MTHFR C677T, MTR A2756G, TS tandem repeat polymorphism) involved in folate metabolism and potentially in sensitivity to 5-FU-based chemotherapy. The genotypes were correlated with tumour response to polychemotherapy, radiochemotherapy and with overall survival. Tumours with the MTR wild-type genotype (2756AA) showed a median survival time of 16 months, whereas tumours with an MTR variant genotype (2756AG/2756GG) showed a median survival time of 42 months (P=0.0463). No prognostic impact could be verified for the genotypes of the MTHFR genes and the TS gene. Among tumours treated with radiochemotherapy and subsequent resection, MTR variant genotype showed higher histopathological response rate than tumours with MTR wild-type genotype (P=0.0442). In contrast, no significant relationship between clinically determined tumour regression after polychemotherapy and polymorphisms of the three genes under analysis was observed. In conclusion, pretherapeutic determination of the MTR A2756G polymorphism may predict survival of multimodally treated oesophageal squamous cell carcinomas. Determination of MTHFR C677T and TS tandem repeat polymorphism has no predictive value.The present study retrospectively examined the correlation between the outcome of patients with locally advanced oesophageal squamous cell carcinoma (cT3-4 cN0-1 cM0) after multimodal treatment , and the presence of genetic polymorphisms in genes involved in folate metabolism. In total, 68 patients who took part in a prospective multicentric trial received 5-fluorouracil (FU)-based radiochemotherapy, optionally followed by surgery. DNA was extracted from pretherapeutic tumour biopsies and was subsequently genotyped for common genetic polymorphisms of three genes ( In recent years, the prognosis of patients with oesophageal squamous cell carcinoma has only slightly improved. The results of surgical therapy have been poor, with 5-year survival rates varying between 9 and 40%, even in resectable stages . TherefoMTHFR gene is highly polymorphic in the general population. The major nucleotide 677 polymorphism (C to T) results in an alanine to valine substitution, which alters enzyme activity  have about 65% enzyme activity (Methionine Synthase (MTR) gene contains a common polymorphism at bp 2756 (MTR A2756G), which causes an amino-acid substitution from aspartic acid to glycine at codon 919 (D919G). Individuals with the 2756GG genotype have lower plasma homocysteine (or higher folate) levels than those with the 2756AA genotype (Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) are key enzymes in folate metabolism. The substrate for MTHFR, 5,10-methylene-tetrahydrofolate (THF) is used for conversion of dUMP to dTMP by the enzyme thymidylate synthase (TS). The enzyme product of MTHFR, 5-methyl-THF, serves as carbon donor for the remethylation of homocysteine to methionine, which is catalysed by the enzyme MTR  Choi et. The MTHactivity . The MetMTHFR C677T polymorphism and/or the MTR A2756G polymorphism may have an effect on the response of cancer cells to 5-fluorouracil (5-FU), a chemotherapeutic agent frequently used in the neoadjuvant treatment of oesophageal squamous cell carcinoma. This is because 5-FU can form a ternary complex involving 5-fluoro-2′-deoyuridine-5′-monophosphate (5-FdUMP), TS, and 5,10-methylene-THF. The formation of this complex inhibits TS activity, which subsequently depletes intracellular thymidylate levels and ultimately suppresses DNA synthesis. Therefore, the MTHFR C677T polymorphism or the MTR A2756G polymorphism, which increase the intracellular concentrations of 5,10-methylene-THF, may increase the cytotoxic effect of 5-FU by increasing the formation of the 5,10-methylene-THF-TS-5-FdUMP ternary complex. The potential relevance of this mechanism has been recently shown in vitro where in colon cancer cell lines the MTHFR 677T polymorphism was associated with increased to sensitivity to 5-FU treatment  responded better to 5-FU-based chemotherapy than patients with the wild-type MTHFR allele , three tandem repeats (3R/3R) or a heterozygous (2R/3R) genotype  or by transthoracal oesophageal resection. Postoperative treatment was not performed.−2, etoposide 100 mg m−2, bolus 5-FU 500 mg m−2, and cisplatin 30 mg m−2 on days 1–3, every 3 weeks (FLEP protocol).Chemotherapy consisted of folinic acid 300 mg m−2 on days 2 and 8, and etoposide 100 mg m−2 on days 4–6.Combined radiochemotherapy was administered on days 22–28 of the last course of chemotherapy. The oesophagus was irradiated using parallel-opposed anterior and posterior fields and photons from a 10- to 15-MV linear accelerator. A total dose of 40 Gy was given in daily fractions of 2 Gy, five times per week. During the first days of irradiation, the following chemotherapy was administered: cisplatin 50 mg mResection of the oesophagus and the proximal stomach was performed by a combined right thoracal and abdominal approach. Resection included excision of the paraoesophageal, paracardial, left gastric, and celiac lymph nodes.Response to chemotherapy was evaluated clinically after the third course and included barium oesophagogram, oesophagoscopy, and computed tomography of the chest and abdomen. Response was categorised as follows: CR – normal barium oesophagogram, no visible tumour by oesophagoscopy, biopsies free of tumour tissue, and normal CT; partial response (PR) – greater than 50% tumour regression as evaluated by CT, and greater than 50% reduction of intraoesophageal tumour extension as assessed by barium swallow and oesophagoscopy; no change (NC) – less than 50% regression of tumour extension, and no evidence of tumour progression; progressive disease (PD) – increasing tumour obstruction indicated by barium swallow or oesophagoscopy, and increasing tumour diameter assessed by CT.Response to radiochemotherapy was evaluated by histopathological examination of cases that underwent transthoracal oesophageal resection. In this evaluation, complete responders (no viable tumour cells in the resection specimens) were separated from cases were incomplete or no tumour regression was detectable.Tumour biopsies that had been endoscopically obtained during pretherapeutic staging procedures were retrieved from the files of pathologic institutes associated with the medical centers that took part in this study. Of the 68 patients, 60 were male and eight were female. The median age was 57 years (range: 37–70). A total of 58 tumours had been clinically categorised as T3 and 10 tumours had been categorised as T4; 15 cases were in category N0 and 53 cases were in category N1. Histologic slides of the biopsy specimens were stained with hematoxylin and eosin for the determination of tumour type and tumour grade according to the WHO criteria. Three tumours were graded as G1, 34 tumours were graded as G2, and 31 tumours were graded as G3.MTHFR C677T genotype was determined by real-time fluorescence PCR and melting curve analysis using a LightCycler instrument . PCR conditions, primers and probes and melting curve analysis were applied as previously described . The MTR A2756G genotype. The primers were forward 5′-GAA CTA GAA GAC AGA AAT TCT CTA-3′ and reverse 5′-CAT GGA AGA ATA TGA AGA TAT TAG A-3′ ; and the amplified fragment of 189 bp is cut into fragments of 159 and 30 bp in the presence of the G allele.A PCR-RFLP assay was used to determine the The promoter region of the TS gene was amplified by PCR using the following primers: forward 5′-AAAAGGCGCGCGGAAGGGGTCCT-3′ and reverse 5′-TCCGAGCCGGCCACCAGGCAT-3′  was performed by means of the two-tailed Fisher's exact test. Therefore, the parameters were dichotomised as follows: tumour response (CR/PR vs NC/PD), MTHFR (CC vs CT/TT), MTR (AA vs AG/GG) and TS (2R/2R vs 2R/3R/3R/3R). The Kaplan–Meier method for analysis of censored data was used for calculating survival rates and tested with the log-rank test and the Wilcoxon test. Probabilities <0.05 were regarded as statistically significant.SAS software package  was used for statistical analyses. Statistical analysis of the correlation between tumour regression following chemotherapy and genotypes (n=20). Among these cases, seven (35.0%) showed complete regression without viable residual tumour cells, whereas in the other 13 (65.0%) cases either incomplete regression or no histological signs of tumour regression were present.Following FLEP chemotherapy, three patients (4.4%) received CR, 28 PR (41.2%), 32 NC (47.1%) and five PD (7.3%). Data on histopathological response evaluation after radiochemotherapy was available only in a subset of patients that underwent transthoracal oesophageal resection (MTHFR gene (98.5%), 64 for the MTR gene (94.1%) and 57 for the TS gene (83.8%).In total, 67 samples could be genotyped successfully for the MTR gene polymorphism. Tumours with an MTR variant genotype (2756AG/2756GG) showed complete histopathological regression in 46.2% of cases whereas tumours with the MTR wild-type genotype (2756AA) showed complete regression in 0% of cases (P=0.0442 according to the χ2 test). No correlation between response to radiochemotherapy and MTHFR and TS genotypes was found.No significant relationship between tumour regression after FLEP chemotherapy and polymorphisms of the three genes under analysis was observed . In contof cases . , 14 of the remaining 67 patients (20.9%) were still alive. The follow-up time for all 67 patients ranged from 4 to 114 months (median: 22 months). The follow-up time for the 14 patients at risk ranged from 29 to 114 months (median: 83 months).MTR wild-type genotype (2756AA) showed a median survival time of 16 months whereas tumours with an MTR variant genotype (2756AG/2756GG) showed a median survival time of 42 months .All patients received complete treatment. Of the 67 patients suitable for survival analyses, 38 received definitive radiochemotherapy and 29 were treated by radiochemotherapy and subsequent oesophageal resection. Analysing all 67 patients, tumours with the 2 months  (P=0.046MTHFR genes and the TS gene either in the group as a whole (No prognostic impact could be verified for the genotypes of the  a whole  or when MTR 2756AG/2756GG genotypes may have better survival than individuals with the MTR 2756AA genotype. Mechanistically, this may make sense taking into account the putative effect of this polymorphism on folate metabolism. Thus, MTR catalyses the transfer of the methyl base from 5-methyl THF to homocysteine, thereby producing methionine and THF. THF is the precursor of 5′-formyl THF, which is an inhibitor of thymidylate synthase in cooperation with 5′-FdUMP, which is a metabolite of 5′-FU. Therefore, an increased activity of the MTR gene may lead to an accumulation of 5′-formyl THF and thereby to a pronounced cytotoxic effect of 5′-FU treatment. The MTR 2756GG polymorphism was initially thought to be associated with lower enzyme activity than the MTR 2756AA genotype (The current study indicates that multimodally treated oesophageal squamous cell carcinoma patients with the MTHFR C677T polymorphism on survival. If the hypothesis is true that accumulation of 5′-formyl THF increases the effect of 5′-FU-based therapy, this observation is difficult to understand. This is because the variant genotype of the MTHFR gene is characterised by less effective catalyzation of 5′10′-methylene THF to 5′methyl THF and thus to an accumulation of 5′-formyl THF. However, one clearly has to take into account that a large variety of other factors than genetic polymorphisms may influence the activity of the key enzymes involved in folate metabolism. Among others, enzyme activities may be influenced by transcriptional regulation, post-transcriptional modification and protein modification. Moreover, 5-FU was not the only cytotoxic drug used in the chemotherapy protocol of the current study. Therefore, the putative effect of 5-FU-associated polymorphisms may be superposed by other effects. Therefore, currently, only empirical studies like the present one may clarify whether a given molecular aberration is associated with clinically important effects or not. Nonetheless, determination of genetic polymorphisms, for example, in peripheral blood, is considered an attractive approach for response prediction because this represents a relatively noninvasive approach for molecular profiling. Other methods, for example, immunohistochemistry and RT-PCR based on endoscopically obtained biopsies, always have the inherent doubt of representativity for the entire tumour. On the other hand, a recent study indicated that determination of genetic polymorphisms in peripheral blood may not be representative for the situation in tumour tissue. Thus, TS gene may have cancers that are homozygous for this polymorphism due to loss of one allele during carcinogenesis. Furthermore, they could show that the response to 5-FU-based chemotherapy in these cases was comparable to cases where the entire individual was homozygous. Therefore, it may be superior to analyse the genotype of polymorphisms in tumour cells than in peripheral blood.On the other hand, we did not observe any effect of the Methylenetetrahydrofolate Reductase C677T polymorphism and the Thymidilate Synthase tandem repeat polymorphism does not provide significant predictive information in multimodally treated oesophageal squamous cell cancer patients. The potential effect of the Methionine Synthase A2756G polymorphism deserves further investigations in similar designed studies.In conclusion, our data indicate that determination of the


A number of studies have examined ways to increase tumour acidity in order to improve tumour-specific targeting of certain drugs and the effectiveness of hyperthermia. However, previous data have shown that exposure of murine tumour cells to acid conditions in culture can enhance their metastatic potential when injected subsequently into mice, raising the concern that deliberate tumour acidification might increase the probability of metastasis. In this study, we examined the effects of in vivo tumour acidification and hypoxia on the spontaneous metastatic potential of the murine KHT-C fibrosarcoma and B16F1 melanoma cell lines. A tumour-specific increase in extracellular acidity, demonstrated by measurements with pH electrodes, was achieved by daily intraperitoneal injections of meta-iodo-benzylguanidine (MIBG) and/or glucose. This method of tumour acidification during tumour growth did not significantly enhance the spontaneous metastatic potential of the two murine cell lines.In addition to hypoxia, acidic extracellular pH (pH Following a two-point calibration , a 21G needle electrode  was inserted into the tumour through a premade 21G needle puncture in the skin and withdrawn in a stepwise manner to record pH values at different depths. A reference electrode  was placed subcutaneously in the same leg. Any drift caused by protein contamination of the needle probe was assessed by placing both of the probes back into pH 7.00 standardising buffer after the measurement of each tumour and by recording the difference that had occurred during the measurement. All of the pH values presented are corrected for their individual drifts, which on average was +0.03 pH units following approximately 10 min measurement interval. This drift increased significantly with increasing measurement time, thus preventing accurate measurements of long-term pHe measurements. The probe was placed initially in a region of the tumour with a pO2 value around 5–10 mmHg and then left in place for a number of hours. Data recording was initiated within 30 min of injection with glucose+MIBG. To obviate the possibility of metastatic contribution by dislodged tumour cells, no needle electrode or pO2 measurements were made in the tumours of mice used to generate the spontaneous metastasis data. All measurements were carried out without general anaesthetics.Measurements of tissue oxygenation were made using the fibreoptic-based OxyLite system  as described previously . Earlier initiation of treatment resulted in a significant inhibition of tumour growth. Mice in the treatment group were injected with 30 mg kg−1 MIBG  in phosphate-buffered saline (PBS) intraperitoneally (i.p.), immediately followed by 3 g kg−1 glucose (i.p.) in distilled water. Owing to different tolerance levels, the drug was administered in different schedules to the two different mouse strains. The C3H-HeJ males with KHT-C tumours received glucose+MIBG on every other day and on the intervening day only glucose was administered at a dose of 3 g kg−1. The C57Bl/CRL females with B16F1 tumours received glucose+MIBG on two consecutive days followed by a day on which only glucose was administered at a dose of 3 g kg−1. Control animals received an equal number of daily PBS injections. This treatment schedule was repeated until a predetermined tumour weight was reached. In two experiments, a separate group of KHT-C tumour-bearing animals were treated with glucose alone, receiving single daily doses of glucose at 3 g kg−1.Treatment to induce tumour acidification was started when the diameter of the tumour-bearing leg reached 7.5 mm . Owing to their higher drug tolerance levels and lower metastatic efficiency, mice with B16F1 tumours were killed when the tumour weight reached 1.7±0.17 g (15.0±0.5 mm) or a maximum of 6 days of tumour-acidifying treatment, whichever end point was reached first. The lungs were then removed for histological assessment of micrometastases where four individual 5 μm sections, separated by at least 200 μm, were obtained from each lung and examined as described previously . The Kruskal–Wallis ranking test was used on the nonparametric data sets with three or more treatment groups with the limit of statistical significance set at P<0.05. A power analysis of the metastasis data was performed to determine its ability to detect a difference. As the data is not normally distributed, it was log-normalized and the pooled variance was calculated. Methods described by P-value of 0.05 and a power of 80%.The Student's e values measured before and approximately 1 h after treatment demonstrate the degree of acidification achieved with various treatments in the KHT-C  was observed following glucose+MIBG injections. This treatment also significantly acidified the B16F1 tumours , as did acute hypoxia , but the B16 F1 tumours were found to become most acidic when these two treatment modalities were combined . In B16F1 tumours, the acute hypoxia+glucose+MIBG-treated group is significantly different from the glucose+MIBG-treated group (P=3.35 × 10−9). In the KHT-C tumours, addition of acute hypoxia to glucose+MIBG injections increased mean tumour pHe as compared to the injections alone (mean difference: 0.08 U). Acute hypoxia alone also increased KHT-C tumour pHe , but the biological significance of this increase remains undetermined. In accordance with previous reports (e: 7.00±0.074 vs 7.07±0.046 s.e.m. 1 h postinjections).Pooled pHhe B16F1  tumours.e values in individual mice showed maximal acidification within 1 h after injections and return of these values near to their pretreatment values within 3 h of injection. The mean degree of KHT-C tumour acidification following glucose+MIBG injections ranged from 0.06 to 0.28 pH units. The mean degree and duration of tumour acidification achieved with less toxic glucose on days 8 and 10 for KHT-C . This moderate tumour sensitivity to glucose suggests that the greater mean degree of tumour acidification (0.26 pH units) achieved with glucose alone in e levels of 7.12. This apparent overcompensation of tumour pHe in response to glucose+MIBG within 24 h of injections was taken into account in generating data for panel B, e in every tumour decreased after each daily treatment, heterogeneity in the responses of individual mice was evident.Examples of individual tumour responses during the first 4 days of tumour-acidification treatment with glucose+MIBG are shown in Figure 2or KHT-C  appears 2 as reported by others . The number of micrometastases in this group did not differ from the groups treated with PBS or glucose+MIBG . Treatment of the KHT-C tumours with acute hypoxia alone led to a nonsignificant increase in microscopic metastases in three of four experimental repeats  and −0.13 pH units for the B16F1 tumours  was less than that seen with glucose+MIBG alone (mean change in pHe: 0.24 U), which is again consistent with the increase in pHe induced by acute hypoxia (0.10 pH units). However, the reason for the increase in pHe due to exposure to acute hypoxia remains unclear. Tumour cell metabolism has been found to be limited primarily by substrate availability, such as glucose for anaerobic glycolysis, rather than the metabolic demands by tumour cells  ranging from 25–100% in individual tumours (median 68%); similar values have been reported in clinical soft tissue sarcomas with HP5 values ranging from 0–76% , as it took approximately 30 min to start pO2 recording after the injections. However, a group of pO2 measurements (panels B and C) were made in a set-up that allowed glucose+MIBG injections to be administered without removing the OxyLite probes and again no consistent changes in pO2 levels were observed. A possible explanation for the lack of a detectable change is offered by e and pO2  even when the pHe is as low as 6.2 (i via an unknown mechanism (2/5% CO2) breathing, which has been reported to result in significant tumour-specific acidification without growth inhibition (e of the KHT-C tumours following 2 h of carbogen breathing (data not shown). To rule out the potential of MIBG-induced increases in tumour oxygenation and subsequent possibility of inhibition of metastasis formation, the tumour-acidifying treatment was also combined with acute hypoxia. The median number of lung metastases in this combination treatment group did not differ from the medians of the groups receiving either treatment alone. We believe that this addition of hypoxia to the tumour-acidifying regime should have been able to compensate any possible increases in tumour oxygenation and thus prevent masking of a positive effect of acidification on metastasis formation. We also examined the power of our data set to detect a difference. Based on this analysis, the data for MIBG+glucose has 80% power to detect a difference greater than 2.7-fold in metastasis formation. We conclude that if the pHe changes induced by the treatment do cause an effect, it is unlikely to be larger than a 2.7-fold difference.The absence of a change in the spontaneous metastatic potential in response to fication  was unexvs 0.24 pH units for KHT-C). Despite this, no increase in metastases was observed contrary to our previous in vitro studies . The acidic conditions were also maintained much longer in vitro  than in vivo (<3 h). Thus, the acidic exposure in vitro was much more severe than could be achieved in vivo, which may explain the differences in the results obtained. This outcome may also be partially due to the low spontaneous metastatic efficiency of the B16F1 cell line. It is well known that growth site of the primary tumour can have a significant impact on the (metastatic) outcome of any given experiments and the predominant view supports an orthotopic rather than nonorthotopic site for maximal spontaneous metastatic effect (Owing to higher tolerance to MIBG, the B16F1 tumour model received tumour-acidifying injections longer and more frequently than the KHT-C model. The maximal degree of acidification was also higher in the B16F1 model (0.35 pH units  studies . The maxin vivo pH quantifying imaging techniques, such as those described In light of our previous data, which showed a significant increase in experimental metastatic potential by KHT-C and B16F1 cells following acidic culture conditions, the absence of an increase in spontaneous metastases underscores the complexity associated with the effect of the tumour microenvironment on malignant disease progression. Together with studies using noninvasive,


A novel tumour system has been used to study the effect of natural killer cells on tumour growth by using agents which modify natural killer cell activity. The tumour cells are hybridoma cells which secrete antibody specific for red blood cells so that tumour growth can be quantitated by a haemolytic plaque assay. Spleen-seeking variants have been derived from original hybrids which are sensitive to natural killer cells. Treatment of mice with polyinosinic-polycytidylic acid substantially enhanced natural killer cell activity and correlated closely with a reduction in the growth of the hybridoma tumour cells in the spleen and life extension. Conversely, a single injection of anti-asialo GM, antibody resulted in a substantial increase in the number of plaque forming splenic tumour cells and virtual elimination of natural killer cell activity. These data demonstrate the important role of natural killer cells in constraining the growth of a tumour of B cell origin and establishes the usefulness of this tumour model in studying the biology of effects on tumour growth.


In 215 patients with operable breast cancer  and no other or previous cancer, presenting to a single breast unit, sufficient tumour was available for the prospective determination of four putative biochemical markers of prognosis: oestrogen receptor (ER) activity, cathepsin D (cath D), epidermal growth factor receptor (EGFR) activity and cyclic AMP-binding proteins (c-AMP-b). There were significant inter-relationships between ER and EGFR (r = -0.26), c-AMP-b and cath D (r = +0.32) and ER and c-AMP-b (r = +0.14). After follow-up (median 36.2 months), a total of 55 recurrences  and 35 deaths were recorded. By univariate analysis, up to 10 of 18 biochemical, clinical and histopathological variables of potential prognostic value were significantly related to disease-free interval or death, but by multivariate analysis only oestrogen receptor concentration and node status contributed significantly to risk of both distant recurrence/death; in addition, tumour size made a small contribution to the risk for a distant recurrence only. Only two parameters, tumour grade and ER concentration, were significantly related to risk of locoregional recurrence by univariate analysis, but by multivariate analysis, only tumour grade was important. It is concluded that tumour ER concentration, axillary nodal status and tumour grade remain as the most important prognostic factors in the early years after presentation of operable breast cancer, with a minor influence of tumour size. At this time, the prognostic significance of quantitative measurements of ER concentration, carefully controlled for the quality of both assay and tumour specimen, is probably greater than is generally appreciated. We have yet to identify other factors, which add significantly to the short-term prognostic value of these key features.


Classical models of tumorigenesis assume that the mutations which cause tumours to grow act in a cell-autonomous fashion. This is not necessarily true. Sometimes tumour cells may adopt genetic strategies that boost their own replication and which also influence other cells in the tumour, whether directly or as a side-effect. Tumour growth as a whole might be enhanced or retarded. We have used mathematical models to study two non-autonomous strategies that tumour cells may use. First, we have considered the production by tumour cells of an angiogenesis growth factor that benefits both the cell from which it originates and neighbouring cells. Second, we have analysed a situation in which tumour cells produce autocrine-only or paracrine-only growth factors to prevent programmed cell death. In the angiogenesis model, stable genetic polymorphisms are likely to occur between cells producing and not producing the growth factor. In the programmed cell death model, cells with autocrine growth factor production can spread throughout the tumour. Production of paracrine-only growth factor is never selected because it is 'altruistic' (that is of no benefit to the cell that makes the growth factor), despite being potentially beneficial to tumour growth as a whole. No polymorphisms can occur in the programmed cell death model. Production of angiogenesis and other growth factors in tumours may be under stable genetic, rather than epigenetic, control, with implications for therapies aimed at such targets. Many of the mutations observed in tumours may have non-autonomous effects.


CCL19 treatment led to a marked reduction in tumour burden with extensive mononuclear infiltration of the tumours compared to diluent treated controls. Flow cytometric analyses showed significant increases in CD4 and CD8T cell subsets as well as DC in the lungs of CCL19-treated mice. Lung tissue cytokine profiles showed a shift towards immune stimulatory molecules with a decrease in the immunosuppressive cytokine TGF-β. Our findings show that CCL19 may serve as a potential immune stimulator and provide a strong rationale for the evaluation of CCL19 in cancer immunotherapy.Epstein–Barr virus-induced molecule 1 ligand chemokine (CCL19) is a CC chemokine that chemoattracts both dendritic cells (DC) and T lymphocytes. We evaluated the antitumour efficacy of CCL19 in a murine model of spontaneous bronchoalveolar cell carcinoma. These transgenic mice (CC-10 TAg) express the SV40 large T antigen under the Clara Cell promoter, develop bilateral, multifocal, pulmonary carcinomas and die at 4 months owing to progressive pulmonary tumour burden. To mimic therapy in late-stage disease, 3-month-old transgenic mice were treated with recombinant CCL19 (0.5  Lung cancer is the leading cause of cancer death in the United States and in the world (Effective antitumour responses require both antigen-presenting cells (APC) and lymphocyte effectors  or normal saline diluent, which contained equivalent amounts of murine serum albumin  as an irrelevant protein for control injections. Starting at 12 weeks of age, CCL19 or control injections were administered three times per week for 4 weeks. At the age of 4 months, mice were killed, and lungs were isolated for quantification of tumour surface area. Tumour burden was assessed by microscopic examination of H&E-stained sections with a calibrated graticule (a 1-cm2 grid subdivided into 100 1-mm2 squares). A grid square with tumour occupying >50% of its area was scored as positive, and the total number of positive squares was determined as described previously  and the results expressed as pg mg−1 of total protein. For the TGF-β ELISA measurements, samples were acidified and hence the active form of TGF-β was measured. The sensitivities of the IL-10, GM-CSF, IFN-γ, TGF-β, IL-12, and CXCL10 ELISA were 15 pg ml−1. The plates were read at 490 nm with a Molecular Devices Microplate reader .The cytokine profiles in lung tumours and spleens were determined in both CCL19 and diluent-treated mice as described previously  according to the manufacturer's instructions as described previously  were used to gate on the CD3 T lymphocytes in the percol purified leucocyte populations from tumour nodules as described previously  or the same concentration of murine serum albumin was injected in the axillary lymph node region beginning at 12 weeks of age, three times per week and continuing for 4 weeks. At 4 months when the control mice started to succumb because of progressive lung tumour growth, mice were killed in all of the treatment groups, and lungs were isolated and paraffin embedded. H&E staining of paraffin-embedded lung tumour sections from control-treated mice revealed large tumour masses throughout both lungs with minimal lymphocytic infiltration (μm2) as compared with diluent-treated control mice (495±40 μm2) (P<0.001).We evaluated the antitumour efficacy of CCL19 in a late-stage spontaneous bronchoalveolar cell carcinoma model. CCL19 (0.5 ltration . In contltration . Mice tr±40 μm2)  , CD8 (50%) and CD11c+DEC205+ DC (70%) and a decrease in CD4+CD25+ Treg (20%) cells at the tumour site . *P<0.00γ, GM-CSF, IL-12, CXCL9, CXCL10, and TGF-β by ELISA, and PGE2 by EIA. Compared to diluent-treated controls, the treatment group receiving CCL19 had a significant increase in type 1 cytokines  and antiangiogenic chemokines  and a decrease in the immunosuppressive cytokine TGF-β at the tumour sites. Compared with lungs from the diluent-treated group, CC-10 TAg mice treated with CCL19 had a significant reduction in TGF-β . This was coupled with an increase in GM-CSF , IFN-γ , CXCL9 , and CXCL10  within the tumour microenvironment , CXCL9 , and CXCL10   .Host APC are critical for the cross-presentation of tumour antigens (CCL19 is produced by a subset of DCs, and possibly by other non-lymphoid cells; in T-cell areas of lymphoid tissue (In most models reported previously, the antitumour efficacy of chemokines was determined using transplantable murine or human tumours propagated at s.c. sites. We embarked on the current studies to determine the antitumour properties of CCL19 in a clinically relevant model of lung cancer in which bronchoalveolar carcinomas develop in an organ-specific manner. The antitumour activity of CCL19 was determined in the spontaneous model for lung cancer by injecting recombinant CCL19 into the axillary lymph node region of the transgenic mice. In many clinical situations, access to lymph node sites for injection may also be more readily achievable than intratumoral administration. The efficacy of injecting immune stimulators in the vicinity of the lymph nodes for the treatment of cancer has been demonstrated previously; vaccination with tumour cell–DC hybrids in the lymph node region led to regression of human metastatic renal cell carcinoma . Althougγ to mediate a range of biological effects that facilitate anticancer immunity. CXCL9/MIG/CXCL9 and CXCL10/IP-10/CXCL10 are antiangiogenic CXC chemokines induced by IFN-γ that chemoattract activated T cells expressing the CXCR3 chemokine receptor (γ, CXCL10/CXCL10/IP-10, CXCL9/CXCL9/MIG, and GM-CSF. The induction of CXCL9/CXCL9/MIG and CXCL10/CXCL10/IP-10 may be responsible in part for the tumour reduction following CCL19 administration. Hence, the tumour reductions observed in this model may be owing to T-cell-dependent immune resposes as well as participation by T cells secreting IFN-γ in inhibiting angiogenesis (γ at the tumour site of CCL19-treated mice could explain the relative increases in CXCL10/IP-10 and CXCL9/MIG. Both CXCL9/MIG and CXCL10/IP-10 are chemotactic for stimulated CXCR3-expressing T lymphocytes that could further amplify IFN-γ at the tumour site (β at the tumour sites; TGF-β is an immune inhibitory cytokine that may potently suppress Ag and presentation, antagonise CTL generation and macrophage activation (β include promotion of antigen presentation and CTL generation (It is well documented that successful immunotherapy shifts tumour-specific T-cell responses from a type 2 to a type 1 cytokine profile (our site . An incrour site . Further+CD25+ Treg population at the tumour site following CCL19 therapy. The decrease in Treg cells may not be physiologically relevant in terms of numbers but may be in terms of function. Further studies are needed to define the functional significance of the decrease in the Treg population at the tumour site.Regulatory T cells have been documented to function as suppressor cells, and may play a role in the progression of cancer . FailureTaken together, the current study indicates that CCL19 injected in the axillary lymph node region in the late-stage spontaneous lung cancer model leads to the generation of effective antitumour responses. The potent antitumour properties demonstrated in this model of spontaneous bronchoalveolar carcinoma provide a strong rationale for additional evaluation of CCL19 regulation of tumour immunity and its use in immunotherapy for lung cancer.


Continuous temperature monitoring was carried out with thermistor probes implanted in breast tumours and in normal breast tissue. The results indicate that an initial rise in tumour temperature induced by the administration of nor-ethisterone acetate is associated with a subsequent objective response to that agent, whilst a fall has the reverse implication. Circadian rhythm has been demonstrated in all of the normal breasts and in all the tumours.It is suggested that skin temperature measurements above the tumour do not accurately relate to temperature within the tumour unless there is skin involvement.Considerable variation with time has been demonstrated in the tumour-normal temperature differential. This casts doubts upon the diagnostic value of the “normal” thermogram when taken as an isolated measurement. It may also tend to obscure any relationship between the degree of tumour temperature elevation and prognosis.We have now extended this investigation to include the effect of radiation to the cervix on temperature patterns in that site.


Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an  Antiangiogenic therapy (AAT) inhibits new blood vessel formation and decreases vessel density (VD) and tumour growth in several tumour models . This ‘nVascular endothelial growth factor (VEGF) is a key growth factor in the angiogenic process, and has a role in endothelial cell migration, proliferation, vascular permeability, and endothelial cell apoptosis. Inhibition of VEGF function by tyrosine kinase inhibitors, or monoclonal antibodies against VEGF or one of its receptors, has a significant antitumour effect. The monoclonal VEGF antibody A4.6.1 significantly decreases VD, tumour growth, and IFP, in several tumour models including colorectal cancer . CPT-11 was a gift from Aventis Pharma Belgium . CPT-11 solution was freshly prepared in 0.9% saline at a concentration of 10 2 in minimal essential medium (MEM) supplemented with 10% foetal bovine serum (FBS), 2 U ml−1 penicillin and streptomycin, 1 mM sodium pyruvate, 2 mML-glutamine, and nonessential amino acids at 37%.The HT29 human colon cancer cell line was obtained from the American Type Culture Collection , and cultured under 5% COad libitum throughout the experiment. HT29 cells  were injected subcutaneously in the right and left flank of the animals. Tumour growth was assessed every 2–3 days. Three orthogonal diameters were measured with vernier callipers, and used to calculate the volume of the tumour using the formula a × b × c × π/6 , and into four groups for the second part of the study, examining the tumour growth delay (10 tumours per group). A measure of 200 μg A4.6.1, diluted in saline, was administered intraperitoneally to each mouse (day 0). The same dose was given on day 4. Control animals were injected with saline. A single injection of 100 mg kg−1 CPT-11 was administered intraperitoneally on day 7. This dose produces a growth delay in our tumour model (data not shown), and is known to be the LD0 in mice  mice, 6–8 weeks old, were used. They were fed a diet of animal chow and water  c × π/6 . When unμl blood sample was taken through an intracardiac puncture and collected in an EDTA tube, and the tumours were resected. Each tumour was halved, and one half was frozen slowly in methylbutane for microscopic evaluation. The other half was snap frozen in liquid nitrogen for the later measurement of tumour CPT-11 concentrations. The blood was centrifuged at 1500 g for 10 min in a swing-out rotor, and 100 μl supernatant plasma was removed and used to measure plasma concentrations of CPT-11 and SN-38.The mice were killed at day 7 by ether inhalation one hour after the CPT-11 injection. A 500 μm (the size of an erythrocyte) was observed, and into large vessels (lumen ⩾7 μm). In the large vessel group, the shortest luminal diameter was measured and noted. The proportion of tumour necrosis, expressed as a percentage, was estimated at a magnification of × 40. All measurements were performed by two independent observers, with interobserver variability less than 10%, and all data were pooled for further analysis. Total VD, endothelial cord VD, and large-vessel VD were calculated.Histological sections were stained for CD34 and CD105 with Biotin conjugated CD34 and CD105 antibody (BD Biosciences), at a dilution of 1 : 40, avidin–biotin complex, and diaminobenzidine as chromogen. Vessel density was counted on CD34 and CD105 immunostained slides, according to conventional stereological methods using an unbiased Gundersen counting frame . Briefly−1 intravenously via the tail vein. The tumours were removed 1 min after dosing. Rather than count the number of H33342-labelled cells microscopically, we decided to determine the global H33342 uptake in the tumour using HPLC. The same HPLC procedure as for the CPT-11 measurements was used (cf. infra).The use of a fluorescent dye, H33342, to visualise and quantify tumour functional vasculature when frozen sections are viewed under ultraviolet light has been described . The mobile phase consisted of 72% triethylamine-acetate buffer (pH 5.2) and 28% acetonitrile at a flow rate of 1.5 ml min−1. CPT-11 and CPT were detected by fluorescence with excitation and emission wavelengths of 369 and 424 nm, respectively. SN-38 and H33342 were detected by fluorescence with respective excitation and emission wavelengths of 376 and 534 nm. The limits of quantitation for CPT-11, SN-38, and H33342 were 0.5, 0.25 and 0.5 ng ml−1, respectively. Within-run and between-run precisions were less than 10% and average accuracies were between 90 and 110%.A high-performance liquid chromatographic (HPLC) method was used and validated for the simultaneous determination of CPT-11, its metabolite SN-38, and H33342, in human plasma and tumour tissue. Camptothecin (CPT) was used as an internal standard. As sample pretreatment, 10 t-test was used to evaluate differences between two independent groups, and data are presented as mean±s.d. The significance level was determined at 0.05. The tumour growth delay calculations were based on the growth increase for a defined period, the tumour volume reached at a defined time point, and the time to grow from the starting volume to a higher defined volume. Since the time points of interest varied for the several questions posed, different parameters were used for the different groups.‘Statistica 5.5’ was used for statistical analysis. Unless indicated, Student's μg g−1vs 10.93±5.39 μg g−1 tumour tissue (P=0.09) . The intratumoral levels of the active metabolite SN-38 were about 50 times smaller, with no significant difference between the anti-VEGF mAb group (0.26±0.10 μg g−1) and the placebo group (0.24±0.09 μg g−1) (P=0.49). The mean tumour volume at day 7, the time of excision, was significantly lower (P<0.001) in the anti-VEGF mAb-treated group (355±123 mm3) compared to the placebo group (492±113 mm3).Pretreatment with anti-VEGF mAb produces a trend to higher intratumoral availability of CPT-11; mean±s.d. 15.98± 11.67 P=0.69). An example of histological sections of anti-VEGF mAb and placebo-treated tumours is shown  than in the placebo group (n=19) (P=0.01)  .in vivo, and this must be achieved with acceptable toxicity to normal host tissues. Hence, it must be possible to deliver the drug in vivo into the tumour tissue in sufficiently high concentrations. Different barriers to successful drug delivery have been recognised, as highlighted by For any cancer treatment to be successful, it needs to meet at least two major criteria. Firstly, it must be potent and effective in destroying or inhibiting cancer cells per se could be a predictive tool for tumour uptake of cytotoxic drugs. Our results clearly show that anti-VEGF mAb definitely does not impair, and may even improve intratumoral uptake of CPT-11 in this tumour model. This is remarkable because anti-VEGF mAb clearly decreases tumour VD in this model. These data are consistent with our previous work where we showed a lack of correlation between melphalan tumour uptake and VD , also vascular permeability (the ‘second’ barrier) could theoretically play a role in the delivery of anticancer agents. In general, the microvasculature of solid tumours is hyperpermeable to macromolecules in comparison to normal vessels. This is presumably due to interactions between vascular endothelial cells and VEGF, also known as the vascular permeability factor (VPF). It has been demonstrated that tumour vascular permeability can be reduced by neutralization of endogenous VEGF with the anti-VEGF mAb A4.6.1  could play a role in the intratumoral availability of small drugs such as CPT-11. The uniformly elevated interstitial fluid pressure in solid tumours leads to negligible convection in the tumour interstitium . With a plasma concentration of 20 μg ml−1 and with approximately 40% of CPT-11 present in erythrocytes, tumoral blood only contains 0.4 μg of a total tumour concentration of 4 μg. Therefore, at most 10% of the intratumoral CPT-11 is derived from blood, and this amount will not greatly affect the measurements.The measurements of CPT-11 only represent concentrations in the whole tumour, not only tumour cells, but also blood vessels and the interstitium. It was not possible to distinguish differences in CPT-11 concentrations within the different compartments in the present study. Plasma and red blood cells in the tumoral vessels also contain CPT-11 . AssuminAs anti-VEGF mAb inhibits the formation of new blood vessels, it is logical that tumour VD decreases. However, most investigators have looked at total VD, which in some tumour types is predominantly determined by endothelial cords that presumably play only a minor role in the delivery and transport of nutrients and drugs, as there is no lumen present. We have found that VEGF mAb diminishes more the number of larger vessels than of endothelial cords in this HT29 human xenograft colorectal tumour model. This effect could be due to inhibition of larger vessel formation, or to a decrease in size of existing larger vessels. In the setting of drug delivery, and in view of transport capacity, it is important to assess the role of large vessels specifically.To evaluate vasculature on tumour sections, various antibodies to endothelial cells (EC) are available. CD34 is one of the classical targets to identify EC, and has been called a ‘panendothelial’ marker. CD105 or endoglin is a more recently recognised endothelial antigen that is strongly expressed on proliferating EC, but little or not on resting EC. Therefore, CD105 represents a powerful marker of neovascularisation . Blood vThe enhanced antitumour effect of chemotherapeutic agents when combined with AAT  in protracted schedules of several weeks (P=0.68). Nevertheless, following CPT-11 administration to the former group at day 7, the growth curves clearly cross, indicating effective growth inhibition in this subgroup.The tumour growth delay with anti-VEGF mAb confirms previous observations in colon cancer and other tumour models  in the xenograft tumours. However, it has been shown that host VEGF (VEGF 164 in mice) can also significantly contribute to tumour growth (−1 (H33342 is used as an index of functional vascular volume, and quantified with a random sampling procedure using the Chalkley method on histological slides (In summary, our results show that the anti-VEGF mAb tends to improve CPT-11 delivery, and this corresponds to an increase in tumour perfusion, even though VD is significantly decreased. This increased perfusion is a plausible cause of the observed increase in CPT-11 uptake, although other phenomena might be involved. Interestingly, the tumour growth delay induced by the combination of anti-VEGF mAb and CPT-11 is at least additive. Tumour VD lacks predictive power for intratumoral delivery of anticancer drugs. Immunohistological staining of endothelial cells with CD34 and CD105, and the separation of endothelial cord and large vessel densities, provide additional information on tumour vascularisation. A new index of functional vasculature and perfusion using HPLC determination of intratumoral H33342 is described, allowing comparison of different treatments in tumour models.


There is a high frequency of diarrhea and vomiting in childhood. As a consequence the focus of the present review is to recognize the different body fluid compartments, to clinically assess the degree of dehydration, to know how the equilibrium between extracellular fluid and intracellular fluid is maintained, to calculate the effective blood osmolality and discuss both parenteral fluid requirments and repair. Water makes up 50-75 percent of the body mass. The most important determinants of the wide range in water content are age and gender: a. the water content of a newborn, an adolescent and an elderly man are approximately 75, 60 and 50 percent; b. after puberty males generally have 2 to 10 percent higher water content than females . Three major forces control the distribution of fluids across the capillary membrane figure : a. the Effective circulating volume denotes the part of the intravascular compartment that is in the arterial system and is effectively perfusing the tissues. The effective circulating volume is biologically more relevant than the intravascular compartment and usually varies directly with the extracellular fluid volume . As a reIn some settings the effective circulating volume is independent of the extracellular fluid volume. Among patients with heart failure the extracellular fluid volume is increased but the patient is effectively volume depleted due to the low cardiac output.20) can be measured directly (via determination of freezing point depression) or estimated from circulating sodium, glucose and urea (in mmol/L [To obtain glucose in mmol/L divide glucose in mg/dL by 18. To obtain urea in mmol/L divide urea nitrogen in mg/dL by 2.8 or urea in mg/dL by 6.0.]) as ) as -9:The effective blood osmolality, known colloquially as blood tonicity, is a further clinically significant entity, which denotes the concentration of solutes impermeable to cell membranes  it becomes an effective extracellular solute.], mannitol) and are therefore restricted to the extracellular compartment . These solutes are effective because they create osmotic pressure gradients across cell membranes leading to movement of water from the intracellular to the extracellular compartment. Solutes that are permeable to cell membranes  are ineffective solutes because they do not create osmotic pressure gradients across cell membranes and therefore are not associated with such water shifts. Since no direct measurement of effective blood osmolality  is possible, following equations are used to calculate this entity:Flame photometry, the traditional assay for circulating sodium, measures the concentration of sodium per unit volume of solution, with a normal range between 135 and 145 mmol/L. In fact, sodium is dissolved in plasma water, which normally accounts for 93% of the total volume of plasma, the remaining 7% consisting of protein and lipid. Ion selective electrodes, that have replaced flame photometry in most laboratories, determine the activity of sodium in plasma water, which ranges between 145 and 155 mmol/L. For convenience, laboratories routinely apply a correction factor so that the reported values still correspond to the traditional normal range of 135-145 mmol/L [The terms extracellular fluid volume depletion and dehydration are mostly used interchangeably. However, these terms denote conditions resulting from different types of fluid losses. Volume depletion refers to any condition in which the effective circulating volume is reduced. It is produced by salt and water loss . Strict sense dehydration refers to water loss alone. The consequence of strict sense dehydration is hypernatremia. The elevation in serum sodium concentration, and therefore effective blood osmolality, pulls water out of the cells into the extracellular fluid. However, much of the literature does not distinguish between the two terms. Although dehydration in general usage means loss of water, in physiology and medicine, the word means both a loss of water and salt. Depending on the type of pathophysiologic process, water and sodium may be lost in physiologic proportion or lost disparately, with each type producing a somewhat different clinical picture, designated as normotonic (mostly isonatremic), hypertonic (mostly hypernatremic), or hypotonic  dehydration. Dehydration develops when fluids are lost from the extracellular space at a rate exceeding intake. The most common sites for fluid loss are 1. the intestinal tract , 2. the skin  and 3. the urine . More rarely, dehydration results from prolonged inadequate intake without excessive losses .Children and especially infants are more susceptible to dehydration than adults. The risk is high for the following causes: a. infants and children are more susceptible to infectious diarrhea and vomiting than adults; b. there is a higher proportional turnover of body fluid in infants compared to adults ; c. young children do not communicate their need for fluids or do not independently access fluids to replenish volume losses -3,10.Dehydration reduces the effective circulating volume, therefore impairing tissue perfusion. If not rapidly corrected, ischemic end-organ damage occurs, leading to serious morbidity.Three groups of symptoms and signs occur in dehydration -3,5-10: When assessing a child with a tendency towards dehydration, the clinician needs to address the degree of extracellular fluid volume depletion. More rarely the clinician will address the laboratory testing and the type of fluid lost (extracellular or intracellular fluid).It is imperative to accurately assess the degree of dehydration since severe extracellular fluid volume depletion calls for rapid fluid resuscitation ,11. DehyAs a result, a pertinent history and a number of findings on physical examination are used to help assess dehydration. Skin turgor, sometimes referred to as skin elasticity, is a sign commonly used to assess the degree of hydration. The skin on the back of the hand, lower arm, or abdomen is grasped between two fingers, is held for a few seconds then released: skin with normal turgor snaps rapidly back to its normal position but skin with decreased turgor remains elevated and returns slowly to its normal position. However, decreased skin turgor is a late sign in dehydration that is associated with moderate or, more frequently, severe dehydration. Like decreased skin turgor, arterial hypotension is a late sign in hypovolemia that is rapidly followed by cardiac arrest . Symptoms and signs of dehydration include dry mucous membranes, sunken eyes, reduced urine output, a sunken open fontanelle, delayed capillary refill, deep respiration with or without increased respiratory rate, and tachycardia. Several attempts have been made to determine a measure of dehydration by using combinations of clinical findings. Very recently, in children < 4 years of age with a diagnosis of acute gastroenteritis, 4 clinical items , which may be summed for a total score ranging from 0 to 8 , were foLaboratory testing can confirm the presence of dehydration . The fra-2 (< 0.5 percent) and the urine spot sodium concentration < 30 mmol/L .is < 0.5 × 102O. The urinary concentration can be measured with an osmometer or fairly estimated, in the absence of proteinuria and glucosuria, from the specific gravity, as determined by refractometry, as follows:Furthermore, in dehydration, the urine is concentrated with an osmolality > 450 mosm/kg HDipstick measurement of specific gravity is very popular but unfortunately unreliable .Furthermore, laboratory testing can detect associated electrolyte and acid-base disturbances but determination of circulating electrolytes and acid-base balance is typically limited to children requiring intravenous fluids. These children are more severely volume depleted and are therefore at greater risk for dyselectrolytemias. Laboratory testing is less useful for assessing the degree of volume depletion.- Bicarbonatemia ≤ 17.0 mmol/L might be the most useful laboratory test to assess dehydration. The blood urea level reflects the severity of dehydration, the decreased glomerular filtration rate and the increased sodium and water reabsorption in the proximal tubule . Unfortu- The sodium concentration varies with the relative loss of solute to water. Changes in sodium concentration play a pivotal role in deciding the type of fluid depletion figure :Hyponatremic and hypotonic dehydration ,6: The dNormonatremic and isotonic dehydration: In this setting, solute is lost in proportion to water loss.Hypernatremic and hypertonic dehydration ,8: This Clinical assessment of dehydration may be difficult, especially in young infants. A large body of evidence suggests the use of a recently developped and validated "4-item 8-point rating scale". Mainly extracellular fluid losses occur in hypotonic dehydration, where signs of dehydration easily become profound; on the contrary, mainly intracellular fluid losses occur in hypertonic dehydration, where signs of dehydration tend to be less evident.The authors declare that they have no competing interests.MGB and AB wrote the first version of the manuscript. GDS revised the manuscript and prepared the figures. All authors have read and approved the paper, have met the criteria for authorship as established by the International Committee of Medical Journals Editors, believe that the paper represents honest work, and are able to verify the validity of the results reported.


In vitro studies explored drug interactions at cellular level on tumour cells (BN-175) and Human umbilical vein endothelial cells (HUVEC). There was a 17% partial response and a 50% arrest in tumour growth when Hi was combined to DXR, without important side effects, against 100% progressive disease with DXR alone and 29% arrest in tumour growth for Hi alone. Histology documented an increased DXR leakage in tumour tissue combined to a destruction of the TAV, when Hi was added to the ILP. In vitro no synergy between the drugs was observed. In conclusion, Hi is a vasoactive drug, targeting primarily the TAV and synergises with different chemotherapeutic agents.Tumour necrosis factor (TNF)-based isolated limb perfusion (ILP) is an approved and registered treatment for sarcomas confined to the limbs in Europe since 1998, with limb salvage indexes of 76%. TNF improves drug distribution in solid tumours and secondarily destroys the tumour-associated vasculature (TAV). Here we explore the synergistic antitumour effect of another vasoactive agent, histamine (Hi), in doxorubicin (DXR)-based ILP and evaluate its antivascular effects on TAV. We used our well-established rat ILP model for  Tumour necrosis factor (TNF)-based isolated limb perfusion (ILP) is an approved and registered treatment for sarcomas confined to the limb in Europe since 1998 and is currently carried out in approximately 30 cancer centres with referral programmes for limb salvage around the continent , an important chemotherapeutic drug in solid tumour treatment . Based oad libitum .Male inbred Brown Norway rats were obtained from Harlan-CPB , weighing 250–300 g and were fed a standard laboratory diet A2 × B) (where B represents the largest tumour diameter and A is the diameter perpendicular to it). When tumour diameter exceeded 25 mm rats were killed by cervical dislocation, under anaesthesia. At the end of the experiment all the rats were killed by the method specified.Small fragments (3 mm) of the syngeneic BN-175 sarcoma were inserted subcutaneously in the right hind leg of the animals as described previously  ; haemaccel + 1000 μg Hi (200 μg ml−1)  or haemaccel with 400 μg DXR and 1000 μg of Hi.The 5 ml total volume perfusate consisted of: haemaccel alone ; haemaccel+400 Tumour dimensions were measured every day for volume calculation. Response was classified as: progressive disease (PD), increase of more than 25%; no change (NC), volume kept in the range of −25 to +25%; partial remission (PR), decrease between −25 to −99% or complete response (CR), no palpable tumour, initial volume as compared to volume on day 9.Limb function was clinically observed as the ability to walk and stand on the perfused limb after ILP. On a scale from 0 to 2, grade 0 is a severely impaired function where the rat drags its hind limb; grade 1, a slightly impaired function ;and finally, grade 2 is an intact function .Locoregional toxicity was also evaluated by clinical observation of limb oedema. Oedema was defined as absent (−), or present, and in this case graded in a scale from + to ++++, according to the extent of increase in volume of the treated limb as compared to the opposite, not treated limb.The studies were carried out in accordance with protocols approved by the Animal Care Committee of the Erasmus University Rotterdam, the Netherlands.Two animals for each group were killed by cervical dislocation directly and 24 h after ILP, and tumours and a piece of underlying muscle were excised, fixed in 4% formaldehyde solution and embedded in paraffin. The slides were stained with haematoxylin and eosin and CD-31 by the Pathology Department of the Erasmus MC. Images were taken on a Leica DM-RXA microscope supplied with a Sony 3CCD DXC camera.Two animals for each group were killed by cervical dislocation 7 days after ILP, and tumours and a piece of adjacent muscle were excised, fixed in 4% formaldehyde solution and embedded in paraffin. The slides were stained by Perl's method, a qualitative technique based on the release of ferric iron from hemosiderin by acid treatment, forming ferric chloride. The ferric iron reacts with potassium ferrocyanide to form ferric ferrocyanide, an insoluble blue compound known as Prussian blue . Images in vivo intratumoral drug distribution, three animals for DXR alone and three for Hi + DXR were submitted to standard ILP plus the addition of 20 μl of the vessel staining FITC-lectin  to the perfusate. Directly after the procedure, the animals were killed by cervical dislocation, tumours were excised, snap frozen in liquid nitrogen and stored at −80°C. Thick sections of 25 μm were mounted with Mowiol and evaluated by confocal microscopy with a Zeiss LSM 510 Meta (488 nm laser with 505–505 band pass filter (FITC) and 543 nm laser with 560 long-pass filter (DXR)). Nine different fields per animal were selected and photographed . Images were further processed, using Image Tool® for Windows 2000. First, the colours of the images were separated, for quantification of vessel density (green) and drug distribution (red). Next, images were binarised, with a lower threshold setting based on the negative control, and percentage of positive pixels was determined. Data were plotted with GraphPad Prism for Windows 2000.To gain insight into For the evaluation of the presence of albumin extravasation into the tumour, two rats submitted to each of the following ILP: (1) sham; (2) DXR; (3) Hi or (4) Hi+DXR were included in this study. Tumours were excised, fixed in 4% formaldehyde solution and embedded in paraffin right after the ILP.M sodium citrate buffer, pH 6.0, at 100°C in a microwave oven for 10 min. Next, the slides were washed and incubated with rabbit-anti rat albumin  at 1 : 1000 in Phosphate buffered saline (PBS), overnight at 4°C. After washing, antibody binding was detected using diaminobenzidine substrate-chromogen  for 5 min at room temperature. Slides were counterstained with Mayer's haematoxylin, dehydrated with ethanol, cleared with xylene and finally mounted with Entellan . Images were taken on a Leica DM-RXA microscope equipped with a Sony 3CCD DXC camera.Sections were deparaffinised, rehydrated and treated with 10 mcirca 10 min); and placed in Methyl blue (Sigma) until collagen was blue . Then, slides were briefly rinsed with 1% aqueous acetic acid, rapidly dehydrated with ethanol, cleared with xylene and finally mounted with Entellan (Merck). Images were taken on a Leica DM-RXA microscope equipped with a Sony 3CCD DXC camera.Two rats submitted to each of the following ILP: (1) sham, (2) DXR, (3) Hi or (4) Hi+DXR were included in this study. Tumours and underlining muscle were excised, fixed in 4% formaldehyde solution and embedded in paraffin right after the ILP. Tumour slides were first deparaffinised and hydrated before being stained by MSB method for fibrin. Shortly, slides were placed in Martius yellow (Sigma) for 2 min, rinsed with distilled water; placed in Crystal scarlet (Ponceau 6R) (Sigma) for 10 min; differentiated with Phosphotungstic acid (Sigma) until only fibrin was red   at a concentration of 10M, pH 9.4). Extinction was measured at 540 nm and the percentage of growth inhibition was calculated according to the formula: percentage of tumour cell growth=(test well/control well) × 100%. The drug concentration leading to 50% reduction in absorbance, as compared to control (IC50), was determined from the growth curve. The experiments were repeated four times.Growth of tumour cells was measured using the sulphorhodamine-B (SRB) assay  were prepared by collagenase treatment of freshly obtained human umbilical veins and cultured in human endothelial–SFM/RPMI medium  supplemented with 10% heat-inactivated human serum , 20% new born calf serum, human EGF, human bFGF and 0.1% penicillin–streptomycin .4 cells ml−1, total volume of 100 μl and allowed to grow as a monolayer. Next, they were cultured for 48 h with Hi, in concentrations ranging from 0 to 200 μg ml−1 and DXR from 0 to 0.5 μg ml−1. The growth and IC50 were determined in the same way as for the tumour cells.Human umbilical vein endothelial cells were plated 24 h before treatment in 96-well plates at 6 × 10U-tests were used to evaluate statistical significance of the results. All statistical tests were two-sided and P-values less than 0.05 were considered as statistically significant. Calculations were performed on a personal computer using Prism v3.0 software  and SPSS v10.0 for Windows 2000.Kruskal–Wallis and Mann–Whitney P<0.01 on day 8 for Hi + DXR as compared to sham; P=0.027 on day 8 for Hi + DXR as compared to DXR alone) . As expected, the best response was seen with the combination of Hi and DXR showing a partial regression in two animals (33%) and arrest of tumour growth for approximately 6 days in three animals (50%)  ; Table 1, leading to a temporary grade 1 toxicity. The oedema compromised the limb as whole, including both the tumour and the normal tissue and was of the soft kind oedema. Isolated limb perfusion with DXR alone also caused a temporary regional toxicity in one (17%) of the treated rats scoring a grade 1 function with limb oedema lasting for 3–4 days.As previously seen in Hi + melphalan ILP, Hi either alone or combined with DXR did not inflict systemic side effects. As for regional toxicity, we observed a very mild (+) oedema in 17% (one out of six) of the rats treated by Hi+DXR and 14% (one out of seven) of those treated by Hi alone ILPImmediately after ILP with Hi + DXR vasodilation was observed, accompanied by tumoral endothelial cell damage and haemorrhage. Next to that some oedema in the tumour was observed. Histamine alone ILP resulted in diffuse oedema, but much less haemorrhage was seen than with the combination of Hi+DXR. Sham (data not shown) or DXR alone ILP had no effect on vasodilation or haemorrhage and predominantly intact tumour cells and few necrotic spots were seen. In accordance to these findings, CD-31 staining clearly showed the Hi-mediated destruction of the TAV-associated endothelial cell lining, with a more striking effect observed in those tumours treated by the combination of Hi+DXR as compared to Hi alone ILP. DXR alone ILP clearly had no effect on endothelial cell lining .In agreement with HE and CD-31 findings, Perl's method documented a haemorrhagic effect linked to Hi administration. Although DXR-alone-treated tumours had some iron deposits 7 days after ILP, mainly in the tumour tissue, these became more abundant after Hi alone ILP. After Hi+DXR ILP though, those iron deposits were clearly bigger and much more pronounced. As expected, iron deposits were mostly observed in the rim of the tumour and close to the vessels. The pattern was exactly the same as the one previously observed after Hi+melphalan ILP (data not shown). Muscle tissue from Hi-treated tumours (both with and without DXR) showed few and much smaller foci. Sham ILP had no iron deposits either in muscle or in tumour tissue. These findings further support the specific TAV-targeting action of Hi .To get a better insight into the pattern of oedema an immunohistochemistry antialbumin staining was carried out. Likewise already observed in the haematoxylin and CD-31 stainings, the vessels looked dilated after Hi treatment either with or without the combination to DXR. Extravasated albumin was observed around, and close to the vessels, once more, this observation was more striking after Hi+DXR ILP .Given the above findings, an additional staining specific for collagen and fibrin was carried out (MSB). Apart from the already expected Hi-related vasodilation, a very interesting observation was the Hi-related disarrangement of the collagen fibres, which could possibly play a role in improving tumour drug distribution . Slides P<0.001, DXR alone compared to DXR combined to Hi). The lectin staining also revealed large areas devoid of functional vessels, mainly in the tumour centre, in which no DXR could be delivered during the ILP.Taking advantage of the natural red fluoresence of DXR, we evaluated drug distribution within tumour and muscle by confocal microscopy of thick slides. When ILP was performed with DXR alone, some extravasation was observed around perfused (lectin-positive) vessels. Increased extravasation of DXR was seen around tumour vessels, when Hi was coadministered, while in muscle no major effects were observed. Moreover, some areas of DXR leakage could be observed in the tumour, with diffuse or even absent lectin staining, indicating severe damage to the endothelial lining of the tumour vasculature. These observations suggest an increased leakage specifically from the tumour vascular bed when Hi was added to the ILP . This fiin vitro cytotoxicity assays were carried out on BN-175 tumour cells and on HUVEC. As shown in 50 of 200 μg ml−1 for Hi and an IC50 of 0.1 μg ml−1 for DXR. While BN-175 tumour cells were effectively killed by DXR with an IC50 of 0.08 μg ml−1, hardly any effect of Hi was noticed with an IC50 as high as 500 μg ml−1. Combining DXR and Hi in vitro had only an additive effect.To evaluate the potential synergistic action between DXR and Hi, In this study, we show, by combining the natural red fluorescence of DXR with lectin–FITC staining of functional vessels during ILP, that the vascular disrupting effect of Hi augments intratumoral delivery of DXR. Based on these observations we hypothesise that both destruction of the tumour-associated vasculature and better tumour drug distribution, when Hi was administered, added to the observed augmented tumour responses. Additional histological stainings, such as CD-31 and Perl's method, further demonstrated Hi-related endothelial lining disruption and tumour haemorrhage, respectively. It is of note that these effects were more intense in tumour than in the muscle tissue, in agreement with the previously reported four-fold increase in melphalan uptake by tumour as compared to muscle , while TNF required the combination with interferon and peripheral blood mononuclear cells, for having an effect on endothelial cells in vitro  for DXR, against 17% partial response and 17% tumour growth arrest for melphalan (A possible explanation for the reduced efficacy of DXR could be its cycle dependency, while melphalan does not have this restriction. Taking into account that during an ILP drugs are delivered in a higher dosage but only once, apposed systemic chemotherapy where drugs are given repeatedly, this difference in activity might play an important role. Furthermore, the relatively poor penetration of DXR has already been reported as probably related to a rapid uptake of this drug by the perivascular cells. DXR binds avidly to the DNA within these cells, thus reducing the amount available for diffusion to distal cells (in vitro on both tumour and endothelial cells as compared to previous results (in vivo. Strikingly, tumour endothelial lining destruction and haemorrhage remained similar after Hi-based ILP, quite comparable to previous results. This observation was well documented by the different histologic stainings used.Specifically in the case of Hi, the batch used in this study was less active An additional important finding of our study is the Hi-related disarrangement of the collagen fibres, which we speculate could play an additional role in improving tumour drug distribution. Further studies, for a better evaluation and understanding of this important step, in improving tumour response rates with the use of vasoactive drugs are underway.In conclusion, the inflammatory mediator Hi acts as a vasoactive drug, targeting the tumour-associated vasculature and is capable of synergising with different chemotherapeutic agents. More importantly, this occurred without systemic side effects and only a mild regional toxicity, with a temporary limb oedema, completely reversible after 3–4 days. These findings support a potential role of Hi in regional treatment and organ perfusions in the clinic.


This is a case report of a 51 year old male who was found to have an incidental left sided non-functioning adrenal mass on routine medical examination and which was confirmed by CT and MRI scans. A laparoscopic left adrenalectomy was done. On gross examination the tumour was a solitary well circumscribed solid-cystic mass with a homogenous pinkish white cut surface. On microscopic examination, the tumour was composed of variably sized tubules and fenestrated channels lined by bland cuboidal cells to epithelioid cells. There was focal extension to capsule and peri-adrenal fat. Immunohistochemically the tumour cells stained with calretinin, Cam5.2, CK7, vimentin and focally with EMA. Ki-67 fraction was <1%. They were negative for ER, CD31, CD34, Factor 8, chromogranin, synaptophysin S100 and inhibin. A diagnosis of an adenomatoid tumour as made. Adenomatoid tumours are rare benign tumours of mesothelial derivation. The adrenal gland is devoid of a mesothelial lining and the most accepted hypothesis for an adenomatoid tumour originating in the adrenal gland is derivation from mesothelial rests. As the adrenal gland is an extremely rare site of occurrence for an adenomatoid tumour, it is frequently mistaken for adrenocortical tumours or a pheochromocytoma clinically and radiologically. Adenomatoid tumours are rare benign tumours of the mesothelial origin. The tumour may pose a diagnostic challenge if encountered at unexpected sites, which are devoid of a mesothelial layer. Here we report a case of an adenomatoid tumour of the adrenal gland, clinically suspicious for a malignant tumour.A 51-year-old male was found to have an incidental left-sided nonfunctioning adrenal mass on routine medical examination. CT and MRI scans revealed solid well-defined left adrenal tumour. Excision was recommended to confirm the nature of the mass, subsequent to which a laparoscopic left adrenalectomy was done. On gross examination the tumour was a solitary well-circumscribed solid-cystic mass with a homogenous pinkish white cut surface . On micrAdenomatoid tumours are rare benign tumours of mesothelial derivation, commonly encountered in male and female urogenital tracts. However, other sites such as intestinal mesentery, omentum, pancreas, heart, and pleura have also been documented. To the best of our knowledge <40 cases of adenomatoid tumour of adrenal gland have been reported in the English literature –20. The The tumour is solid yellow to grey on macroscopy sometimes, associated with a cystic component. Macroscopic calcification is occasionally present. A tubulocystic pattern is seen on histology, lined by epithelioid to flat cells. Signet ring-like cells can be seen , 8–10. LAs the adrenal gland is an extremely rare site of occurrence for an adenomatoid tumour, it is frequently mistaken for adrenocortical tumours or a pheochromocytoma clinically and radiologically and hence excised. The tumour can sometimes be seen to extend to the periadrenal tissues, raising the clinical suspicions of an infiltrative process. Signet ring cells, if present, can lead to a misinterpretation of metastatic adenocarcinoma. The commonly encountered tubulocystic pattern with lymphoid follicles can also lead to an erroneous diagnosis of lymphangioma or other vascular neoplasms. However immunohistochemically the tumour is alike to its extra-adrenal counterparts, and diagnosis can be accurately established. Surgical excision is curative, with no known recurrences.Patient consent is not required for this case report because the information are sufficiently anonymised. The figures used include macroscopic image of resection specimen, and pathology slide images do not require consent as they are anonymised by the removal of any identifying marks and are not accompanied by text that could reveal the patient's identity through clinical or personal detail.Competing Interest: “none to declare.”


We have studied loss of heterozygosity at the BRCA1 and BRCA2 loci in 992 normal cell clones derived from topographically defined areas of normal tissue in four samples from BRCA1/BRCA2 mutation carriers. The frequency of loss of heterozygosity in the clones was low (1.01%), but it was found in all four samples, whether or not a tumour was present. Topographical mapping revealed that the genetic changes were clustered in some breast samples. Our study confirms the previous finding that a field of genetic instability can exist around a tumour, suggesting that sufficient tissue must be removed at surgery to avoid local recurrence. We also demonstrate that such a field of genetic change can exist in morphologically normal tissue before a tumour develops and, for the first time, we demonstrate that the field is of a size greater than one terminal duct-lobular unit. The genetic changes are not identical, however, which suggests that genetic instability in these regions may play an early role in tumour development. We also confirm and extend our original observation of loss of the wild-type BRCA1 allele in some clones, and loss of the mutant allele in others, demonstrating that loss of either allele is a stochastic event. In the second case, the whole mastectomy specimen was required for pathological diagnosis; however, samples from the contralateral prophylactic mastectomy were available for this study. In the third case, tissue was available from both the tumour-carrying breast and the contralateral unaffected breast. In total, therefore, we used a total of four separate breast specimens: two mastectomies and two prophylactic mastectomies. Fresh unfixed breast specimens were sliced and examined by a pathologist and pieces of normal tissues from multiple sites throughout the breast were selected and their positions recorded. The rest of the specimen was examined and processed routinely. Paraffin-embedded samples of tumour-free lymph node removed at surgery were used as normal controls. Paraffin embedded tumour samples were used to investigate LOH in the tumour and, by inference, establish which were the mutant and wild-type alleles, the loss in the tumour being assumed to be wild-type.−1 collagenase  in Leibovitz L-15 medium plus 5% foetal calf serum. The tissue fragments were collected by centrifugation and then passed sequentially through a 70 μm and a 40-μm cell strainer  to collect the epithelial fragments and remove the single cells consisting predominantly of fibroblasts, endothelial and blood cells. The epithelial fragments were plated in RPMI medium plus 10% FCS, 5 μg ml−1 hydrocortisone, 5 μg ml−1 insulin and 100 ng ml−1 cholera toxin . The epithelial cells were allowed to mobilise from the fragments for approximately 5 days, and were then trypsinised to form a single-cell suspension. The cells were plated in 15 cm Petri dishes (1000 cells per dish), which had been preseeded with 106 lethally irradiated (40 Gy) mouse 3T6 fibroblasts. The clones were grown in Ham's F12 medium plus 10% FCS, 1 μg ml−1 hydrocortisone, 5 μg ml−1 insulin, 10 ng ml−1 human recombinant EGF and 100 ng ml−1 cholera toxin . This medium results in the clonal growth of both luminal and myoepithelial cells, but does not allow the growth of primary tumour cells under clonal conditions  and cytokeratin 14 . Two antibodies were used to identify luminal cell clones because, in our hands, up to 30% of luminal clones will have only weak or absent expression of one or other of the cytokeratins in culture (unpublished observation). The primary antibodies were applied as a cocktail as follows: LL002  1:200, DC10  1:200, LP2K  1 : 5. These antibodies were detected with a cocktail of goat-anti-mouse subclass-specific secondary antibodies (Southern Biotechnology) as follows: biotinylated anti-IgG3, FITC anti-IgG1, Texas Red anti-IgG2b, followed by a tertiary layer of streptavidin AMCA, resulting in CK18 seen as green, CK19 blue and CK14 red. The clones were examined using a Zeiss Axiovert fluorescence microscope and individually scored for the expression of each antigen to identify the cell type of origin.The clones were labelled by triple immunofluorescence for the expression of cytokeratins 18 and 19  were analysed for the presence of LOH. Of these, two samples had tumour present and two were prophylactic mastectomies. For the prophylactic mastectomies, in each case the contralateral breast contained tumour. A summary of the results of these analyses are presented in The rate of LOH in each case was low (0.739–1.579% of clones) but it was detected in all samples, both in the presence and absence of tumour. In sample 1, two luminal clones had LOH at D13s1293, both losses of the peak from the wild-type allele (hereafter referred to as loss of wild-type). In sample 2, one luminal and one myoepithelial clone showed LOH at D17s806 (loss of wild-type). Sample 3 had a total of three LOH all in luminal epithelial cells. Two of the clones were adjacent to the tumour, one showing LOH in D17s1814 (loss of wild-type) and the other in D17s1323 (loss of mutant). The third clone with LOH at D17s1814 (loss of wild-type) appears to be distant from the tumour and the other clones showing LOH , but it χ2 test, and the difference did not reach significance (P=0.22).A summary of the losses from the wild-type and mutant alleles is presented in In this study we have investigated the frequency and distribution of LOH in normal breast tissue from patients carrying a mutation in the BRCA1 or BRCA2 genes, using a single cell cloning technique.It might be argued that the cloning process may produce LOH, particularly in cells with BRCA1/2 mutations. If the alterations occurred during the growth of the clone, however, only a proportion of the cells would carry the LOH, and this would be reflected in the levels of each peak on the Genescan analysis. In this study, each clone showed complete loss of one peak, indicating that the LOH was present in all the cells, and therefore must have occurred before the growth of the clone.In our previous study only one LOH was found in three reduction mammoplasty samples (These studies are particularly labour intensive, making it impractical to examine many samples in order to produce statistically significant results. Nevertheless, some interesting observations of distribution and allele loss have still arisen from this study. We have shown that LOH is found close to tumours, and is sometimes found in other areas of the breast. In particular, we have shown that genetic alterations at the single-cell level may be clustered, and extend to an area that consists of more than one TDLU. Such clustering suggests that a change may have occurred in a precursor cell, which gave rise to that area of the breast. This result complements the findings of Our finding of clusters of LOH has clinical implications. Firstly, our results may help to account for some local tumour recurrences following apparent surgical clearance. The ‘recurrence’ may actually be a new tumour formed in the area of underlying genetic changes close to the original tumour. If this is the case, it implies that the rate of ‘recurrence’ may be reduced by removing more tissue at surgery. Secondly, one patient had two clusters of LOH, one close to the tumour and the other in the contralateral breast. It is impossible to know whether the area of LOH in the non-diseased breast might have gone on to form a clonal proliferation/tumour.et al demonstrated only loss of the wild-type allele in their normal breast samples (Our studies have demonstrated a second novel observation: loss of either the mutant or wild-type allele at microsatellite markers in the BRCA1 region (We have shown in this study that areas of genetic alterations are present in normal breast tissues of BRCA1/2 carriers, and that LOH at BRCA1/2 is possibly an early, but not necessarily the earliest event. Further studies using alternative techniques such as SNP arrays will be required to fully understand the sequence of events that leads to tumour formation in these tissues.


Techniques are described for hapten attachment to the cell membranes of mouse tumour cells. Dinitrophenylation and tyrosylation could be achieved without substantial loss of viability as measured by dye exclusion. In addition hapten coated tumour cells were capable of initiating new tumour formation in syngeneic hosts. Pre-immunization of recipient mice with hapten coated tumour cells did not increase their resistance to tumour formation upon subsequent challenge with graded doses of untreated tumour cells.


The function of the reticulo-endothelial system in mice bearing Lewis lung carcinomas has been measured by(1) the rate of clearance of carbon particles from the circulation in vivo and calculation of the phagocytic index K; (2) chemotactic locomotion of macrophages in vitro in the presence or absence of serum or tumour supernate. The ability of the bone marrow to develop macrophage colonies in vitro in the presence or absence of sera from tumour-bearing mice has also been tested. A clear depression of macrophage locomotion and macrophage colony formation in vitro was found in the presence of sera or tumour supernates from tumour-bearing mice as early as 24 to 72 h after tumour inoculation. Similarly, tumour-bearing mice showed marked depression of carbon clearance in tests repeated throughout the first 72 h after tumour inoculation. This early depression of macrophage function may be an important step in allowing escape of tumour cells from host resistance.


Resistance of tumour-bearing mice to a second tumour challenge, that is concomitant resistance, was evaluated in euthymic and nude mice using nine tumours with widely different degrees of immunogenicity. Two temporally separate peaks of concomitant resistance were detected during tumour development. The first one was exhibited only by small immunogenic tumours; it was tumour specific and mediated by classical immunological T-cell-dependent mechanisms. The second peak was shared by both immunogenic and non-immunogenic large tumours; it was non-specific, thymus independent and correlated with the activity of a serum factor (neither antibody nor complement) that inhibited the in vitro proliferation of tumour cells. This factor was eluted from a Sephadex G-15 column at fractions corresponding to a molecular weight of approximately 1000 Da and it was recovered from a high-performance liquid chromatography column in one peak presenting maximum absorption at 215 and 266 nm. The data presented in this paper suggest for the first time, to our knowledge, that in spite of the differences between immunogenic and non-immunogenic tumours, a common serum-mediated mechanism seems to underlie the concomitant resistance induced by both types of tumours at late stages of tumour development.


Activities of glucose-6-phosphatase, fructose 1,6-diphosphatase, ornithine transcarbamylase, arginase and xanthine oxidase were measured in thioacetamide induced primary hepatoma and its tumour cell suspension. It was observed that the percentage decrease in the activities of all the enzymes in tumour cell suspension was far more than that observed in tumour tissue. However, in these studies no qualitative difference was observed between the parenchymal cells and the tumour cells.