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27-hydroxycholesterol (27HC), synthesized from cholesterol by the enzyme CYP27A1, differentially impacts estrogen receptor positive (ER+) breast cancer (BC) cell growth depending on estrogen levels. This study examined the association between CYP27A1 expression and prognosis in a cohort of 193 premenopausal patients with lymph node-negative primary BC with limited exposure to adjuvant systemic cancer treatments. In multivariable analyses among patients with ER+ tumors, high CYP27A1 protein and mRNA expressions were associated with four- and eight-fold reductions in the incidence of distant recurrence-free survival events: HRadj = 0.26, 95% CI = 0.07–0.93 and HRadj = 0.13, 95% CI = 0.03–0.60, respectively. In vitro studies revealed that 27HC treatment potently inhibited ER+ BC cell proliferation under lipid-depleted conditions regardless of estradiol levels, transcriptionally mediated through the downregulation of ER signaling with a concomitant upregulation of cholesterol export. Importantly, if validated, these results may have implications for adjuvant treatment decisions in premenopausal patients, especially when de-escalation of therapy is being considered.

INTRODUCTION: Endometrioid endometrial carcinoma is a cancer type with generally excellent prognosis when diagnosed at an early stage, but there is a subset of patients with relapsing disease in spite of early diagnosis and surgical treatment. There is a need to find prognostic markers to identify these patients with increased risk of relapse. Depth of myometrial invasion, histological grade, and presence of lymphovascular invasion are known risk factors. DNA content (ploidy) and proliferation measured as S-phase fraction (SPF) have been discussed as prognostic markers but need additional evaluation. MATERIAL AND METHODS: We evaluated relapse-free survival (RFS) with respect to ploidy and SPF, which was analyzed by flow cytometry on fresh tumor tissue, in a cohort of 1001 women treated for stage I endometrioid endometrial carcinoma in northern Sweden during the period of 1993-2010, with a median follow up time of 12.0 years. Data were obtained from historical records. RESULTS: In simple analysis, both aneuploidy and high SPF were associated to increased risk of relapse with hazard ratios (HR) 2.37 (95% CI 1.52-3.70) and 1.94 (95% CI 1.24-3.02), respectively. Our data also confirmed stage, tumor grade, and ploidy as independent prognostic markers in an age adjusted cox regression multivariable analysis but we did not find SPF to contribute to prognosis. However, the combination of aneuploidy and high SPF identified a group of patients with increased risk of relapse, HR 2.02 (95% CI 1.19-3.44). CONCLUSION: In this study, which is the largest study of ploidy and SPF in stage I endometrioid endometrial carcinoma using fresh frozen tissue, aneuploidy was shown to be an independent prognostic marker. Furthermore, the combination of aneuploidy and high SPF could be used to identify patients with increased risk of relapse.

Mithilfe hoch-auflosender DNA-Durchflusszytophotometrie wurden DNA-Ploidie und S-Phasenfraktion (SPF) bei 279 Speicheldrusentumoren bestimmt. Alle 229 benignen Neoplasien waren diploid; 12 von 50 malignen Tumoren wiesen Zellpopulationen mit aneuploidem DNA-Gehalt auf. Die SPF-Werte der diploiden Malignome waren signifikant hoher als bei pleomorphen Adenomen, unterschieden sich jedoch nicht von denen der Zystadenolymphome (Warthin-Tumoren). Wahrend Aneuploidie ein Malignitatsmerkmal darstellt, ist der SPF-Wert fur die Dignitatsdiagnostik bei Speicheldrusentumoren nur von eingeschranktem Nutzen.

DNA ploidy is an important prognostic parameter in paediatric B-ALL, but the significance of the S-phase fraction is unclear. In present study, DNA ploidy was assessed in 40 pediatric B-ALL cases by flow cytometry. The DI (DNA index) and percentage of cells in S-phase were calculated using Modfit software. Aneuploidy was noted in 26/40 (65%) cases. A DI of 1.10-1.6 (hyperdiploidy B) was noted in 20/40 (50%) and 6/40 (15%) had a DI1.60 (triploid and tetraploid range). Some 14/40 (35%) cases had a diploid DI between 0.90-1.05. None of the cases had a DI0.90 (hypodiploid) or in the 1.06-1.09 (hyperdiploid A) range. The mean S-phase fraction was 2.6%, with 24/40 (60%) having low and 16/40 (40%) high S-phase fractions. No correlation was noted with standard ALL risk and treatment response factors with DI values or S-phase data, except for a positive correlation of low S-phase with high NCI risk category (p=0.032). Overall frequency of hyperdiploidy in our cohort of B-ALL patients was very high (65%). No correlation between hyperdiploidy B and low TLC or common B-phenotype was observed in our study as 42% cases with DI 1.10-1.6 had TLC50 x 109 and 57.1% CD 10 negativity. The study also highlighted that S-phase fraction analysis does not add any prognostic information and is not a useful parameter for assessment in ALL cases. However, larger studies with long term outcome analysis are needed to derive definitive conclusions.

Female Taiwanese breast cancer patients are younger than their Western counterparts. This study examined the predictors of axillary lymph node metastases in Taiwanese women with T1 breast cancer.Data from 394 Taiwanese women with T1 invasive breast carcinoma were retrospectively reviewed.The data contained 6 T1a, 51 T1b, and 337 T1c breast tumors. The patients' ages ranged from 23 to 82 years (mean +/- SD, 48.2 +/- 11.4 years; median, 46.4 years). Axillary nodal metastases were present in 38.3% of the patients (16.7% in T1a, 35.3% in T1b, and 39.2% in T1c tumors). The patients with nodal metastases had significantly greater body weights and S-phase fractions than those without nodal metastases. Univariate analysis revealed that unfavorable pathology, lymphovascular invasion, S-phase fraction7%, and nondiploid DNA ploidy were significantly associated with lymph node metastases. Lymphovascular invasion was the only significant variable as the independent predictor in the multiple logistic regression analysis. In the Cox proportional hazards regression analysis, axillary nodal status and lymphovascular invasion were significantly associated with survival.Taiwanese women with small breast cancer displayed a relatively higher incidence of axillary lymph node metastases than Western women. Axillary lymph node dissection or sentinel lymph node biopsy should be conducted on Taiwanese patients with small invasive breast carcinomas, particularly when risk factors exist.

Background S-phase fraction (SPF) measurement by flow cytometry is a clinically useful prognostic factor in patients with breast carcinoma. Standardized SPF determination is essential. As part of a multicenter study, we evaluated the influence of the choice of software and histogram resolution (256, 512, or 1,024 channels) on SPF quantification. Methods One hundred thirty-three DNA histograms were analyzed in three laboratories with Modfit 5.2, Modfit LT, and Multicycle AV software. Strict rules for histogram interpretation and software management were applied. The following five options were compared: MF 5.2 1024, MF 5.2 256, MF LT 256, MC AV 256, and MC AV 512. Results In the DNA diploid and aneuploid groups, SPF distributions were not statistically different among the five options. Excellent quantitative correlations were obtained between pairs of options. When using tertiles as cutpoints for SPF classification, concordance rates ranged from 79.7% to 93.2% for DNA diploid samples and from 87.8% to 95.9% for DNA aneuploid samples, the best results being obtained with software working with a similar histogram resolution. Conclusions Standardized use of commercially available software, including the choice of histogram resolution, provides comparable SPF results. Cytometry 48:66–70, 2002. © 2002 Wiley-Liss, Inc.

Background: The prevalence of locally advanced esophageal squamous cell carcinoma (ESCC) remains high despite technological advancements in its diagnosis. This leads to prolonged multimodality treatment often resulting in poor response eventually leading to poor prognosis. Currently, there are no biomarkers available to predict response to neoadjuvant chemotherpy. Aldehyde dehydrogenase 1 (ALDH1), human epidermal growth factor receptor-2 (HER2), p16INK4A, ploidy and S phase fraction are considered significant biomarkers in various solid malignancies. However, there is paucity of data on their clinical significance in ESCC. In the present study, we investigated their significance in predicting the response to neoadjuvant chemotherapy (NACT) in ESCC patients. Methods: Immunohistochemisty of ALDH1, HER2, p16INK4A and propidium iodide based cell cycle analysis through flow cytometer was performed in pre treatment biopsy sample collected from 108 ESCC patients who were presented at a comprehensive cancer centre in northeast India and all of them subsequently received neo adjuvant chemotherapy. Results: ALDH 1, HER2 and p16INK4A were found positive in 65.7%, 7.4% and 22% of pre treatment ESCC specimens respectively. ALDH1 expression correlates with poor response to neo adjuvant chemotherapy (P Citation Format: Rajeev Kumar, R. Ravi Kannan, Akalesh Kumar Verma, Anuradha Talukdar, Monoj Kumar Deka, Ritesh Tapkire, Litika Vermani, Sankar Kumar Ghosh. High ALDH1, S phase fraction, p16INK4A in esophageal squamous cell carcinoma could predict response to neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2788. doi:10.1158/1538-7445.AM2017-2788

Prognostic factors which can predict response to imatinib in patients with chronic myeloid leukemia (CML) have been of much interest. Identification of patients who are likely to fail to imatinib t...

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.

Women with estrogen-receptor (ER)-positive breast cancer and no axillary lymph-node involvement are considered to have excellent overall prognosis. However, this population is not homogeneous with regard to risk of recurrence; in fact, some of these patients have a prognosis no better than that of many women with ER-negative tumors or positive axillary nodes. Consequently, better tumor markers and better use of those currently available are needed to distinguish patients who would benefit from more aggressive therapy from those for whom such therapy is unnecessary. A well-defined cohort of over 4000 breast cancer patients from National Surgical Adjuvant Breast and Bowel Project (NSABP) Protocol B-14 who had ER-positive tumors and no axillary lymph-node involvement was analyzed to ascertain the usefulness of tumor cell S-phase fraction for prognosis. The significance of clinical tumor size, patient age at surgery, ER and progesterone receptor (PgR) expression, and nuclear grade was also explored. Statistical methods based on smoothing splines were used to relate treatment failure and mortality rates to patient and tumor characteristics. Models for 5- and 10-year disease-free survival (DFS) and overall survival were developed and summarized. The attenuation of the prognostic importance of covariates over time was investigated. After other characteristics were accounted for, a strong association was found between S-phase fraction and DFS, as well as survival. Tumor size, patient age at surgery, and PgR status were also significantly associated with outcome. The diversity of risk in the B-14 population was more extreme than is generally recognized. The prognostic capabilities of S-phase, tumor size, and PgR status were sharply attenuated as the time from surgery increased.

In many different solid tumours the DNA content of cells has been reported to be associated with malignancy. With one exception, studies using flow cytometry to estimate cellular DNA in uveal melanoma have used archival material. We report the findings of a prospective study using fresh uveal melanoma where the DNA index (DI) and percentage of cells in S phase (%SPF) have been determined by flow cytometry in 51 patients with a minimum follow-up of 5 years. Forty-one (80%) of the tumours were large, 9 (18%) were medium and only 1 was small. Eight (16%) were epithelioid tumours, 41 (80%) were mixed cell and only 2 were spindle B cell tumours. Twenty-seven tumours were diploid, of which 10 (37%) developed metastases. Of the 24 aneuploid tumours, metastases developed in 6 (25%). The median %SPF was significantly reduced in patients with diploid tumours compared with aneuploid tumours (p0.0001), but there were no significant differences in %SPF between tumours associated with or without metastatic disease. The DI and %SPF were not significantly associated with the operation performed, cell morphology, tumour size or location. Neither %SPF nor aneuploidy was associated with a poor prognosis. Our results suggest that the estimation of DI or %SPF by flow cytometry is of little value in predicting survival from uveal melanomas after a minimum follow-up of 5 years.

One hundred and fifty-eight histologically verified mammary carcinomas with known mammographic doubling time (DT) were studied with special emphasis on a morphologic classification proposed by Linell et al. [8, 12, 14, 15]. The hypothesis that Linell classification of ductal carcinomas into comedo, tubuloductal and tubular carcinomas is easy to perform with small inter-observer variations, was not fully confirmed. The Linell classification was found to correlate well with conventional WHO malignancy grading, S-phase fraction and DNA-ploidy. The Linell classification also correlated to surgical stage, lymph node status and DT, but not at all to tumour size. Using distant disease-free survival as an endpoint, the Linell classification gave prognostic information comparable to conventional histologic grading, seeming to be a simple, cheap and reliable method well worth trying on a larger scale.

BACKGROUND Young patients with colorectal carcinomas are considered to have a worse prognosis than older patients. It was the goal of this study to assess if biologic characteristics of tumors in young patients differ from those observed in 2 different groups of patients with the same clinical characteristics but ranging in age either from 41 to 60 years or 61 years and older, respectively. METHODS Colorectal carcinoma tumor samples were obtained from storage from patients age 40 years and younger and examined for tumor ploidy and S-phase fraction. For each younger patient, a control was selected among patients matched for Dukes stage, site of primary tumor, and sex, with the two age groups. RESULTS Thirty-one of 1361 patients (2.2%) with colorectal carcinoma treated at our institution between 1984 and 1994 age 40 years or younger. Tumor aneuploidy was present in 3 younger patients, in 5 patients in the 41 to 60 years age group, and in 5 patients in the 61 years and older age group. S-phase fraction was 27.67 +/- 13.62 in patients younger than 40 years, 25.35 +/- 11.6 in the 40 to 61 years age group, and 22.45 +/- 8.48 in the 61 years and older age group. These differences were not statistically significant. CONCLUSIONS It appears that there are no significant differences in S-phase fraction and tumor aneuploidy in patients younger or older than 40 years, suggesting that colorectal tumors arising in young people do not have different biologic properties.

Purpose: To assess the predictive value of pretreatment proliferative activity of epidermoid cervix carinoma cells with respect to short- and long-term results of radiotherapy. Methods and Materials: The proliferative activity of 25 epidermoid cervix carcinomas was evaluated as the immunofluorescent labellng index (LI) by rabbit antithymidine antibodies reacting specifically with single-stranded DNA of replication forks in S-phase cells. The short-term clinical outcome was estimated at 3–6 months after treatment by visual and palpatory examination. Three-year follow-up data were obtained through hospital charts adn correspondence with referring physicians for only 19 patients. Results: There was no statistically significant association between LI and such conventional prognostic factors as clinical stage. The LI value of cervix carcinomas was significantly associated with complete regression at 3–6 months after radiotherapy and 3-year disease-free survival. Complete regression at 3–6 months was observed in 87.5% patients with fast proliferating tumors (LI > 7.0%), and only in 41.2% patients with slowly proliferating tumors ( p = 0.03). Probability of 3-year disease-free survival was 85.7% in patients with fast proliferating tumors and 50.0% in those with slowly proliferating tumors ( p = 0.05). Conclusion: The immunofluorescent LI of epidermoid cervix carvix is able to provide prognostic information on short-term tumor response to radiotherapy and disease-free survival.

Studies on the cell kinetics of the human gallbladder are difficult because of epithelial degeneration by bile. Using the epithelial isolation technique, however, we were able to determine the degree of degeneration and to examine the cell kinetics of gallbladder lesions in freshly resected surgical specimens. Normal and neoplastic epithelia were isolated nonenzymatically from freshly resected gallbladder. The nuclear DNA content and S-phase fraction were estimated in 110 patients with gallbladder lesions by flow cytometry (FCM). Normal tissues and all lesions except carcinomas were diploid. The S-phase fraction of gallstone cases was significantly higher (1.47 +/- 0.70%; mean +/- SD) than normal (0.79 +/- 0.39%) (P < 0.0006). All gallbladder carcinomas were multiploid, and their S-phase fraction was 11.63 +/- 3.65%. Cell renewal of normal gallbladder is low. In the gallstone cases, the S-phase fraction was increased, possibly correlated with carcinogenesis.

Background: We evaluated the association of the immunohistochemical Ki-67 expression, and S-phase fraction with clinicopathological variables and patient outcome. Patients and methods: Histological samples from 100 primary Libyan breast carcinoma patients were retrospectively studied with monoclonal antibody to Ki-67. S-phase fraction was determined by DNA image cytometry. Results: The median Ki-67 percentage for all tumors was 27.5%, ranging from 1 to 80% and the median S-phase fraction (SPF) was 11%, ranging from 0 to 62 %. Tumors with high Ki-67 expression were found in 76% of patients and with high SPF values in 56%. Ki-67 expression was more frequent in tumors with high SPF than low SPF. High Ki-67 and high SPF were associated with advanced stages, poor differentiation of tumors, positive lymph nodes, and distant metastasis. The Ki-67 was associated with hormone receptor negative tumors. The SPF was higher in young patients (

OBJECTIVE: Our purpose was to determine the usefulness of deoxyribonucleic acid ploidy and S-phase fraction as prognostic factors in patients with cervical cancer. STUDY DESIGN: Paraffin-embedded tumor specimens were obtained from 123 patients with cervical cancer (mean age 51 years, range 21 to 87 years). The mean follow-up period was 6.4 years. Deoxyribonucleic acid ploidy and S-phase fraction were determined by flow cytometry. RESULTS: A total of 119 patients were evaluable for deoxyribonucleic acid ploidy and 92 were evaluable for S-phase fraction. The overall rate of deoxyribonucleic acid aneuploidy was 60%, and the median S-phase fraction was 12.5%. Neither factor was significantly related to stage of disease. In univariate survival analyses patients with deoxyribonucleic acid aneuploid tumors had slightly better early survival than did patients with deoxyribonucleic acid diploid tumors (median survivals 2.7 and 1.4 years, respectively, p = 0.08 [Wilcoxon]), but the overall survival was not significantly different, p = 0.37 (log-rank). In multivariate analyses deoxyribonucleic acid ploidy was not a significant predictor of survival. S-phase fraction was not statistically significant in either analysis. CONCLUSION: Deoxyribonucleic acid ploidy and S-phase fraction are not useful prognostic factors for patients with cervical cancer.

The quality of life in postmenopause is seriously affected by the symptoms related to vaginal atrophy. To evaluate in a 3-month, prospective, randomized, double blind, study whether vaginal suppositories containing genistein might improve genital symptoms, colposcopical and cytologic findings or modify DNA cytometric features in postmenopausal women affected by vaginal atrophy, in comparison with vaginal suppositories containing hyaluronic acid (HA). A total of 62 postmenopausal women were randomly assigned to receive intravaginally 97 μg of genistein (group A, n = 31) or 5 mg of HA (group B, n = 31) daily for 15 days continuously/month for 3 months. Vaginal and cervical smear, colposcopy, vaginal biopsy were performed before and at the end of the study. Maturation value (MV) was calculated. Flow cytometric analysis of DNA ploidy (DI) and S-phase fraction (SPF) were performed. After 90 days of study, a significant improvement was obtained in genital symptoms, colposcopy scores and MV (p

Despite the relatively good prognosis of node negative breast cancer (NNBC) it is necessary to determine prognostic factors to identify subgroups of high and low risk situation for recurrence and death. In 111 patients with primary NNBC we have examined the impact of two new prognosticators on disease-free intervall (DFI) and overall survival (OAS): S-phase fraction (SPF) as a proliferative marker (n = 107 P) and epidermal growth factor receptor (EGF-R) as a marker for differentiation (n = 62 P). Only fresh frozen tissue was used. SPF was detected by flow cytometry and EGF-R by a radioimmunoassay There was a strong correlation between grade III tumours and high SPF (>5%) (p 5% had a significant shorter DFI (p = 0.009) and OAS (p = 0.01) than with SPF 15 fmol/mg (p = 0.07 resp. p = 0.05). However, in a Cox regression model only trends have been observed for independency of the new factors. Using a combination of both factors, a group of patients with good risk could be identified, if at least one factor was expressed favourably. If both factors were elevated, clinical outcome was poor (DFI: p

The immunohistochemical expression of HER-2/neu and cytofluorimetric data were retrospectively analyzed in a group of primary advanced ovarian cancers. Thirty-three out of 94 (35%) cases showed a specific p185/neu immunoreaction. No correlation between p185/neu expression and any of the clinico-pathologic parameters examined was observed. As far as cytofluorimetric data are concerned, 38 out of 69 (55%) of the tumors were diploid (DNA index = 1) while 31 (45%) were aneuploid (DNA index from 1.10 to 2.50 with a median value of 1.50). Ovarian tumors were defined as of low and high S-phase fraction in 68% and 32% of the cases, respectively. Tumor ploidy and S-phase fraction did not correlate with the clinico-pathologic characteristics or p185/neu oncoprotein expression. Aneuploid tumors had a higher S-phase fraction (mean: 15.81 ± 13.44) than diploid tumors (mean: 8.89 ± 7.98) (P< 0.01). p185/neu expression failed to affect significantly both overall and progression free survival. On the other hand tumor ploidy was found to be related to the prognosis of advanced ovarian cancer patients although the difference was not statistically significant. As far as progression free survival is concerned, the median time to recurrence was not reached for diploid cases whereas it was 21 months for aneuploid cases (P< 0.05). The 5-year survival for patients with a low S-phase fraction (58%) was significantly higher than for patients with high S-phase fraction tumors (28%) (P< 0.01). Median time to recurrence was 48 and 17 months for low and high S-phase fraction tumor patients, respectively (P< 0.05). However, in a multivariate analysis both tumor ploidy and S-phase fraction did not retain their prognostic value. The assessment of the role of the parameters examined in improving the prognostic characterization of ovarian cancer patients should be investigated in large multicenter clinical trials.

BACKGROUND The functional definition of aplastic anemia (AA) is the failure of hematopoietic stem cells to proliferate. The aim of the present study was to analyze the S-phase fraction (SPF) (proliferative activity) in patients with AA at diagnosis to explore its relationship with disease characteristics and its value in discriminating among patients with different prognoses. We also investigated whether the SPF value influenced the response to immunosuppressive therapy in AA patients. PATIENTS AND METHODS The analysis of SPF at the time of diagnosis was carried out by flow cytometry on peripheral blood samples from 53 consecutive patients with AA and 30 age- and sex-matched controls. All patients were given cyclosporine and followed up periodically to determine response to therapy. RESULTS Based on the median SPF, AA patients were divided into two groups: patients with SPF ≤ 0.59% (n = 27) and patients with SPF > 0.59% (n = 26). An SPF > 0.59% was associated with advanced age (P = .02) and elevated serum LDH level (P = .01). Patients with an SPF > 0.59% also had a higher incidence of paroxysmal nocturnal hemoglobinuria and cytogenetic abnormalities. During a median follow-up of 18 months, 3.7% of patients with SPF ≤ 0.59 and 11.5% of patients with SPF > 0.59% developed dysplasia and one patient with SPF > 0.59% converted into AML. A significantly higher (P = .018) overall response rate of 53.9% was found in patients with SPF > 0.59% versus 22.2% of patients with SPF ≤ 0.59% at 6 months. CONCLUSIONS Independently of the peripheral blood count, the SPF at diagnosis may provide information on the expected response to immunosuppressive therapy and the propensity for disease to evolve into MDS/AML. Hence, SPF may serve as an early indicator for the evolution of MDS/AML in patients with AA and thus contribute to therapeutic decisions.

The nuclear DNA content of head and neck tumors (57 malignant, 20 benign) was determined by flow cytometory (FC M).We used fresh specimens and analyzed the ploidy pattern, S phase fraction and G2M phase fraction. Twenty-six of the 57 (45.6%) malignant tumors were aneuploid and all the benign tumors were diploid. Recurrent tumors tended to be aneuploid.DNA indices of the recurrent tumors were higher than those of the fresh tumors (p

For therapeutic decisions it is essential to have an evaluation of the tumor volume and the grade of dedifferentiation. Measurement of PSA gives a good additional guide to the tumor volume and to dissemination of the disease. Investigation of the DNA ploidy values can offer further important information on the aggressiveness of the tumor and be helpful for our understanding of the process of tumor propagation. However, DNA studies can still not be regarded as being standard in the clinical work-up of these patients. They are optional but they have a definite place in the research on prostatic cancer. The various methods to study tumor growth by analysis of the S phase fraction are interesting new contributions but still belong to the research laboratories. When we consider prognostic indicators we have to take into account the biologic character of carcinogenesis. Modern research has shown that the development and the progression of cancer is not an instantaneous and solitary reaction. It is a series of events and a net-work reaction between growth-regulating factors, stimulating and inhibiting, a step-wise alteration of the genome. We must recognize that what we are observing is the condition at the present time, and, of course, the observation must be evaluated together with the whole clinical scenario, the man's age, his general condition etc. But still the series of diagnostic procedures presented here will give a rather solid ground for both our therapeutic decisions and for evaluation of the results of treatment.

Between 1971 and 1974, 1665 women with primary operable breast cancer were randomized into a National Surgical Adjuvant Breast and Bowel Project (NSABP) trial (B-04) conducted to evaluate the effectiveness of several different regimens of surgical and radiation therapy. No systemic therapy was given. Cells from archival paraffin-embedded tumor tissue taken from 398 patients were analyzed for ploidy and S-phase fraction (SPF) using flow cytometry. Characteristics and outcome of patients with satisfactory DNA histograms were comparable to those from whom no satisfactory cytometric studies were available. In patients with diploid tumors (43%), the mean SPF was 3.4% +/- 2.3%; in the aneuploid population (57%), the SPF was 7.9% +/- 6.3%. Only 29.9% +/- 17.3% of cells in aneuploid tumors were aneuploid. Diploid tumors were more likely than aneuploid tumors to be of good nuclear grade (P less than 0.001) and smaller size (P equals 0.03). More tumors with high SPF were of poor nuclear grade than were tumors with low SPF (P equals 0.002). No significant difference in 10-year disease-free survival (P equals 0.3) or survival (P equals 0.1) was found between women with diploid or aneuploid tumors. Patients with low SPF tumors had a 13% better disease-free survival (P equals 0.0006) than those with a high SPF and a 14% better survival (P equals 0.007) at 10 years than patients with high SPF tumors. After adjustment for clinical tumor size, the difference in both disease-free survival and survival between patients with high and low SPF tumors was only 10% (P equals 0.04 and 0.08, respectively). Although SPF was found to be of independent prognostic significance for disease-free survival and marginal significance for survival, it did not detect patients with such a good prognosis as to preclude their receiving chemotherapy. The overall survival of patients with low SPF was only 53% at 10 years. These findings and those of others indicate that additional studies are necessary before tumor ploidy and SPF can be used to select patients who should or should not receive systemic therapy.

It is often difficult and challenging task to differentiate aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (HMDS) among the patients with bone marrow aplasia. This is possibly because of the considerable clinical, cytological and histological similarities between these two disorders. As prognostic and therapeutic approach to AA and HMDS are different, it is imperative to differentiate them at the time of initial diagnosis. Various attempts have been made in the past to differentiate AA from HMDS. In the present study, we explored the value of certain new parameters i.e. S-phase fraction (SPF) and aneuploidy that could be used for this purpose.The study included 46 consecutive patients with aplastic anemia, 15 patients with HMDS along with 32 age and sex-matched control subjects. S-phase fraction and aneuploidy analysis was carried out by flow cytometry using Mod Fit-LT V3.0 software.The mean SPF value was significantly lower (p=0.02) in patients with AA and higher (p=0.01) in HMDS as compared to that of the control. Aneuploidy was present in 15.2% patients with AA and in 33.3% HMDS cases. During follow-up, 4 patients with AA developed MDS, out of these, three patients had aneuploidy as well as high SPF value at the time of diagnosis. Two patients with HMDS who had aneuploidy and high SPF, converted into AML. Eleven patients died during the study, in whom 8 had aneuploidy and high SPF value.We conclude that high SPF value and presence of aneuploidy favour the diagnosis of HMDS rather than AA. SPF and aneuploidy may be important parameters in patients with AA to predict their propensity to evolve into myelodysplastic syndrome and acute myeloid leukemia. SPF value may also be useful in the early diagnosis of HMDS before morphologically evidence of dysplasia is apparent.

予後の判明している大腸癌切除症例122例を対象として, 切除材料のパラフィンブロックを用いてflow cytometryによりDNA ploidy patternを求め, Ploidy patternが大腸癌の進展, 予後を反映する因子となるか, retrospective studyを行った.その結果, DNA異常を示すaneuploid症例は, over allと治癒切除症例において5年生存率がDNA異常のないdiploid症例に比べ有意に低く (p

The nuclear DNA content of archival paraffin-embedded bladder cancer samples (70 patients) of WHO grades I-III has been measured by flow cytometry. The female/male ratio was 15/55. The mean follow-up time was 13 years (range 9.6-22.0 years). 37 of 70 (53%) patients had DNA index 1.0 (diploid DNA content), and the remaining 33 (47%) patients had an aneuploid tumor. There was no significant difference in the age (mean +/- SD) of the patients having a diploid (66 +/- 9 years) or an aneuploid tumor (68 +/- 11 years) at the time of diagnosis. 47 deaths occurred during the follow-up period; 24 (51%) of these were due to bladder cancer (12 diploid, 12 aneuploid tumors). No significant difference was found after radical treatment during the disease-free interval (mean +/- SD) between diploid (48 +/- 45 months) and aneuploid (35.5 +/- 35 months) groups of patients. Recurrences during the follow-up period were equally common among aneuploid and diploid tumors. A statistically significant relation between histological grade and survival could be demonstrated, but DNA ploidy and S phase fraction had little prognostic value in this respect. There was no statistically significant difference in survival between aneuploid (30%) and diploid (35%) groups of tumors during the follow-up period. The study suggests that flow cytometric determination of nuclear DNA ploidy from paraffin-embedded samples in bladder tumors does not add to the prognostic power of subjective histological grading.

Histological assessment for prognostication of myxofibrosarcomas remains challenging. We previously reported independent prognostic value of Skp2, an oncoprotein promoting S-phase progression (Clin Cancer Res 2006;12:487–98). We evaluated S-phase fraction (SPF) and ploidy of myxofibrosarcomas and the association between SPF and Skp2. Flow cytometric findings were analyzed for 75 cases and correlated with clinicopathological factors, Skp2 labeling index (LI), metastasis-free survival (MeFS), and overall survival (OS). Forty-seven and 28 cases were classified as diploid and nondiploid, respectively. High SPF (≥20%) was detected in 32 of 61 interpretable cases. Skp2 overexpression (LI ≥ 10%) was seen in 36 of 72 cases with scoring. Nondiploidy correlated with higher French Federation of Cancer Centers (FNCLCC) grades (P = .006), remarkable necrosis (P = .010), and Skp2 overexpression (P = .018). Noticeably, SPF was significantly related to Skp2 LI (P

Purpose: To determine whether quantitative flow cytometric androgen receptor density expression (MFC ratio) in prostate cancer was associated with S-phase fraction. Methods: Flow cytometry was performed to determine DNA aneuploidy, S-phase fraction, percentage of androgen receptor (AR)-positive cells, and MFC ratio in prostate cancer patients. Results: MFC ratio showed distinct clustering. Eight patients had a low MFC ratio of 1.78–2.74, while 10 patients had high MFC ratios between 4.99 and 6.48. The S-phase fraction had average values of 11.05 vs 4.92 in tumors with high vs low MFC ratio (P

From 1981 to 1995, 1755 patients aged 70 years or over who had nonmetastatic unilateral breast carcinoma received curative local or regional treatment in our institute. Median follow-up was 8 years. The median age of these patients was 75 years (range: 70-94), and 86% were under 81 years of age. Tumors were classed as T3-4 in 24% of them; 18% had N1b/N2 tumors, and in 12% grade 3 disease was present. Only 19% were both ER and PR negative. The S phase fraction was5% in 79% of patients. In 1046 patients (60%) modified radical mastectomy was performed, while 20% underwent lumpectomy and in 20% radiotherapy was the only treatment administered. Adjuvant endocrine therapy was given in 463 (26%) cases, and only 3% of patients received chemotherapy. The median overall survival time was 121 months. The overall cancer-related death rate was 49%. The 10-year disease-free survival (DFS) rate was 64%, and the 10-year local relapse rate was 14%. Prognostic factors determined on univariate analysis were tumor size, clinical nodal status (ER and PR), and grade. No significant difference in outcome was observed between mastectomy and conservative treatment. Parameters for which correlations with DFS were found on multivariate analysis were clinical nodal status (P0.0001), tumor size (P0.0001), ER (P0.0001), and PR (P = 0.04). Breast cancer in elderly women is frequently hormone-dependent (81%) with a low proliferation index. Prognostic factors are the same as in younger postmenopausal patients. More than 50% of these patients died from a cause other than their breast cancer.

Aims—To investigate the prognostic value of recently proposed flow cytometric S-phase fraction (SPF) variables (average SPF and SPF tertiles) compared with conventional SPF, and to compare the one with the best predictive value with the immunohistochemical Ki-67 index in breast carcinoma. Methods—A short term follow up study (median, 39.6 months) of a large series of patients (n = 306) was conducted. DNA ploidy was analysed on fresh/frozen tumour samples by flow cytometry, and the SPF was calculated from the DNA histogram using an algorithm. The Ki-67 index was assessed on paraffin wax embedded material by immunohistochemistry (cut off point, 10%). The two methods were compared by means of κ statistics, and the prognostic significance of both in relation to disease free survival (DFS) and overall survival (OS) was determined. Results—SPF and Ki-67 analysis was performed on 234 (76.5%) and 295 (96.4%) tumours, respectively. The two assessments were simultaneously available in 230 cases. All SPF variables analysed in the whole series significantly correlated with disease evolution, with the conventional median SPF (cut off point, 6.1%) showing the highest predictive value in relation to both DFS (p = 0.0001) and OS (p = 0.0003). SPF tertiles and median SPF evaluated according to DNA ploidy status had no prognostic significance. The Ki-67 index showed a trend in relation to DFS (p = 0.086) that did not reach significance, and no correlation with OS was found (p = 0.264). The comparative analysis of SPF and Ki-67 revealed some agreement between the two methods (agreement, 69.13%; κ statistic, 0.3844; p < 0.001), especially in the subgroup of diploid tumours. Conclusions—Flow cytometric SPF is a better prognosticator than the Ki-67 index, but only SPF variables applied in the whole series show potential clinical usefulness. Key Words: breast carcinoma • DNA flow cytometry • immunohistochemistry • S-phase fraction • Ki-67 • prognosis

The aim of this study was to analyze the flow cytometric S-phase fraction (SPF) in rectal tumors before and after preoperative radiotherapy (15x2 Gy) and to compare the findings to the clinical outcome. Archival specimens from 84 cases, treated from 1980 to 1988 with S-phase data and complete follow-up were reviewed. There was no significant correlation between SPF and clinicopathological factors. The median SPF for the 26 diploid tumors before irradiation was 6.6%+/-3.1, compared to a significantly higher median for the 58 preirradiated aneuploid tumors (20.3%+/-6.1; p0.0001). With a median follow-up of 6 years, there was a significant difference in the number of recurrences for aneuploid tumors with a pretreatment SPFand20.3 (51.7% vs. 20.7%; p=0.029), which also led to a significant difference in recurrence-free survival (p=0.05). For diploid tumors, a reduction in the percentage of cells in S-phase after radiation resulted in a borderline significant lower number of recurrences (p=0.06). It is concluded that pretreatment S-phase measurements may be of predictive value especially for aneuploid tumors. An alteration in SPF after radiotherapy may also be helpful in predicting outcome and planning therapy.

BACKGROUND The prognostic value of DNA ploidy, S-phase fraction (SPF) and K-ras-2 mutations in gastric carcinoma (GC) has not yet been clearly defined. The aim of this study was to clarify the association between biomolecular variables, tumor characteristics, and clinical outcome in GC patients. METHODS Resected specimens from a consecutive series of 69 patients with GC who underwent potentially curative surgery were studied prospectively. DNA ploidy and SPF were assessed by flow cytometry on multiple frozen tumor samples, whereas K-ras-2 mutations were detected by polymerase chain reaction followed by single-strand conformation polymorphism. All the patients involved in this study were followed up for a mean of 95 months. RESULTS DNA aneuploidy was present in 72% of the cases (50 of 69), whereas 10% of these (5 out of 50) showed multiclonality. Mutations of K-ras-2 were detected in 8% of the tumors (5 of 63). Both DNA ploidy and SPF were associated with TNM stage (American Joint Committee on Cancer [AJCC] staging system) and node status. Moreover, DNA aneuploidy was significantly related to high SPF. K-ras-2 mutations were not associated with clinicopathologic variables or flow cytometric indicators. At univariate analysis, advanced TNM stage, node involvement, diffuse histotype, depth of invasion, DNA aneuploidy, and high SPF proved to be significantly related to quicker tumor relapse and to shorter overall patient survival. With multivariate analysis, DNA aneuploidy, high SPF, and depth of invasion were related to risk of tumor relapse and patient death, whereas diffuse histotype was independently related to patient risk of tumor relapse. CONCLUSIONS DNA ploidy and SPF, when associated with clinicopathologic staging, might be useful for the identification of GC patients who have different risks for death or relapse of disease. Cancer 2001;92:294–302. © 2001 American Cancer Society.

Flow cytometric DNA analysis with assessment of S-phase fraction and DNA ploidy was compared to Nottingham histologic grade. The study population consisted of 654 patients who presented between 1987 and 1996 with primary operable breast cancer and whose tumours had been analysed for S-phase fraction and DNA ploidy at the time of surgery. Grade, tumour size, node status, steroid receptor status, age, S-phase fraction and DNA ploidy were analysed univariately and multi-variately in a Cox proportional hazard analysis. In the univariate analyses all parameters were statistically significantly associated with breast cancer mortality during the follow-up period of 2-11 years. The most powerful predictor of death from breast cancer in the multiple regression analysis was grade. Patients with grade 1 tumours have excellent prognosis. We conclude that tumour grade is a strong prognostic indicator applicable to all breast cancer patients, regardless of size and nodal status, and advocate its general use.

The S-phase fraction (SPF) in flow cytometric DNA histograms in soft tissue sarcoma (STS) can be calculated in various ways. The traditional planimetric method of Baisch has been shown to be prognostic, but is hampered by a failure rate of around 40%. We therefore tested other models to see if this rate could be decreased with retained prognostic value. In 259 STS of the locomotor system the SPF was calculated according to Baisch and with commercial parametric MultiCycle software using different corrections for background. Using the Baisch model, 159 histograms could be evaluated for SPF. The 5-year metastasis-free survival rate (MFSR) was 0.94 for the low-risk group (defined with SPF), and 0.53 for the high-risk group. In the low-risk group, four of the seven patients who developed metastasis did so after 5 years. Using the MultiCycle software, SPF could be calculated in 253 tumours. Depending on type of background correction used, the 5-year MFSR varied between 0.67 and 0.82 for the low-risk group, and between 0.47 and 0.53 for the high-risk group. The late metastasis pattern in the low-risk group was never seen using the MultiCycle software. We conclude that in paraffin archival material, calculation of SPF according to Baisch is preferable in clinical use due to better separation between low-risk and high-risk groups, and also the possibility to identify patients who metastasize late. © 1999 Cancer Research Campaign

A key prognostic parameter for vulvar carcinoma is the presence of lymph node metastases. Determination of proliferation markers has been suggested as a method to predict lymph node metastases in several tumor types. If this were true in vulvar carcinomas, reduced surgical therapy for patients with low-risk vulvar carcinoma could be considered. The authors analyzed whether the proliferation-associated markers silver nucleolar organizer region (Ag-NOR) and Ki-67 are predictors for inguinofemoral lymph node metastases in women with vulvar carcinoma. The authors also analyzed whether these proliferation markers are interrelated. Data were obtained from samples of 145 patients with T1/T2 squamous cell carcinoma of the vulva who were treated with vulvectomy and bilateral lymphadenectomy. None of these patients received preoperative therapy, and the invasion depth of the tumors was more than 1 mm. The median age was 71 years. The group consisted of 67 patients with differentiation grade 1, 64 with grade 2, and 18 with grade 3; 22% (15 of 67) of the patients with grade 1, 45% (29 of 64) with grade 2, and 43% (six of 14) with grade 3 had lymph node metastases. Formalin-fixed, paraffin-embedded sections were stained for proliferation markers Ag-NOR and MIB-1 (an equivalent of Ki-67 for fixed material). Both parameters were scored at the tumor stroma interface. AS NOR number and area were quantified by interactive image analysis and Ki-67 index was scored microscopically with a grid. No relation was found between Ki-67 or Ag-NOR and lymph node metastases. A relation was found between Ki-67 and mitotic index (MI), but not between Ag-NOR and MI or Ki-67 index. Therefore, it is questionable whether Ag-NOR is, indeed, a marker for proliferation. The authors conclude that quantitation of Ki-67 and Ag-NOR does not contribute to the prediction of inguinofemoral lymph node metastases in squamous cell carcinoma of the vulva. Copyright (C) 1996 by W.B. Saunders Company.

Twenty-five patients with cervical carcinoma were treated with combined external beam and high dose rate afterloading radiotherapy. Biopsies obtained at different time points in the course of therapy were analysed with respect to cell proliferation and cytogenetic damage. The fraction of cells with an S-phase DNA-content as well as the frequency of micronuclei were determined. These two parameters were then related to treatment outcome, in particular patient survival. Neither S-phase fraction nor the micronucleus frequency before radiotherapy were predictive of treatment outcome in this small group of patients. However, when changes in response to therapy were considered, patients whose S-phase fraction decreased and patients whose micronucleus frequency increased tended to have a better prognosis. Although statistical significance was not achieved with either criterion alone, when applied together the combination predicted patient survival quite reliably; the 5-year survival rate of those patients who showed a decrease in S-phase fraction as well as an increase in micronucleus frequency was about 90% in contrast to less than 30% for the non-responders (p < 0.03).

AIMS--To predict 5 year survival in patients with uveal malignant melanomas DNA indices were studied. METHODS--Using 45 paraffin embedded uveal malignant melanomas, the DNA index and S phase fraction of each tumour were the predictor variables recorded. RESULTS--Using the Cox proportional hazards model, aneuploid tumours and tumours which had an S phase fraction greater than 4% were significant predictors of early death. In order to demonstrate a biological gradient between a larger DNA index and shorter survival time, linear regression and transformed linear regression models were used. However, no such gradient could be demonstrated. CONCLUSION--Although this study shows promise for the use of DNA studies in the prognosis of uveal malignant melanoma, the exact role of these techniques remains to be determined.

Eighty-five male breast carcinomas limited to the breast, diagnosed between 1958 and 1967 in Sweden, were investigated by flow cytometry using paraffin-embedded tissue. DNA ploidy and S-phase fraction (SPF) were studied in relation to lymph node status, tumour size and malignancy grade, and with respect to clinical outcome. Median age at diagnosis was 65.5 years. Median survival time related to breast cancer was 78.7 months, and 37 patients died from the disease. Forty-nine (57.6%) carcinomas were aneuploid or had multiple stemlines. Median SPF was 8.9% for all tumours, for diploid and tetraploid (euploid) together 6.6%, and for aneuploid tumours 14.3% (p0.001). Ploidy was significantly related to the tumour size (p = 0.03), but no significant correlations with node status or malignancy grade were observed. In univariate survival analysis, node status, tumour size and malignancy grade predicted breast cancer mortality significantly. In a multivariate Cox's model, only node status and tumour size were independent prognostic factors. In contrast to females with breast cancer, ploidy and SPF had no significant relation to prognosis in males. This lack of predictive value of DNA analysis needs further evaluation. Studies based on extended materials are necessary to further investigated the role of DNA analysis as a predictor of outcome in male breast cancer patients.

Flow cytometry was performed on frozen specimens from the primary tumour of 184 women with recurrent breast cancer. No significant association was seen between DNA ploidy and the other prognostic factors investigated. Patients with a high S-phase fraction had more often a negative estrogen receptor (ER) status and a short disease-free interval. A shorter survival after disease recurrence was seen both in patients with DNA aneuploid tumours and among those with a high S-phase fraction. Patients with DNA tetraploid tumours showed the longest survival after recurrence. In this subgroup, half of the patients survived more than 3 years after recurrence and the estimated survival rate at 10 years was 17%. In a Cox's regression analysis including 116 patients, site of recurrence, number of positive nodes at time of primary operation, size and ER content of the primary tumour as well as DNA ploidy showed additional prognostic value.

We conducted neoadjuvant chemotherapy with CDDP-UFT for Borrmann 4 type gastric carcinoma and report here our findings on its usefulness. The study included an investigation of the association between measured DNA ploidy pattern and chemotherapeutic efficacy.We administered two three-week course of CDDP 100mg/m2 (day1) UFT400g/m2 (on consecutive days) preoperatively to 13 cases of Borrmann 4 type gastric carcinoma judged to be curatively unresectable. In addition, the DNA contents in the cancer tissue cell nuclei was mesured by flow cytometry before and after the chemotherapy. The response rate was 53.8%(7/13) and there was no difference found in therpeutic efficacy with difference in the DNA ploidy pattern. Ten cases were resectable. Cases exhibiting more marked histological efficacy were those an accumulation of S phase fraction among the diploid cases and thosewith a tendency toward flattening of the aneuploid peak among aneuploid cases.

The authors studied a series of 97 consecutive cases of salivary gland tumors to investigate the correlation between the biologic parameters DNA ploidy and S-phase fraction (SPF) and the presumptive behavior of the neoplasms, as well as the potential clinical utility of these parameters. Histopathologic classification and grading of the tumors were evaluated according to 1991 World Health Organization criteria. DNA analysis was performed by flow cytometry in fresh material after propidium iodide staining. Clinical data and follow-up information were obtained from the clinical charts. All the 71 benign salivary tumors showed a DNA diploid pattern. Seven carcinomas (7.2%) exhibited DNA aneuploidy. Eleven (42.3%) of 26 malignant tumors were considered low grade carcinomas, all of them being DNA diploid. Of the remaining 15 tumors, classified as high grade carcinomas, 7 showed DNA aneuploidy. SPF values ranged from 0.6% to 27.7%. A statistically significant difference was found between the mean SPF values of benign and malignant tumors, diploid and aneuploid tumors, and low grade and high grade carcinomas. When a cutoff value of 3% was used to discriminate histopathologic subgroups with prognostic impact, a significant difference was found between benign and malignant salivary tumors, high grade and low grade carcinomas, and high grade and benign tumors (P