterparts. Histological grade was the most useful prognostic factor in this population.     

DNA flow cytometric analysis and prognosis of axillary lymph node-negative breast carcinoma.

METHODS. The prognostic significance of flow cytometric analysis in patients with node-negative invasive breast carcinoma was evaluated in a retrospective series of 158 patients with a minimum follow-up study of 9 years. RESULTS. The ploidy status could be assessed in 147 specimens (93%), and the proliferative phase or S-phase fraction (SPF) could be assessed in 136 tumors (86%); 70 tumors (48%) were diploid, 49 tumors (33%) were aneuploid, and 28 tumors (19%) were tetraploid. Ploidy status and SPF were correlated significantly with tumor size, histologic grade, nuclear grade, and mitotic rate. By itself, ploidy was not a statistically significant prognostic factor, although all of the patients with multiploid and hypertetraploid tumors had recurrence of disease. The SPF was related significantly to recurrence of disease (P = 0.04). However, when multivariate analysis of various histopathologic variables was performed, SPF ceased to be a significant prognostic determinant, whereas peritumoral lymphovascular invasion was the most important variable. The combination of tumor size and flow cytometric parameters permitted stratification into three groups with different prognoses at the 9-year follow-up review (P less than 0.001). In the low-risk group (diploid tumors less than or equal to 2 cm in diameter with a low SPF or small tetraploid tumors), the recurrence rate was 12%. In the intermediate-risk group (diploid tumors greater than 2 cm in diameter with a low SPF or aneuploid tumors with a low SPF), the recurrence rate was 21%. In the high-risk group (diploid or aneuploid tumors with a high SPF or large tetraploid tumors), the recurrence rate was 49%. The high-risk group status remained a significant variable in the Cox proportional hazards multivariate analysis model. CONCLUSIONS. These results indicate that flow cytometry in breast carcinoma contributes useful but limited prognostic information and stress the importance of using multiple prognostic factors to improve prognostication and optimize patient management.     

DNA-synthesizing enzymes in breast cancer (thymidine kinase, thymidylate synthase and thymidylate kinase): association with flow cytometric S-phase fraction and relative prognostic importance in node-negative premenopausal patients

S-phase fraction (SPF) is a reference for cell-kinetic analysis. In this study, the links between SPF and the essential enzymes participating in the pyrimidine synthesis were investigated in breast cancer and their relationships with the natural history of the disease were compared. We measured thymidine kinase (TK) for salvage synthesis, thymidylate synthase (TS) for de novo synthesis and thymidylate kinase (TMK), which is required for both pathways. Our study population consisted of 211 premenopausal women with node-negative tumors. SPF was assessed prospectively by flow cytometry, whereas enzyme activities were measured retrospectively in cytosols using radioenzymatic methods. Among the enzymes analyzed, only TK demonstrated a strong correlation with SPF (r(s) = 0.59). In univariate analysis, high SPF and high levels of TK were associated with increased risk of developing distant recurrences (p < 0.001). Correlations with other prognostic factors (histological grade, steroid receptors, DNA ploidy status, urokinase plasminogen activator and plasminogen activator inhibitor type 1) confirmed a parallel association of SPF and TK with the most aggressive tumors. In contrast, TS and TMK were not associated with prognosis. After adjustment for SPF, the risk of relapse increased significantly with TK values. Subgroup analysis showed that additional information was provided by TK in the tumors with low SPF. When urokinase plasminogen activator (uPA) was a candidate variable in multivariate analysis, TK remained significant. Combined with SPF and uPA, TK could be useful to define premenopausal node-negative patients with rapidly proliferating tumors at a high risk of metastatic disease.     

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

PURPOSE: To assess the significance of S-phase fraction (SPF) and DNA ploidy evaluated by DNA flow cytometry as prognostic markers in stage I or II breast cancer. PATIENTS AND METHODS: A series of 271 patients, treated by surgery, radiotherapy+/-systemic therapy was analysed (median follow up: 64 months). Standardized flow cytometry cell preparation from frozen samples and consensus rules for data interpretation were followed. Three SPF classes were defined on the basis of tertiles after adjustment for ploidy. Four groups were defined based on combinations of DNA ploidy (DIP: diploid; ANEUP: aneuploid) and SPF: DIP and low SPF (DL, N=37), DIP and medium or high SPF (DMH, N=76), ANEUP and low SPF (AL, N=24), ANEUP and medium or high SPF (AMH, N=68). Local control rate (LCR), disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS) were correlated with DNA ploidy, SPF, DL to AMH groups, T and N stages, SBR grading, age, and hormonal status on univariate and multivariate analysis (Cox model). RESULTS: On univariate analysis, DFS and LCR were higher for DIP tumours. High SPF values were associated with shorter DFS. LCR, MFS, DFS, and OS rates were significantly different with an increasingly poorer prognosis from DL to AMH. On multivariate analysis, groups DL to AMH, histological node involvement and T stage were independently associated with MFS, and DFS. In N- patients, DL to AMH remained independent for MFS and DFS. For SBR III tumours, MFS and OS were significantly different in DL to AMH groups. These results strongly support the use of combined evaluation of DNA ploidy and SPF as independent parameters in clinical trials for N- stage I and II breast cancer.     

Low proliferative rate of invasive node-negative breast cancer predicts for a favorable outcome: a prospective evaluation of 669 patients

This study was designed to compare outcome in terms of disease-free survival (DFS) in women with histologically negative axillary lymph nodes and documented low proliferative rate cancer to other well-defined prognostic factors including type of adjuvant treatment. Between 1988 and 1998, we studied 669 patients with invasive node-negative breast cancer up to 5 cm in size and low proliferative rate measured by flow cytometry to determine S-phase fraction (SPF) or by histochemistry (Ki67/MIB1). At a median follow-up of 53 months, 5-year DFS for the entire group was 94% and did not differ significantly by type of systemic adjuvant treatment: none (133 patients, 95% DFS), tamoxifen (441 patients, 94% DFS), or chemotherapy with doxorubicin and cyclophosphamide (95 patients, 92% DFS). In a multivariate prognostic factor analysis, only tumor size was significant; 5-year DFS was 96% for T1N0 cancer versus 89% for T2N0 cancer (P = 0.01). We have prospectively confirmed that a low rate of proliferation as measured by SPF or MIB1 determination confers an excellent prognosis in invasive node-negative breast cancer up to 5 cm in size, regardless of adjuvant treatment.     

人表皮生长因子受体2表达对腋淋巴结阳性和阴性乳腺癌患者预后的不同影响

目的 探讨人表皮生长因子受体2(Her-2)表达在腋淋巴结阳性和阴性乳腺癌患者预后中的意义.方法 采用抗Her-2单克隆抗体CB11进行免疫组化,检测981例原发性乳腺癌肿瘤组织Her-2蛋白的表达,分析其与乳腺癌预后的关系.结果 981例原发性乳腺癌Her-2蛋白的阳性表达率为19.7%(193/981),Her-2表达水平与乳腺癌患者的发病年龄、雌激素受体(ER)和孕激素受体(PR)表达状态明显相关(P<0.05).单因素分析显示,在腋淋巴结阳性的乳腺癌患者中,Her-2表达与预后显著相关,Her-2阳性者的5年无病生存(DFS)率和5年总生存(OS)率分别为48.8%和55.2%,Her-2阴性者分别为66.9%和76.4%,差异均有统计学意义(P<0.01).而在腋淋巴结阴性的患者中,Her-2表达与患者的5年DFS率和5年OS率均无显著相关性(P>0.05).多因素分析显示,在腋淋巴结阳性的乳腺癌患者中,Her-2表达水平是影响乳腺癌OS的独立因素,但不是影响乳腺癌DF5的独立因素.结论 在腋淋巴结阳性乳腺癌患者中,Her-2表达水平是重要的预后因素.     

Prognostic potential of flow cytometric S-phase and ploidy prospectively determined in primary breast carcinomas.

In a prospective study of a consecutive breast cancer series accumulated in the period 1978-82, the S-phase fraction (SPF) and ploidy status were determined by flow cytometry performed on cell nuclei derived from samples of 580 primary tumors. Sixty percent of the tumors were non-diploid. After correction for debris the median SPF values were 7.3% overall, 12% for non-diploid tumors, and 2.9% for diploid tumors (2.6% when nodal subsets N2 and N3 and cases with metastases at presentation were excluded). The SPF values correlated both to tumor size (p = 0.008) and to the number of positive axillary lymph nodes (p = 0.03). At clinical follow-up in 1986, 467 unilateral breast cancer patients who had undergone radical treatment for cure could be evaluated with respect to the prognostic value of both the SPF value and ploidy status. The median duration of follow-up was then 59 months (range 2-90), and the median time-to-recurrence 24 months (range 2-69, n = 137). At follow-up in 1991, 201/467 of the patients had died, the median duration of follow-up being 50 months (range 2-126) for the decreased, and 119 (range 6-148) for the survivors. In multivariate analysis (Cox's proportional hazards models), the strongest independent predictors of distant recurrence-free survival (DRFS) were the number of positive axillary lymph nodes (p less than 0.0001), the debris-corrected SPF value alone (p = 0.003, versus p = 0.05 for uncorrected value), and ploidy status combined with the corrected SPF value (p = 0.0002). When age was taken into account, both the corrected SPF value and the ploidy-SPF combination were predictors of crude survival (p = 0.006 and p = 0.002, respectively). In univariate life-table analysis, the 5-year DRFS rate was 93% in node-negative (N0) cases with an SPF less than 7.3%, as compared to 80% in those with an SPF greater than or equal to 7.3% (p = 0.005). Among node-positive cases, the prognostic value of the SPF was confined to those with 1-3 positive nodes, the 5-year DRFS rate being 68% in cases with an SPF less than 7.3%, as compared to 40% in cases with an SPF greater than or equal to 7.3% (p = 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Prognostic significance of DNA image cytometry in libyan breast cancer

Background: We evaluated the relation of nuclear DNA content and clinicopathological features and prognosis in primary breast cancer of female Libyan patients with variable stage and grade and different treatment regimes. Patients and Methods: Histological samples from 104 patients of breast carcinoma were retrospectively studied by computerized nuclear DNA cytometry. Isolated nuclei from paraffin sections were stained with Feulgen stain and DNA was measured using a computer-assisted image analysis cytometry system. In each case, 200 nuclei were measured and the DNA histograms, S phase fraction (SPF) and number of cells above 5c and 9c were determined. We applied different approaches in the analysis of DNA to compare the DNA histograms with different clinicopathological features and survival. Results: The mean of DNA ploidy mode for all tumors was 3.43; 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF was 3.5% for DNA diploid and 13.5% for DNA aneuploid tumors. DNA aneuploid tumors and high SPF were associated with advanced stage, distant metastasis, high histological grade and lymph node involvement. The SPF was also associated with large tumor size and with younger patients (<50 years). In the overall population (median follow-up 51 months), patients with aneuploid DNA histograms and high SPF values had shorter survival times than those with diploid DNA histograms and low SPF values (p = 0.001, p < 0.0001, respectively). Also, short survival was associated with a multiploid DNA histogram and with DNA aneuploid cells ≥5 cells (p < 0.0001, p = 0.001, respectively). In a Cox multivariate analysis, DNA ploidy (p = 0.010), age (p = 0.038) and clinical stage (p = 0.001) were independent predictors of overall survival, and DNA ploidy (p = 0.018) and clinical stage (p = 0.001) also proved to be independent predictors of disease-specific survival. The SPF cutoff point of 11% might be applied to separate patients into good and poor prognosis groups. Conclusions: DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in Libyan breast cancer, with potential clinical implications in patient management, particularly in predicting the patients at high risk for metastasis and recurrence who should be considered as candidates for combined adjuvant therapy.     

DNA-flow cytometry (ploidy and s-phase fraction) as prognostic factor in a retrospective series of 515 primary breast-cancer

Paraffin-embedded tissues are used in retrospective studies to evaluate the prognostic significance of DNA-flow cytometry (DNA-FCM) in human breast cancer. Although paraffin-embedded samples yield information on disease-free survival (DFS) and overall survival (GAS) of homogeneously selected patients, the resulting DNA-histograms have a lower resolution of aneuploid subpopulations and higher debris levels than those of fresh tumor samples. The aim of this study was to evaluate, retrospectively, the prognostic value of ploidy and the S-phase fraction (SPF) using 515 samples of paraffin-embedded consecutive primary breast cancer tissue (median follow-up: 75.4 months). Ploidy was detectable in 89% cases (34% diploid and 66% aneuploid) and SPF in 77%. The optimal cut-off for SPF was 6%. High SPF values were significantly correlated with shorter DFS (p=0.028) and OAS (p=0.018); aneuploidy was significantly correlated only with a shorter OAS (p=0.0058). Using the Cox proportional hazards regression model to evaluate the independence of DNA-FCM derived parameters, only high SPF was able to predict both a shorter DFS (p=0.02) and OAS (p=0.002). Furthermore, high SPF values were found correlated to aneuploidy (p<0.00001), tumor necrosis (p<0.015) and high histopathological grade (p<0.03). The data reported confirm that SPF is a valuable single independent prognostic factor in human breast, cancer and strongly support the use of archival tumor specimens to study the prognostic role of DNA-FCM in human cancer.     

Elevated expression of podocalyxin is associated with lymphatic invasion, basal-like phenotype, and clinical outcome in axillary lymph node-negative breast cancer

Lymphatic invasion (LVI) is associated with disease recurrence in axillary node-negative (ANN) breast cancer. Using gene expression profiling of 105 ANN tumors, we found that podocalyxin (PODXL) was more highly expressed in tumors with LVI (LVI+) than in those without LVI (LVI?). Differences in PODXL expression were validated using real-time polymerase chain reaction as well as by immunohistochemistry in an independent set of 652 tumors on tissue microarrays. Disease-free survival (DFS) analyses were conducted for association of high PODXL protein expression with risk of distant recurrence overall and within breast cancer subtypes using both Cox and cure-rate models. High PODXL expression was associated with poor prognosis features including large tumor size, high histological grade, estrogen and progesterone receptor negativity, and with clinical alterations characteristic of the basal-like breast cancer phenotype. Surprisingly, despite having other poor prognosis characteristics, women with high PODXL expressing tumors had better long-term DFS in multivariate analysis with traditional clinicopathologic factors including LVI and HER2 status (P = 0.001). PODXL has the potential to be a useful biomarker for identifying good prognosis patients in characteristically poor prognosis breast cancer groups and may impact treatment of women with this disease.     

Prognostic potential of flow cytometric S-phase and ploidy prospectively determined in primary breast carcinomas

In a prospective study of a consecutive breast cancer series accumulated in the period 1978–82, the S-phase fraction (SPF) and ploidy status were determined by flow cytometry performed on cell nuclei derived from samples of 580 primary tumors. Sixty percent of the tumors were non-diploid. After correction for debris the median SPF values were 7.3% overall, 12% for non-diploid tumors, and 2.9% for diploid tumors (2.6% when nodal subsets N2 and N3 and cases with metastases at presentation were excluded). The SPF values correlated both to tumor size (p=0.008) and to the number of positive axillary lymph nodes (p=0.03).At clinical follow-up in 1986, 467 unilateral breast cancer patients who had undergone radical treatment for cure could be evaluated with respect to the prognostic value of both the SPF value and ploidy status. The median duration of follow-up was then 59 months (range 2–90), and the median time-to-recurrence 24 months (range 2–69, n=137).At follow-up in 1991, 201/467 of the patients had died, the median duration of follow-up being 50 months (range 2–126) for the deceased, and 119 (range 6–148) for the survivors. In multivariate analysis (Cox's proportional hazards models), the strongest independent predictors of distant recurrence-free survival (DRFS) were the number of positive axillary lymph nodes (p<0.0001), the debris-corrected SPF value alone (p=0.003,versus p=0.05 for uncorrected value), and ploidy status combined with the corrected SPF value (p=0.0002). When age was taken into account, both the corrected SPF value and the ploidy-SPF combination were predictors of crude survival (p=0.006 and p=0.002, respectively).In univariate life-table analysis, the 5-year DRFS rate was 93% in node-negative (N0) cases with an SPF<7.3%, as compared to 80% in those with an SPF7.3% (p=0.005). Among node-positive cases, the prognostic value of the SPF was confined to those with 1–3 positive nodes, the 5-year DRFS rate being 68% in cases with an SPF<7.3%, as compared to 40% in cases with an SPF7.3% (p=0.01).Ploidy status and SPF were combined to form four groups: diploid & SPF<2.6% (DL), diploid & SPF2.6% (DH), non-diploid & SPF<12% (NDL), and non-diploid & SPF12% (NDH). Among node-negative patients, the DRFS rate fell from 95% in the DL group to 87% in the NDL group, with the DH group at an intermediate level, as compared with 74% (p=0.03) for the NDH group which accounted for the bulk of the early distant recurrences. Among patients with 1–3 positive lymph nodes, the 5-year DRFS rate was 68% in both the groups with low SPF values (DL and NDL), as compared with 45% in the DH group (p=0.03), and 37% in the NDH group (p=0.006).In this study, the flow cytometry SPF value, alone or in combination with ploidy status, yielded the most profound additional prognostic information, enabling both node-negative patients with a high probability of cure and patients at risk of early relapse to be identified. Among node-positive patients, the prognostic value of the SPF value was confined to those with 1–3 positive axillary lymph nodes (the predominant node-positive subgroup), enabling a high and a low DRFS rate subgroup to be distinguished – a useful distinction where selection for adjuvant drug treatment is concerned. As the predictive strength of the SPF value was enhanced when correction was made for debris, we would recommend that the effect of such factors as debris be minimized as far as possible when flow cytometry-derived SPF values are to be used for prognostic purposes.     

S-phase fraction and urokinase plasminogen activator are better markers for distant recurrences than Nottingham Prognostic Index and histologic grade in a prospective study of premenopausal lymph node-negative breast cancer.

PURPOSE: Histologic grade, Nottingham Prognostic Index (NPI), estrogen receptor (ER) and progesterone receptor (PgR) status, and tumor size have previously been shown to be important prognostic indicators for distant recurrence of breast cancer. The purpose of this study was to compare the prognostic value of these factors with flow cytometric S-phase fraction (SPF), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1) in premenopausal patients with lymph node-negative breast cancer. PATIENTS AND METHODS: In 237 consecutive premenopausal patients with lymph node-negative breast cancer and freshly frozen tumor material available, SPF, ER and PgR status, uPA and its inhibitor PAI-1, histologic grade, and NPI were evaluated. RESULTS: SPF was univariately the most powerful prognostic factor for distant recurrence, followed by uPA, histologic grade, PgR, age, ER, NPI, and PAI-1, the latter being nonsignificant. Multivariate analysis revealed that neither NPI nor histologic grade was significant after adjustment for SPF, a fact that may be explained by the strong association between these factors. uPA was, however, an independent prognostic factor in addition to SPF, NPI, or histologic grade. CONCLUSION: In this prospective study, SPF and uPA were found to be independent prognostic factors in premenopausal women with lymph node-negative breast cancer. We suggest that SPF, if performed under standardized conditions, can replace histologic grade as a selection instrument for adjuvant medical treatment. The value of the combination of SPF and uPA needs to be confirmed in an independent prospective trial.