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.     

Prognostic value of DNA ploidy and S-phase fraction in relation to estrogen receptor content and clinicopathological variables in primary breast cancer

Tumors from 472 women with primary breast cancer were analyzed by flow cytometry. Divided into four categories, DNA ploidy showed significant association with disease recurrence and mortality. When allowance was made for its correlation with nodal status and estrogen receptor (ER) content, DNA ploidy did not add prognostic information. S-phase fraction was estimated in 290 DNA histograms. In contrast, it was significantly related to recurrence and mortality when controlling for nodal status, tumor size and ER content. When the follow-up was divided into two periods DNA ploidy and S-phase fraction showed association with disease recurrence in the first period only (<2.5 years), while the association with mortality was valid for both periods. Light scatter was measured in 234 samples. A low light scatter variability for the stemline nuclei was related to a high recurrence rate during the early follow-up period. In conclusion, DNA flow cytometry adds prognostic information concerning breast cancer patients.     

S-phase fraction and breast cancer - a decade of experience

During the past decade, more than 300 articles, abstracts, and book chapters have been published about S-phase fraction (SPF) determined by DNA flow cytometry and its clinical utility for patients with breast cancer. However, the use of SPF for making treatment decisions for breast cancer patients remains controversial.After reviewing 273 published articles, we conclude: 1) Despite different techniques and cutpoints, correlations between SPF and other prognostic markers are relatively consistent across studies; higher SPF is generally associated with worse tumor grade, absence of steroid receptors, larger tumors, and positive axillary lymph nodes. 2) Higher SPF is generally associated with worse disease-free and overall survival in both univariate and multivariate analyses; SPF values from laboratories that have conducted validation studies can be used, in combination with other factors, to estimate the prognosis of patients with primary breast cancer. 3) There is considerable variability among laboratories regarding assay methodology, cell- cycle analysis techniques, and cutpoints for classifying and interpreting SPF; use of SPF values from different laboratories is problematic, and there remains a need for standardization of these processes and well-designed confirmation studies.We conclude that measurement of SPF does have clinical utility for patients with breast cancer, but standardization and quality control must be improved before it can be routinely used in community settings.     

Significance of immunohistochemical assessment of steroid hormone receptor status for breast cancer patients

The assessment of steroid hormone receptors in resected breast cancer tissues is essential to decide whether endocrine therapy is indicated and to select the best treatment for each patient on the basis of receptor status. Both enzyme immunoassay (EIA) and immunohistochemistry (IHC) have been generally used as methods for examination of estrogen receptor (ER) and progesterone receptor (PgR). In some patients, receptor status cannot be examined for various reasons. A questionnaire survey in Japan clarified that ER status is not examined in approximately 40% of patients receiving breast conserving surgery. To eliminate "receptor unknown" cases, IHC examination on paraffin-embedded tissue is useful to assess the in situ receptor status. The concordance rate of ER and PgR status between EIA and IHC is very high and a study of 88 cases revealed a 97.7% concordance for ER and 92.0% for PgR at a cutoff point of 10%. The cutoff point of IHC is controversial and some studies demonstrated that patients showing 1% ER positive cancer cells would benefit from endocrine therapy. On the other hand, immunohistochemical expression of receptors is heterogeneous and some patients with ER negative invasive tumors have ER positive intraductal components. A study of 65 breast cancers demonstrated that ER positive intraductal components were detected in 3.1% cases of ER negative invasive lesions. According to these results and the recommendation of the St. Gallen International Conference, IHC is thought to be more useful than EIA in the assessment of steroid hormone receptor status for breast cancer patients.     

S期细胞比率对乳腺癌新辅助化疗敏感性的预测价值

目的 探讨乳腺癌细胞S期细胞比率（SPF）能否作为乳腺癌新辅助化疗敏感性的预测因子.方法 通过流式细胞仪检测2006年1月至2010年11月贵阳医学院第一附属医院乳腺外科收治的66例乳腺癌患者新辅助化疗前后乳腺癌组织中细胞SPF的变化,并比较SPF与临床病理特征之间的关系,统计分析采用配对资料的t检验及秩和检验.结果 在66例患者中,新辅助化疗疗效评价为6例病理完全缓解（pCR）,41例部分缓解（PR）,17例病情稳定（SD）,2例疾病进展（PD）.41例PR患者的SPF新辅助化疗前为（7.69±4.67）％,新辅助化疗后为（5.58±4.61）％,差异有统计学意义（t=2.314,P=0.026）.19例化疗无效的患者（包括SD、PD）新辅助化疗前SPF为（4.52±3.30）％,新辅助化疗后SPF为（4.19±3.01）％,差异无统计学意义（t=0.50,P=0.623）.47例客观缓解的患者（包括pCR、CR、PR）新辅助化疗前的SPF为（7.26±4.53）％,与19例化疗无效的患者新辅助化疗前的SPF[（4.52±3.30）％]比较,差异有统计学意义（t=2.394,P=0.020）.新辅助化疗前癌灶的SPF与患者年龄、月经状况无明显关系（Z=-1.461,-1.097;P＞0.05）,而与肿瘤直径、临床分期、淋巴结转移有关（x2=8.258,11.920;Z=-2.194;P＜0.05）.结论 新辅助化疗对乳腺癌细胞S期的影响是明显的,可以降低乳腺癌细胞的增殖,SPF值较大者对化疗敏感.     

S-phase fraction and survival benefit from adjuvant chemotherapy or radiotherapy of breast cancer.

Cancer chemotherapy interacts with cell proliferation, but data on the relationship between cancer cell replication and the effect of adjuvant chemotherapy are scarce. We have investigated the S-phase fractions of the primary tumour from premenopausal breast cancer patients who participated in a randomised trial comparing 12 cycles of polychemotherapy (CMF) with post-operative radiotherapy. DNA flow cytometry was performed on frozen tissues from 208 primary breast carcinomas, of which the S-phase fraction was estimated in 176 cases. There was a significantly higher benefit from CMF among patients with a high S-phase fraction (P = 0.0033). The relative risk of distant recurrence or death in the chemotherapy group as compared with the radiotherapy group was 0.19 for patients whose tumours had an S-phase fraction of 10% or over (95% CI 0.07-0.51) and 1.55 (0.88-2.73) for patients whose tumours showed lower S-phase levels. The interaction was still significant in multivariate analysis (P = 0.0057), including lymph node metastases, tumour size and oestrogen receptor content. We conclude that the benefit from adjuvant chemotherapy compared with radiotherapy is largely confined to patients with highly proliferative tumours.     

The relationship between c-erbB-2 expression, S-phase fraction and prognosis in breast cancer.

The relationship between c-erbB-2 gene expression (assessed immunohistochemically), S-phase fraction (SPF) and prognosis has been analysed in 172 women with primary breast cancer. c-erbB-2 staining was independent of age, tumour size, number of nodes involved, tumour grade and DNA ploidy, but was more common in oestrogen receptor (ER) negative tumours (P = 0.02) and progesterone receptor (PgR) negative tumours (P = 0.03). A weak correlation between c-erbB-2 staining and SPF was observed (r = 0.18). Amongst women with node negative disease, SPF was significantly related to relapse free survival (RFS, P = 0.04) while c-erbB-2 staining was not (P = 0.2). In contrast, both SPF (P = 0.002) and c-erbB-2 staining (P = 0.016) provided significant prognostic information on RFS for women with node positive disease. Multivariate analysis showed that c-erbB-2 staining and SPF gave independent information on RFS for women with node positive disease.     

Association of c-erbB-2 expression and S-phase fraction in the prognosis of node positive breast cancer.

An immunohistochemical study was performed on 211 primary breast carcinomas for c-erbB-2 expression. All patients had involvement of axillary lymph nodes and all were randomised onto one of the Ludwig Breast Cancer Trials I-IV between July 1978 and August 1981. c-erbB-2 overexpression significantly correlated with high S-phase fraction, four or more positive axillary nodes involved, estrogen receptor negative primaries, progesterone receptor negative primaries, high grade tumours and DNA aneuploidy. With a nine year median follow-up c-erbB-2 positive tumours had worse disease-free survival (p = 0.0002) and overall survival (p less than 0.0001). Multivariate analyses using proportional hazard regression models demonstrated that c-erbB-2 positivity continued to predict a poor outcome even when accounting for the effects of other prognostic factors.     

Significance and management of micrometastases in patients with breast cancer

The routine use of sentinel lymph node biopsy in the care of patients with invasive breast cancer has led to an increase in the identification of micrometastases in the axillary lymph nodes. Currently, the clinical relevance of this small-volume disease is debated. This article reviews the most current literature with respect to detection of micrometastases and isolated tumor cells in sentinel lymph nodes, the prognostic significance of these findings, and recommendations for locoregional and systemic treatment.     

Significance and management of micrometastases in patients with breast cancer

The routine use of sentinel lymph node biopsy in the care of patients with invasive breast cancer has led to an increase in the identification of micrometastases in the axillary lymph nodes. Currently, the clinical relevance of this small-volume disease is debated. This article reviews the most current literature with respect to detection of micrometastases and isolated tumor cells in sentinel lymph nodes, the prognostic significance of these findings, and recommendations for locoregional and systemic treatment.     

DNA index, S-phase fraction, histological grade and prognosis in breast cancer

DNA index and S-phase fraction (SPF) were measured by flow cytometry on paraffin embedded tissue from 140 primary breast tumours. The results of DNA analysis were compared with the size, degree of axillary node involvement, histological grade and steroid receptor content of the tumours, as well as with the patients' subsequent clinical course. Forty-four (31.4%) of the 140 tumours were diploid. S-phase fraction was evaluable for 134 (95.7%). The median SPF of the whole population was 7.1%, with diploid tumours having a significantly lower median SPF (3.2%) than aneuploid (10.1%, P less than 0.001). Both aneuploidy (P = 0.002) and high SPF (P less than 0.001) were strongly associated with high histological grade. There was no significant association between either DNA ploidy or SPF and tumour size, nodal status or steroid receptor content. An SPF below the median was strongly associated with better relapse-free survival (P = 0.008), overall survival (P = 0.004) and survival after relapse (P less than 0.001). Ploidy did not correlate significantly with clinical course. Multivariate analysis using the Cox model suggested that, while SPF gave prognostic information independent of tumour size or nodal status, this independent significance was lost when histological grade was included in the analysis.     

DNA ploidy, S-phase fraction and mitotic indices as prognostic predictors of female breast cancer.

DNA ploidy, S-phase fraction (SPF), mitotic index (MI), volume corrected mitotic index (M/V index) and standard prognostic factors were related to disease outcome in a series of 363 women with breast cancer followed-up for over 10 years in our clinic. DNA ploidy and SPF were significantly related to histological type, tumour grade and mitotic indices (p < 0.001). In univariate survival analysis, pN status (p < 0.0001), tumour diameter (p < 0.0001), MI (p = 0.001), M/V index (p = 0.0003) and SPF (p = 0.015) predicted survival. In pN(-) tumours. MI (p = 0.059) was related to survival. In pN(+) tumours, tumour diameter (p = 0.0004), M/V index (p = 0.023) and SPF (p = 0.045) predicted survival. In multivariate survival analysis, tumour diameter (p < 0.001). M/V index (p < 0.007), pN status (p = 0.014) and patient age (p = 0.09) were independently related to survival. In pN(-) tumours, tumour diameter independently predicted survival (p = 0.033). In pN(+) tumours, tumour diameter (p < 0.001), M/V index (p = 0.006) and the year of treatment (p = 0.08) were independent predictors. The results show that tumour diameter, pN status and proliferative activity of cancer cells are important prognostic factors in breast cancer. Of the proliferation indices, M/V index and SPF are equally powerful predictors, and the use of M/V index is advocated due to simplicity of the assessment.     

S-phase fraction after gating on epithelial cells predicts recurrence in node-negative breast cancer

We have compared the prediction of distant recurrence for S-phase fraction (SPF) and DNA-ploidy, as estimated by flow cytometry, on an epithelial cell population and an unselected cell population from 268 node-negative breast-cancer patients diagnosed between 1985 and 1988. The tumor tissue was mechanically disintegrated and divided for flow cytometric analysis using both gated cells containing cytokeratin 8 and 18 and ungated cells treated with a detergent-trypsin solution. The relationship to distant recurrence was investigated for flow cytometric data, tumor size and estrogen and progesterone receptor content in univariate and multivariate Cox's regression analysis. The regression analyses were performed on 209 cases with S-phase fractions estimated by both methods. In 11 cases, DNA-ploidy classification differed, reflecting increased sensitivity to minor aneuploid peaks but a decreased ability to separate peaks in the near-diploid region for the gated populations. When SPF were used in univariate analysis as a continuous parameter or the upper tertile was used as cut-off value, SPF from the cytokeratin-gated cell population were most closely associated with recurrence and contributed additional prognostic information to SPF from the unselected cell population in the multivariate analysis. Out of the following variables:tumor size, ER and PR status, SPF and DNA ploidy, only SPF from immunoselected cells contributed prognostic information in multivariate analysis. These results indicate that SPF from immunoselected cell populations improves the prediction of recurrence in node-negative breast cancer.     

Steroid hormone receptor expression in male breast cancer.

AIMS: To investigate expression of the steroid hormone receptors estrogen receptor (ER)-alpha and -beta, progesterone receptor (PR) and androgen receptor (AR) in male breast cancer. METHODS: Specimens from 16 male breast cancers were immunostained for ERalpha, ERbeta, PR and AR. FINDINGS: Eighty-seven percent of tumours expressed ERalpha, 93% PR, 87% ERbeta and 87% AR. Staining for ERalpha and PR was confined exclusively to the nuclei of epithelial cells with some heterogeneity. Nuclear immunoreactivity was also observed with AR. Again this was restricted to epithelial cells but tended to be more uniform. ERbeta was seen in the nuclei of epithelial cells and also in stromal fibroblasts and lymphocytes. Analysis of serial sections revealed a similar pattern of staining with ERbeta and AR in epithelial cells. CONCLUSIONS: In addition to expression of the better known steroid receptors, ERalpha, PR and AR, we have demonstrated a high rate of expression of ERbeta in male breast cancer. This is in keeping with the generally high steroid receptor expression seen in males. However, the abundance of ERbeta expressed in this small series of male breast cancer is in contrast to female breast cancer where ERbeta expression is often reduced.     

Comparison of PCNA/cyclin immunohistochemistry with flow cytometric S-phase fraction in breast cancer

Kinetic index determined by enumeration of neoplastic cells positive for proliferative cell nuclear antigen (PCNA) in 70 breast carcinomas (avidin-biotin immunoperoxidase technique) was compared to synthesis-phase fraction (S-phase, or SPF) values obtained by flow cytometry (FCM) using a multiparametric, 2 color method (dual-label propidium iodide/cytokeratin-FITC). The percent PCNA positive tumor cells (12.5% mean, range 1–28%) was significantly greater in aneuploid tumors (14.2% mean, N=35) compared to diploid range tumors (10.7% mean, N=35) (p<0.05), and was correlated with SPF derived from ungated DNA histograms (12.5% mean ± 5.5%, r=0.45, p<0.001). Marginally stronger statistical correlations were observed between the PCNA index and SPF values calculated from cytokeratin-gated (15.8% mean, r=0.53, p<0.001) DNA histograms or from SPF values obtained following linear baseline debris subtraction (mean=8.1%, r=0.48, p<0.001). Significant associations were identified between PCNA index and prognostically important clinicopathologic parameters including nuclear grade (p=0.014), presence of necrosis (p=0.005), and angiolymphatic invasion (p=0.003). We conclude: 1) PCNA index is comparable to FCM SPF and correlates with factors of known prognostic importance in carcinoma of the breast; 2) baseline debris and contaminating events derived from non-epithelial cells both represent significant artifacts in proliferative fraction estimates derived from FCM DNA histograms; and 3) multiparametric analysis may represent one means of improving the specificity and clinical value of FCM SPF determinations.     

The relationship between hormone receptor content and the effect of adjuvant tamoxifen in operable breast cancer

The relationship between hormone receptor status and the effect of adjuvant tamoxifen in early breast cancer remains controversial. This article presents the results of a randomized trial of adjuvant tamoxifen (40 mg daily for 2 years) versus no adjuvant endocrine therapy in postmenopausal patients. During 1976 to 1984, 1,407 patients were included in the study. Of these, 427 (30%) had high-risk tumors (pN + or pT greater than 30 mm) and were included in a concurrent randomized comparison of postoperative radiotherapy versus adjuvant polychemotherapy. The mean follow-up time was 61/2 years. Tamoxifen improved the recurrence-free survival (RFS) (P less than .01), but the overall survival difference in favor of the tamoxifen-allocated patients was not significant. Data on estrogen (ER) and progesterone receptor (PgR) content were available in 750 patients. Their mean follow-up time was 41/2 years. The effect of tamoxifen was significantly related to ER level (P less than .01). No benefit with tamoxifen was observed among ER-negative patients. The relation to PgR level was of borderline significance (P = .06). Multivariate analysis indicated that most of the interaction between treatment and receptor content was explained by the interaction with ER (P less than .01). The PgR status appeared to modify the effect of tamoxifen among the ER-positive patients and the greatest effect was observed among patients who were positive for both receptors. However, the additional predictive information provided by the PgR assay did not help to identify an unresponsive subgroup of patients.     

Uptake of 11C-methionine in breast cancer studied by PET. An association with the size of S-phase fraction.

L-[methyl-11-C]methionine (11C-methionine) uptake of seven primary breast cancers, four soft tissue metastases of breast cancer, and three other breast lesions was studied by positron emission tomography (PET). 11C-methionine accumulation was assessed by calculating the standardised uptake value (SUV). The mean SUV for breast cancer was 8.5 +/- 3.3 (s.d.), while the maximal uptake in the liver was 12.4 +/- 1.6, in the bone marrow 5.8 +/- 0.7, and in the myocardium 3.4 +/- 0.6. All eight malignant tumours larger than 30 mm in diameter accumulated clearly 11C-methionine, whereas none of the three smaller cancers (from 12 to 15 mm in diameter) were visualised. Strong uptake of 11C-methionine was associated with a large S-phase fraction (SPF) measured with flow cytometry (r = 0.77, P = 0.01), and the non-visualised cancers had all a small SPF (less than 5.5%). One benign tumour (an abscess) accumulated slightly 11C-methionine. The results indicate that both primary and metastatic breast cancer can be effectively imaged with 11C-methionine by PET, and that the accumulation of 11C-methionine may correlate with the proliferation rate of breast carcinoma.