The therapeutic efficacy of gemcitabine, a new nucleoside analogue, was assessed in a variety of well-established human soft tissue sarcoma and ovarian cancer xenografts grown s.c. in nude mice. Tumour lines selected had different histological subtypes, growth rates and sensitivities to conventional cytostatic agents. The three different doses and schedules designed on the basis of a mean weight loss between 5% and 15% were i.p. injections of daily 3.5 mg kg-1 x 4, every 3 days 120 mg kg-1 x 4, and weekly 240 mg kg-1 x 2, which ultimately resulted in 19%, 10% and 4% toxic deaths, respectively. The weekly schedule induced > or = 50% growth inhibition in 2/4 soft tissue sarcoma and 4/6 ovarian cancer lines, while in three ovarian cancer lines > or = 75% growth inhibition was obtained. The anti-tumour effects of gemcitabine appeared to be similar or even better than previous data with conventional drugs tested in the same tumour lines. In comparison with the every 3 days schedule, the weekly and the daily schedule were less effective in 5/7 and 3/3 tumour lines (P < 0.001), respectively. In another experiment in three human tumour lines selected for their differential sensitivity to gemcitabine, weekly injections of 240 mg kg-1 x 6 did not result in a significant increase in the percentages of growth inhibition when compared to lower doses of 120 mg kg-1 or 60 mg kg-1 in the same schedule. However, the 240 mg kg-1 weekly x 6 schedule showed superior effects in 2/3 tumour lines in comparison with the same dose given every 2 weeks x 3 (P < 0.05). The preclinical activity of gemcitabine suggests that the drug can induce responses in soft tissue sarcoma and ovarian cancer patients. Our results further indicate that clinical trials of gemcitabine in solid tumour types should be designed on the basis of a schedule rather than a dose dependence.


The effect of cancer cachexia on the oxidative metabolism of lipids has been studied in mice transplanted either with the MAC16 adenocarcinoma, which induces profound loss of body weight and depletion of lipid stores, or the MAC13 adenocarcinoma, which is the same histological type, but which grows without an effect on host body weight or lipid stores. While oxidation of D-[U-14C]glucose did not differ between animals bearing tumours of either type and non-tumour bearing controls, oxidation of [1-14C]triolein administered by intragastric intubation was significantly (P less than 0.05) higher in animals bearing the MAC16 tumour than in either non tumour-bearing controls or in animals bearing the MAC13 tumour. Intestinal absorption of [14C]lipid was significantly (P less than 0.05) reduced in animals bearing the MAC13 tumour when compared with either non tumour-bearing animals or MAC16 tumour-bearing animals, but was not significantly different in the latter two groups. The level of labelled lipids in heart and adipose tissue after an oral [14C]lipid load was significantly lower in animals bearing the MAC16 tumour compared with the other two groups. The level of tumour lipids was also higher in the MAC16 than in the MAC13 tumour after both an oral [14C]lipid load or by direct injection of [U-14C]palmitate complexed to albumin into epididymal fat pads. Oxidation of [U-14C]palmitate was also significantly enhanced in liver and heart homogenates from animals bearing the MAC16 tumour. These results suggest that in cachectic tumour-bearing animals mobilisation of body lipids is accompanied by an increased utilisation.


Ge(IV) phthalocyanine (GePc) with two axially ligated cholesterol moieties was prepared by chemical synthesis and incorporated in a monomeric state into small unilamellar liposomes (CGP 55398). Upon photoexcitation with light wavelengths around its intense absorption peak at 680 nm, GePc shows an efficient photosensitising activity towards biological substrates through a mechanism which largely involves the intermediacy of singlet oxygen. GePc injected systemically into mice bearing an intramuscularly implanted MS-2 fibrosarcoma is quantitatively transferred to serum lipoproteins and localises in the tumour tissue with good efficiency: at 24 h post injection the GePc content in the tumour is 0.74 and 1.87 micrograms per g of tissue with a tumour/peritumoral ratio of 4.35 and 5.67 for injected doses of 0.76 and 1.52 mg kg-1 respectively. At this time the red-light irradiation of the GePc-loaded fibrosarcoma causes a fast and massive tumour necrosis involving both malignant cells and blood vessels.


A tumour model consisting of the highly metastatic mammary 13762 parental line, the non-metastatic and 6-thioguanine-resistant (TgR) variant line, and two TgR revertant lines  that were occasionally metastatic, were used to determine whether the release of N-acetylneuraminic acid (NANA) was related to tumour metastasis. For comparative purposes, the occasionally metastatic R3230AC and the nonmetastatic DMBA8 tumour lines were studied. The NANA was considered to be in bound form, because acid hydrolysis was required to release it for high-pressure liquid chromatographic analyses. Sera of animals bearing the 13762 and R3230AC tumours had high levels of bound NANA. No differences were found in serum NANA levels in animals bearing metastatic or non-metastatic R3230AC tumours. Low levels of bound NANA were found in animals bearing the other tumour lines regardless of whether metastasis occurred or not. The experiments in vitro substantiated the in vivo findings. The phenotypic expression of bound NANA shedding did not correlate well with metastatic potential of the mammary tumour line. Our analyses suggest that this phenotypic marker cannot be used as a reliable indicator of metastasis.


Animals bearing a cachexia-inducing tumour, the MAC16 adenocarcinoma, showed a progressive decrease in blood glucose levels with increasing weight loss, while animals bearing a histologically similar tumour, the MAC13 adenocarcinoma, showed no change in either body weight or blood glucose levels with growth of the tumour. The effect of the MAC16 tumour on blood glucose levels appeared to be unrelated to food intake, glucose consumption by the tumour, or to the production of increased levels of IGF-I and IGF-II mRNA by the tumour cells. The relationship between the induction of cachexia and alteration in blood glucose levels remains unknown.


The aim of this study was to evaluate whether food intake modulates experimental tumour growth by acute alterations in the energy state and blood flow of the tumour, and if so whether such changes are related to alterations in the enzyme ornithinedecarboxylase (ODC) and DNA synthesis. Inbred mice (C57BL/J) bearing a syngeneic undifferentiated and rapidly growing tumour were used. The tumour levels of high energy phosphates were measured in vivo by 31-P-NMR spectroscopy and biochemically following tissue extraction. DNA synthesis was estimated by measuring the incorporation of bromodeoxy-uridine into tumour DNA. Difluoro-methylornithine (DFMO) was used to inhibit ODC-activity. Tumour blood flow was estimated by a 132Xe local clearance technique. Tumour progression was associated with a significant decrease in tumour tissue high energy phosphates. Acute starvation decreased DNA-synthesis and tumour energy charge as well as its PCr/Pi which were rapidly normalised during subsequent refeeding. These changes were related to similar alterations in tumour blood flow. The inorganic phosphate (Pi) resonance and the resonances in the phosphomonoester (PME) region were considerably increased in tumour tissue. Inhibition of ODC-activity by DFMO decreased DNA-synthesis, which was associated with a secondary increase in tumour high energy phosphates probably due to a lowered energy demand for tumour cell division. The results demonstrate that host undernutrition was translated into retarded tumour growth associated with a decrease in the energy state and blood flow of the tumour. The results have bearing for the evaluation and planning of all treatment protocols with potential influence on food intake in experimental tumour-bearing animals.


Nebulisation chemotherapy, a chemotherapeutic method for the treatment of lung cancer that involves the administration of anticancer agents through the inhalation of nebulised aerosols, has been found to be highly effective . We confirmed that 5-FU administered by this method accumulates in the trachea, bronchi and regional lymph nodes of patients treated before surgery, along with 5-FU metabolites, FUR and FUdR, indicating that 5-FU is directly incorporated and metabolised in the respiratory tract. Parallel result were obtained using mongrel dogs. The 5-FU levels in other organs, such as the heart and liver, were found to be extremely low. Only a trace of 5-FU was found in the serum of both the patients and the dogs. We further investigated the anti-tumour effect of this therapy in ten selected patients and observed a satisfactory anti-tumour response of 60.0%. These results, along with our previous finding that the retention time of isotope tracers inhaled as aerosol is considerably longer in tumour tissues than in normal parts  explain the high antitumour action of this therapy and the absence of adverse effects of administered 5-FU.


Outpatient drugs are dispensed through both community and mail order pharmacies. There is no empirical evidence that substitution of community pharmacy with mail order reduces overall drug expenditures. The need for evaluating the potential effects on utilization and costs of the possible extension of mail order services in Medicaid provides the rationale for conducting this study. This study compares drug utilization and drug product cost in community vs. mail order pharmacy dispensing services in a Medicaid population.This study is a retrospective cohort study comparing utilization and cost patterns in community vs. mail order pharmacy. A simulation model was employed to assess drug utilization and cost in mail order pharmacy using community pharmacy claim data. The model assumed that courses of drug therapy (CDT) in mail order pharmacy would have utilization patterns similar to those found in community pharmacy. A 95% confidence interval surrounding changes in average utilization and average cost were estimated using bootstrap analysis. A sensitivity analysis was performed by varying drug selection criteria and supply, fill point, and medication possession ratio (MPR). Sub-analyses were performed to address differences between mail order and community pharmacy related to therapeutic class and dual-eligible patients.Data for the study derived from pharmacy claims database of Ohio Medicaid State program for the period January 2000-September 2004. Drug claims were aggregated to obtain a set of CDTs representing unique patient IDs and unique drug products. Drug product cost estimates excluded dispensing fees and were used to estimate the cost reduction required in mail order to become cost neutral in comparison with community pharmacy.The baseline model revealed that the use of mail order vs. community pharmacy would result in a 5.5% increase in drug utilization and a 5.4% cost reduction required in mail order to become cost neutral. Results from Ohio Medicaid drugs for chronic use revealed a 5.1% increase in utilization and a 4.9% cost reduction required to become cost neutral in comparison with community pharmacy.The results of the simulation model indicate that mail order pharmacy increases drug utilization and can also increase drug product cost if the cost per unit is not reduced accordingly. Prior consideration should be given to the patient population, day-supply, disease, therapy, and insurance characteristics to ensure the appropriate use of mail order pharmacy services. Outpatient drugs are dispensed through both community and mail order pharmacies. In 2004, mail order pharmacies dispensed 214 million prescriptions, representing 6.5% of the prescriptions dispensed in the U.S. outpatient pharmaceutical sector during that year [Initially, mail order pharmacy was perceived as an alternative for patients who use a high volume of prescription drugs for chronic conditions -6 or forIncreases in utilization could result in higher overall prescription drug expenditures for mail order pharmacy in spite of a lower cost per unit. Previous studies found that drug utilization in pharmacy benefit managers (PBM) commercial populations was higher in mail order pharmacy than in community pharmacy,9,10. HiIn addition, drug therapy discontinuation is frequent regardless of the therapy utilized -18, and The scope of this study is a Medicaid population. Mail order services have been implemented in the Medicaid programs of Maine and Washington , and mulMail order services in Medicaid have not been examined previously in the scientific literature. Medicaid differs from traditional comercial plans in that benefits are controlled at both the federal and state levels. Moreover, Medicaid programs are managed by the state departments with PBM support for claims processing and other services. Also, state Medicaid programs define their own pharmacy reimbursement structures, including the estimated drug acquisition cost and dispensing fees. Finally, Medicaid drug pricing is specially influenced by pharmaceutical company rebates, which are regulated at the federal level, and most states have a supplemental rebate program.The objectives of this study are: 1) to compare drug utilization in a Medicaid population using community pharmacy services with the results of a model simulating the effect of mail order pharmacy in the same population; and 2) to estimate the mail cost reduction required to become cost neutral in comparison with community pharmacy.This study is a retrospective cohort study comparing utilization and cost patterns in community and mail order pharmacy. Drug utilization is defined in this study as the day supply of drug product reimbursed by the Ohio Medicaid program A novel simulation model using community pharmacy claim data was employed to assess drug utilization in mail order pharmacy and mail order cost reduction required to become cost neutral in comparison with community pharmacy. The model used the course of drug therapy (CDT) representing unique patient IDs and unique drug products (i.e. generic name(s), formulation, and strength(s)) as the unit of analysis. The cost was estimated from the perspective of the Medicaid program.Pharmacy claims data from the Ohio Medicaid program for the period January 2000-September 2004, including 93.0 million unduplicated claims for patients who were not institutionalized at any point during the study period were used for the analysis Table . The datAll CDTs initiated between August 2000 and July 2001 were selected for inclusion in the study. CDTs initiated between January and July 2000 were excluded to ensure that the drug therapy was newly provided (i.e. that patients had not taken the therapy during the six months prior to selection). Therapies initiated after July 2001 were excluded to allow for a follow-up period of at least three years and two months and to minimize right censoring data issues. The baseline analysis excluded chronic drugs for short-term use and included acute drugs for chronic use.The criteria for selecting the baseline sample from the final data set was that, for each CDT, patients should have utilized a supply of at least 90 days and should have maintained a Medication Possession Ratio (MPR) of at least 0.5. The MPR was estimated using the number of days supply reimbursed by Ohio Medicaid according with the information available in the claims data. Although the MPR does not assess actual consumption, it is a proxy measure of adherence that determines the proportion of days a patient has medication available to be taken . The MPRThe mail order simulations did not include the first fill, because mail order pharmacy starts typically with the first refill. The effect of mail order on drug utilization was calculated for each CDT by dividing the MPR-adjusted community pharmacy day supply by 90 and rounding that result to the next highest integer. Incremental changes in drug utilization attributable to the effect of mail order were estimated.The methodology utilized in the mail order simulation model can be illustrated by the following example Table : In the The mail order pharmacy cost reduction required to become cost neutral in comparison with community pharmacy were estimated by first calculating the drug product cost per claim in community pharmacy; that is, by deducting the Ohio State Medicaid dispensing fee of $3.70 per claim. Next, community pharmacy drug product cost per day of supply was calculated for each CDT by dividing the drug product cost by the total number of days the drug was supplied. It was assumed that drug product cost in community and mail order pharmacy would be the same. This assumption allowed for the estimation of cost differences between both channels and the assessment of the cost reduction that mail order should achieve to become cost neutral. Hence, drug product cost in mail order pharmacy was estimated by multiplying the mail order day supply figure by the community pharmacy drug product cost per day supply. Costs were not adjusted for inflation or discounted. As with the estimates of drug utilization, estimates of differences in drug product cost did not take into account the day supply associated with patients' first fill.Once the incremental changes in utilization attributable to the use of mail order were modeled and the mail order cost reduction required to become cost neutral in comparison with community pharmacy, a 95% confidence interval surrounding these changes was constructed. Specifically, bootstrap analysis, a non-parametric approach used to avoid distributional assumptions  was usedSub-analyses were performed in relation to drugs for chronic conditions (as determined by State Medicaid drug classifications), dual-eligible patients (Medicare/Medicaid patients), and that subset of dual-eligible patients using drugs for chronic conditions. The sub-analyses utilized a baseline sample based on the selection criteria described above. Similar to the main analysis, sub-analyses also calculated incremental changes in utilization attributable to mail order and the mail order cost reduction required to become cost neutral in comparison with community pharmacy.Multivariate sensitivity analysis was performed in relation to the baseline model by varying the following parameters: 1) point where mail order filling was initiated , 2) minimum acceptable MPR for a CDT being eligible for mail order, and 3) minimum day supply utilized in community pharmacy for a CDT being eligible for mail order.The baseline model included 258,412 patients, 680,277 CDTs, and 869 drugs in 26 therapeutic classes. Demographic characteristics were estimated at the patient and CDT levels Table .The distribution of number of day supply per CDT was right-skewed for both community and mail order pharmacy Figures  and 2, rThe final sample of 680,277 CDTs yielded a mean of 640.0 ± 515.0 day supply in community pharmacy and the model for mail order resulted in 675.0 ± 518.0 day supply .Thus, a 5.5% increase in drug utilization was associated with the use of mail order. This increase in utilization would lead to an estimated 5.4% mail order cost reduction required to become cost neutral in comparison with community pharmacy over the follow-up period that started in January 2000 and ended in July 2004  regardless of the sample utilized . Nevertheless, an increase in total day supply per CDT was observed in the sub-analyses, which resulted in a lower relative difference in utilization in mail order vs. community pharmacy. This reduction in the utilization relative difference has implications for mail order's impact on cost: The lower the overall relative difference in utilization, the lower the overall difference in unit cost must be for mail order pharmacy to become cost-neutral.The results of the sensitivity analysis indicate that a reduction in the minimum number of day supply the CDTs were utilized in community pharmacy to become eligible for mail order increased the difference in utilization between mail order and community pharmacy. This result was the expected because the reduction in the number of day supply allowed for inclusion in the model of medications used for short term purposes. Finally, increases in the MPR reduced utilization relative differences, indicating that mail order is more appropriate for patients with high compliance.Information about the unit cost of mail order in Medicaid is not available therefore, we assumed that the cost would be the same in mail and in community pharmacy regarding the estimation of the breakeven point; that is the cost reduction required in mail order to become cost neutral in comparison with community pharmacy. The results of our study indicate that mail order cost should be reduced in 5.4% to breakeven with the cost in community pharmacy.Mail order pharmacy has cost advantages and disadventages over community pharmacy. First, dispensing a 90 day supply prescription instead of three 30 day supply prescriptions reduces dispensing costs for mail order operations. However, this reduction in dispensing costs could also be expected in community pharmacy if a 90 day supply were dispensed instead of a 30 day supply. Second, large mail order operations may have lower drug product acquisition cost than the average community pharmacy. With commercial plans, the cost per unit is lower in mail order than in community pharmacy due to a reduction in drug product cost, lower dispensing fees and higher rebates. However, mail order pharmacy could experience higher overall cost per CDT for the Medicaid program if drug product costs, supplemental rebates and/or dispensing fees are not adjusted accordingly with the increase in utilization. In addition, wholesaler distribution and inventory costs may also be lower in mail order pharmacy than in community pharmacy. However, mail order pharmacy has costs that are not found in community pharmacy, including packaging, mailing, and special handling costs, and the cost of drugs lost in mail.Third, plan benefits may be structured according to pharmacy characteristics and geographical area. For example, pharmacy reimbursement in the Medicaid programs of Maine and Washington is lower for mail order than for community pharmacy Table . An examThe CDT selection criteria used in the study were designed to restrict the sample to those CDTs that would be more appropriate for mail order. First, only CDTs in which the patient had utilized the drug for at least 90 days during the study period were selected. In practice, this is an ex-post criterion, as mail order pharmacists would not have this information prior to any decision to initiate mail order dispensing; this criterion is also consistent with mail order in that those medications discontinued immediately after the first refill were excluded. Second, the study utilized only those CDTs in which the patient maintained a minimum MPR of 0.5 during the study period. The study's CDT selection criteria allowed for the exclusion of drugs that were used during multiple periods of time  and for short-term purposes (e.g. antibiotics). Third, the model assumes that the MPR associated with mail order pharmacy is the same as that observed in community pharmacy. In fact, however, mail order could increase the MPR given that patients have more medication available when 90-day supplies (vs. the 30-day supplies used in community pharmacy) are dispensed. Another assumption of the model is that mail order begins with the second fill; initiation of mail order at the first fill would further increase utilization given the high rate of therapy discontinuation that occurs before the second fill is dispensed. Fourth, the model also assumes that the drug supplies dispensed in mail order pharmacy are MPR-adjusted 90-day supplies. In fact, mail order programs can dispense supplies of 120 days or more. Increases in day supply would increase the difference in utilization in mail order vs. community pharmacy. And fifth, the baseline sample included all medications suitable for mail order, regardless of their classification and therapeutic category. To assess the validity of the criteria used to select these drugs, a sub-analysis was performed in relation to those drugs classified by the Ohio State Medicaid program as drugs for chronic use. A sub-analysis related to the dual-eligible population was also performed as this population of Ohio Medicaid patients are eligible for the new Medicare prescription drug program that began in 2006. A final sub-analysis included the subset of dual-eligible patients using drugs for chronic conditions. The three sub-analyses conducted in this study yielded similar increases in utilization derived from the use of mail order pharmacy.Patients included in the study could have gaps in coverage; exclusion of these patients would misrepresent the potential effect of mail order pharmacy in the Medicaid program. Given a high turnover rate, periods of fluctuating coverage, and therapy discontinuation for enrollees associated with the Medicaid program, caution should be applied when generalizing these findings to other populations. In addition, the study only included drug reimbursement (drug product costs and dispensing fees); non-health care costs, such as patient costs to pick up medication at a local pharmacy, acquisition of medications through channels outside the scope of this study, and indirect costs were excluded from the analysis.This study did not discount drug costs or adjust them for inflation. Discounting would have increased the cost differences resulting from mail order in that mail order payments are for three-month supplies as opposed to the one-month supplies used in community pharmacy. Adjusting drug costs for inflation would have reduced cost differences for the same reason. However, the study results would not change had these adjustments occurred.Finally, this study does not evaluate the effect of increasing utilization on patients' outcomes. While higher utilization could result in higher patient compliance with drug treatments and increased access to drugs, this link has not been established in the available mail order pharmacy literature. Future research is needed to examine therapy discontinuation and estimation of drug wastage, resulting from medications that were not used because of tolerability issues, medication changes, ineffectiveness, and wastage attributed to other factors such as mail mishaps, spoilage, and patient failure to pickup rates, and an analysis of the impact of mail order pharmacy utilization on patient health outcomes. Future studies should also evaluate if the increase in utilization associated to mail order pharmacy could improve medication adherence and if adherence rates vary in different groups of Medicaid beneficiaries.While the scope of this study is the Medicaid population, the results of the model related with increases in utilization can be also applicable to PBM commercial populations. Evaluation of mail order in a commercial population should evaluate if the reduction in costs per unit justifies the increase in utilization.The results of the simulation model used in this study indicate that mail order pharmacy increases drug utilization and can increase drug product cost if the cost per unit is not reduced accordingly. These results were achieved with CDT selection criteria that were designed to restrict the sample to only those CDTs for which the dispensing of 90-day supplies was appropriate.Although increases in utilization may be beneficial for certain patients and may be linked to increases in medication adherence, policymakers should evaluate the utilization and cost effects that result from the implementation of mail order pharmacy programs. In addition, any program that increases the number of day supply dispensed to the patients should carefully target specific populations and therapies whose potential savings and/or health outcomes overcome the potential extra costs that result from increased utilization and wastage. Although, mail order is expected to have a large role in the Medicare Part D program, data are not yet available for analysis. Projections from this study reveal an increase in utilization attributed to mail order should be expected, and mail order services would be more appropriate for patients using maintenance drug therapies for chronic conditions for long periods of time (more than 2 years). In addition, mail order pharmacy services could facilitate access to medications for underserved areas and for elderly and disabled populations. This potential impact on access should be considered when evaluating the overall effect of mail order pharmacy.This study was funded by the Ohio Department of Job and Family Services and the Ohio Commission to Reform Medicaid as part of a larger analysis of the Ohio Medicaid pharmacy benefit. The authors developed the study's methodology, analyzed the data, and wrote this manuscript independently of the Ohio Medicaid program.The author(s) declare that they have no competing interests.All authors actively participated in every step of the research process and in the development of this manuscript.The pre-publication history for this paper can be accessed here:


A previously described mathematical model designed to evaluate resistance and tumour-kill for individual patients, and to predict changing tumour sizes, has been applied to patients with small cell lung cancer. The model requires tumour volume measurements, and these were obtained via computed tomography scans of the chest. The model fitted the data well, and was able to predict later tumour volumes using earlier ones, as well as suggesting times at which to change or abandon treatment for individual patients. The model gave estimates for resistance and tumour-kill which may provide additional useful outcome measures for clinical trials, and help in the design of future studies.


A simple technique for maintaining low oxygen concentrations (0.1-20%) is described. These conditions were then used to study the effect of oxygen on colony growth of neoplastic cells in soft-agar. Physiologically low oxygen concentrations (0.1-10%) compared to 20% O2 were found to enhance plating efficiency and colony size of tumour cells. The optimal oxygen concentration for plating efficiency varied with tumour studied and may be as low as 0.1%. Having established that tumour cell colonies will grow better at 0.1-10% O2 compared to 20% O2, the mechanism by which this enhancement occurs was investigated. Observations on the effect of free radical scavengers and superoxide dismutase on plating efficiency of Ehrlich's ascites tumour cells suggests that this phenomenon occurs through oxygen toxicity mediated by superoxide anion.


In a population-based study serum sialic acid level was examined in relation to subsequent development of central nervous system (CNS) tumours (229 cases). Significantly increased sialic acid concentration was found in men with a malignant CNS tumour diagnosed within 8 years of analysis, compared with corresponding matched controls. These findings suggest that the tumour existed at the time of examination which is supported by a negative linear association between sialic acid level and the time from screening to tumour diagnosis.


Various immunization assays were used to demonstrate the lack of immunogenicity of three BALB/c tumours of spontaneous origin and of a fourth one resulting from foreign body tumorigenesis. All four tumours inhibited the growth of a second implant of the same tumour into the contralateral flank. In our tumour models "concomitant immunity" (1) was not mediated by macrophage or T-cell dependent immune reactions: both thymectomized BALB/c and nude mice (treated or untreated with silica) gave the same results as intact mice; (2) showed some degree of non-specificity, inhibiting the growth of a different tumour in 3/4 cases; though, the existence of a specific component could not be discarded; (3) was proportional to the volume of the primary tumour at the time of the second challenge; (4) was dependent on actively growing primary tumour, not being obtained with progressively increasing daily inocula of irradiated tumour cells; (5) was detectable in an actively growing secondary tumour; recurrent growth after partial surgical excision was inhibited and (6) involved cytostasis of the secondary tumour: a syngeneic graft of the overlying skin led to tumour growth while histological studies revealed the presence of viable tumour cells. It is postulated that "concomitant immunity" or resistance can be generated without the active participation of the immune system and that tumour-related factors are, in certain cases, responsible for blocking the growth of secondary tumours.


The combination of heat and chemotherapy was studied in an intraperitoneal tumour model. Rats bearing peritoneal CC531 tumours (2-6 mm) were treated i.p. with cDDP or CBDCA [maximal tolerated dose (MTD)] in combination with regional hyperthermia  of the peritoneal cavity. The addition of hyperthermia to the i.p. treatment led to a decrease in the MTD of cDDP by 33.3% at 41.5 degrees C. This was due to increased nephrotoxicity. The MTD of CBDCA did not change as a result of hyperthermia treatment. The chemo-hyperthermia treatment resulted in more cDDP or CBDCA DNA adducts in peritoneal tumours after the combined treatment than after chemotherapy alone. The increased tumour platinum concentrations, rising from 1.3 micrograms Pt g-1 tumour at 37 degrees C to 5.4 micrograms Pt g-1 tumour at 41.5 degrees C for cDDP and from 0.2 microgram Pt g-1 tumor to 0.7 microgram Pt g-1 tumour at 41.5 degrees C for CBDCA, contributed considerably to the enhanced numbers of cDDP or CBDCA DNA adducts. As a result of the latter, i.p. chemotherapy combined with regional hyperthermia led to an increase in tumour growth delay (TGD) after increasing the temperature to 41.5 degrees C for cDDP and CBDCA . These data were in agreement with the in vitro findings, i.e. that higher temperatures led to increased cytotoxicity.


A chelating derivative of alpha-melanocyte stimulating hormone (MSH) has been synthesised, in which two molecules of the hormone are cross-linked by diethylenetriamine pentaacetic acid (DTPA). This compound, bisMSH-DTPA, was equipotent with MSH in an in vitro tyrosinase assay with Cloudman S91 melanoma cells. When DBA/2 mice bearing the same tumour were injected with bisMSH-DTPA labelled with the gamma-emitting isotope indium-111 (111In), the radioactivity became rapidly associated with the melanoma tissue. By 24 h post-injection, radioactivity in tumour tissue was significantly higher (P less than 0.001) than in spleen, lung, brain, eye and skin. Uptake of radioactivity by the tumours was inhibited by a 200-fold molar excess of MSH, whereas uptake by liver, kidney, spleen, lung, brain, eye and skin was unaffected. We conclude that bisMSH-DTPA may offer an alternative to antibody targeting in the imaging of malignant melanoma.


A previous publication  showed that administration of basic fibroblast growth factor  to mice bearing xenografted DLD-2 human colon cancers would increase treated tumour growth rates as compared to control neoplasms. Additionally, at the end of the 7 day treatment period, clonogenic excision assays showed that the percentage of hypoxic cells in tumours from mice receiving FGF-2 administration was significantly decreased as compared to control neoplasms (from about 42 to about 19%). The present study was undertaken to better define the kinetics of changes in hypoxic percentages as a function of tumour volume and FGF-2 treatment. In sham-injected control tumours, the hypoxic percentage increased from about 14% at day 15 postimplantation, (i.e. when sham- or FGF-2 injections were begun) to about 42% by day 22, and to about 75% at 29 days postimplantation . In contrast, the hypoxic percentages in mice treated with FGF-2 remained at the levels seen in control mice on day 15, not only throughout the 7 day FGF-2 treatment schedule, but for at least 1 week after the cessation of growth factor administration. The hypoxic percentage was 16% on day 29 postimplantation, even though average tumour volumes were about 4325 mm3. These data show that the effect of FGF-2 administration on tumour growth rate and hypoxic percentages in xenografted DLD-2 neoplasms is rapid, and continues for some period of time even after administration is ended. Studies of tumour perfusion with injected 86RbCl at equivalent tumour volumes of about 1800 mm3 indicated that the percentage of cardiac output to FGF-2 treated tumours was 33% greater than in sham-injected control neoplasms.


Murine L5178Y-ML cells, when transplanted subcutaneously into the flank of (BALB/c x DBA/2)F1 mice, grew locally and always formed spontaneous metastases in the liver. Even after surgical removal of the primary tumour mass 5 or 7 days after tumour cell inoculation, all mice died due to liver metastases within 18 days. Using this model of tumour metastasis, we examined whether serine protease or deoxyribonuclease I (DNase I) would affect metastasis. Spontaneous liver metastasis of L5178Y-ML cells was enhanced by systemic administration of alpha-chymotrypsin at 3, 4 and 5 days or at 5, 6 and 7 days after tumour cell inoculation. This result was consistent with a previous report on blood-borne lung metastasis. In contrast, systemic administration of DNase I at 3, 4 and 5 days or at 5, 6 and 7 days after tumour cell inoculation inhibited liver metastasis. Neither treatment affected primary tumour growth. An influence of DNase I on tumour cell arrest in the microvasculature of the liver was suggested by scanning electron microscopy. DNase I treatment resulted in a statistically significant prolongation of the survival period, however, the effect was not satisfactory. A more striking anti-metastatic treatment resulting in a greater prolongation of the survival period was achieved by combining surgical removal of the primary tumour mass with DNase I treatment. These results suggest that DNase I could be a potential therapeutic agent used in conjunction with surgery to prevent clinical blood-borne metastasis.


Restriction fragment length polymorphism (RFLP) of the L-myc gene was examined in DNAs from primary tumour tissues and peripheral blood cells (PBC) of 76 Indian patients with squamous cell carcinoma of the oral cavity, and PBC from 101 normal healthy volunteers. The patients and the normal healthy volunteers were classified into three genetic types according to the polymorphic patterns defined by the two alleles . DNA isolated from the PBC of each patient always exhibited the same pattern of L-myc alleles as that observed for the corresponding tumour DNA. However, a striking correlation was found between the RFLP pattern and the stage of differentiation of the tumours, as well as the size of the tumour. Thus, a preponderance of the S fragment was observed in the poor to moderately differentiated tumours and the larger (greater than 4 cm) sized tumours. Further, analysis of L-myc RFLP with the clinical pattern of the malignancy showed no significant correlation with nodal metastasis, TNM staging or recurrence of the tumour. The relative ratios of the three genotypes  in the oral cancer patients were not significantly different from those seen in the healthy Indians, implying no predisposition to oral cancer by either allele. However, our results showed that oral cancer patients with a genotype including an S fragment are more likely to develop a poor to moderately differentiated tumour or a larger tumour than a patient without an S fragment. The L and S alleles were equally distributed in the population, with the frequency of each allele being 0.50, consistent with Hardy-Weinberg's law.


The effect of 45 min systemic heating at 41 degrees C on plasma and RIF-1 tumour pharmacokinetics of intraperitoneally administered melphalan (MEL) was studied in C3H mice. This heat dose causes greater potentiation of MEL in tumour than in marrow cells, resulting in a therapeutic gain for the combined therapy . MEL (7.5 mg kg-1) was administered at the start of heating and concentrations assayed from 20-90 min by high-performance liquid chromatography (HPLC). With or without heat peak concentrations were achieved by 20 min and were 3 to 4 micrograms ml-1 in plasma and 1-3 micrograms g-1 in tumour. Higher MEL concentrations in both plasma and tumour were found in heated animals at times after 20 min from injection, but the effect was greater in plasma (2.5-4 fold) than in tumour (1.5-2 fold) where differences were not always significant. At 40 min after a dose of 7.5 mg kg-1, plasma and tumour concentrations in heated animals were equivalent to those after 12.5 mg kg-1 and 8.5 mg kg-1, respectively, without heating. Tumour/plasma ratios were usually lower in heated than in unheated animals where they often exceeded 100%. The apparent plasma elimination half-life (t1/2) was 17.5-25 min in unheated and 24-44 min in heated animals. The area under the curve (AUC) was increased by a factor of 1.2-1.5 in heated animals, at least partly due to a decrease in volume of distribution. The heat induced increase in MEL exposure may be involved in the enhanced response to the drug, but does not appear to explain the therapeutic gain compaired to MEL alone.


Inhibition of angiogenesis through blocking of growth factors involved in this process could be a novel therapeutic approach in several important pathologies, neoplasia among them. Suramin has recently been described to possess antineoplastic activity in animals and humans, and it has been proposed that an important role in this activity is played by antagonism of growth factors and especially bFGF. To investigate this hypothesis in vivo, we used gelatin sponges loaded with bFGF and implanted subcutaneously in mice. Suramin showed an inhibitory activity on bFGF-induced angiogenesis, whereas it was inactive in the case of heparin-complexed bFGF. Suramin was also studied in an in vivo model of tumour-induced angiogenesis using the murine M5076 reticulosarcoma, a tumour producing significant levels of bFGF. Suramin was able to reduce tumour growth and tumour induced angiogenesis, and exogenous administration of bFGF countered suramin effects.


The aim of the present study was to show the feasibility of determining radiotherapeutically relevant hypoxia and early tumour response by  and vinorelbine (30 mg/m2) followed by concurrent radio-  and chemotherapy with gemcitabine (300–500 mg/m2) every two weeks. FMISO PET and FDG PET were performed in all patients 3 days before and 14 days after finishing chemotherapy.Eight patients with non-small-cell lung cancer underwent PET scans. Tumour tissue oxygenation was measured with FMISO PET, whereas tumour glucose metabolism was measured with FDG PET. All PET studies were carried out with an ECAT EXACT 922/47FMISO PET allowed for the qualitative and quantitative definition of hypoxic sub-areas which may correspond to a localization of local recurrences. In addition, changes in FMISO and FDG PET measure the early response to therapy, and in this way, may predict freedom from disease, as well as overall survival.These preliminary results warrant validation in larger trials. If confirmed, several novel treatment strategies may be considered, including the early use of PET to evaluate the effectiveness of the selected therapy. At present, combined modality treatment (chemotherapy followed by surgery or radio-/chemotherapy) for patients with locally advanced non-small-cell lung cancer (NSCLC) is being studied extensively. It is clear, however, that for different reasons, a substantial number of patients had lesser benefits from such intensive treatment. For example, tumour anaemia and tumour hypoxia are considered as multifactorial causes of tumour treatment resistance ,21. The With positron emission tomography (PET), radio-labelled hypoxia-avid compounds can be applied to evaluate the oxygenation status in experimental or human tumours . FluorinConventional techniques used to monitor therapeutic effects in oncology, such as CT and MRI, are based on morphologic changes and show limited accuracy ,14. SeveThis study evaluates the impact and feasibility of determining tumour hypoxia by FMISO PET, and of one factor related to tumour metabolism, namely tumour glucose use, through FDG PET in patients with NSCLC in relation to response to radio-/chemotherapy.The Medical Ethical Committees of the University of Aachen approved the study. The study was conducted according to the Helsinki Declaration. All patients gave written informed consent before they were enrolled in the study.2) and vinorelbine (30 mg/m2) given on day 1, 8 (cycle 1A and 1B) and on day 22, 29 (cycle 2A and 2B). Gemcitabine was administered first in a 30-min i.v. infusion, followed by vinorelbine, which was given as a 5-min i.v. infusion. The change in haemoglobin concentrations during chemotherapy are shown in Figure All eight patients had histologically proven and unresectable NSCLC. The detailed patient characteristics are listed in Table 2 in 100 mg/m2 increments. At the end of radio-/chemotherapy one of these patients developed a perpetual arrhythmia caused by hyperthyroidism, followed by a histologically proven atypical pneumonia caused by cytomegalovirus without leucopenia or neutropenia during the treatment. He died because of cardiopulmonary insufficiency.Six patients were included in a phase I study to identify the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of gemcitabine when administered in a 14-day interval (three times) in combination with radiotherapy which began 14 days after the induction chemotherapy. Radiotherapy was administrated with 10- or 15-MV photons. The total radiation dose to the initial volume was 50.0 Gy in 2.0 Gy fractions over 5 weeks. A boost dose of 10 to 16 Gy to the gross tumour volume (defined by FDG PET after induction chemotherapy) was given in the same dose fractionation. The gemcitabine dose ranged between 300 and 500 mg/mOne patient outside the phase I study suddenly died of a lung embolism after two cycles of chemotherapy, and one patient was initially excluded from the phase I study because of distant lymph node metastasis detected by FDG PET. The latter was being treated with two cycles of chemotherapy followed by radio-/chemotherapy, due to left primary bronchus compression. Following chemotherapy, one patient had decreased FDG- and FMISO uptakes in the primary tumour, whereas he had an increased FDG- and nearly unchanged FMISO-uptake in one large mediastinal lymph node metastasis. Here, both tumour localisations were evaluated separately as patient 7a (lymph node metastasis) and 7b (primary) Tables  and 3. AAll PET studies were carried out with a Siemens ECAT EXACT 922/47 scanner with an axial field of 16.2 cm of view . FMISO and FDG were produced at the Research Centre Juelich . Quality assurance was done according to the GMP principles. FMISO PET and FDG PET were performed in all patients within 3 days before as well as 14 days after finishing induction chemotherapy. The relevant field of view was defined with the help of computed tomography. FMISO PET consisted of one static scan of the relevant region, performed 180 min after intravenous administration of 329 ± 36 MBq FMISO. The acquisition and reconstruction parameters were as follows: Acquisition consisted of 30 min emission scanning and 4 min transmission scanning with 68-Ge rod sources. The emission data were corrected for attenuation through the use of a segmented μ-map and empirical attenuation coefficients.Following normalization and scatter correction, the emission scan was reconstructed with and without attenuation correction using a weighted iterative OSEM algorithm . In a final step, a three-dimensional isotropic Gauss-filter was applied (FWHM 8 mm). Transversal, coronal, and sagittal slices of 7 mm thickness were reconstructed with and without attenuation correction.Three venous blood samples were taken at minute 0, 5, and 10 of the emission scans. The radioactivity concentrations were measured with a well counter and corrected for decay. An FDG PET of the tumour region was performed 60 ± 12 min after intravenous administration of 250 ± 60MBq FDG, using a whole-body acquisition protocol with 8 min emission scanning and 4 min transmission scanning. The data were also corrected for attenuation and reconstructed with the same parameters as for FMISO PET above.For FMISO PET, tumour-to-muscle ratios of the regional radioactivity concentrations were calculated, using the transverse plane with the maximum FMISO uptake and manually drawn regions of interest of the tumour and the ipsilateral muscles. The tumour was defined according to the image data of FDG PET.Mean and maximum standardized uptake values (SUV) of the FMISO and FDG uptake of the tumour were calculated after normalization of the radioactivity concentration to the injected radioactivity and the body weight. Additionally, the mean SUV was corrected for partial volume effects by applying recovery coefficients obtained from phantom studies .Tumour response was evaluated by computed tomography (CT) of the chest and FGD PET , according to standard WHO criteria .Before and two weeks after chemotherapy treatment, mean and maximum standardized uptake values (SUV) of the FMISO and FDG as well as tumour-to-muscle ratios of FMISO were calculated for the tumour regions Table . The metUsing FDG PET, the metabolic responses 2 weeks after completing induction chemotherapy (two cycles gemcitabine/vinorelbine) were as follows: All five patients with a decreased FDG- and FMISO uptake had a partial remission (PR) after chemotherapy. One patient died after chemotherapy, due to a lung embolism. Evaluating tumour response 6 weeks after radio-/chemotherapy three patients had a complete remission (CR), and one had a PR after radio-/chemotherapy. There was a discrepancy between FDG PET and CT response in one patient, who had a CR in FDG PET and PR in CT after 6 weeks, but had a CR in both methods of diagnostic examination after 18 weeks. One patient developed tumour oxygenation near to muscle oxygenation reaching CR after radio-/chemotherapy. There was a discrepancy between the FMISO and FDG PET in one patient with a decreased FMISO uptake. However, there was an increase in the mean SUV and a decreased maximum SUV in FDG PET. This patient had a local PR after induction chemotherapy and after radio-/chemotherapy, but developed histologically proven kidney metastasis, as well as an early local tumour progression. One patient showed a decrease in FMISO uptake, but a high increase in the FDG uptake. This patient reached a stable disease (SD) after chemotherapy and a local PR, but in the same way, disseminated lung metastases and a solitary cerebral metastasis after radio-/chemotherapy Table .The patient (patient7a/b) who had different changes in FDG and FMISO uptakes in the primary tumour and in one large mediastinal lymph node metastasis after chemotherapy also showed a discrepant tumour response. Whereas there was a histologically proven CR (by bronchoscopy) of the primary tumour (decrease of FMISO and FDG uptake), there was no change in the extension of lymph node metastasis, where we found an increase of FDG values and only a small reduction in the FMISO tumour to muscle ratio with central accumulation of FMISO Figure .One patient with initial high FMISO uptake and decreased FMISO and FDG uptakes after chemotherapy showed a focal enhancement of FMISO at the chest wall, representing the localisation of early local recurrence.There was no association between tumour hypoxia detected by FMISO PET and tumour glucose use measured by FDG PET.A recent meta-analysis of 83 randomized clinical trials demonstrated a significant overall improvement in local control and survival with hypoxia modification . ExperimIt is recognized that nitroimidazoles bind selectively to hypoxic cells , leadingOur study was conducted in order to evaluate the feasibility of FMISO PET for the depiction of tumour oxygenation in NSCLC. Furthermore, we wanted to examine whether there is a reoxygenation after two-cycle chemotherapy, and whether there is an association between tumour metabolism or tumour hypoxia and tumour response or tumour recurrence. After chemotherapy, we were able to show a decrease in the FMISO uptake, reflecting reoxygenation in most patients, which resulted in a good tumour response. On the other hand, an increased (patient 6) or a nearly unchanged, high tumour to muscle ratio (patient 7a) corresponded to worse local tumour outcomes. But no association between initial high FMISO uptake and treatment outcome or tumour response could be detected.Although there was a significant decrease in Hb concentrations due to chemotherapy  influencing tumour oxygenation, we did not find a direct association between the reduction of the Hb concentration and FMISO or FDG uptake. Even the patient with an increased FMISO uptake showed stable Hb concentrations with an Hb of 13.4 g/dl at the time of second PET examinations (data not shown). Another patient showed a focal enhancement of FMISO at the chest wall after chemotherapy, representing the localisation of early local recurrence.Mac Manus et al. prospectively studied the capacity of FDG PET and computed tomography (CT) to determine the response soon after radio- or radio-/chemotherapy in patients with NSCLC. Both CT and PET responses were individually significantly associated with survival duration; but in the multifactor analysis, this included the known prognostic factors of CT response, performance status, weight loss, and stage; only the PET response was significantly associated with the duration of survival (p < 0.0001) .In another study, FDG PET imaging before and after induction therapy was prospectively evaluated in patients with oesophageal cancer to determine whether changes in PET images could measure the response to therapy. A comparison of the percentage decrease in SUV with the percentage of treatment effects through pathological examinations of oesophagectomy specimens indicated a correlation between large decreases in SUV and pathologic measurements of treatment effect .In our study, all five patients with a clear decrease in the FDG uptake had a PR after chemotherapy; two had a PR and even three a CR after radio-chemotherapy. On the other hand, patients with nearly unchanged or increased FDG values showed worse tumour control or disease-free survival; therefore, changes in tumour metabolism detected by FDG PET may predict the tumour response and outcome in NSCLC.The results of this prospective study elucidate that in patients with NSCLC changes in FDG PET may represent early response to therapy, and in this way, may predict freedom from disease and overall survival. In addition a qualitative and quantitative definition of hypoxic sub-areas is possible on the basis of FMISO PET, and that these sub-areas may correspond to the localization of local recurrences, making the definition of a biological target volume possible. Nevertheless, data on the role of FMISO PET scans for NSCLC are limited. In addition, the problem of image-fusion and the low spatial resolution restrict the use for target definition. Our feasibility study included only 8 patients. Therefore, these preliminary results warrant validation in larger trials.Financial competing interestsThe pharmaceutical company Hoffmann- La Roche partially financed the costs for FMISO, a substance used to carry out PET scans.The authors declare that there are no other competing interests.BG has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; PR has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; CD has made substantial contributions to conception and design; HJK has been involved in acquisition of data, analysis and interpretation of data; MZ has made substantial contributions to conception and design, acquisition of data; MP has been involved in acquisition of data; MP has been involved in acquisition of data; SS has been involved in analysis and interpretation of data; BA has been involved in acquisition of data; KH has been involved in analysis and interpretation of data and has made contributions to conception and design; HHC has been involved in analysis and interpretation of data and has made contributions to conception and design; UB has been involved in analysis and interpretation of data and has made contributions to conception and design; MJE has been involved in analysis and interpretation of data and has made contributions to conception and design.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG  = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent , unlabelled MAb E48 IgG  or 800 microCi 131I-labelled isotype-matched control MAb . A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.


Nitroimidazoles have been shown to be potent sensitisers of certain clinically active chemotherapeutic agents. This process of chemopotentiation has been shown to be hypoxia-mediated. The present studies evaluated whether increasing the level of hypoxia in the tumour tissue, by treatment with the vasoactive agent hydralazine, could modify the chemosensitising ability of nitroheterocyclics. Administering either misonidazole or RSU 1164 before, or hydralazine after, the chemotherapeutic agents melphalan, cyclophosphamide or the nitrosourea CCNU, increased the extent of cell kill in both the KHT sarcoma and RIF-1 tumour. However, even greater enhancements could be achieved when hydralazine was used in treatment protocols in which a nitroimidazole was combined with chemotherapy. For example, a 5.0 mg kg-1 dose of hydralazine given 30 min after melphalan, or a 2.5 mmol kg-1 dose of misonidazole administered 30 min before melphalan, increased, compared to melphalan alone, the resultant tumour cell kill by factors of approximately 1.9 and approximately 1.3, respectively. By comparison, when hydralazine was given after the melphalan plus misonidazole combination, treatment efficacy was enhanced approximately 3-fold compared to melphalan alone. Yet in contrast to the results of the tumour response studies, the inclusion of hydralazine did not increase the bone marrow toxicity associated with the chemotherapeutic agent when used alone or in conjunction with a nitroimidazole. The results, therefore, imply that the addition of hydralazine to the chemotherapy, or chemotherapy-sensitiser protocol, led to a therapeutic advantage.


Estraumustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, has been shown to exert cytotoxic effects on glioma cells in vitro. The drug uptake is assumed to depend on a specific estramustine binding protein (EMBP). One of the main difficulties in achieving cytotoxic effect in malignant brain tumours is believed to be due to the poor penetration of cytotoxic drugs into tumour tissue. In patients with malignant supratentorial brain tumours we have analysed the uptake of EMP metabolites in tumour tissue after oral administration and demonstrated EMBP in the same tissue specimens. Sixteen patients were given 280 mg EMP orally 14 h prior to surgery. Specimens from brain tumour tissue, cystic fluid, and serum were collected during surgery. Using gas chromatography the metabolites of EMP, estramustine (EaM) and estromustine (EoM), were quantified, EMBP was demonstrated by immunohistochemistry. The mean concentrations of EaM and EoM, expressed in ng g-1, were 60.3 and 38.4 in tumour tissue and 3.5 and 56.3 in serum, respectively. An accumulation of EaM in tumour tissue was found with a mean concentration gradient of 16.1 versus serum, while the gradient for EoM was 0.76. EMBP was demonstrated with a high degree of staining in all but one tumour. The high concentrations of EaM and EoM found in malignant brain tumour tissue correspond to potentially cytotoxic levels. The present results as well as the earlier in vitro demonstrated cytotoxic effects on glioma cells strengthen the possibility of a therapeutic effect of EMP in the treatment of malignant brain tumours.


SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species  representing four different cell lineages , and using four different drug classes . By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers . Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules.


An immunoperoxidase technique has been used to detect the in vivo binding of a 2-nitroimidazole hypoxia marker in histochemical sections of a variety of excised canine tumours. The binding occurred 10-12 cell diameters away from tumour blood vessels, consistent with the expected location of hypoxic cells in tissues in which oxygen concentration gradients are established by diffusion. Hypoxic fractions ranging from 4 to 13% have been estimated on the basis of morphometric analysis of multiple tumour sections. The binding of the marker was restricted to the cytoplasm of the cells. The marker appeared in regions adjacent to necrosis but also in regions free of necrosis. As in earlier autoradiography studies, binding was occasionally observed in cells adjacent to tumour blood vessels. Generally, binding to normal tissues was not observed. However, binding to smooth muscle cells surrounding arterioles in some sections of normal tissue and tumour tissue was observed.


DNA-synthetizing lymphocytes were identified in the lymph nodes regional and more distal to the site of developing P-815 tumours by incorporation of [3H]-thymidine followed by autoradiography of lymph node sections. It appeared that not only T but also B cell areas of draining and to a lesser extent of distal lymph nodes were stimulated by the growing tumour. This result was unexpected since neither humoral nor tumour cell-bound antibody could be identified so far as a functional correlate of B cell stimulation. In general the proliferative response of lymphocytes followed a biphasic pattern with an early peak of reactivity on days 2-3 and a second peak around day 12-15 after tumour cell inoculation. In the draining (axillary) lymph node the second peak of reactivity was suppressed, possibly as a consequence of metastatic tumour cells in this node when tumour cells were inoculated in the flank. The pattern of lymphocyte stimulation revealed larger individual variations after tumour cell inoculation in the flank than the foot pad. These results were associated with a slower and less regular drainage of carbon particles from the flank to the axillary and exceptionally the brachial lymph node than from the foot pad to the popliteal node after injection of India ink.


Tumour localisation of anti-tumour antibodies varies greatly between patients. Factors which may be responsible for this have been investigated in 56 patients with colorectal carcinoma with a view to improving radioimmunotherapy. Thirty-seven to seventy-four MBq of 125-I labelled mouse monoclonal antibody to CEA, was given intravenously and tumour resected 70-480 h later. Percentage injected activity kg-1 (% inj.act kg-1) in tumour, was inversely correlated with the time interval between injection and operation (P = 0.004). To assess the influence of other parameters on localisation, patients were divided into two time groups according to time interval between injection and operation, 70-120 h (n = 33) and 144-480 h (n = 23). In neither group was there a significant correlation of % inj.act kg-1 with time. The % inj.act kg-1 in tumour showed a significant correlation with that in the blood for both groups (P = 0.005 and P = 0.01). There was no significant correlation for either time group between % inj.act kg-1 in tumour and serum CEA values, the per cent of tumour cells positive for CEA and vascularity. Tumour to blood ratios varied considerably (range 0.3-28.5:1) suggesting that factors other than time and persistence of activity in the blood contribute to efficient targeting. Tumour to blood ratios were inversely correlated with % inj.act kg-1 in blood for the 70-120 h group (P = 0.007), and were positively correlated with % inj.act kg-1 in tumour (P = 0.012). Autoradiography showed that antibody localised predominantly on tumour cells but was distributed heterogeneously, was not solely related to the expression of antigen and in some cases accumulated in necrotic more than viable areas of tumour. Penetration of antibody into malignant acinar structures was poor and CEA-positive cells closer to the blood supply were targeted to a greater extent than distant cells. Preoperative administration of radiolabelled antibody to CEA may be helpful in selecting patients with favourable localisation for radioimmunotherapy.


The effect of photodynamic therapy (PDT) on interstitial fluid pressure (IFP), tumour volume and water content was measured in melanomas grown in hamsters. Unlike control tumours, treated tumours exhibited a 40-60% increase in volume at 1, 3 and 6 h post PDT. IFP also increased at 1 and 3 h after PDT, but decreased to 50% of control value after 24 h, presumably as a result of PDT-induced microcirculatory impairment.


The characteristics of a method for measuring cyclic AMP binding proteins in cytosols of human breast cancer are described. Using the assay, binding proteins were demonstrable in all of 100 tumour cytosols. Levels of binding in individual tumours varied from 0.8 to 15 pmol mg-1 cytosol protein  and the dissociation constant ranged from 0.5 to 5.2 X 10(-8)M (mean 1.73 X 10(-8)M). Whilst replicate measurements within a single portion of tumour were reproducible (intra-assay coefficient of variation was between 4.5 and 7.8% and that for inter-assay variation was between 2.1 and 4.0%) there were often considerable differences in levels of binding proteins between different portions of the same tumour. Similar intra-tumour variations have been reported for other binding proteins and steroid receptors. The inter-relationships with such parameters may elucidate whether the differences are associated with variations in cellularity, cell type, or other specific factors.


Esophageal Doppler provides a continuous and non-invasive estimate of descending aortic blood flow (ABF) and corrected left ventricular ejection time (LVETc). Considering passive leg raising (PLR) as a reversible volume expansion (VE), we compared the relative abilities of PLR-induced ABF variations, LVETc and respiratory pulsed pressure variations (ΔPP) to predict fluid responsiveness.We studied 22 critically ill patients in acute circulatory failure in the supine position, during PLR, back to the supine position and after two consecutive VEs of 250 ml of saline. Responders were defined by an increase in ABF induced by 500 ml VE of more than 15%.p = 0.15). A PLR-induced increase in ABF of more than 8% predicted fluid responsiveness with a sensitivity of 90% and a specificity of 83%. Corresponding positive and negative predictive values  were 82% and 91%, respectively. A ΔPP threshold value of 12% predicted fluid responsiveness with a sensitivity of 70% and a specificity of 92%. Corresponding PPV and NPV were 87% and 78%, respectively. A LVETc of 245 ms or less predicted fluid responsiveness with a sensitivity of 70%, and a specificity of 67%. Corresponding PPV and NPV were 60% and 66%, respectively.Ten patients were responders and 12 were non-responders. In responders, the increase in ABF induced by PLR was similar to that induced by a 250 ml VE (16% versus 20%; The PLR-induced increase in ABF and a ΔPP of more than 12% offer similar predictive values in predicting fluid responsiveness. An isolated basal LVETc value is not a reliable criterion for predicting response to fluid loading. In sedated, mechanically ventilated patients, fluid responsiveness can be efficiently predicted by assessing the respiratory changes in arterial pressure. However, in some patients it might be attractive to use a reversible maneuver that mimics a fluid challenge and to assess its hemodynamic consequence directly.Esophageal Doppler (ED) provides a continuous measurement of the descending aortic blood flow (ABF), which constitutes a reliable indicator of global cardiac output . This dePassive leg raising (PLR) is a simple reversible maneuver that mimics a rapid fluid loading. It transiently and reversibly increases venous return by shifting venous blood from the legs to the intrathoracic compartment ,7. PLR iTherefore, the aim of our study was to evaluate and compare the relative ability of variations of ABF during PLR, LVETc and ΔPP to predict fluid responsiveness in mechanically-ventilated patients with acute circulatory failure requiring VE.The study patients were (1) intubated, mechanically ventilated in a volume-controlled mode, and fully sedated, (2) in acute circulatory failure, (3) receiving stable doses of vasopressive drugs, (4) monitored by ED and a radial or femoral arterial catheter and (5) requiring VE according to the attending physician.Acute circulatory failure was defined as (1) a systolic blood pressure lesser than 90 mmHg (or a decrease of more than 50 mmHg in previously hypertensive patients) or the need for vasopressive drugs, (2) a urine output below 0.5 ml/kg/minute for at least two hours, (3) tachycardia (heart rate > 100 bpm) and (4) the presence of skin mottling.Fluid loading requirements were based on the presence of at least one clinical sign of acute circulatory failure and/or associated signs of visceral hypoperfusion, including signs of renal dysfunction, hepatic dysfunction, and/or increased arterial blood lactate, in the absence of a contraindication for a fluid challenge. Contraindication for a fluid challenge was defined as a life-threatening hypoxemia and by the evidence of blood volume overload and/or of hydrostatic pulmonary oedema. Patients with spontaneous breathing activity, cardiac arrhythmias or patients having contraindication for the use of ED  were excluded, as were patients with incapacity to practice PLR.ED measurements were obtained by using the Hemosonic 100™ device . This device enables continuous measurement of descending thoracic aorta blood velocity (Doppler transducer) and measures the real aortic diameter (M-mode echo transducer) . ABF is max) and minimum (PPmin) values were determined over a whole respiratory cycle. ΔPP was calculated as previously described [max ΔPPmin)/([PPmax + PPmin]/2)}. Three respiratory cycles were used and averaged to calculate ΔPP.Pulse pressure is the difference between systolic and diastolic arterial pressure. Maximum (PPescribed : ΔPP (%)The protocol sequence is shown in Figure 1. Twice over two minutes at one-minute intervals, in the supine position (Base 1).2. Four times over four minutes at one-minute intervals, during PLR (lower limbs were lifted in a straight manner by an assistant to a 45°).3. Twice over two minutes at one-minute intervals, in the supine position (Base 2).4. Once after a fluid loading of 250 ml of 0.9% sodium chloride (VE 250 ml).5. Once after a second fluid loading of 250 ml of 0.9% sodium chloride (VE 500 ml), which was begun immediately after the first 250 ml of fluid loading.The ED probe was repositioned if aortic blood velocity or aortic diameter signals were lost during the procedure. No treatments (such as ventilatory settings or vasopressive drugs dosage) that might alter hemodynamic status were given during the study period.Because ED monitoring and VE are part of routine care in patients with acute circulatory failure treated in our unit, French law authorizes the conduct of this kind of observational study without informed consent. Whenever possible, each patient or next of kin was informed and consented to the use of registered data.After completion of the study protocol, patients were divided into two groups: responders and non-responders to fluid loading. A patient was classified as a responder when an increase of more than 15% in ABF was induced by fluid loading between Base 2 and VE 500 ml. Base 1, PLR and Base 2 sequences contain two or four consecutive hemodynamic measurements. An average of these multiple measurements was calculated for each sequence and used in the statistical analysis.U test. Linear correlations were tested with the Spearman rank method. Receiver operating characteristic (ROC) curves were generated for PLR-induced changes in ABF, LVETc and ΔPP by varying the discriminating threshold of each parameter. The area under each ROC curve (AUC) was calculated and expressed as AUC ± SD. p < 0.05 was chosen as being significant. Statistical analysis was performed with SPSS version 12.0 .Given the small number of patients, results are expressed as median (interquartile range) or number and percentage. Non-parametric tests were used for comparisons. The Friedman test followed by the Wilcoxon rank sum test was performed to detect changes over time (before and after PLR and VE) within the same group (responder or non-responder). The comparisons of hemodynamic parameters between responders and non-responders were assessed with the Mann–Whitney n = 6), ischemic cardiomyopathy (n = 8), dilated cardiomyopathy (n = 1), chronic obstructive pulmonary disease (n = 4), chronic renal failure (n = 1) and diabetes (n = 2).Twenty-three patients were screened for inclusion, and 22 were studied; their clinical characteristics are reported in Table Ten patients were responders (increase in ABF of more than 15% induced by VE between Base 2 and VE 500 ml), and 12 were non-responders. Hemodynamic data for responders and non-responders are presented in Tables p = 0.03) and increased significantly during PLR  l/minute versus 4.2 3.3 to 4.9) l/minute; .3 to 4.9r2 = 0.5, p < 0.0001; Figure p = 0.15). The most discriminant value of changes in ABF that was able to predict the absence or presence of fluid responsiveness was assessed with the ROC curve  versus 9% (5 to 10%),  to 17% vp = 0.10; Figure Base 1 LVETc was not statistically different between responders and non-responders (232 ms (209 to 272 ms) versus 259 ms (242 to 289 ms), As shown in Figure Our study demonstrates mainly that fluid responsiveness in mechanically ventilated patients with acute circulatory failure can be efficiently predicted by assessing the effects of PLR on ABF monitored by ED. We found that the PLR maneuver had a predictive value similar to that of a respiratory variation in pulse pressure greater than 12%. In contrast, an isolated basal LVETc value is not a reliable criterion for predicting response to fluid loading.As underlined by recent recommendations, dynamic criteria are better predictors of volume responsiveness than static criteria . In thisWe found that a ΔPP threshold value of 12% offers the best sensitivity:specificity ratio to predict fluid responsiveness . However, the PPV and NPV of ΔPP  were not as high as previously described: Michard and colleagues  found a r = 0.52) than between thermodilution-measured cardiac output and pulmonary capillary wedge pressure (r = 0.2). More recently, Seoudi and colleagues [r = 0.49) than between pulmonary artery diastolic pressure and end-diastolic short-axis area (r = 0.10). Our findings are in accordance with those of Singer and colleagues.For several authors, LVETc may constitute an index of left ventricular preload. Singer and colleagues ,21 inveslleagues  also suglleagues  found a In responder patients, LVETc increased during PLR and VE but did not change in non-responders. However, we found an isolated basal LVETc value to be a poor predictor of ABF response to fluid loading. In a recent study of critically ill patients with acute circulatory failure, Monnet and colleagues  showed tOur study does have some limitations. The results were obtained from only a small number of patients and the study is underpowered to permit a definitive conclusion. With regard to the power of our fluid challenge to discriminate between responders and non-responders, we infused only 500 ml of crystalloids; other authors have used larger amounts of fluids. In addition, considering that vasopressors were used in all these patients, several of them might have been classified as non-responders just because they did not receive sufficient fluids to affect their preload . It is pOur data support the non-invasive assessment of ABF variations provoked by a PLR manoeuver in the prediction of fluid responsiveness in mechanically ventilated patients with acute circulatory failure. The predictive value of this test is comparable to that of ΔPP. In contrast, an isolated static LVETc value furnished by ED devices is not a reliable criterion for predicting response to fluid loading.• PLR is a simple reversible maneuver that mimics a rapid fluid loading.• We found that a PLR-induced increase in ABF greater than 8% predicted the response to subsequent VE with a sensitivity of 90% and a specificity of 83%.• An isolated basal LVETc value is not a reliable criterion for predicting response to fluid loading.• Fluid responsiveness in mechanically ventilated patients with acute circulatory failure can be efficiently predicted by assessing the effects of PLR on ABF monitored by ED.ABF = aortic blood flow; AUC = area under the receiver operating characteristic curve; ΔPP = respiratory pulse pressure variation; ED = esophageal Doppler; LVETc; left ventricular ejection time corrected for heart rate; NPV = negative predictive value; PLR = passive leg raising; PPV = positive predictive value; ROC = receiver operating characteristic; SV = stroke volume; VE = volume expansion.The authors declare that they have no competing interests.AL and AC designed the study. AL, FP, CG, AD, VM and GC realized the experiments. JDC, JPM and AC contributed to data collection. AL, FP and AC wrote the manuscript. All authors participated in its critical revision. AC had full access to all data in the study and had final responsibility for the decision to submit for publication. All authors read and approved the final manuscript.


Photofrin II  was labelled with indium-111 and its biodistribution in tumour bearing mice compared with that of 111In chloride. The uptake and clearance of 111In labelled DHE differed markedly from that of indium-111 chloride in that the former was not taken up by the tissues as much as the latter. Scintillation scanning with a gamma-camera showed marked uptake of both 111In agents at the site of the tumour, but a much lower tissue background  for the mice given 111In DHE. Tumour:muscle ratios of dissected tissues were 2-3 times higher in 111In DHE treated animals as compared to the uptake of 111In chloride. There was a distinct difference in the pattern of distribution of the two 111In preparations in the tissues. The major accumulation of 111In chloride was in the kidneys, whereas the highest uptake of 111In DHE was in the liver, the organ in which unlabelled porphyrins accumulate. Extraction and testing of materials from tumours of 111In DHE treated animals indicated that most of the tumour extractable 111In had remained associated with the porphyrin in vivo up to 4 days after injection.


As yet, there is no reported study of chromosome allele loss in fibrolamellar carcinoma (FLC), a distinct, rare variant of hepatocellular carcinoma (HCC). We searched for evidence of allele loss in FLC using 18 DNA probes for 10 chromosomes and compared the pattern of loss with our series of HCC. Two of the probes, lambda MS32 (1q42-43) and cMS621 (5p) showed allele losses in one tumour, while other probes showed no loss. The frequency of allele loss in FLC was much lower than in HCC, which may be associated with their different prognoses.


Four patients underwent intraoperative photodynamic therapy after surgery with meso-tetra-(hydroxyphenyl)-chlorin (mTHPC-PDT) for diffuse malignant mesothelioma. Preliminary procedures were performed in two patients in order to establish the efficacy of mTHPC-PDT and to optimise its tumoricidal effect. The tumoricidal effect was related to the mTHPC dose, light dose and the time interval between sensitation and activation. 0.3 mg kg-1 mTHPC activated after 48 h with 10 Joules cm-2 of non-thermal laser light at 650 nm resulted in a 10 mm deep tumour infarction, due to tumour vessel necrosis and thrombosis. The mTHPC tissue concentration was up to 14 times higher in the tumour than in normal tissues. Skin photosensitivity was mild, dose dependent and occurred 3 to 10 days after administration of mTHPC. According to the results obtained, intraoperative mTHPC-PDT was performed following pleuropneumonectomy in two, pleurectomy and lobectomy in one and pleurectomy in one patient. Ten Joules cm-2 were delivered to the diaphragm and the costophrenic sulcus and 5 Joules cm-2 to the remaining thoracic cavity. The postoperative course was marked by loss of appetite, fluid retention, hypoproteinemia and severe chest pain. One patient succumbed from aspiration pneumonia. The remaining patients developed no neural or vascular alterations and no bronchial stump insufficiency during follow-up. mTHPC-PDT following surgical tumour resection deserves further evaluation in good risk patients with diffuse malignant mesothelioma.


Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in vitro and in vivo. BCA has a major absorption peak at 760 nm where tissue penetration is optimal. This property, together with rapid tissue clearance promises minor skin photosensitivity. The tissue localising and photodynamic properties of BCA were studied using isogeneic RMA mammary tumours, transplanted into subcutaneous tissue in transparent 'sandwich' observation chambers on the back of WAG/Rij rats. The fluorescence kinetics following an i.v. administration of 20 mg kg-1 BCA was assessed in blood vessels, tumour and normal tissue. Subsequently, the development of vascular- and tissue damage after a therapeutic light dose  was observed. Fifteen minutes post injection (p.i.), the fluorescence of BCA in the tumour reached a plateau value of 2.5 times the fluorescence in the normal tissue. From 1 h post injection the tumour fluorescence diminished gradually; after 24 h, the tumour fluorescence signal did not exceed that of the normal tissue. Following photodynamic therapy (PDT), 24 h p.i., complete vascular stasis was observed 2 h post treatment in the tumour only, with subsequent recovery. The presence of viable tumour cells following PDT was assessed by histology and re-transplantation of treated tumour tissue from the chamber into the flank immediately or 7 days after treatment. In both cases tumour regrowth was observed. BCA-PDT  1 h after BCA administration, an interval which gives the optimal differential between tumour and normal tissue, was sufficient to prevent tumour regrowth. However, this only occurred when re-transplantation was performed 7 days after PDT. During PDT, 1 h p.i., vascular damage in tumour and normal tissue was considerable. Complete vascular shut-down was observed in the tumour 2 h after therapy and in the surrounding tissues at 24 h. Circulation damage was associated with vascular spasm and occlusion probably due to thrombi formation. Oedema was notable, especially following PDT with 600 J cm-2 at 24 h p.i.


The deuterated benzaldehyde derivative zilascorb(2H), 5,6-O-benzylidene-d-L-ascorbic acid, was administered once daily by i.v. injection in nude mice with grafted tumours of a human malignant melanoma (E.E.) and ovarian carcinoma (OVCAR-3) origins. Like benzaldehyde, zilascorb(2H) has been shown to induce protein synthesis inhibition at otherwise non-toxic doses in cells grown in vitro, and acts reversibly in the sense that protein synthesis returns to normal shortly after removal of the drug. The present data indicate that daily injections with zilascorb(2H) induce a tumour volume growth inhibitory effect in both tumour xenografts studied. Furthermore, from histological examinations of each single tumour it was found that tumours of drug-treated animals, although smaller than those of placebo-treated (i.e. control) animals, had, on average, a higher necrotic fraction than control tumours. Thus, it is concluded that zilascorb(2H) induces tumour necrotisation and not just inhibition of the rate of tumour cell production. Continued measurement of tumour volume after ended treatment with zilascorb(2H) indicated that surviving tumour cells resumed their normal growth rate immediately. The reversibility of the effect induced by this compound, earlier observed in vitro only, is therefore here confirmed to be valid also in two different tumour xenografts in vivo. The present data accords well with the assumption that protein synthesis inhibition is the primary cellular effect of zilascorb(2H) in vivo. We therefore conclude that zilascorb(2H)-induced cancer cell lethality in tumour xenografts probably comes as a secondary consequence of prolonged protein synthesis inhibition.


The presence of cancer makes it difficult to predict the progress of pregnancy and can be deleterious to the maternal-foetal relationship. Apoptosis may affect a range of placental functions and result in the retardation of foetal growth. In this work, we investigated the placental alterations produced by tumour growth and the effects on the expression of apoptotic factors in placental tissue.th, 19th or 21st day of pregnancy. Placental tissues were analysed using biochemical and histochemical assays.Adult female Wistar rats  were allocated to control (C), tumour-bearing (W), or ascitic fluid-injected (A) groups and were killed on the 16The placental protein content and glutathione-S-transferase activity were decreased in groups W and A. Histochemical analysis showed an increase in the number of cells with cleaved PARP, caspase 3 and cytochrome-c in groups W and A, indicating that the tumour growth clearly damaged placental tissue and affected the levels of apoptotic factors. These results were confirmed by western blotting.Since trophoblastic cells are responsible for maintaining a normal placental function, the uncontrolled death of these cells in response to tumour cell growth or substances derived from ascitic fluid could have a negative impact on foetal development. Further knowledge of these events may help to preserve the foetus and placenta during development. Cancer is the second most common cause of death during the reproductive years  and compThe foetal nutrient supply depends on the mother's reserves and food intake, as well as on the placental function. Placental growth increases during gestation, concomitant with or ahead of foetal growth , and a fThe placenta-foetal metabolic relationships are complex, dynamic processes that control many aspects of foetal development. The exchange between maternal and foetal blood occurs across the labyrinth layers of the placental barrier. In rats, the placenta, which consists of three trophoblast layers in the labyrinth zone, is the rate-limiting permeability barrier to substrate exchange between the maternal and foetal compartments .Programmed cell death has been implicated in normal and pathological processes in several human tissues and diseases, including cancer ,12. PlacDuring apoptosis, the activation of caspases by nuclear, metabolic or external stimuli occurs in a cascade fashion, and results in nuclear engulfment and cell death ,22. SincAdult female Wistar rats  were obtained from the animal facilities of the State University of Campinas (CEMIB-UNICAMP) and were housed with adult males  for 12 h . Mating 5 in approximately 0.5 mL of 0.9%, w/v, NaCl), originally obtained from the Arthur D'Little Hospital, USA, were injected subcutaneously on the second day of pregnancy. Control rats received 0.5 ml of 0.9% NaCl solution. Since we have previously shown that ascitic fluid from tumour-bearing rats produces foetal death and resorption  goat polyclonal antibody  followed by incubation with biotinylated rabbit anti-goat secondary antibody  for 1 h at room temperature and further incubation with streptavidin horseradish peroxidase solution (Vector Laboratories). The sections were reacted with diaminobenzidine  and H2 each) in one slide from each of at least six dams per group.The cells positive for cleaved PARP, caspase-3 and cytosolic cytochrome-c were counted using Image Pro-Plus software  after capturing the image on a Leica microscope  using a 100× magnification for cleaved PARP images and a 40× magnification for caspase-3 and cytochrome-c images. The number of positive cells was determined by counting 20 fields  were homogenised in buffer  and centrifuged at 10,000 × Bradford . The homBradford . WesternThe results were expressed as the mean ± SEM. Inter- and intragroup comparisons were done using one-way analysis of variance (ANOVA)  followedThe foetal and placental ratio was altered by tumour growth and the injection of ascitic fluid. Figure The placental protein levels of the control groups were similar, regardless of the gestational stage . In the tumour groups, the protein content was significantly lower (P < 0.05) than in the corresponding control only for W21 . A significant decrease in the protein content  was seen in all groups injected with ascitic fluid .th day may be related to the maximal placental function at this time interval, as previously reported [Glutathione-S-transferase (GST) activity is associated with the cell cycle and is responsible for the conjugation and detoxification of intermediates produced by oxidative stress in rat placenta . There wreported . Tumour-reported ,30.th and 21st days  compared to the control group. These data suggest that the presence of ascitic fluid decreases the protective action of anti-oxidative enzymes such as GST.Similar data were obtained in the ascitic fluid group, in which GST activity was reduced on the 19th day of pregnancy). The labyrinth zone is not shown since the alterations in this zone were the same as those in the junctional zoneThe placental histological layer investigated here was the junctional zone, which contains cytotrophoblastic cells, trophoblastic giant cells and vesicular glycogen cells   during apoptosis . Immunohcy Table .Cytochrome-c showed a similar distribution in the cytosol of placental cells during different stages of pregnancy in the control groups   in placental tissue was significantly enhanced on the 21y Figure , Table 1y Figure . The prid Figure , suggests Figure .The nutritional demand of tumour cells is extremely high  and may An optimal maternal-foetal exchange is necessary for a successful pregnancy. Complications associated with pregnancy, including limited intrauterine growth and pre-eclampsia, have been related to poor trophoblast invasion and/or placental insufficiency that may result in placental ischemia and oxidative stress . HoweverAs shown here, there was a marked reduction in the GST activity of placental tissue in groups W and A. Assuming that tumour growth during pregnancy induces foetal resorption and death  and jeopTumour growth clearly damaged the placental tissue by altering the foetal/placental ratio and the levels of apoptotic signalling molecules such as cleaved PARP, cytochrome-c and caspase 3. Tumours can damage foetuses  through competition for nutrients and by harming the foetus and placenta indirectly through substances synthesized by the tumour and/or host cells present in ascitic fluid . ApoptosNormal placental development depends upon the differentiation and invasion of the trophoblast, the main cellular component of the placenta. Apoptosis in the trophoblast increases in normal placentas as gestation proceeds, with a higher incidence in pregnancies complicated by preeclampsia or IUGR . ApoptosIn agreement with these findings, we observed increased caspase 3 expression in groups W and A that may have been initiated by the release of cytochrome-c into the cytosol . Indeed,Since trophoblastic cells are responsible for maintaining a normal placental function, the association to tumour growth and/or substances produced by tumour cells, such as cytokines and factors present in ascitic fluid, could be involved in the uncontrolled death of trophoblastic cells that have a negative impact on foetal development. Further work are underway in our laboratory to improve our understanding of the events that occur at the maternal-foetal interface in the presence of tumours, especially with regard to the role of cytokines present in ascitic fluid. Such knowledge may help to improve the welfare of the foetus and placenta during tumour development.The author(s) declare that they have no competing interests.Author 1 M.T.T. carried out the experiments, biochemical and immunohistochemical assays and performed the statistical analysis.Author 2 G.V. carried out the biochemical assays and performed the statistical analysis.Author 3 M.C.C.G.M. conceived of the study and participated in its design and coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The mechanism of tumour necrosis photosensitised by liposome-delivered Zn(II) phthalocyanine (Zn-Pc) has been studied in mice bearing a transplanted MS-2 fibrosarcoma. Ultrastructural analyses of tumour specimens obtained at different times after red light-irradiation  indicate an early (3 h) photodamage of malignant cells especially at the level of the mitochondria and rough endoplasmic reticulum. The cellular damage becomes more evident between 6 h and 15 h after photodynamic therapy. On the other hand, the capillaries supplying the tumour tissue are modified at a much slower rate and appear to be severely damaged only after 15 h from irradiation, when the whole tissue becomes necrotic. Occasionally, mildly damaged capillaries are observed even at 72 h after irradiation. These findings support the hypothesis that low density lipoproteins (LDL) play a major role in the delivery of Zn-Pc to the tumour tissue; the photosensitiser is released specifically to malignant cells as a consequence of a receptor-mediated endocytosis of LDL.