High S-phase Fraction Research Articles

Combination of aneuploidy and high S-phase fraction indicates increased risk of relapse in stage I endometrioid endometrial carcinoma

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.

Analytic study of hormone receptor status of breast cancer patients visiting in a tertiary care hospital of North-West India

Background: Increasing evidence shows the importance of young age, estrogen receptor (ER), progesterone receptor (PR) status, and HER-2 expression in patients with breast cancers. Breast tumors in younger age groups were more likely to be of higher grade, hormone receptor– negative, poorly differentiated, and aneuploid, and to have high S-phase fraction, abnormal expression of P53, greater extent of lymphovascular invasion, and over-expression of human epidermal growth factor receptor 2 (HER-2) than breast tumors in older age groups. Methods: We organised a retrospective study of 600 women diagnosed with breast cancer, who have been operated from 2016 to 2020. We evaluated age, size, hormone receptor status, HER-2 receptor status. Estrogen receptor and progesterone receptor status were evaluated by immunohistochemistry. Results: Total 600 patient data was evaluated in this study. Rate of hormone positivity is more in older patients than in younger patients. Conclusions: In this study we conclude that the hormone positivity in younger patient is less as compared to older patients.

Detection of genetic instability and proliferative activity in urinary bladder intraepithelial flat lesions using UroVysion fluorescence in-situ hybridization and flow cytometry

Background Chromosomal abnormalities play a key role in neoplastic potential for flat urothelial lesions, irrespective of whether cytologic atypia is present or not. Having information about lesions with malignant potential in the urinary bladder can help clinicians in taking logical actions concerning management after urinary bladder-conserving resections.Aim The aim of the study was to differentiate various flat urothelial lesions by detecting its genetic abnormalities and proliferative activity using UroVysion fluorescence in-situ hybridization and flow cytometry.Patients and methods Ninety-eight paraffin-embedded tissue sections from 35 patients with infiltrative bladder cancer and flat urothelial lesions [reactive atypia, hyperplasia, dysplasia, carcinoma in situ (CIS), squamous metaplasia, glandular metaplasia, histologically normal urothelium in patients with tumor and control] were assessed by flow cytometry and fluorescence in-situ hybridization using UroVysion targeted against the band 9p21 locus and pericentromeric areas of chromosomes 3, 7, and 17.Results Gain of at least one of the chromosomes was observed in 60% of samples being more than 90% in CIS, dysplasia, and infiltrating bladder tumors. The mean percentage of cells with polysomy in reactive atypia, flat urothelial hyperplasia, glandular metaplasia, keratinizing squamous metaplasia, and histologically normal urothelium in patients with tumors ranged from 12 to 20%, but in CIS and dysplasia it was 67 and 44%, respectively. Deletion of the P16 locus was detected in 49% of lesions. Flow cytometry displayed aneuploidy pattern in 60% of lesions with higher percentage in CIS, dysplasia, and infiltrating bladder tumors. High S-phase fraction (SPF) was observed in 44% of samples. High S phase showed significant association with polysomy and DNA ploidy (P=0.02 and 0.01, respectively). In addition, there was association between polysomy expression with del 9p21 (P=0.03) and between del 9p21 with DNA ploidy and SPF (P=0.003 and 0.02).Conclusions Our analysis has shown that combined evaluation of flat urothelial lesions using UroVysion, DNA ploidy, and SPF help in the recognition of these lesions, especially those with higher genetic abnormalities and having higher S phase because follow up of these lesions showed a higher tendency for malignant transformation. Furthermore, estimation of continuing chromosomal instability such as polysomy, DNA aneuploidy, and high SPF might deliver further interpretation of urinary bladder cancer risk assessment away from the position and histopathology of precancerous lesions.

Lifestyle chemical carcinogens associated with mutations in cell cycle regulatory genes increases the susceptibility to gastric cancer risk.

In the present study, we correlated the various lifestyle habits and their associated mutations in cell cycle (P21 and MDM2) and DNA damage repair (MLH1) genes to investigate their role in gastric cancer (GC). Multifactor dimensionality reduction (MDR) analysis revealed the two-factor model of oral snuff and smoked meat as the significant model for GC risk. The interaction analysis between identified mutations and the significant demographic factors predicted that oral snuff is significantly associated with P21 3'UTR mutations. A total of five mutations in P21 gene, including three novel mutations in intron 2 (36651738G > A, 36651804A > T, 36651825G > T), were identified. In MLH1 gene, two variants were identified viz. one in exon 8 (37053568A > G; 219I > V) and a novel 37088831C > G in intron 16. Flow cytometric analysis predicted DNA aneuploidy in 07 (17.5%) and diploidy in 33 (82.5%) tumor samples. The G2/M phase was significantly arrested in aneuploid gastric tumor samples whereas high S-phase fraction was observed in all the gastric tumor samples. This study demonstrated that environmental chemical carcinogens along with alteration in cell cycle regulatory (P21) and mismatch repair (MLH1) genes may be stimulating the susceptibility of GC by altering the DNA content level abnormally in tumors in the Mizo ethic population.

DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy.

ObjectiveWe investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents.Patients and methodsFifty-three patients with peritoneal carcinomatosis from ovarian cancer were enrolled. Frozen tumor tissue was dissociated by a detergent–trypsin method, and the resulting cell suspension was stained with RNase A and propidium iodide. Samples were then analyzed for ploidy and SPF by flow cytometry. Fresh tumor tissue was dissociated by enzymatic digestion, and cells were exposed to different concentrations of cisplatin, adriamycin, carboplatin, gemcitabine and taxol for 72 hours. In vitro drug sensitivity was then measured using the sulforhodamine B assay.ResultsNo significant correlation was found between ploidy or SPF and patient characteristics, even though primary carcinomas were mainly hyperdiploid and more proliferative than recurrent tumors. SPF differed significantly among ploidy categories (P=0.01), and high SPF was associated with short-term survival (P=0.48). Patients with multiploid tumors were the most resistant to platinum-based chemotherapy, whereas those with hyperdiploid tumors were the most responsive. In vitro multiploid tumors were the least sensitive, while hypodiploid samples showed the highest sensitivity to the tested drugs. Sensitivity to adriamycin was significantly correlated with ploidy (P=0.03), whereas sensitivity to taxol was correlated with SPF (P=0.04).ConclusionOur results indicate that ploidy and SPF could facilitate the choice of therapy for patients with peritoneal carcinomatosis.

Podocalyxin as a Prognostic Marker in Gastric Cancer.

BackgroundPodocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein associated with aggressive tumor phenotype and poor prognosis in several forms of cancer. The aim of this study was to investigate PODXL expression in gastric cancer by use of two different antibodies.MethodsBy tumor-tissue microarrays and immunohistochemistry we evaluated PODXL expression in tumor specimens from 337 patients who underwent surgery for gastric adenocarcinoma at Helsinki University Hospital. We used two different antibodies: HPA2110, which is a polyclonal antibody and an in-house monoclonal antibody called HES9, to investigate the association of PODXL expression with clinicopathologic variables and patient survival.ResultsPODXL staining was positive by the polyclonal antibody in 153 (57.5%) cases and by the monoclonal antibody in 212 (76%). Polyclonal antibody expression was associated with intestinal cancer type (p<0.001). Monoclonal antibody staining was associated with age over 66 (p = 0.001), with intestinal cancer (p<0.001), and with small tumor size (≤ 5 cm; p = 0.024). Both antibodies were associated with high S-phase fraction (p = 0.022; p = 0.010), and high tumor proliferation index (Ki-67; p = 0.003; p = 0.001). PODXL positivity by the polyclonal antibody indicated reduced gastric-cancer-specific 5-year survival of 24.0% (95% CI 16.9–31.1), compared to 43.3% (95% CI 33.7–52.9) for patients with PODXL negativity (p = 0.001). The result remained significant in multivariable analysis (HR = 3.17; 95% CI 1.37–7.34, p = 0.007).ConclusionIn gastric cancer, PODXL expression by the polyclonal antibody HPA2110 is an independent marker of poor prognosis.

DNA Ploidy and S-phase Fraction Analysis in Paediatric B-cell Acute Lymphoblastic Leukemia Cases: a Tertiary Care Centre Experience.

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 DI>1.60 (triploid and tetraploid range). Some 14/40 (35%) cases had a diploid DI between 0.90-1.05. None of the cases had a DI <0.90 (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 TLC> 50 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.

A prospective, multicenter validation study of a prognostic index composed of S-phase fraction, progesterone receptor status, and tumour size predicts survival in node-negative breast cancer patients: NNBC, the node-negative breast cancer trial

BackgroundIn a retrospective study on node-negative breast cancer, a prognostic index consisting of a proliferation factor, S-phase fraction (SPF), progesterone receptor status (PR), and tumour size identified one-third of patients as high risk, with a sixfold increased risk of breast cancer death. This prospective multicenter cohort study was set up to validate the index. Patients and methodsIn 576 T1-2N0 patients <60 years, prospective analyses of PR and SPF were carried out. High risk was defined as ≥2 of the following: size >20 mm, PR-negativity, and high SPF (in the absence of SPF, Bloom–Richardson grade 3). Median follow-up was 17.8 years. ResultsThirty-one percent were high risk. In univariate analysis, the index was prognostic for breast cancer-specific survival after 5 years [hazard ratio (HR) = 4.7, 95% confidence interval (95% CI) 2.5–8.9], 10 years (HR = 2.2, 95% CI 1.5–3.3), and 15 years (HR = 1.7, 95% CI 1.2–2.5), and remained significant after adjustment for adjuvant medical treatment and age. In the 37% of patients with no risk factors, only one patient died of breast cancer the first 5 years. ConclusionsThis prospective study validates a prognostic index consisting of a proliferation factor, PR-status, and tumour size. The index may be helpful for prognostic considerations and for selection of patients in need of adjuvant therapy.

Flow Cytometric Measurement of Aneuploid DNA Content Correlates with High S-Phase Fraction and Poor Prognosis in Patients with Non-Small-Cell Lung Cancer

Cellular DNA content (ploidy) and proliferation activity (e.g., S-phase fraction) measured by flow cytometry have been usually related to the biologic aggressiveness of various neoplasms. In this study, these parameters were analyzed in paraffin-embedded tumor specimens from 43 cases of resected non-small-cell lung cancer (NSCLC). Additionally, the correlation of them with both prognosis and a variety of clinic-pathological features were investigated. The stage and the appearance of both local recurrence and metastasis were related to overall survival of patients. Twenty-two tumors (51.2%) had a diploid DNA distribution, while 21 were aneuploid (48.8%). The mean of aneuploidy was 1.6% ± 0.3%. A correlation was found between ploidy and survival as well as with the appearance of local recurrence and/or metastasis. The mean values of S-phase fraction of diploid and aneuploid tumors were 16.7 ± 11.3% and 32.9 ± 12.1%, respectively, which were significantly different (P=0.0022). Similar results were obtained analyzing the proliferation index (sum of cells in S and G2/M phases of cell cycle) (P=0.0040). However, no correlation between these parameters and both overall survival of patients and clinicopathological features was observed. Our results could suggest the potential use of ploidy analysis as a useful complement of TNM stage in NSCLC.

Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy.

The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy.

A Ploidia de ADN é um Biomarcador Prognóstico Independente no Carcinoma Ductal Invasivo da Mama

To evaluate 'classic' prognostic parameters, as well as DNA ploidy and S-phase fraction, in relation to disease-free and overall survival in breast invasive ductal carcinoma with long-term follow-up. The study involved 400 patients with breast invasive ductal carcinoma and median follow-up of 134 months (50-240). Histological grading, tumour size, axillary nodal involvement, pathological staging and hormone-receptor status were assessed as established prognostic markers. Ploidy and S-phase fraction were determined prospectively by DNA flow cytometry using fresh/frozen tissue. A Cox regression model was used for statistical analysis of the prognostic variables. There were 106 deaths (26.5%) and 141 disease recurrences (35.2%) during follow-up. Two hundred thirty-five (58.7%) tumours were aneuploid. High S-phase fraction and aneuploidy were associated with tumours with higher grade of differentiation, greater size and negative hormonal receptors. In univariate analysis, all the clinicopathological and cytometric features (including patients < 40 years and a subgroup presenting hipertetraploid/multiploid tumours), but S-phase fraction and estrogen receptors for disease free survival, significantly correlated with clinical outcome. In multivariate analysis, advanced disease stage, DNA aneuploidy and lack of progesterone receptors retained statistically significant association with shorter survival. In the subgroup of patients with intermediate differentiation tumours (G2), aneuploidy associated with worse prognosis. In the subset of node-negative patients, only estrogen receptors showed significant correlation with disease evolution. In node-positive patients, greater size tumours and aneuploidy (in relation to overall survival) were indicators of worse prognosis. Along with disease staging and hormone-receptor expression, DNA ploidy is an independent prognostic biomarker of long-term survival in breast invasive ductal carcinoma.

DNA Ploidy is an Independent Predictor of Survival in Breast Invasive Ductal Carcinoma: A Long-term Multivariate Analysis of 393 Patients

To evaluate "classic" prognostic parameters, as well as DNA ploidy and S-phase fraction (SPF), in relation to disease-free (DFS) and disease-specific (DSS) survival in breast invasive ductal carcinoma (IDC) with long-term follow-up study. The study involved 393 patients with IDC and median follow-up of 134 months (50-240). Histological grading, tumor size, axillary nodal involvement, pathological staging and hormone receptor status were considered as established prognostic markers. Ploidy and SPF were determined prospectively by DNA flow cytometry using fresh/frozen tissue. A Cox regression model was used for statistical analysis of the prognostic variables. There were 105 (26.7 %) deaths and 140 (35.6 %) disease recurrences during follow-up. Two hundred thirty-one (58.8 %) tumors were aneuploid. High SPF and aneuploidy were associated with tumors with higher grade of differentiation, greater size and negative hormone receptors. Higher SPF and advanced disease stage are correlated. In univariate analysis, all the clinicopathological and cytometric features, including patients <40 years and a subgroup presenting hypertetraploid/multiploid tumors, are significantly correlated with clinical outcome, apart from SPF and estrogen receptors for DFS. In multivariate analysis, nodal involvement, DNA aneuploidy and lack of progesterone receptors (for DSS) retained statistically significant association with shorter survival. In node-negative patients, ploidy (for DFS) and estrogen receptors (for DSS) significantly predicted survival. In both subgroups of node-positive patients and those (n = 195) with intermediate differentiation tumors (G2), aneuploidy was an indicator of worse prognosis. Along with nodal status and hormone receptor expression, DNA ploidy is an independent predictor of long-term survival in IDC.

Effects of DNA Ploidy and S-Phase Fraction on Fluorine-18 FDG Uptake of Primary Breast Cancer Lesions

BackgroundFlow cytometry (FCM) evaluating DNA content is emerging as the tool to monitor cell proliferation and malignant potential in several cancers such as stomach, lung, and salivary gland tumor. The purpose of this study was to correlate 18F-FDG uptake of dual–time-point (DTP) positron emission tomography (PET) imaging with DNA ploidy and S-phase fraction (SPF) in primary breast cancer lesions. Patients and MethodsSeventy-two consecutive female patients (mean age ± SD, 52.7 ± 11.1 years; range, 28-81 years) had undergone 18F-FDG DTP PET/computed tomography (CT) imaging for staging of breast malignancy in our institution during a 5-month period. FCM was performed on fresh-frozen samples of specimens obtained from surgery. 18F-FDG uptake was then compared with DNA content. ResultsForty-four malignant lesions were included in this study. On FCM, DNA aneuploidy was detected in 14 lesions (31.8%). The maximum standardized uptake values (SUVmax1 and SUVmax2) (SUVmax1, 6.8 ± 4.6 vs. 3.4 ± 2.4; P = .017), (SUVmax2, 7.9 ± 5.7 vs. 3.6 ± 2.7; P = .015) and retention index (RI) (12.8 ± 11.6 vs. 2.4 ± 10.8; P = .010) were significantly higher in DNA aneuploidy cancer than in DNA diploidy cancer. The value of RI (11.3 ± 11.5 vs. 2.6 ± 11.2; P = .022) was significantly higher in high SPF (> 15%) breast cancer than in low SPF (≤ 15%) breast cancer. ConclusionsHigh 18F-FDG uptake in breast cancer might be an indicator of DNA aneuploidy and high SPF. We propose that 18F-FDG PET/CT imaging may be a noninvasive and useful tool for predicting the DNA content in breast cancer.

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.

Prognostic value of proliferation in pleomorphic soft tissue sarcomas: a new look at an old measure

Though proliferation has repeatedly shown a prognostic role in sarcomas, it has not reached clinical application. We performed a comprehensive evaluation of the prognostic role of 5 proliferation measures in a large series of soft tissue sarcomas of the extremities and the trunk wall. One hundred ninety-six primary soft tissue sarcomas of the extremities and the trunk wall were subjected to DNA flow cytometry for quantification of S-phase fraction and to immunohistochemical evaluation of Ki-67, Top2a, p21, and p27Kip1. In univariate analysis, positive expression of Ki-67 (hazard ratio = 4.5, CI = 1.6-12.1), Top2a (hazard ratio = 2.2, CI = 1.2-3.5) and high S-phase fraction (hazard ratio = 1.8, CI = 1.2-3.7) significantly correlated with risk for metastasis. When combined with currently used prognostic factors, Ki-67, S-phase fraction and Top2a fraction contributed to refined identification of prognostic risk groups. Proliferation, as assessed by expression of Ki-67 and Top2a and evaluation of S-phase fraction and applied to statistical decision-tree models, provides prognostic information in soft tissue sarcomas of the extremity and trunk wall. Though proliferation contributes independently to currently applied prognosticators, its role is particularly strong when few other factors are available, which suggests a role in preoperative decision-making related to identification of high-risk individuals who would benefit from neoadjuvant therapy.

S-phase fraction and ploidy as predictive markers in primary disease and recurrence of papillary thyroid carcinoma

To investigate the prognostic influence of DNA ploidy and S-phase fraction (SPF) on disease-free (DFS) and overall survival (OS) of patients with primary disease and loco-regional lymph node recurrence of papillary thyroid carcinoma (PTC). A large prospective study with long-term follow-up (median, 117 months). Two series of patients with primary PTC (n = 305) and lymph node recurrence metastasis (LNM) (n = 39) were involved in the study. Patient's age and gender, histological variant, pathological tumour-node-metastasis (pTNM) staging, extrathyroidal extension, vascular and lymphatic invasion and tumour bilateral growth were the clinical and pathological characteristics evaluated. DNA flow cytometry was performed on fresh/frozen surgical tumour samples. Cox regression models were estimated for prognostic analyses. Seventeen (5·6%) primary tumours and five (12·8%) LNMs were aneuploid, while mean SPF was 2·7% and 3·7%, respectively (P = 0·022). High SPF was significantly associated with lymphatic invasion and tall cell and diffuse sclerosing variants. In univariate analysis, all the clinico-pathological variables, but tumour bilateral growth and gender, were significantly correlated with survival. SPF showed borderline significance (P = 0·051) in relation to OS. In multivariate analysis, older age (≥48 years), lymph node spread and high SPF were significantly adverse prognostic factors. Extrathyroidal extension and distant metastasis for OS, as well as tumour size for DFS, were also found as unfavourable prognostic features. In LNM, the Kaplan-Meier curves showed significant differences for older age and DNA aneuploidy (recurrence; P = 0·011). The results indicate that SPF and ploidy can provide additional predictive information in patients with PTC.

Tumourigenic characteristics of embryonal carcinoma cells as a model for studying tumour progression of human embryonic stem cells

The objective of this study was to compare tumourigenic characteristics of human embryonic stem cells (HESCs) and embryonal carcinoma cells (ECCs) to identify a robust and simple model for studying certain aspects of cell transformation and tumourigenesis, in tumour progression of HESCs. SSEA-3 positive ECCs (NTERA-2) cells were identified and compared to HESCs (ch HES-20) in terms of pluripotency and differentiation capacity, growth characteristics, gene expression profiles and signalling pathways. Our results showed that NTERA-2 cells shared similarities in expression markers of pluripotency to ch HES-20 cells. However, NTERA-2 cells also expressed some markers of differentiation and had a tendency to differentiate towards ectodermal endpoints. We identified NTERA-2 cells with higher S-phase fraction in cell cycle distribution, anti-apoptosis markers and robust self-renewal ability, compared to ch HES-20 cells. Microarray analysis and real-time PCR results showed that some oncogenes were up-regulated and tumour-suppression genes were down-regulated, whereas pluripotency-related genes were up-regulated and differentiation-related genes were down-regulated, and that Wnt and Notch signalling pathways were activated during progression from ES cells to EC cells. Tumourigenic characteristics of ECCs may provide a valuable insight into possible tumour progression of HESCs.

Prognostic significance of ploidy and S-phase fraction in primary intraoral squamous cell carcinoma and their corresponding metastatic lymph nodes

BackgroundDespite improvements in diagnosis and therapy of oral and oro-pharyngeal carcinomas during the past 30years the 5-year disease-free survival is still poor. Patient’s prognosis is affected by cervical lymph node metastasis rather than primary tumors. The DNA ploidy and S-phase fraction (SPF) are associated with tumor aggressiveness and patient outcome in many solid tumors. PurposeAnalysis of DNA ploidy and SPF in primary oral squamous cell carcinoma (OSCC) and corresponding node metastasis as prognostic markers in relation to conventional prognostic factors and disease-free survival (DFS). MethodsPloidy status and SPF (mean value) of 37 formalin-fixed paraffin embedded (FFPE) primary OSCC tumors and their corresponding lymph node metastasis were assessed by flow cytometry (FCM) and correlated with clinicopathologic prognostic parameters and DFS. ResultsMost of OSCC tumors (86.5%) were Grade II. Among primary OSCC the incidence of aneuploidy was 19%, 51.4% showed high SPF (>10.62%) and 48.6% had low SPF (<10.62%). Border line significance (P=0.10) was detected between ploidy status and SPF in primary tumors. In lymph node metastases all tumors were diploid, 78.4% of metastatic tumors revealed low SPF and only 21.6% showed high SPF. There was a statistically significant correlation (p=0.02) between site of tumors and DFS and a highly statistically significant correlation (p=0.01) between SPF of primary tumors and DFS. ConclusionsHigh SPF of primary OSCC tumors assessed by FCM was significantly associated with decreased disease free survival rates. DNA ploidy showed no relationship to bad prognostic indicators in either primary OSCC or their metastatic tumors.

Prognostic Value of Proliferation Markers: Immunohistochemical Ki-67 Expression and Cytometric S-Phase Fraction of Women with Breast Cancer in Libya

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 (<50 years) than in older patients. In the overall population (median follow-up 49 months), patients with high Ki-67 and high SPF had shorter survival time and predicted recurrence than patients with low Ki-67 and low SPF. In a Cox multivariate analysis, high SPF (p= 0.007), hormonal status (p= 0.001) and clinical stage (p=0.005) were independent predictors of disease-specific survival. The Ki-67 (p=0.065) in borderline significance proved to be independent predictor of disease-free survival. The SPF showed more statistically significance with a high grade of malignancy and survival time than Ki-67.Conclusions: The SPF value is useful cell proliferation marker to assess tumor prognosis. These markers may reflect the aggressive behavior of Libyan breast cancer and predict of the recurrence. It is therefore important to take these markers into consideration to select a high risk subgroup of the patients for intensive treatment.

P2-12-22: A Prognostic Index Composed of Progesterone Receptor Status, Tumour Size and S-phase Fraction, Predicts Survival in Node-Negative Breast Cancer Patients in a Large Multicentre Prospective Cohort Study.

Abstract Background The importance of the added prognostic value of proliferation, either single factors such as Ki67 or flow cytometric S-phase fraction (SPF), or as the main common denominator in the majority of gene expression profiles, has been highlighted over the last years in node-negative breast cancer. There are however few published prospective studies. In an earlier retrospective study on node-negative breast cancer from our group, a prognostic index consisting of PR status, tumour size and a proliferation factor, SPF, identified one third of the patients as high risk, with a fourfold increased risk of distant recurrence. The present study was set up to validate this index in a large prospective multicenter cohort study with long term follow-up. Material and Methods: 596 patients from 3 regions in Sweden were between 1991–1995 included in the study. Inclusion criteria were: 10mm≤tumour size≤50mm, node-negativity, ≥5 lymph nodes removed, age &amp;lt;60 years, and radical surgery. Patients with bilateral breast cancer or previous malignancy were excluded. Prospective analyses of ER, PR, and flow cytometric SPF were performed. High- risk was defined as 2 or more of the following: 1. size &amp;gt;20mm 2. PR- (in the absence of PR status, ER-) and 3. high SPF (in the absence of SPF, Bloom Richardson grade 3). 82% of the patients received no adjuvant medical treatment. Cox proportional hazards regression, stratified for centre, was used to model the impact of the index on breast cancer specific survival (BCSS). Median follow-up was 16 years for the 452 patients alive at last follow-up. Analyses were done after 5 and 10 years follow-up, during which 42 and 95 patients, respectively, died of breast cancer. Results: 31% of the patients were identified as high-risk. In univariate analysis, the index was prognostic for BCSS after 5 years (HR 5.1, 95%CI: 2.7−9.8,) as well as 10 years (HR 2.2, 95%CI: 1.5−3.4). The prognostic impact remained significant after adjustment for adjuvant medical treatment and age. The 5- and 10-year BCSS (95%CI) was 97% (94-98) and 87% (83-90) for low risk patients, compared with 85% (79-89) and 76% (69-81) for high risk patients. In the group with no risk factors (n=218 ), no patient died of breast cancer during the first 5 years. Proliferation was the strongest factor for BCSS followed by PR and tumour size, both in uni- and multivariate analyses. In multivariate analysis, adjusted for adjuvant treatment and age, the HR for proliferation was 7.5 (95%CI: 3.2-18) after 5 years and 2.5 (95%CI: 1.6−4.0) after 10 years. Discussion: This large prospective multicenter cohort study validates the results from an earlier retrospective study, that a prognostic index consisting of PR status, tumour size and a proliferation factor, SPF, reliably identifies one third of the node-negative patients with a high risk of relapse. The index also identifies an extreme low risk group; patients with no risk factors, with 100% 5-year survival. This group could be spared adjuvant medical treatment, especially chemotherapy. Taken together, this index may be clinically helpful for prognostic considerations and for selection of adjuvant treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-22.