Cell Kinetic Measurements Research Articles

2D Kinetic Analysis of TCR and CD8 Coreceptor for LCMV GP33 Epitopes.

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.

LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal.

Context:Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production.Objective:The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover.Design:This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7).Interventions:Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3).Main Outcome Measures:A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured.Results:LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers.Conclusion:LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.

Critical Appraisal of In Situ Cell Kinetic Measurements as Response Predictors in Human Tumors.

The purpose of this article is to review the potential advantages of measuring cell kinetic parameters, to indicate in which settings they are likely to be most useful and to point out some deficiencies in present methodologies. This will be done by attempting to answer a series of fundamental questions. Cell kinetics crucially affect many factors related to the timing of response after therapy. These include the time and rate of cell death, the rate of regression, the rate of reoxygenation, and the time to recurrence. Proliferation of surviving cells can also occur during gaps in treatment, whether the treatment be radiotherapy or chemotherapy, and whether the gaps are short (eg, between daily treatments) or longer (eg, a weekend; weeks between treatment courses; weeks between surgery and adjuvant radiotherapy or chemotherapy), l The end result of such proliferation is the partial repopulation of a tumor before treatment is ended. In other words, repopulation can compete with treatment-induced cell killing, reducing the effectiveness of the treatment. There

Gallbladder carcinoma: the role of p53 protein overexpression and Ki‐67 antigen expression as prognostic markers

The overexpression of p53 protein and the expression of Ki-67 antigen may affect the survival of patients with gallbladder carcinoma. This association has been tested in a series of 41 patients with relatively early carcinoma of the gallbladder. Forty-one surgical specimens from patients with a postoperative histological diagnosis of gallbladder carcinoma were studied. All patients were operated by simple cholecystectomy only because the tumours were not advanced and/or their general condition was poor. Patients submitted to radical operations were excluded. p53 expression was calculated from nuclear staining according to the intensity and extent of positive cells, as graded on a scale from 1 to 3; a combined score of >3 was considered as overexpression. Ki-67 expression was calculated by the MIB-I index: the percentage of positively stained tumour cell nuclei out of the total tumour cells counted (n = 1000); >20% of stained cells was considered positive. Twenty-nine gallbladder carcinomas (71%) overexpressed p53 protein in the cell nuclei. No significant differences were found in relation to cell differentiation on the level of tumour infiltration through the gallbladder wall. Five-year survival of patients with gallbladder carcinoma overexpressing p53 was 17.2%, while survival of patients without p53 overexpression was 30% (not significant). Twenty-four cases (58.5%) were considered positive for the MIB-I index. There were no differences between the grade of cell differentiation and wall infiltration. Five-year survival of the patients with a MIB-I positive index was 9.2% as opposed to 27.7% for those with a negative index (not significant). p53 protein nuclear overexpression and Ki-67 protein expression in gallbladder carcinoma were not related to histological differentiation, level of gallbladder wall invasion or patient survival.

In vivo cell kinetic measurements in a randomized trial of continuous hyperfractionated accelerated radiotherapy with or without mitomycin C in head-and-neck cancer

Purpose: Tumor cell repopulation is still considered to be a major cause of failure in radiotherapy. In this study, we investigated the influence of cell kinetic parameters on the outcome of patients treated in a randomized trial of accelerated fractionation, with or without mitomycin C, vs. conventional fractionation. Methods and Materials: Sixty-two patients were studied using administration of bromodeoxyuridine (BrdUrd), and cell kinetic parameters were measured using flow cytometry. The patients were treated with either 70 Gy for 7 weeks or 55.3 Gy for 17 continuous days (V-CHART) with or without 20 mg/m 2 mitomycin C on day 5. Results: The potential doubling time (Tpot) and labeling index (LI) failed to provide any prognostic information with regard to local control or survival. However, the duration of the S phase (Ts) revealed patients whose tumors had a long Ts had significantly worse local control ( p = 0.028) and survival ( p = 0.034) irrespective of treatment. A similar trend was evident within the different treatment arms particularly associated with overall survival. Conclusions: The Ts values of head-and-neck squamous cell cancers provided prognostic information that predicted clinical outcome irrespective of treatment schedule in this study. This neglected parameter of the Tpot method might provide information related to redistribution of cells during fractionated radiotherapy.

Is there a future for cell kinetic measurements using IdUrd or BdUrd?

Purpose: The analysis of causes of radiation failure both in retrospective series of patients with head and neck cancer and in several randomized clinical trials suggests a loss of local control as the overall treatment time increases for the same total dose. This is attributed to tumor cell proliferation during fractionated radiotherapy. As longer treatment times lead to loss of local control, it has been suggested that shorter treatment times could lead to an increase in local control. For this reason accelerated treatment regimens have been and are being designed. However, these treatments may cause severe acute reactions. Due to this, lower total doses are sometimes given. Slowly proliferating tumors, therefore, may do worse when treated with accelerated schedules compared with conventional schedules. In addition, it is not desirable to subject all patients to the more intense acute reactions of accelerated schedules. It would thus be useful to predict which tumors will rapidly proliferate during treatment and are likely to benefit from accelerated radiotherapy. The potential doubling time (Tpot) is defined as the time within which the cell population of a tumor would double if there were no cell loss. The hypothesis is that the median Tpot measured before treatment might correlate with the effective doubling time (Tp) during treatment. Conclusion: Tpot can be calculated knowing the labeling index (LI; proportion of cells incorporating the DNA precursor IdUrd or BdUrd) and Ts (the DNA synthesis time) measured by flow cytometry. A recent multicenter analysis has shown that the only pretreatment kinetic parameter for which some evidence is found for an association with local control is LI, not Tpot. Pitfalls associated with cell kinetic measurements such as assay variability, intratumor and intertumor variability, interlaboratory variability and the problem of an admixture of normal and malignant cells make Tpot not accurate and reproducible enough for a robust predictive assay. It therefore appears that pretreatment Tpot measurements using flow cytometry, provide only a relatively weak predictor of outcome after radiotherapy in head and neck cancer. Immunohistochemistry allows a simple measure of LI and may give additional independent information from labeling patterns, suggesting that this method is the (short term) future for clinical cell kinetic measurements using BdUrd or IdUrd.

Four A6-TCR/Peptide/HLA-A2 Structures that Generate Very Different T Cell Signals Are Nearly Identical

The interactions of three singly substituted peptide variants of the HTLV-1 Tax peptide bound to HLA-A2 with the A6 T cell receptor have been studied using T cell assays, kinetic and thermodynamic measurements, and X-ray crystallography. The three peptide/MHC ligands include weak agonists and antagonists with different affinities for TCR. The three-dimensional structures of the three A6-TCR/peptide/HLA-A2 complexes are remarkably similar to each other and to the wild-type agonist complex, with minor adjustments at the interface to accommodate the peptide substitutions (P6A, V7R, and Y8A). The lack of correlation between structural changes and the type of T cell signals induced provides direct evidence that different signals are not generated by different ligand-induced conformational changes in the αβTCR.

The value of pretreatment cell kinetic parameters as predictors for radiotherapy outcome in head and neck cancer: a multicenter analysis

Purpose: The aim of this study was to assess the potential of pre-treatment cell kinetic parameters to predict outcome in head and neck cancer patients treated by conventional radiotherapy. Materials and methods: Data from 11 different centers were pooled. Inclusion criteria were such that the patients received radiotherapy alone, and that the radiotherapy was given in an overall time of at least 6 weeks with a dose of at least 60 Gy. All patients received a tracer dose of either iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) intravenously prior to treatment and a tumor biopsy was taken several hours later. The cell kinetic parameters labeling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were subsequently calculated from flow cytometry data, obtained on the biopsies using antibodies against I/BrdUrd incorporated into DNA. Each center carried out their own flow cytometry analysis. Results: From the 11 centers, a total of 476 patients conforming to the inclusion criteria were analyzed. Median values for overall time and total dose were 49 days and 69 Gy, respectively. Fifty one percent of patients had local recurrences and 53% patients had died, the majority from their disease. Median follow-up was 20 months; being 30 months for surviving patients. Multivariate analysis revealed that T-stage, maximum tumor diameter, differentiation grade, N-stage, tumor localization and overall time correlated with locoregional control, in decreasing order of significance. For the cell kinetic parameters, univariate analysis showed that only LI was significantly associated with local control ( P=0.02), with higher values correlating with a worse outcome. Ts showed some evidence that patients with longer values did worse, but this was not significant ( P=0.06). Tpot showed no trend ( P=0.8). When assessing survival in a univariate analysis, neither LI nor Tpot associated with outcome ( P=0.4, 0.4, respectively). Surprisingly, Ts did correlate with survival, with longer values being worse ( P=0.02). In the multivariate analysis of local control, LI lost its significance ( P=0.16). Conclusions: The only pretreatment kinetic parameter for which some evidence was found for an association with local control (the best end-point for testing the present hypothesis) was LI, not Tpot, and this evidence disappeared in a multivariate analysis. It therefore appears that pretreatment cell kinetic measurements carried out using flow cytometry, only provide a relatively weak predictor of outcome after radiotherapy in head and neck cancer.

Relationship between potential doubling time ( Tpot), labeling index and duration of DNA synthesis in 60 esophageal and 35 breast tumors: is it worthwhile to measure Tpot?

Background: In recent years T pot (potential doubling time) has been measured before treatment in human tumors in an attempt to estimate the proliferation taking place during a course of irradiation. T pot is defined as T s/ LI, where T s is the duration of DNA synthesis and LI is the labeling index (proportion of cells synthesizing DNA). T s is more difficult to measure than LI, so the question arises whether variation introduced during the determination of T s is compensated by the theoretically better relevance of the quotient T pot than of LI alone. It is not clear from comparisons with clinical outcome whether T pot is a useful indicator of proliferation or whether LI is more prognostic, as suggested by a currently ongoing multicenter analysis elsewhere. Therefore, we investigated intercomparisons between T pot and its components LI and T s in two in their proliferation rates contrasting types of tumor where multiple biopsies were taken from each tumor. Materials and methods: Sixty patients with esophageal carcinoma and 57 patients with breast cancer were included in this study. All patients were injected with IUdR 6–8 h before surgery. From each tumor three to five biopsies were taken at surgery. Using flow cytometry, LI and T s were measured on all biopsies in order to calculate T pot. Logarithmic transformations of the distributions were used to examine correlations. Kappa-tests were used to assess how reliable an LI value could be in predicting the corresponding T pot. Results: T s and LI were not completely independent, based on the within-tumor coefficients of variation ( CV w). The ratio of between-tumor coefficient of variation ( CV b) to the CV w suggested that the discriminative power of T pot was higher than LI for esophagus, but the reverse in breast tumors, which had a larger range. Pearson correlation coefficients were high for log T pot versus log LI in both types of tumor, but the predictive power was low, as shown by κ-values of only 0.3–0.41 starting with LI and trying to predict the corresponding value of T pot. Increasing widths of a central `gray zone' were investigated for improved discrimination between fast and slow proliferation. Multiples of the within-tumor standard deviation, equally on each side of the median, were used to vary the width of the gray zone. Without a gray zone no more than 70% successful matching was obtained in esophagus tumors, compared with 80% in breast tumors. However, by excluding about half of the esophageal tumors an 80% success rate was achieved. In breast tumors over 90% matching was obtained more easily, keeping 80% of the tumors classifiable. For both tumor types correlations between T s and T pot were weak, with a trend towards short T s associated with short T pot and also with low LI. The latter correlation was significant for esophageal tumors and resulted in T pot values having a smaller range than the LIs. Conclusion: Although there were good correlation coefficients between T pot and LI, the predictive power of either from the other was not reliable, except by excluding a significant number of tumors close to the medians. The predictive value of LI for T pot was higher for breast tumors because the spread in cell kinetic measurements was wide. Until more clinical data become available on outcome in comparison with LI or T pot, it is still worthwhile to measure T pot and to assess the prognostic value of both LI and T pot in relation to outcome.

Foreword

The rapid developments in image cytophotometry in the late 1950s and early 1960s and the in flow cytometry a decade later roused great expectations among researchers in biological and medical sciences. Image cytophotometry made it possible for the first time to measure many different parameters both qualitatively and quantitatively in combination with morphological studies. A second major step forward occurred when flow cytometers, originally developed in a few research laboratories, became commercially available in the early 1970s, permitting automated cellular analysis and sorting at extremely high speed. Although the flow systems, sources of excitation light, and methods for detecting emitted light are basically the same today as 20 years ago, the almost exponential development in certain related fields of research has led to entirely new opportunities for this technology. Through modern computer technology, the tasks of data acquisition, handling, and analysis have been enhanced enormously. In particular, multiparameter analysis during cell measurement and sorting, as well as the ability to analyze listmode data, have given new dimensions to the field. The number and scope of immunological and immunohistochemical techniques utilizing monoclonal and other antibodies have greatly expanded, and at the same time these techniques have become widely available as everyday tools for any cytometric researcher. Molecular biology and genetics have likewise added another dimension for applications of cytometric methodology. Perhaps the greatest impact has been in the application of flow cytometry to cell biology. Improvements in cell kinetic measurements—e.g., using BrdU and analysis of cellular DNA—combined with other techniques for identifying subpopulations of cells have helped make cytometry an important tool for anyone involved in studies of cell growth. At the other end of the cellular life cycle, automated measurements of apoptosis have given insight into the complex equilibrium between cell proliferation and cell loss in any tissue. In parallel with the development of all these new, directly applicable methods, flow cytometers have become more reliable and user friendly, opening up this powerful methodology to anyone who studies eukaryotic and prokaryotic cells. One important result has been that clinical applications of cytometry as an adjunct to pathology, cytology, and hematology are expanding rapidly. As the breadth of both the technology and applications of cytometry expanded, however, the need for standardized methods for image and flow cytometry became increasingly crucial. Experimenters newly drawn to cytometry needed guidance in applying it to their particular research areas; moreover, the cytometric literature has expanded so rapidly that even for those actively working in the field, keeping track of recent developments has become a nearly unmanageable task. In response to this problem, in 1990 the International Society for Analytical Cytology (ISAC) first took the initiative to assemble the original Handbook of Flow Cytometry Methods. The continuing pace of change has necessitated several revisions. The latest descendent of that handbook is the present more complete and updatable volume, Current Protocols in Cytometry, which presents an abundance of advanced methodology that will be of great utility to a wide circle of readers. The combination of introductory chapters and general overviews on the one hand and specific methods on the other makes this a valuable resource for newcomers to the field and experienced researchers alike. Perhaps most importantly, the manual is designed to evolve over time along with the methods it describes: its looseleaf format and quarterly updates allow new techniques in emerging areas to be added and existing material to be revised as procedures are improved and expanded. Moreover, this volume as well as the updates are also available on CD-ROM, a format that permits readers to conveniently access the numerous cross-references within the manual by hypertext links, perform context-based searching, and make individualized notebooks containing frequently used protocols. More than ever, education and training are vitally important tasks in modern scientific disciplines. In addition to fulfilling these goals, this volume also presents a forum for standardization and exchange of methodology in an area of research that is essentially interdisciplinary. Not least, the continuous updating and expansion of Current Protocols in Cytometry will make it an important tool in the forefront of this type of science now and in the future.

Cell kinetic measurements in prostate cancer

Two approaches have been suggested for escalating the total dose in radiotherapy treatment of prostate cancer. One is conformal radiotherapy; the other is hyperfractionation using many small fractions. Both imply some possible prolongation in overall treatment time. To judge whether prolonged treatment schedules would be detrimental, it is necessary to know the proliferation rates in human prostate tumors, specifically, the potential doubling time (Tpot). There is a lack of data on this parameter in the literature. Seven patients with adenocarcinoma of the prostate were studied. A tracer dose of 100 mg/m2 of IUdR was infused intravenously 4-12 h before biopies were taken. Biopsies were fixed in 70% ethanol, stored at 4 degrees C, and later prepared and stained by standard methods for flow cytometry, using the red fluorescence signal for DNA and the green fluorescence signal (fluorescein isothiocyanate) for 5-iodo-2'-deoxyuridine. The duration of DNA synthesis (Ts) was determined by the relative movement (RM) method, knowing the interval between tracer administration and biopsy. Tpot was calculated as the quotient of Ts by labeling index (LI). In two of the seven tumors the LI was too low (<0.6%) for a reliable estimate of RM to be made, so no determination of Tpot was possible for these tumors. The mean LI values in the other five tumors were 2.4%, 1.4%, 1.0%, 3.0%, and 0.9%. The durations of Ts were 13.2, 9.5, 10.0, 11.7, and 12.7 h, respectively. The resulting values of Tpot were 23, 28, 42, 16, and 61 days, respectively. The low labeling indices in prostate tumors, also reported by others, made estimation of Ts by RM impossible in about a third of these tumors. However, five tumors yielded long estimates for Tpot, implying that prolongation from 6 to about 8 weeks should not be detrimental.

Enrichment of tumor cells for cell kinetic analysis in human tumor biopsies using cytokeratin gating

Purpose: To determine the feasibility of using cytokeratin antibodies to distinguish normal and malignant cells in human tumors using flow cytometry. The goal was ultimately to increase the accuracy of cell kinetic measurements on human tumor biopsies. Material and methods: A panel of four antibodies was screened on a series of 48 tumors from two centres; 22 head and neck tumors (Amsterdam) and 26 esophagus carcinomas (Leuven). First, screening was carried out by immunohistochemistry on frozen sections to test intensity of staining and the fraction of cytokeratin-positive tumor cells. The antibody showing the most positive staining was then used for flow cytometry on the same tumor. Results: The two broadest spectrum antibodies (AE1/AE3, E3/C4) showed overall the best results with immunohistochemical staining, being positive in over 95% of tumors. Good cell suspensions for DNA flow cytometry could be made from frozen material by a mechanical method, whereas enzymatic methods with trypsin or collagenase were judged failures in almost all cases. From fresh material, both collagenase and trypsin produced good suspensions for flow cytometry, although the fraction of tumor cells, judged by proportion aneuploid cells, was markedly higher for trypsin. Using the best cytokeratin antibody for each tumor, two parameter flow cytometry was done (cytokeratin versus DNA content). Enrichment of tumor cells was then tested by measuring the fraction of aneuploid cells (the presumed malignant population) of cytokeratin-positive cells versus all cells. An enrichment factor ranging between 0 (no enrichment) and 1 (perfect enrichment, tumor cells only) was then calculated. The average enrichment was 0.60 for head and neck tumors and 0.59 for esophagus tumors. Conclusions: We conclude that this method can substantially enrich the proportion of tumor cells in biopsies from carcinomas. Application of this method could significantly enhance accuracy of tumor cell kinetic measurements.

408 Enrichment of tumor cells for cell kinetic analysis in human tumor biopsies using cytokeratin gating

The feasibility of using cytokeratin antibodies to distinguish nonnal and malignant cells in human tumor biopsies using flow cytometry was tested for increasing the accuracy of cell kinetic measurements. Four different antibodies were screened on a 48 tumors from two centres; 26 head and neck (Amsterdam) and 22 oesophagus (Leuven). First screening was done by immunohistochemistry (ICC) on frozen sections to test staining intensity and fraction of cytokeratin-positive (CK+) tumor cells. The antibody showing the most positive staining was then used for flow cytometry: Two broad spectrum antibodies (AE1/AE3, E3/C4) showed the best results with ICC (>90% tumors positive). Cell suspensions for flow cytometry could be made from frozen material by a mechanical method. Enzymatic methods failed for frozen material but were best for fresh material. Tumor cell enrichment was tested by gating on CK+ cells in flow cytometry using the best antibody. Average enrichment was 0. 58 for head and neck tumors and 0.75 for oesophagus tumors; overall enrichment 0.65 (0 = no enrichment, 1 = pure tumor population). In several cases, 100% enrichment was achieved. We conclude that this method can significantly reduce errors in tumor cell kinetic measurements by reducing nonnal cell “contamination”.

Rodent carcinogenicity bioassay: past, present, and future.

Toxicity/carcinogenicity studies in rodents have played a pivotal role in identifying chemicals that are potentially hazardous to humans. In fact, nearly all of the known human carcinogens are also carcinogenic in 1 or more rodent species. During the past 20 yr the quality and consistency of rodent studies has improved considerably, and much has been learned about mechanisms whereby chemicals initiate or promote the carcinogenic process in rats and mice. The process of identifying chemicals that cause toxicity or carcinogenicity in rodents is quite well established, but the procedures for extrapolating this data for risk management decisions in the protection of human health have lagged far behind. While many would accept the assumptions that genotoxic chemicals that cause cancer in animals pose a cancer risk to humans and that genotoxic chemicals causing cancer at high doses pose a risk at lower doses, there is much less certainty with respect to nongenotoxic chemicals. The confusion about risk extrapolation for nongenotoxic chemicals has often lead to criticism of the hazard identification process for chemicals in general. There is increasing awareness of the complexity of the carcinogenic process that has made species extrapolation and dose extrapolation from rodent studies to humans more complex. Although newer molecular biological techniques and cell kinetic measurements offer exciting possibilities for better risk assessment, it is the combination of well-designed rodent studies with appropriate mechanistic studies that offers the best hope for regulatory decisions based on sound scientific principles.

In vivo cell kinetic measurements in human oesophageal cancer: What can be learned from multiple biopsies?

The importance of intratumour variability of cell kinetics was studied in 60 patients with cancer of the oesophagus. Five biopsies per tumour were taken. The labelling index, S-phase duration and potential doubling time (T pot were measured using flaw cytometry. The mean T pot value was 5.56 ± 4.43 days (± 1S.D.) for adenocarcinomas and 4.40 ± 2.45 days (± 1S.D.) for squamous cell carcinomas. These values were statistically significantly different. Although intratumour variation in T pot measurements occurred, the intertumour variability was more important ( P < 0.00001). This feature permits classification of tumours into slow and fast proliferating groups, leaving an intermediate group of tumours that could not be unequivocally categorised. The relative distribution of tumours into these three categories depends on the intratumour and intertumour variability of T pot and on the cut-off values used. Increasing the number of biopsies from one to five reduces the number of non-classifiable tumours.

Proliferative Activity in Non-Hodgkin’s Lymphomas: A Comparison of the Bromodeoxyuridine Labeling Index with PCNA Immunostaining and Quantitative Image Analysis

Cellular proliferation is being evaluated as an independent prognostic indicator in a variety of tumors, including non-Hodgkin's lymphomas (NHL). Although the labeling index (LI), using either tritiated thymidine or monoclonal antibodies to bromodeoxyuridine, is considered to be the gold standard of cell kinetic measurements, recent advances in the use of antibodies specific to cellular proliferating antigens and computer-based image analysis have potentially established a new technology for evaluating the growth fraction of NHL. Proliferating cell nuclear antigen (PCNA), a protein associated with DNA polymerase that is expressed only in the nuclei of cells actively synthesizing DNA, was quantitated in 46 formalin-fixed, paraffin-embedded samples of various types of NHL using the Cell Analysis Systems 200 image analyzer. The results were expressed as the percentage of nuclear area (%NA) positive for PCNA. These results were compared with the bromodeoxyuridine LI, and a correlation coefficient of 0.78 was calculated using first-order linear regression. An average positive NA of 8.2% for low-grade NHL, 32% for intermediate-grade, and 43% for high-grade NHL was observed. The potential advantages PCNA quantitation by image analysis may offer over LI are discussed.

Critical appraisal of in situ cell kinetic measurements as response predictors in human tumors

The purpose of this article is to review the potential advantages of measuring cell kinetic parameters, to indicate in which settings they are likely to be most useful and to point out some deficiencies in present methodologies. This will be done by attempting to answer a series of fundamental questions. Cell kinetics crucially affect many factors related to the timing of response after therapy. These include the time and rate of cell death, the rate of regression, the rate of reoxygenation, and the time to recurrence. Proliferation of surviving cells can also occur during gaps in treatment, whether the treatment be radiotherapy or chemotherapy, and whether the gaps are short (eg, between daily treatments) or longer (eg, a weekend; weeks between treatment courses; weeks between surgery and adjuvant radiotherapy or chemotherapy), l The end result of such proliferation is the partial repopulation of a tumor before treatment is ended. In other words, repopulation can compete with treatment-induced cell killing, reducing the effectiveness of the treatment. There

Tumour growth rates in squamous carcinoma of the head and neck measured by in vivo bromodeoxyuridine incorporation and flow cytometry.

The cell kinetics of 82 squamous cell carcinomas of the head and neck were studied by in vivo administration of the thymidine analogue, bromodeoxyuridine (BrdUrd). Ploidy, BrdUrd labelling index (LI), duration of S-phase (Ts), potential doubling time (Tpot) and S-phase fraction (SPF) were measured by flow cytometry on 50 microns paraffin embedded sections. The range of values obtained compared well with other in vivo cell kinetic studies of head and neck cancer. Aneuploid tumours had a significantly higher BrdUrd labelling index and SPF, and a short Tpot than diploid tumours. To validate the use of 50 microns sections for measuring cell kinetic parameters by flow cytometry a comparison of values obtained by 50 microns sections and small blocks of tissue was made. No significant difference was found between the two methods. Reproducibility of values between two consecutive thick sections was also good. We conclude that reproducible cell kinetic measurements can be made in tumour samples using 50 microns sections of BrdUrd labelled tissue.

Tumor cell kinetics, local tumor control, and accelerated radiotherapy: A preliminary report

The local tumor control achieved in patients treated in a pilot study of continuous, hyperfractionated, accelerated radiotherapy has been related to the tumor cell kinetics evaluated by in vivo administration of bromodeoxyuridine and flow cytometry. In 42 of 50 patients with advanced squamous cell carcinomas in the head and neck region it was possible to sample the primary tumor prior to treatment. In three further cases, involved regional nodes were studied: in the remaining five, tissue was obtained subsequently either from a local recurrence or from a distant metastasis. Successful cell kinetic measurements were made in 38 (90%) of the 42 primary tumors. The median values of the labelling index, the duration of the DNA synthetic phase and, thus, the potential doubling time for all primaries were 7.1%, 9.8 hr, and 3.9 days, respectively. Complete regression was achieved in 28 (74%) of the primary tumors and in 23 (61%) this was maintained to the time of observation for this report. There was no significant influence of any of the cell kinetic parameters upon the immediate or longer term local tumor control. This result is compatible with the overcoming of cellular repopulation by the acceleration of radiotherapy.

DNA ploidy and cell kinetic measurements as predictors of recurrence and survival in stages B2 and C colorectal adenocarcinoma

DNA content and cell proliferation were measured by flow cytometry on paraffin-embedded Stage B2 or C colorectal adenocarcinomas from 694 patients enrolled in adjuvant trials conducted by the North Central Cancer Treatment Group. Patients with diploid tumors had a higher survival rate than those with nondiploid tumors (P less than 0.001). The proliferation index (the sum of the percent of cells in S-phase plus those in G2M phase) was also a strong prognostic factor (P less than 0.001). The ploidy and proliferation data were combined, and the patients in the favorable group (diploid and low proliferative index) had a 5-year survival of 74% compared with 54% for the unfavorable group (high proliferative index or nondiploid, P less than 0.001). This grouping was prognostic for survival in B2 (P less than 0.001), C (P = 0.013), colon (P less than 0.001), and rectal (P = 0.026) patient subsets. This study indicates that cell kinetic parameters are important and independent prognostic factors for Stages B2 and C colorectal cancer.