Search

The main goal of this study was to evaluate the possible effects of electromagnetic field (EMF) of transmission lines exposure on testis tissue of mice. Forty adult BALB/c mice were divided into two groups. One group (twenty mice) has been done by daily exposures of 24 hours, and the other sham-exposed group (twenty mice) served as the control. After seven weeks of exposure, two groups of the mice were sacrificed. The testis tissue was assessed by serum biochemical assay, histopathological examination and flow cytometric analysis (FCM). EMF exposure did not significantly affect the distribution of DNA ploidy (1n, 2n and 4n cells), but significantly decreased the S phase fraction and the Proliferation Index of testis germ cells.

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.

DNA ploidy studies on salivary gland tumours have shown that the proportion of aneuploid cases, although confined to the malignant entities, is considerably lower than for other solid malignancies. To analyse whether the S-phase fraction (SPF) may contribute to discrimination of diploid malignant from benign tumours, DNA flow cytometric data from 45 different malignant salivary gland tumours was compared with that of 121 pleomorphic adenomas. All benign tumours were diploid. Twelve malignant tumours contained aneuploid cell populations. The SPF values for diploid malignancies ranged between 0.9% and 11.0% (mean 3.9%), and between 0.5% and 7.9% (mean 2.7%) for pleomorphic adenomas. A 4% cut-off value gained statistical significance for discriminating diploid malignant tumours from pleomorphic adenomas (P0.01). The sensitivity for SPF4% was 46% and the positive predictive value was 40%. A sensitivity of 60% and a positive predictive value of 54% was achieved by combining aneuploidy and SPF4%. These results show that DNA flow cytometry may contribute to diagnostic assessment in salivary gland tumours.

Objectives: To investigate the prognostic significance of DNA ploidy and silver-stained nucleolar organizer regions (AgNOR), their relation to the cytological grades of cervical dysplasia and correlation of cells in the S-phase of the cell cycle and DNA ploidy with AgNOR count. Methods: Multiparametric DNA flow-cytometric analysis and AgNOR count were performed on cervical smear samples, cytologically diagnosed as normal (n = 196), atypical squamous cells of unknown significance (ASCUS, n = 98) and various grades of cervical dysplasia (n = 127). Results: Among the cytological grade, aneuploid cases from low-grade squamous intraepithelial lesion (mild) and high-grade squamous intraepithelial lesion (moderate and severe) groups were showing high AgNOR counts (ranges 4.33–5.86 and 5.91–7.42, respectively). Aneuploid cases from the ASCUS group also presented high AgNOR (range 3.72–4.93). The high percentage of cells in the S-phase (>12%) correlated with these 2 parameters in the ASCUS and dysplasia groups. After grouping the cases into those with high (>4.0) and low (Conclusions: Thus, the study shows that high AgNOR count, S-phase fraction and aneuploid DNA have prognostic significance in the early detection of cervical cancer.

Resume Objectif de l'etude. – Etudier la signification pronostique du contenu en ADN et de la fraction de cellules en phase S de cancers du sein de stades I et II. Patientes et methodes. – Une etude a ete realisee chez 271 patientes traitees par chirurgie suivie ou non de chimiotherapie, d'hormonotherapie et de radiotherapie. La duree mediane de suivi etait de 64 mois. Une preparation cellulaire standardisee de cytometrie en flux a ete utilisee a partir d'echantillons congeles avec des regles consensuelles pour l'interpretation des donnees. Trois classes de fraction de cellules en phase S ont ete definies sur la base de terciles apres ajustement au contenu en ADN. En combinant le contenu d'ADN et la fraction de cellules en phase S, quatre groupes pronostiques ont ete definis : tumeurs diploides a fraction de cellules en phase S basse (n = 37), tumeurs diploides a fraction de cellules en phase S intermediaire et/ou haute (n = 76), tumeurs aneuploides a fraction de cellules en phase S basse (n = 24), tumeurs aneuploides a fraction de cellules en phase S intermediaire et/ou haute (n = 68). Une correlation a ete recherchee entre les taux de controle local, de survie sans rechute, de survie sans metastase et de survie globale d'une part, le contenu en ADN, la fraction de cellules en phase S, ces deux criteres combines, les stades T et N, le grade SBR, l'âge et a la presence de recepteurs hormonaux, d'autre part, avec une analyse uni- et multifactorielle (modele de Cox). Resultats. – Apres analyse unifactorielle, les taux de controle local et de survie sans rechute etaient significativement meilleurs en cas de tumeur diploide. Le taux de survie sans rechute etait significativement plus bas en cas de fraction de cellules en phase S elevee. Les taux de controle local, de survie sans rechute, de survie sans metastase et de survie globale etaient moins bons lorsque la tumeur etait diploide et la fraction de cellules en phase S intermediaire et/ou haute que lorsqu'elle etait diploide et la fraction de cellules en phase S basse. D'apres analyse multifactorielle, le groupe defini a partir de la combinaison du contenu d'ADN et la fraction de cellules en phase S etait un facteur independant de survie sans metastase, de meme que l'atteinte ganglionnaire (N+) et le stade T. Chez les patientes dont le cancer n'atteignait pas l'aisselle (N–), le groupe defini en combinant le contenu d'ADN et la fraction de cellules en phase restait le seul facteur pronostique independant de survie sans metastase et de survie sans rechute, alors qu'il n'avait aucune influence en cas d'atteinte axillaire. Lorsque la tumeur etait de grade SBR III, il y avait une relation significative entre le groupe defini en combinant le contenu d'ADN et la fraction de cellules en phase S et la survie sans metastase et la survie globale. Conclusion. – Ces resultats soulignent l'interet de l'utilisation de la combinaison du contenu en ADN et de la fraction de cellules en phase S chez les patientes atteintes d'un cancer du sein de stades I et II, sans atteinte axillaire.

ToprospectivelyevaluatetheprognosticsignificanceofTP53,H-,K-,andN-Rasmutations,DNA-ploidyandS-phasefraction(SPF) in patients affected by locally advanced laryngeal squamous cell carcinoma (LSCC). Eight-one patients (median follow-up was 71 months) who underwent resective surgery for primary operable locally advanced LSCC were analyzed. Tumor DNA was screened for mutational analysis by PCR/SSCP and sequencing. DNA-ploidy and SPF were performed byflow cytometric analyses. Thirty-six patients (44%) had, at least, a mutation in the TP53 gene. Of them, 22% (8/36) had double mutations and 3% (1/36) had triplemutations.Intotal,46TP53mutationswereobserved.Themajority(41%)oftheseoccurinexon5(19/46),whilethemutations inexons6,7,and8wererepresentedin14,7,and6patients,respectively(31%,15%,and16%).FiveLSCCpatients(6%)showeda mutation in H-Ras gene. Sixty-three percent of the cases (51/81) were DNA aneuploidy, 14% of these (7/51) were multiclonal. Thirty-ninepatients(48%)hadanhighSPFvalue.AtUnivariateanalysis,theDNAaneuploidy,highSPF(>15.1%),TP53mutations and, in particular, the mutations that occur in exons 5 and 8 were significantly related to quicker disease relapse and short OS. At Multivariate analysis, the major significant predictors for both disease relapse and death were high SPF and any TP53 mutations. While histological grade G3 was an independent factor only for relapse. In conclusions, any TP53 mutations and high SPF are importantbiologicalindicatorstopredicttheoutcomeofLSCCpatients. J.Cell.Physiol.206:181‐188,2006.2005Wiley-Liss,Inc.

DNA ploidy and S phase fraction analysis by flow cytometry on breast and ovarian tumor cells continuously exposed to aloin, a natural anthraquinone, at two concentrations (20-60 microg/ml) was done. Untreated breast and ovarian tumor cells (control) showed an aneuploid pattern, with a mean DNA index of 2.10+/-0.10 and S phase fraction of 28.46+/-1.5 and 17.40+/-0.75%, respectively. Treatment of breast and ovarian tumor cells with aloin showed a persistent aneuploid pattern and a significantly dose-dependent increase in the percentage of S phase fraction and in the proportion of cells cycling at a higher ploidy level (G2M). The polyploidization indicates that aloin does not inhibit initiation of DNA synthesis and that cells replicated a full complement of DNA but had difficulty in M phase.

Background The aim of the present work was to study the prognostic impact of ploidy and S-phase fraction (SPF) assessed according to recently described methods. These methods of analysis combine different ploidy groups and separate euploid (good) prognostic groups from noneuploid (bad) prognostic groups. The definition of euploidy varied according to the author; some of them even included aneuploid peaks with few events. A comparison was also drawn to the average SPF and the diploid peak SPF observed in aneuploid histograms. Methods From January 3, 1990 to January 7, 1999, 1,984 previously untreated, invasive breast carcinoma samples were snap-frozen and processed for FCM. The present study evaluated all nondiploid and nonmultiploid histograms, using different analysis patterns and the values of the average SPF and diploid SPF. Results SPF is a salient prognostic factor even after multivariate analysis for DFS and MFS. Using several methods of analysis of ploidy and SPF shows that the classical method of analysis involving separation of ploidy according to diploidy versus aneuploidy and analysis of SPF restricted to the aneuploid peak in nondiploid and nonmultiploid histograms is as relevant as other recently proposed patterns of analysis, and that the average SPF or the diploid SPF of aneuploid tumors does not add significant prognostic information. Conclusions SPF is a valuable predictor of survival and can be confidently assessed in a simple way by restricting the analysis to the peak of interest (except for multiploid tumors). © 2004 Wiley-Liss, Inc.

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.

Different opinions about flow cytometric estimates of DNA aneuploidy and/or S‐phase fraction (SPF) as supplementary prognostic markers in colorectal cancer are to some degree associated with methodology. Using univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival. To improve detection of DNA aneuploid subpopulations and particularly estimation of their SPF's we investigated a method for bivariate DNA/cytokeratin analysis on fine‐needle aspirates of 728 frozen biopsies from 157 colorectal tumors. Unfixed aspirates were stained with propidium iodide and FITC‐conjugated anti‐cytokeratin antibody in a saponin‐buffer. A significant association between SPF and debris was observed. There were no substantial difference in DNA ploidy patterns between univariate and bivariate measurements (concordance was 92–95%). No new DNA aneuploid subpopulations were detected in cytokeratin‐gated compared to ungated or univariate histograms. Debris‐adjusted SPF's of cytokeratin‐gated histograms were significantly higher than of ungated histograms, also for subpopulations with DI>1.4 (p < 0.0001). There was no significant association between SPF and survival.

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

Multidrug resistance (MDR) and more specifically the expression of P-glycoprotein (Pgp) have been studied extensively in vitro. Unfortunately, it appears that the predictive value of MDR recognized in vitro is mostly an incorrect measure to determine the responsiveness of a particular tumour in the clinic. This misunderstood or overvalued role of MDR might explain the failure of strategies to reverse Pgp function by the use of modulators in solid tumours. To obtain more insight in in vivo drug resistance we investigated a panel of 15 human ovarian cancer xenografts consisting of the most common histological subtypes known in ovarian cancer patients. The response rate to cisplatin, cyclophosphamide and doxorubicin in the xenografts resembled the results of phase II trials with these agents in ovarian cancer patients. This resemblance justifies drug resistance studies in this experimental in vivo human tumour system. We determined the expression levels of MDR 1, MRP 1, LRP and topoisomerase IIα mRNA by the RNase protection assay and the presence of MRP1 and LRP proteins by immunohistochemistry. The S-phase fraction was investigated as a separate parameter by flow cytometry. In none of the 15 ovarian cancer xenografts was MDR 1 expression detectable. The expression levels of MRP 1 and LRP were low to moderate and resembled the presence of the MRP1 and LRP proteins. There was a weak, inverse relationship between the expression levels of LRP and sensitivity to cisplatin and cyclophosphamide (r = –0.44 and –0.45), but not to doxorubicin. The levels of topoisomerase IIα varied among the xenografts (0.73–2.66) and failed to correlate with doxorubicin resistance (r = 0.14). The S-phase fraction, however, showed a relation with the sensitivity to cisplatin (r = 0.66). Among the determinants studied in ovarian cancer in vivo, LRP mRNA and the S-phase fraction were the best predictive factors for drug response and most specifically for the activity of cisplatin. © 2000 Cancer Research Campaign

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...

Factors reflecting two major aspects of tumour biology, invasion (urokinase-type plasminogen activator (uPA), plasminogen activator inhibiter (PAI-1), cathepsin D) and proliferation (S-phase fraction (SPF), Ki-67, p53, HER-2/neu), were assessed in 125 node-negative breast cancer patients without adjuvant systemic therapy. Median follow-up time was 76 months. Antigen levels of uPA, PAI-1 and cathepsin D were immunoenzymatically determined in tumour tissue extracts. SPF and ploidy were determined flow-cytometrically, Ki‴-67, p53, and HER-2/neu immunohistochemically in adjacent paraffin sections. Their prognostic impact on disease-free (DFS) and overall survival (OS) was compared to that of traditional factors (tumour size, grading, hormone receptor status). Univariate analysis determined PAI-1 (P < 0.001), uPA (P = 0.008), cathepsin D (P = 0.004) and SPF (P = 0.023) as significant for DFS. All other factors failed to be of significant prognostic value. In a Cox model, only PAI-1 was significant for DFS (P < 0.001, relative risk (RR) 6.2). In CART analysis for DFS, the combination of PAI-1 and uPA gave the best risk group discrimination. For OS, PAI-1, cathepsin D, tumour size and ploidy were statistically significant in univariate, but PAI-1 was the only independently significant factor in Cox analysis (P < 0.001, RR 8.9). In particular, this analysis shows that PAI-1 is still a strong and independent prognostic factor in node-negative breast cancer after extended 6-year median follow-up. © 1999 Cancer Research Campaign

The role of DNA flow cytometry in the prediction of prognosis for patients with stomach cancer remains to be defined. Thus we studied prospectively the role of DNA flow cytometry as a prognosis indicator in stomach cancer patients in a high-incidence area. Between November 1990 and December 1992, primary stomach cancer tissues were obtained from the surgical specimens from 217 patients (148 male, 69 female). DNA flow cytometric analyses of DNA ploidy and S-phase fraction were performed and the results were correlated with patient survival. The median age of the patients was 55 years (range 24–78). Aneuploid cell population was found in 114 of 217 samples (53%). Tumour S-phase fraction was obtained in 96 of 103 diploid tumours (93%) and 61 of 114 aneuploid tumours (54%). After median follow-up of 66.1 months, the patients with tumours with an S-phase fraction over 17% had significantly worse survival rates than patients with tumours with S-phase fractions of lower than 8% or 8–17% (45% vs 59% and 63% of patients surviving, P = 0.007). Tumour ploidy status did not correlate with patient survival. Multivariate analyses showed that the TNM stage remained the most important prognostic indicator. The tumour S-phase fraction was also an independent prognostic indicator (relative risk 2.300, 95% CI, 1.252–4.223). Tumour S-phase fraction obtained by DNA flow cytometry is an independent prognostic indicator for the survival of the patients with stomach cancer. © 1999 Cancer Research Campaign

The nuclear DNA content of 20 anembryonic pregnancies was studied by flow cytometry from paraffin embedded tissue blocks. An abnormal amount of DNA content was found in 8 of the cases. This was a significantly higher percentage than encountered in spontaneous abortions studied by the same population (40 and 9%, respectively, p < 0.05). The S-phase fraction in anembryonic pregnancies was lower than in spontaneous abortions (22.4 ± 12.7 and 35.4 ± 6.8, respectively, p < 0.01). The results indicated that abnormal embryogenesis with grave chromosomal aberrations may play a major role in the etiology of anembryonic pregnancy.

S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in breast cancer. There are, however, some inherent difficulties in the estimation of SPF, such as the influence of debris, aggregates, and normal cells. Most of the available SPF calculation principles try to consider these difficulties, but so far no consensus has been reached with regard to which principle is to be recommended. The aim of the present study was to investigate the prognostic impact of SPF when estimated with different calculation methods in frozen breast cancer samples from 350 patients. Two nonparametric (Rman, Rmin/both rectangle) and three parametric (ACAS/DNA-base, ModFit, and MultiCycle) calculation methods, with and without correction for debris and aggregates, were used. The mean values for SPF varied from 4.3% (ACAS/DNA-base with correction for debris and aggregates) to 9.4% (MultiCycle without any correction for background). The pairwise correlation between methods varied considerably (R = 0.72-0.98). After categorization of SPF values into low SPF (lower two tertiles) and high SPF (upper tertile), all methods yielded statistically significant Pvalues for recurrence-free survival (median follow-up time 67 months), both univariately (0.0004-< 0.0001) and multivariately (0.048-0.0004), after adjusting for nodal status, tumor size, and estrogen receptor status. SPF with background correction did not yield lower P values than SPF without. Regardless of which method was used, SPF showed similar correlations with lymph node involvement, tumor size, and estrogen receptor content. In conclusion, as the mean value of SPF for different calculation methods varies, each laboratory must be restricted to use only one method. Background correction does not seem to improve the prognostic impact of SPF in DNA histograms. Based on the experiences obtained in the present study, S-phase calculation methods without background correction may therefore be the most suitable for routine evaluation of DNA histograms of fresh frozen breast cancer material (ModFit, MultiCycle, and Rman [the latter only for experienced operators]). The nonparametric Rmin, with an automatic setting of the region used for SPF calculation, may be an alternative, but suffers from the disadvantage of not being commercially available yet.

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.

Seventy-one histologically malignant brain tumors, 52 histologically benign brain tumors, and 14 cerebral metastases were characterized according to DNA content and proliferative capacity. DNA ploidy, DNA index (DI), S-phase fraction (SPF), 5-bromo-2'-deoxy-uridine (BrdUrd) labelling index (LI), duration of S-phase (Ts), and potential doubling time (Tpot) were assessed by flow cytometry (FCM). In histologically benign tumors, a high percentage of DNA diploid tumors and a low proliferative capacity in DNA diploid tumors were found. Histologically malignant tumors and cerebral metastases were both found to be characterized by a low percentage of DNA diploid tumors and a high proliferative capacity in DNA diploid tumors. The proliferative capacity of DNA aneuploid benign tumors and that of DNA aneuploid malignant tumors, however, appeared not to differ significantly. The number of DNA aneuploid tumors was small. Duration of S-phase was short (range 3.9-4.7 hr) and appeared not to differ between the three groups. From this, the observed differences in Tpot values should be accredited mainly to differences in LI. High-grade as well as low-grade gliomas both appeared to be characterized by malignant (FCM) features, i.e., 1) a high percentage DNA aneuploidy, 2) a high mean DI (for DI > 1), and 3) a high proliferative capacity.

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: Histopathological grading of oral dysplastic lesions is the method of choice for evaluating malignant and potentially malignant disorders. Owing to inter- and intra-observer variability, determination of the DNA ploidy status of lesions may serve as an adjunct in the prediction of malignant transformation. Aim: To correlate histopathological grading and ploidy status in potentially malignant and malignant disorders of the oral mucosa. Settings and Design: A pilot study was done with 30 patients (10 patients with oral potentially malignant disorders predominantly leukoplakia, 10 patients with oral malignant lesions and 10 patients with normal mucosa). Materials and Methods: Incisional biopsy was done after isolating the biopsy site with 1% Toluidine blue staining. Two sections of the tissue were removed and sent for histopathological and Flow-cytometric analysis respectively. Histopathological diagnosis was obtained and compared with Flow-cytometric results which were graded as diploid and aneuploid. Further, the S - phase fraction, DNA index were also calculated to evaluate the severity of malignant transformation or malignancy. Statistical Analysis: The results were analyzed using Pearson Chi-Square Test. Results: There exists a significant correlation between histopathology and ploidy status in both potentially malignant and malignant group. ( P = 0.002). Conclusion: The data from this study has shown that DNA Ploidy analysis can be used as a valuable tool in assessing the carcinomatous progression of potentially malignant and malignant lesions.

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.

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.

Clinical use of flow cytometric (FCM) DNA analysis requires effective quality controls. Thirty-two laboratories with various degrees of FCM experience participated in the first phase of a quality control program organized by the Association Francaise de Cytometrie. All received diskettes containing ten list-mode files and ten histogram files that were derived from FCM analysis of various unfixed tumor specimens. A total of 610 responses on DNA ploidy and cell cycle were obtained with three different DNA analysis softwares: CellFit used by (44% of responses), MultiCycle (44%), and ModFit (12%). After statistical analysis, 31% of the responses were excluded from the final analysis for precise reasons. The groups were too small to carry out a valid analysis of the slight differences in the percentage of cells in the DNA synthesis phase (S%) between CellFit and MultiCycle. To estimate the influence of gating on the final cell-cycle results, five of the histogram files were derived from corresponding list-mode files, but the participating laboratories were unaware of this. A good correlation (r = 0.98) was obtained for S% values in the five paired files. The fact that 31% of the responses had to be excluded clearly reflects inadequate training in the use of these analysis softwares and, in some cases, a failure to grasp the biological meaning of the results. In contrast, the laboratories fulfilling consensus recommendations obtained remarkably homogeneous results, showing that standardization is feasible.

Cell proliferation of transitional cell bladder cancer (TCC) was determined by MIB-1 immunolabeling, volume-corrected mitotic index (M/V index) and S-phase fraction measurement in 207 patients with superficial (Ta-T1) bladder cancer. The results were compared to T category, WHO grade and DNA-ploidy. The MIB-1 score was related to T category (P

In 79 patients with uveal melanoma, the tumours were investigated by DNA flow cytometry and immunohistochemical staining of PCNA and Ki-67. S-phase as a continuous variable was significantly correlated with Ki-67 (P = 0.033), but not with PCNA. DNA ploidy was not correlated with either of the two antigens. Ki-67 was significantly correlated with histopathological type (P0.001) and tumour size (P0.001). Large tumours and epithelioid cell type were associated with a high frequency of Ki-67 positive cells. A high level of Ki-67 positivity (or = 6.5%) was also associated with a shorter survival (P = 0.0037), and when adjusted for DNA ploidy, histopathological type and tumour size, Ki-67 in the multivariate analysis remained an important prognostic factor (P = 0.017).

The dna ploidy status and S-phase fraction (spf) of benign proliferative lesions (bpl) and malignant tumours (mt) in the mammary glands of dogs were determined by flow cytometric analysis and the results were related to their clinical and histological features. Seven (14·3 per cent) of 49 bpl and 16 (48·5 per cent) of 33 primary mt had aneuploid G0,1 peaks (P