Cell kinetics.

Cell kinetic concepts have pervaded radiation therapy since the early part of the 20th century and have been instrumental in the development of modern radiotherapy. In this review, the fundamental radiobiological concepts that have been developed based on cell kinetic knowledge will be revisited and discussed in the context of contemporary radiation therapy. This will include how the proliferation characteristics, variation in sensitivity during the cell cycle and the extent of radiation-induced cell cycle delay translate into a variable time for the expression of damage, how cell kinetics interacts with hypoxia and how the response to fractionated radiation schedules is influenced by cell kinetics in terms of repair, redistribution, reoxygenation and repopulation. The promise of combining radiation with new biologically targeted agents and the potential of non-invasive positron emission tomography imaging of proliferation are areas where cell kinetics will continue to influence radiotherapy practice.

Ki-67 expression in melanoma. A potential method of risk assessment for the patient with a positive sentinel node.

Melanoma patients with a positive sentinel node biopsy generally proceed to regional lymph node dissection, though ultimately only around 20% have evidence of tumour in their "non-sentinel" nodes. A means to identify patients at high risk of non-sentinel node involvement could potentially spare a large number of patients a procedure with significant morbidity. The proliferation marker Ki-67 has been associated with tumour progression in primary melanoma but has not been extensively studied in metastases. The study aims to investigate Ki-67 in primary melanoma and lymph node metastases and investigate any relationship with disease progression. Tissue Arrays of primary melanoma (n=79) and lymph node metastases (n=92) were constructed from paraffin embedded tissue and Ki-67 expression examined by immunohistochemistry. Staining positivity and intensity were assessed and correlated with standard staging criteria and clinical outcome. High Ki-67 expression was associated with both Breslow thickness (chi(2)=8.54, p=0.035) and presence of ulceration (Fisher's Exact test p=0.003) in primary melanoma. In lymph node metastases high Ki-67 expression correlated with Nodal (N) Stage (chi(2)=8.193, p=0.0 17). High Ki-67 expression is associated with melanoma progression and multiple lymph node involvement. This might potentially form the basis of a risk analysis for patients with positive sentinel nodes.

Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks.

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.

Long term follow-up of c-myc, p53 and proliferation measurements in malignant melanoma.

AIMS: We report a prospective study examining the prognostic significance of the c-myc oncoprotein, p53 tumour suppressor gene and proliferation rate measurements in malignant melanoma. METHODS: Flow cytometry (FCM) was used to measure the expression of c-myc, p53 and proliferation parameters in patients who had received an injection of the thymidine analogue bromodeoxyuridine prior to surgery. RESULTS: Sixty-seven patients had successful FCM measurements of the three parameters. c-myc was detected in 97% of patients with a median cell positivity of 62%. The median p53 positivity was 13%. The median potential doubling time (T(pot)) of the tumours wasf 9.4 days. In univariate analysis, each of the parameters showed an association with survival in metatstatic disease with rapid proliferation (p=0.006) or overexpression of c-myc (p=0.038) related to poor survival whereas increased positivity for p53 predicted better survival (p=0.013). CONCLUSIONS: These data indicate that laser cytometric technology can be used to obtain quantitative data on oncoproteins expression and cell proliferation rates in clinical samples of malignant melanoma.

Effect of prolactin on lactalbumin production by normal and malignant human breast tissue in organ culture.

To determine whether lactalbumin production by normal and neoplastic human mammary tissue is under the same control, the effect of prolactin treatment was studied in organ culture. Of 9 premenopausal normal breast samples, 6 produced lactalbumin in culture, and all 6 responded to prolactin treatment over 4 days. One biopsy of pregnant breast tested also responded to prolactin treatment, producing 200 times more lactalbumin in culture than did normal breast. Two of 4 normal postmenopausal biopsies produced lactalbumin, and one increased synthesis and release after prolonged exposure to prolactin. Of 10 scirrhous carcinomas, 6 produced lactalbumin, but none responded to prolactin treatment. In 2 premenopausal patients, normal breast tissue responded to different concentrations of prolactin, which were without effect on malignant tissue from the same breast. In summary, lactalbumin production in the samples that we have studied can be stimulated in normal but not in malignant breast tissue. This may indicate an absence or deficiency of prolactin receptors in malignant tissue.

Combined analysis of DNA ploidy index and N-myc genomic content in neuroblastoma.

The aim of the study was to assess, in a group of nonselected patients with neuroblastoma, the prognostic value of both N-myc gene amplification and DNA ploidy index, taking into account potential confounding factors such as age and stage. Of 59 patients studied, 23 were younger than 1 year at diagnosis, 31 presented with stage IV, 10 with stage III, 5 with stage II, 8 with stage I, and 4 with stage IV-S. N-myc genomic content was analyzed by Southern blot hybridization technique and N-myc amplification (greater than or equal to 3 copies/haploid genome) was present in 6 stage IV, 2 stage III, and 1 stage IV-S. The DNA ploidy index was analyzed by flow cytometry. Of the 59 neuroblastomas, 26 were diploid (DNA index, 1) and 33 were aneuploid (DNA index, greater than 1). The majority of the aneuploid tumors (28 of 33) were near-triploid with DNA indexes between 1.25 and 1.68, 4 were near-diploid (DNA index up to 1.18), and 1 was hypotetraploid (DNA index, 1.85). The proportion of near-triploid tumors was significantly greater among patients under 1 year of age and among patients presenting with stages I, II, and IV-S. Interestingly, 0 of 28 near-triploid neuroblastomas exhibited N-myc gene amplification, compared to 9 of 31 in the group of diploid, near-diploid, and hypotetraploid tumors (Fisher's exact test, P less than 0.001). Four factors were significantly related to a high risk of relapse in univariate analysis, i.e., age, stage, DNA index, and N-myc amplification. In multivariate analysis, only N-myc amplification and the DNA index remained significantly associated with a high risk of relapse. The 2-year disease-free survival rate was 94% (95% confidence interval, 77-98%) for patients with near-triploid neuroblastoma, compared to 45 and 11% (95% confidence interval, 32-70 and 4-23%) for patients with diploid or near-diploid tumors, without and with N-myc amplification, respectively. We concluded that the combination of N-myc and DNA index should be included in routine management of neuroblastoma.

Hypoxia-inducible expression of tumor-associated carbonic anhydrases.

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays.

Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bromodeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G2 for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours.

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and Tpot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's ( < 10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. Tpot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.

Cell kinetic changes in human squamous cell carcinomas during radiotherapy studied using the in vivo administration of two halogenated pyrimidines.

The aim of this study was to investigate cell cycle changes during radiation treatment and establish whether treatment intervention could be considered if these changes helped to predict outcome. 33 patients with head and neck cancer were administered iododeoxyuridine (IdUrd) prior to treatment and a second administration of bromodeoxyuridine (BrdUrd) prior to the fifth fraction of 2 Gy. Biopsies were taken several hours after each injection and flow cytometry was used to calculate changes in the cellular kinetics and cell cycle delay in vivo. The kinetic response of the tumour cells was variable; some showed an increase in proliferation during the first week of treatment, whilst the majority showed an inhibition of proliferation. Reduction in the labelling index (LI) and the pretreatment DNA ploidy status and not delays in G2 were the only parameters to correlate with clinical outcome. A lack of reduction in the LI after 1 week of radiotherapy and DNA aneuploidy predicted a group of patients where radiotherapy failed. This information could be helpful in planning future treatment interventions.

Flow cytometric characterisation of proliferating cell nuclear antigen using the monoclonal antibody PC10.

Anti-PCNA antibodies have aroused considerable interest recently as potential immunohistochemical markers of proliferation for use on clinical samples. PC10 is a monoclonal antibody which has been shown to recognise its epitope on formalin-fixed, paraffin-embedded, archival material. However, whilst PC10 gives the expected labelling pattern for growth fraction in normal tissues and some tumours, discrepant results have been obtained, for example, in carcinoma of the breast. By means of flow cytometry, we have attempted to characterise the different staining patterns that can be obtained with PC10. Intact fixed cells from proliferative mammalian cultures show 100% labelling, consistent with a growth fraction estimate. In contrast, detergent-extracted nuclei show S-phase specific staining. Nuclei extracted by treatment of fixed cells with pepsin show a different staining pattern again, with many G1 cells weakly stained and staining intensity increasing through S-phase into G2. The results demonstrate that multiparametric flow cytometry can define the cell populations which label with proliferation-related antibodies, such as PC10, under a variety of experimental conditions.

Predictive value of potential doubling time in head and neck cancer patients treated by conventional radiotherapy.

PURPOSE: The goal of this study was to investigate the clinical utility of pretreatment measurements of tumor cell kinetics to predict the outcome of patients with squamous cell carcinoma of the head and neck receiving conventional radiotherapy. METHODS AND MATERIALS: All patients received between 64 and 70 Gy as 2 Gy fractions, five fractions per week. Cell kinetics were assayed rapidly and quantitatively using flow cytometric evaluation of iododeoxyuridine (IdUrd) incorporation, in vivo, from a biopsy removed several hours after the administration of the DNA precursor to the patient prior to the start of treatment. RESULTS: The measured proliferation parameters were not related to the clinicopathological features of the tumors, emphasizing the independent nature of these parameters. In univariate analysis, nodal involvement was the most important clinical feature of the tumors related to local control followed by Tpot, DNA aneuploidy, and attainment of complete regression at 6 weeks. Of these only Tpot and nodal status maintained significance in multivariate analysis, with respect to loco-regional control. In subgroup analysis, Tpot was able to stratify patients into high or low rate of loco-regional control in node negative patients, in aneuploid tumors and in patients who did achieve complete regression at 6 weeks. For cause specific survival, N-stage was the only parameter that significantly discriminated the prognosis in these patients. CONCLUSIONS: The conclusion of this study is that Tpot provides clinically important information that can predict patients with a low probability of achieving long-term local control with conventional fractionation. Further improvements to the methodology to address the shortcomings of analyzing diploid tumors may increase the predictive power of the measurement.

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.

Enhancement of tumor radiosensitivity and reduced hypoxia-dependent binding of a 2-nitroimidazole with normobaric oxygen and carbogen: a therapeutic comparison with skin and kidneys.

To evaluate the therapeutic potential of normobaric oxygen and carbogen as hypoxic-cell sensitizers, both radiosensitization in a mouse mammary carcinoma, mouse skin and kidneys, and the reduction in the proportion of hypoxic tumor cells were quantified in mice breathing air, oxygen, or carbogen. Local tumor control, acute skin reactions, reduced renal clearance, and hematocrit were used as assays. X rays as 10 fractions in 5 days were given to skin and tumors and 10F/12 days to kidneys. In the tumor study, the pre-irradiation breathing time was varied from 2 to 20 min. Hypoxic cells, before and during a 10F/5 day schedule, were quantified using a 2-nitroimidazole with a theophylline side chain. Bioreductively reduced metabolites of this probe were localized in hypoxic cells that were then stained using an immunofluorescent technique and analyzed by flow cytometry. The fraction of cells with high fluorescence intensity was 19% in air, 9% in oxygen, and 3% in carbogen-breathing mice. For all three gases, hypoxia-dependent binding was similar in non-irradiated tumors and those treated with four or nine fractions. Both gases significantly enhanced tumor radiosensitivity (ER = 1.3 to 1.6) and carbogen was slightly more effective than oxygen. With carbogen, maximum sensitization was observed with a 5 min pre-irradiation breathing interval. With oxygen, pre-irradiation breathing times of 2-20 min gave similar sensitization. In skin an enhancement ratio of 1.2 was observed, whereas enhancement ratios for both renal endpoints were significantly lower (1.0 to 1.07). Relative to both tissues, there was therefore a substantial therapeutic gain by irradiating CaNT tumors under both gases, especially with carbogen.

Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single kidney in the pig.

PURPOSE: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed. METHODS AND MATERIALS: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy gamma rays. Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd). At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions. The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically. The kidneys were also assessed histologically. RESULTS: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 +/- 0.32% and 1.04 +/- 0.13%, respectively, were seen 4 weeks PI, significantly greater (P < 0.001) than the preirradiation value of 0.18 +/- 0.01%. The LI values then declined with time, but remained greater than those seen prior to irradiation. A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically. The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI. The irradiated kidney exhibited diffuse, progressive glomerular alterations. In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening. The contralateral unirradiated kidney appeared unchanged. CONCLUSION: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys. Renal irradiation results in significant alterations in glomerular and tubular cell proliferation and morphology within 2-4 weeks of irradiation; glomerular changes appear predominant.

Predictive assays of radiation response in patients with head and neck squamous cell carcinoma: a review of the Institute Gustave Roussy experience.

PURPOSE: The aim of the study was to present the updated Institut Gustave Roussy experience of the predictive value of three biological parameters in patients with squamous cell carcinoma of the Head and Neck (HNSCC) treated with radiation therapy. METHODS AND MATERIALS: Three parameters have been investigated independently: tumor cell kinetics (TS, Tpot and LI), oxygen tension measurements (PO2) and intrinsic radiosensitivity (SF2Gy). RESULTS: No relationship has been found between local-regional control and Tpot or LI in a series of 74 patients. Our data also support that the surviving fraction at 2 Gy, (SF2) was unlikely to predict the clinical outcome in a series of 92 patients. Differences in PO2 measurements have been observed between tumors, and tumor oxygenation was lower than that of normal tissue for the majority of patients. However PO2 measurements did not predict clinical outcome, but further investigations are needed to draw definitive conclusions, given the limited number of patients entered in our study (35 patients). In addition, we were able to measure the three parameters in 10 patients showing no correlation between PO2, SF2 and Tpot. CONCLUSIONS: The method used to evaluate Tpot and SF2 did not provide clinically relevant predictive parameters for this type of cancer. Further investigations are needed to assess the predictive value of PO2 measurements and of new biological parameters in a multiparametric approach, taking into account other possible clinical and biological confounding factors.

bcl-2 expression in head and neck cancer: an enigmatic prognostic marker.

PURPOSE: The role of bcl-2 overexpression in cancer presents a paradox. In some tumor types, it is associated with favorable outcome, whereas in others the reverse is true. The purpose of this study was to explore the influence of bcl-2 in a large series of head and neck cancer patients treated in the CHART randomized trial. METHODS AND MATERIALS: Histologic material was obtained from 400 patients; bcl-2 expression was assessed by immunohistochemistry as either positive or negative cytoplasmic staining. RESULTS: Positivity of bcl-2 was recorded in 12.8% (9.5-16.5%, 95% confidence limits) of tumors. There were significant differences in positive tumors within different sites with nasopharynx showing the highest incidence (46.2%). A multivariate logistic regression analysis showed that bcl-2 was strongly associated with histologic dedifferentiation, as well as increasing N stage and female gender. In univariate analyses, bcl-2 positive patients had a lower locoregional relapse rate (RR 0.57, p = 0.02) and improved survival (RR 0.49, p = 0.004) compared to bcl-2 negative patients; this became more significant in multivariate analysis. CONCLUSION: These data demonstrate that bcl-2 overexpression is a marker of what is considered to be more advanced and aggressive disease yet it is associated with a more favorable outcome irrespective of the treatment schedule.

Proliferation measurements with flow cytometry Tpot in cancer of the uterine cervix: correlation between two laboratories and preliminary clinical results.

PURPOSE: To assess the prognostic value of the pretreatment potential doubling time (Tpot) in carcinoma of the uterine cervix, relative to other established clinical factors. METHODS AND MATERIALS: Fifty-two patients with cervical cancer were studied prospectively from March 1991 to October 1993. Pretreatment evaluation included examination under anesthesia and tumor biopsy 6 h following the intravenous administration of bromodeoxyuridine (200 mg). Tpot was determined by deriving the labeling index (LI) and S-phase synthesis time (Ts) using flow cytometry. Six patients were not evaluable and excluded. The remaining 46 patients (average age 55 years) were treated uniformly with radical radiation therapy. There were 39 squamous carcinomas and 7 adenocarcinomas. Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stages were: Ib and IIa, 12 patients; IIb, 18 patients; III and IV, 16 patients. The median external beam dose was 50 Gy (range, 45-52.8 Gy) delivered in 25 fractions. The median intracavitary dose was 40 Gy (range. 25.5-40 Gy) delivered with a single line source to a point 2 cm lateral of the midline, with a mean dose rate of 0.71 Gy/h. The median overall treatment time was 45 days (range, 34-73 days). As of July 31, 1994, 12 patients had died of disease, and the average follow-up for alive patients was 1.4 years (range, 0.5-3.3 years). RESULTS: There were 27 tumors with diploid deoxyribonucleic acid (DNA) content and 19 tumors were aneuploid. The median and mean Tpot for the 46 patients were 5.5 and 6.6 days, respectively [range, 2.0-25.6 days; coefficient of variation (CV), 74%]. For 25 patients where Tpot measurements were performed at two separate laboratories, there was a fair correlation (r = 0.74), but systematic differences were detected suggesting that the lack of agreement was not simply due to intratumoral variation. To date, 30 patients remained disease free, while 8 patients had pelvic failure and 9 patients developed distant metastases as the first failure site (1 patient developed both at the same time). In univariate analysis, the only significant prognostic factor for disease-free survival was tumor size (p = 0.004). A short Tpot (or high LI) and long overall treatment time (OTT) were weakly associated with poorer disease-free survival, although not statistically significant (1/Tpot, p = 0.14; LI, p = 0.23; OTT, p = 0.04). Age, FIGO stage, hemoglobin level, S-phase fraction, DNA ploidy, and Ts were not associated with disease-free survival. Multiple regression analysis was not performed because of the relatively small number of patients and short follow-up. CONCLUSIONS: Tpot values determined with current techniques by different laboratories cannot be used interchangeably for the purpose of therapy decisions. Vigorous quality assurance and standardization of the laboratory procedures and analysis methods are important to reduce interlaboratory variation. In this uniformly treated group of patients with cancer of the uterine cervix, traditional clinical prognostic factors remain the most important. Preliminary data suggest that the flow cytometry-determined Tpot and labeling index predict for disease-free survival, although a larger number of patients with longer follow-up is required to assess the true prognostic significance of these assays and to determine if their effect is independent of other clinical factors.

Bioreductive fluorescent markers for hypoxic cells: a study of 2-nitroimidazoles with 1-substituents containing fluorescent, bridgehead-nitrogen, bicyclic systems.

The oxygen-sensitive bioreductive binding of 2-nitroimidazoles labeled with fluorescent side chains has been used to stain hypoxic mammalian cells selectively. Several novel compounds were synthesized with a 1-substituent containing a fluorescent, bicyclic system having a bridgehead-nitrogen atom. Additional amine and secondary alcohol substituents were also included in the link between the fluorophor and the nitroimidazole to improve water solubility. Their ability to discriminate between hypoxic and oxic cells was compared by flow cytometric analysis. A wide range of cellular fluorescence and hypoxic-oxic differentials in fluorescence was observed when compounds with indolizine fluorophors were incubated with cells, and one such compound was considered suitable for further evaluation in vivo. Two compounds with bimane fluorophors gave very little cellular fluorescence when incubated with hypoxic cells.

Fluorescent markers for hypoxic cells: a study of nitroaromatic compounds, with fluorescent heterocyclic side chains, that undergo bioreductive binding.

Several novel compounds having both a 2-nitroimidazole nucleus and a fluorescent ring system in their molecular structure were prepared and evaluated as potential fluorescent probes for hypoxia. Bioreduction of nitroimidazoles, which is inhibited by oxygen, is known to lead to binding of bioreductive metabolites to cellular macromolecules and this provides a mechanism for binding the fluorescent moiety to hypoxic cells. These compounds can incorporate a wide range of fluorophors and can therefore be designed to suit the laser-line wavelengths available for excitation of fluorescence in the flow cytometer. Several nitroimidazoles with naphthalimide side chains were rapidly taken up into cells and became concentrated in the cells, thus reducing their concentration in the extracellular medium. This suggests a potential microscopic bioavailability problem with probes of this type when used in vivo as they would become progressively depleted in the extracellular fluid as they diffused from blood vessels, through layers of packed cells in tumors, to the hypoxic cells where they could undergo hypoxia-specific metabolism. Synthesis of nitroimidazoles with coumarin fluorophors led to several potentially useful probes for hypoxia; substituents on the coumarin fluorophor had a marked effect on the cellular fluorescence of these compounds.