The tumours in 22 out of 23 animals given therapeutic amounts showed dose-dependent, rapid complete remission. The diagnostic amount (0.5 MBq [177Lu-DOTA0-Tyr3]-octreotate) did not influence tumour growth and was rapidly excreted. In contrast, the therapeutic amount (30 MBq [177Lu-DOTA0-Tyr3]-octreotate) induced rapid tumour regression and entrapment of 177Lu so that the activity concentration of 177Lu remained high, 7 and 13 days after injection. The entrapment phenomenon increased the absorbed dose to tumours from 1.6 to 4.0 Gy MBq−1 and the tumours in animals treated with 30 MBq received 120 Gy. Therapeutic amounts of [177Lu-DOTA0-Tyr3]-octreotate rapidly induced apoptosis and gradual development of fibrosis in grafted tumours. In conclusion, human midgut carcinoid xenografts can be cured by receptor-mediated radiation therapy by optimising the uptake of radioligand and taking advantage of the favourable change in biokinetics induced by entrapment of radionuclide in the tumours.Somatostatin receptor (sstr)-mediated radiation therapy is a new therapeutic modality for neuroendocrine (NE) tumours. High expression of sstr in NE tumours leads to tumour-specific uptake of radiolabelled somatostatin analogues and high absorbed doses. In this study, we present the first optimised radiation therapy via sstr using [ Each kit contained 40 μg of [DOTA0-Tyr3]-octreotate. The compound was stored at −25°C until use. 177LuCl3  was diluted in 0.05 M HCl and mixed with [DOTA0-Tyr3]-octreotate to a specific activity of 30 MBq μg−1. For the biokinetic studies with 0.5 MBq [177Lu-DOTA0-Tyr3]-octreotate, the specific activity was 0.5 MBq μg−1. The mixture was incubated for 30 min at 80°C .[DTPA]-octreotide was labelled with 177Lu and 111In was assessed by instant thin layer chromatography  with 0.1 M sodium citrate  as mobile phase. The fraction of peptide-bound 177Lu and 111In was more than 99%.The peptide-bound fraction of The exact amount of radiopharmaceutical administered was determined by measuring the activity in the syringes before and after administration of the radiopharmaceutical with a well-type ionisation chamber .177Lu-DOTA0-Tyr3]-octreotate, five groups of tumour-bearing animals were injected with a single i.v. injection of 7.5, 15, 30, 60 and 120 MBq of 177Lu-DOTA-Tyr3-octreotate (30 MBq μg−1), respectively . The animals were killed 24 h later and tumour tissue and multiple organs were collected for measurement of radioactivity and histopathological analyses. The 177Lu activity was measured using a Wallac 1480 gamma counter . Correction was made for detector background and radioactive decay. The activity concentration (C) of the radionuclide was expressed as the fraction of injected activity per unit mass of the tissue (%IA g−1).For studies of the biodistribution of -octreotate, that is, diagnostic and therapeutic amounts of radionuclide, respectively. All animals were injected with a single i.v. injection and were killed in groups of five at 1, 3, 7 and 13 days post injection (p.i.), respectively. Control animals (n=5) were untreated. Tumour tissue and multiple organs were collected for measurements of radionuclide uptake and histopathological analyses.For studies of biokinetics, tumour-bearing animals were injected with 0.5 MBq (1 −1) was determined for tumour tissue (CT) and normal tissue (CN). The tumour-to-normal tissue activity concentration ratio (TNC) was calculated according to the formula TNCN=CT/CN.The activity concentration , 15 , 30 , 60  or 120  MBq of [177Lu-DOTA0-Tyr3]-octreotate, respectively. Control animals (n=11) were given 4 μg of [DOTA-Tyr3]-octreotate. Tumour size was measured and the volume was calculated according to the formula V=4πab2/3 (a is the longest radius and b is the transverse radius). The animals were followed for 2–7 months. Complete response (CR) was defined as >99% reduction of tumour volume. Partial response (PR) was defined as 50–99% reduction and minor response (MR) as <50% reduction of tumour volume.The therapeutic effect of n=5) was injected i.v. with 4 MBq (0.2 μg) of [111In-DTPA]-octreotide 24 h before killing. Tumours and blood samples were collected and weighed, and the 111In activity in tumour and blood was measured using the gamma counter. The TNCBlood was calculated as described above  in PBS at pH 7.4 for 24 h. Specimens were subsequently dehydrated and embedded in paraffin wax. Parallel sections were counterstained with haematoxylin (htx) and eosin for morphological analysis, and with Ladewig staining to facilitate the detection of fibrin thrombi in tumour vessels. In all animals given a therapeutic amount of -octreotate, respectively. The maximum uptake of [177Lu-DOTA0-Tyr3]-octreotate in GOT1 tumours 24 h p.i. was seen both in animals given 7.5 and 15 MBq and reached 17±3% IA g−1 (mean±s.e.m.). In animals given larger amounts of activity, the uptake declined, indicating saturation of sstr in tumour tissue.Five groups of tumour-bearing animals were given 7.5, 15, 30, 60 and 120 MBq [−1)  . For all177Lu-DOTA0-Tyr3]-octreotate after diagnostic and therapeutic amounts, two groups of animals were injected with 0.5 and 30 MBq [177Lu-DOTA0-Tyr3]-octreotate, respectively. The specific activity of [177Lu-DOTA0-Tyr3]-octreotate in the diagnostic group was lower, that is, the amount of peptide (1 μg) was identical in both groups.In order to compare the biokinetics of [177Lu-DOTA0-Tyr3]-octreotate in tumours was similar for both groups of animals 24 h p.i. However, the biokinetics of the activity concentration differed significantly between the two groups. In animals given 0.5 MBq [177Lu-DOTA0-Tyr3]-octreotate, which did not retard tumour growth, the activity concentration declined over time and was less than 50% at 7 days and less than 20% at 13 days p.i. compared to the initial concentration. In contrast, in animals given 30 MBq, the activity concentration remained high over time and the biokinetic curves differed significantly both at 7 days p.i. (P=0.00044) and 13 days p.i. (P=0.00081) (The uptake of [0.00081) . The tum177Lu-DOTA0-Tyr3]-octreotate were determined up to 13 days after a single injection of 30 MBq. In general, the TNC value increased compared to the situation at 24 h p.i., which indicates a favourable change over time for the radiation to the tumour in relation to the radiation received by liver, kidneys and bone marrow. The increase in TNC value at 7 days p.i., compared to 24 h p.i., was significant for the bone marrow (P=0.0031), liver (P=0.033) as well as for the kidneys (P=0.00015)  .−1, when the absorbed dose was calculated using biokinetic data for the therapeutic amount of 30 MBq. For normal tissues in animals given 30 MBq, the absorbed doses were 0.15 Gy to bone marrow, 0.68 Gy to liver and 8.7 Gy to kidneys, respectively. The diagnostic amount 0.5 MBq, with clearly different biokinetics, resulted in 1.6 Gy MBq−1 to the tumours.Tumours treated with 30 MBq received about 120 Gy, corresponding to 4.0 Gy MBq177Lu-DOTA-Tyr3-octreotate, respectively. Control animals were given 4 μg of unlabelled DOTA-Tyr3-octreotate. The tumours in control animals grew rapidly and doubled their volumes after 2 weeks. Treatment with [177Lu-DOTA0-Tyr3]-octreotate resulted in a rapid and dose-dependent reduction of tumour volume . The TNCBlood for [111In-DTPA]-octreotide was then 410±70 (mean±s.e.m.).The activity concentration of [177Lu-DOTA0-Tyr3]-octreotate, a significant increase in apoptotic cell count, compared to controls, was observed both at 1 day p.i. (P=0.0000030) and 3 days p.i. (P=0.00000059). In animals killed 3 days p.i., all five tumours had large confluent necroses and intercellular oedema. In animals, killed 7 and 13 days p.i., the number of tumour cells was clearly reduced as was the apoptotic cell count and the oedema and necroses were replaced by fibrosis. The typical appearance of a tumour 13 days p.i. was that of almost complete fibrosis and absence of tumour cells  and Vesicular Mono Amine Transporters (VMAT1 and 2). In animals treated with 30 MBq [ur cells . The apo177Lu-DOTA0-Tyr3]-octreotate, revealed brownish nodules, 1–2 mm in diameter. The microscopic analysis demonstrated a thin rim of fibroblasts in the periphery and a central part consisting of crystalline structures surrounded by inflammatory cells including macrophages and giant cells (In the therapy groups (7.5–120 MBq), all tumours, or tumour residues, were morphologically examined at the end of the observation period. Gross examination of the small residual tumour tissue, found in animals with CR in response to [nt cells . Specifi177Lu-DOTA0-Tyr3]-octreotate therapy revealed two animals with reactive inflammatory response in the portal fields of the liver. These inflammatory infiltrates were composed both of CD 3-positive T cells and CD 20-positive B cells and had a Ki-67 proliferative index of <1%. In one animal, the kidney contained a fibrous scar. The bone marrow of all animals was normal with preserved haematopoietic cells.Morphologic analysis of the liver, kidneys and bone marrow after [177Lu-DOTA0-Tyr3]-octreotate of a human midgut carcinoid xenografted to nude mice. The first part of the study demonstrated that saturation of sstr in the tumours after a single i.v injection of [177Lu-DOTA0-Tyr3]-octreotate was evident between 0.5 μg (15 MBq) and 1 μg (30 MBq). It is important to define this level, since suboptimal therapeutic amounts will not lead to maximal uptake and maximal absorbed doses in the tumours. The uptake, and hence the absorbed dose, in the kidneys, liver and bone marrow increased proportionally to the amounts administered. Therefore, amounts in excess of the tumour saturation level will increase adverse effects of radiation therapy.This study presents the first systematic optimisation of sstr-mediated radiation therapy with [177Lu-DOTA0-Tyr3]-octreotate, given in diagnostic vs therapeutic amounts. The activity concentration in tumours after being given diagnostic amounts  was reduced over time. On the other hand, in tumours from animals given a therapeutic amount , the concentration remained high over time and significantly differed from the animals given a diagnostic amount both at 7 and 13 days p.i. The preserved high activity concentration in the therapy group was unexpected and significantly contributed to the high absorbed doses in the tumours, and thereby a very high efficacy of the radiation therapy. The difference in biokinetics can to a large extent be explained by tumour shrinkage, but other factors may also be of importance, for example, upregulation of sstr expression, radionuclide sequestration, increased intratumoural pressure and changed tumour blood flow initiated by the radiation.The second part of the study described the markedly different biokinetics of -octreotate, the absorbed doses were low and CR was seen only occasionally. These tumours started to grow again after 2 weeks of reduced volume. In animals treated with 30–120 MBq [177Lu-DOTA0-Tyr3]-octreotate, the absorbed doses were high; all, but one, of these animals showed CR. It is very unusual to obtain CR in animal tumour models using radionuclide therapy. Besides the present study and our previous study on [177Lu-DOTA0-Tyr3]-octreotate therapy in the human small-cell lung cancer cell line NCI-H69 xenografted to nude mice, CR is very unusual (131I-MIBG radiation therapy of NE tumours was proposed some 10 years ago (The third part of the study showed dose-dependent tumour responses. In animals treated with 7.5 and 15 MBq [ears ago . The pre177Lu-DOTA0-Tyr3]-octreotate in clinical series of patients with large tumour burden has not resulted in high rates of CR, most probably due to the administration of suboptimal amounts of radiopharmaceutical (Sstr-mediated radiation therapy using -octreotate has the potential to induce almost complete remission (111In-DTPA]-octreotide. The biological characteristics of GOT1 closely resemble the tumour of the original patient, that is, preserved neuroendocrine differentiation and molecular markers, preserved sstr expression and a TNCBlood value of about 400 for [111In-DTPA]-octreotide uptake (The present study not only showed a high cure rate of human midgut carcinoids but also revealed an unexpected entrapment of


The present study evaluated serial serum measurements of tissue polypeptide-specific antigen (TPS) in comparison with prostate-specific antigen (PSA) for assessment of tumour progression in patients with advanced prostate cancer receiving intermittent androgen suppression therapy (IAS). Twenty-three men were recruited into an IAS trial consisting of an initial 8 months of androgen suppression, followed by cycles of treatment cessation and resumption of therapy upon increases of PSA > 20 ng ml(-1) to prolong the hormone responsiveness of the tumour cells. Periods of androgen suppression resulted in reversible reduction in serum testosterone (< 1.8 nmol I(-1)) and PSA (< 4 ng ml(-1)) and decreases in tumour volume (mean reduction for first cycle 24 +/- 10%), indicating partial growth arrest and apoptotic regression of the tumours. In contrast to PSA values, non-specifically elevated TPS values were found in 8 of 23 patients. In 15 of 23 patients, TPS fell during periods of apoptotic tumour regression and increased simultaneously with testosterone and preceded the increases in PSA by 2 months during the period of treatment cessation. Although TPS represents a highly sensitive marker of tumour proliferation in this IAS clinical model of controlled tumour regression and regrowth, its low specificity compared with PSA limits its usefulness to monitoring of prostate cancer patients with proven absence of non-specific elevations of this marker.


Estimation of tumour proliferation may allow the design of individualised radiotherapy schedules to optimise response. This prospective study correlates the tumour proliferation rate of cervical carcinoma with response to conventional radiotherapy. The potential tumour cell doubling rate (Tpot) was estimated following flash labelling of the tumours in vivo using the DNA precursor, bromodeoxyuridine (BrdUrd); samples were analysed by flow cytometry. Tumour ploidy, DNA index and mitotic count were also assessed as was histological grade and type. Multiple biopsies from each tumour were obtained from 121 women. The median Tpot was 4.0 days, median S-phase duration 12.8 h and median adjusted labelling index 9.8%. Higher BrdUrd labelling was seen in patients who developed pelvic tumour recurrence following radiotherapy. This was the only biological/histological parameter with univariate and multivariate significance in relation to locoregional recurrence (P = 0.006 and P = 0.034 respectively). This study represents the first assessment of Tpot in relation to long-term response of cervical tumours treated by radiotherapy treatment. The association of high BrdUrd labelling and poor pelvic disease-free survival indicates the need for further research into the potential of radiotherapy schedule alteration to reflect tumour proliferation. The predictive value may be enhanced by combination with other biological parameters.


C. parvum 2 days before tumour inoculation. Intraperitoneal injection of C. parvum gave similar results but subcutaneous injection was less effective. Incubation of tumour cells with heterospecific antitumour globulin (ATG) in the absence of complement before inoculation sometimes but not always resulted in moderate inhibition of tumour growth. When pre-incubation of tumour cells with ATG and treatment of the host with C. parvum were combined the inhibitory effect was much greater than that produced by either procedure alone, and was roughly equivalent to reducing the dose of viable cells in control animals by a factor of 100. Since A/HeJ mice lack the fifth component of complement (C′5) it was expected that this serum would be ineffective as a source of C′ in in vitro cytotoxic tests but effective in opsonisation tests. This has been confirmed. The possible significance of this finding in relation to the synergistic effect of ATG and C. parvum is discussed.It has been confirmed that the growth of intrastrain transplants of a mammary carcinoma in A/HeJ mice is inhibited to a moderate extent by giving the prospective recipient an intravenous injection of killed


A primary renal tumour of childhood with histological appearances different from the nephroblastoma is described. This neoplasm, predominantly seen in boys, has a tendency to metastasize to bone. Such metastases are considered to be rare in nephroblastoma and this aspect in studies of Wilms' tumour series is thought to be due, for the most part, to the inclusion of a particular bone-metastasizing tumour in the material.


The reproducibility of measurements of oestrogen-receptor activity has been examined in multiple specimens from a rabbit uterus, a rat mammary tumour and human breast tumours. The relationship between receptor concentration and tumour histology has also been investigated in 11 large primary tumours. In the animal tissues, receptor measurements were relatively reproducible  but in human breast tumours receptor activity varied considerably . In addition to these variations in receptor activity within tumours, there was a difference between tumours, as demonstrated by an analysis of variance (P less than 0.01). In the 11 primary breast cancers selected for study, the level of receptor activity was related to menopausal status and the tumour content of the specimen. We conclude that the receptor activity detected varies within a tumour and depends upon the tumour content of the biopsy specimen. Predictions based on precise quantitation of receptor concentrations may therefore necessitate replicate tumour sampling and correction for the fraction of non-tumour tissue in each sample.


Equimolar doses of 7-methylbenz(a)anthracene and 3 of its derivatives were given to newborn male and female Swiss mice. All 4 substances tested increased the risk of tumour development compared with that seen in control mice given the vehicle, arachis oil, only.7-Methylbenz(a)anthracene itself was the most actively tumorigenic of the compounds studied, giving rise to subcutaneous sarcomata at the site of injection, and multiple lung tumours and liver tumours. 7-Bromomethyl-12-methylbenz(a)-anthracene was similarly active in the lung and liver but evoked fewer subcutaneous sarcomata. 7-Bromomethylbenz(a)anthracene was seemingly slightly less active than either 7-methylbenz(a)anthracene or 7-bromomethyl-12-methylbenz(a)anthracene. 4-Chloro-7-bromomethylbenz(a)anthracene exhibited only marginal activity in that it slightly increased the risk of liver tumour development in male mice.


Patients received one or two doses of131I-Lipiodol via hepatic arterial injection. The mean total administered activity was 668 (SD 325) MBqin CCA and 953 (SD 477) MBq in HCC. One patient with CCA retained 131I-Lipiodol. The cumulativeradiation dose was 9.6 Gy to tumour, 6.4 Gy to liver and 1.5 Gy to lung. The patient remainedasymptomatic with no evidence of tumour 30 months from the start of treatment, whereas the remaining7 patients exhibited tumour progression. The mean survival in CCA was 11.6 (SD 14.5) months. All 15patients with HCC retained 131I with tumour: liver ratios of up to 30:1. The mean cumulative radiationdose was 34.7 (SD 32.4) Gy to tumour, 3.3 (SD 1.5) Gy to liver and 4.4 (SD 2.3) Gy to lung. The meandose per administered activity was 3.8 (SD 4.1) cGy/MBq. Partial response (reduction in tumour size >50%) was observed in 6 patients (40%). The mean survival was 7.1 (SD 6.0) months.The therapeutic potential of         131I-Lipiodol can deliver highly selective internal irradiation to foci of HCC with evidence of objectiveresponse and may be the treatment of choice for patients with cirrhosis and a small tumour.


Following injection into the portal venous or vena caval systems, tumour cells are held up almost exclusively in the liver or lung respectively, and subsequent outgrowth of tumour only occurs in these organs. Following systemic arterial injection, cells are distributed, and subsequently grow, in a variety of organs. However, the adrenal gland supports tumour growth from much fewer cells than the lung, and this is partly due to the fact the rate of tumour cell loss in the initial 48 h is very high in the latter compared to the former organ.


BRAF gene is present in a high proportion of cutaneous pigmented lesions. Until recently this mutation had however only been identified in one case of posterior uveal melanoma. Despite this apparent lack of the BRAF mutation, inappropriate downstream activation of the Ras/Raf/MAPK pathway has been described in posterior uveal melanoma. Based on the already recognised morphological and cytogenetic heterogeneity in uveal melanoma, we hypothesised that the BRAF mutation may be present in uveal melanoma but only in some of the tumour cells. In this study, we analysed 20 ciliary body and 30 choroidal melanomas using a nested PCR-based technique resulting in the amplification of a nested product only if the mutation was present. This sensitive technique can identify mutated DNA in the presence of wild-type DNA. The mutation was identified in 4 of 20 (20%) ciliary body and 11 of 30 (40%) choroidal melanomas. Further analysis of separate areas within the same choroidal melanoma demonstrated that the mutation was not present in the entire tumour. In conclusion, the T1799A BRAF mutation is present in a proportion of posterior uveal melanomas but within these tumours the distribution of the mutation is heterogeneous.An activating mutation in exon 15 of the  BRAF gene (a member of the Raf family that encodes a serine/threonine protein kinase) have been shown to occur in the majority of cutaneous melanomas  were obtained from the pathology report. A total of 30 choroidal melanomas and 20 ciliary body melanomas were selected for this study. This project received the full approval of the West Ethics Committee, North Glasgow Hospitals, and adhered to the tenets of The Declaration of Helsinki. Normal tissue adjacent to the tumour was used as a negative control. The SKmel-28 cell line was used as a positive control. Human foreskin fibroblasts (HFF) were used as a negative control for developing the technique.μm section was examined from each case and appropriate areas of tumour and normal tissue identified. A 15 μl droplet of proteinase K (1% solution in 10 mM Tris (pH 8.5) and 1 mM EDTA) was added to the relevant areas on the section and the cells scraped with a fresh scalpel blade until suspended in the droplet. The solution was then pipetted into a 0.2 ml PCR tube. The samples were incubated at 55°C for 20 h. Following the addition of 15 ml of water, proteinase K was inactivated by incubation at 95°C for 8 min. The isolated DNA was stored at 4°C until further use in PCR. For all choroidal and ciliary body melanomas one area of tumour was sampled for investigation. To investigate heterogeneity within the tumour additional areas of tumour were sampled from 12 of the choroidal melanoma cases.One unmounted haematoxylin and eosin- (H&E) stained 4 g, 4°C, for 10 min, and washed with PBS. The cells were incubated for 3 h at 50°C in 1 volume lysis solution, . The lysate was treated by phenol and chloroform extraction, and the aqueous layer centrifuged at 12 000 g for 15 min at 4°C. After isopropanol/0.2 M NaCl precipitation at room temperature for 10 min, the sample was centrifuged at 12 000 g for 10 min at 4°C. Pellets were washed three times with 75% ethanol, air dried, and dissolved in H2O.For control DNA SKmel-28 and HFF cells were harvested and pelleted by centrifugation at 1500 μl reaction using ReddyMix  containing 5 pmol of forward (5′-TCATAATGCTTGCTCTGATAGGA-3′) and reverse (5′-GGCCAAAAATTTAATCAGTGGA-3′) primer and 5 μl of gDNA template isolated from tumour cells. Reactions consisted of an initial denaturation at 95°C for 3 min followed by 28 cycles of denaturation at 95°C for 20 s, annealing for 30 s at 55°C and extension at 72°C for 45 s. A final extension was carried out at 72°C for 10 min. Three reactions were carried out per DNA extraction.Primary PCR amplification reactions were performed in a 25 μl reaction using ReddyMix (ABgene) containing 5 pmol of mutation-specific forward (5′-GATTTTGGTCTAGCTACACA-3′) primer, which was optimised to anneal only at the mutation site, if present. Also included were 5 pmol of wild-type forward primer (5′-GCTCTGATAGGAAAATGAGATC-3′) and reverse primer (5′-GTGGAAAAATAGCCTCAATTC-3′), with 2 μl of template from the primary PCR. Reactions consisted of an initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation at 95°C for 20 s, annealing for 30 s at 50°C and extension at 72°C for 45 s. A final extension was carried out at 72°C for 10 min. All reactions were carried out in triplicate.Secondary PCRs were performed in a 20 Precast polyacrylamide gels (6%)  were immersed in TBE buffer. Samples were loaded on to the gel and electrophoresed for 1 h at 100V.M NaOH containing 0.007% formaldehyde until bands appeared. Finally, the gel was rinsed in de-ionised water and washed two times in fixing solution. A band of 200 nucleotides (nt) was obtained in each sample representing the wild-type gene. A further band at 100 nt was present in cases that contained the T1799A mutation. When the wild-type reverse primer was used the second band was approximately 140 nt. In addition, in reactions from DNA extracted from archival paraffin-embedded sections, occasional additional bands were present. This artefact was considered unavoidable as DNA extracted from the paraffin tissue is highly fragmented, and some of the small fragments can lead to self-priming within the reaction.Following electrophoresis the gels were immersed in fixing solution  for 5 min. A further 50 ml of fixing solution containing 0.2 g silver nitrate was added, and gels stained for 10 min. Gels were washed two times in de-ionised water for 20 s and 1 min, respectively. The gel was developed by immersing it immediately in 150 ml of 0.75 BRAF mutation, sex, cell type and vascular loops was tested one at a time by a Kaplan–Meier analysis and the relationship of survival time with age and tumour diameter were tested using the ‘Regression with Life Data’ function of Minitab 13.1 . The relationship between known survival time and the other variables was also considered in a single multivariate survival analysis. When several tumour areas were sampled from a single tumour association with cell type were assessed by Fisher's exact test.For choroidal melanoma the relationship of known survival time with BRAF mutation was assessed by a two-tailed 2-sample t-test.It was not possible to do this analysis for ciliary body melanoma due to the smaller number of cases and absence of survival data. The relationship between age and the presence of the BRAF mutation controls were used to ensure the optimisation of the method. Two products of 200 and 100 base pairs (bp) were obtained for SK-mel28, which contains the T1799A BRAF mutation and one product of 100 bp for HFF negative control . Two of the four patients were alive and well, one had died from an unrelated cause. We were unable to obtain clinical follow up information on the fourth patient. The pathology report and original sections were available for review in all cases. The average largest dimension of the tumour was 15.5 mm . Three tumours were composed of spindle cells and one was of mixed spindle and epithelioid cells. Three of the tumours contained closed vascular loops.BRAF negative cases: Of the 16 cases out of 20 negative cases there were eight enucleations and eight local resection specimens from seven men and nine women with an average age of 59.6 years . One patient had died of metastatic disease and six patients were alive and well at last follow up. We were unable to obtain clinical follow up information on the remaining nine patients. The pathology report and original sections were available for review in all cases. The average largest dimension of the tumour was 12.7 mm . Seven tumours were composed of spindle cells and nine were mixed spindle and epithelioid cells. Seven tumours contained closed vascular loops.BRAF mutation and clinical and pathological features.The clinical and pathological details are summarised in BRAF-positive cases: Of the 11 positive cases out of 30 there were six enucleations and five local resection specimens from six women and five men with an average age of 51 years . Clinical follow up was available in all cases. Eight of the patients had died from metastatic tumour and three were alive and tumour free at last follow up. The pathology report and original sections were available for review in all cases. The average largest dimension of the tumour was 14.5 mm (range 10–18 mm). Five tumours were composed of spindle cells, one of epithelioid cells and five were mixed spindle and epithelioid cells. Nine tumours contained closed vascular loops.BRAF-negative cases: Of the 19 positive cases out of the 30 negative cases there were 12 enucleations and seven local resection specimens from 11 women and eight men with an average age of 57.4 years . Clinical follow up was available in all cases. Eight of the patients had died from metastatic tumour and 11 were alive or had died from other causes. The pathology report and original sections were available for review in all cases. The average largest dimension of the tumour was 14.7 mm . Eleven of the tumours were composed of spindle cells and eight were of mixed spindle and epithelioid cells. Twelve tumours contained closed vascular loops.BRAF mutation is shown in BRAF-positive tumours to show a shorter survival but this was not statistically significant. There were no statistically significant associations between any other clinical or pathological characteristics and the presence of the BRAF mutation. Furthermore, in tumours where several areas of the tumour were dissected there was no association between cell type in the individual areas and presence of the mutation.The clinical and pathological details are summarised in BRAF gene have been identified in many human cancers, with the highest frequency of mutations found in cutaneous melanomas  ciliary body melanomas and 11 of 30 (40%) choroidal melanomas.Activating mutations in the BRAF mutation in other tissues including cutaneous melanoma they have not been applied to uveal melanoma (The majority of other studies have used some form of direct sequencing to identify this mutation (RASSF1a within individual uveal melanomas. It is therefore plausible that the BRAF mutation could also be distributed heterogeneously. To address this problem we studied several separate tumour areas in 11 choroidal melanomas positive for the T1799A mutation. In six of the tumours positive for the BRAF mutation it was observed that the mutation was present in some areas of the tumour but not in others. However, it did not show any association with the cell types in the different areas.The sensitivity of the PCR technique utilised would make it possible to identify mutant DNA in the presence of wild-type DNA. As separate samples from the same tumour did not always reveal the mutant band, this supports the theory that this mutation is only present in some of the tumour cells. Clonal heterogeneity, particularly morphological heterogeneity is well recognised in uveal melanoma  and the BRAF mutation is heterogeneously distributed in uveal melanoma the frequency of this mutation still appears considerably lower than in cutaneous melanoma. This may be due to insufficient tumour sampling. Both cutaneous and uveal tumours share a common embryological origin from the neural crest but there are considerable differences between the cutaneous and uveal environment. For example, ultraviolet light is considered to be an important risk factor in cutaneous melanoma. It has also been suggested that exposure to ultraviolet light may be a key factor in melanomas with the T1799A point mutation(BRAF mutation frequency is lower in melanoma arising in sites protected from sun exposure compared with those from sun-exposed sites(BRAF mutation is not a UV-signature mutation, it has been suggested that it could still arise due to error-prone reduplication of UV-damaged DNA (BRAF mutation is an infrequent event in uveal melanoma and the observed, inappropriate downstream activation of the MAPK component is due to genetic alterations in other components of this pathway.Although the results of this study confirm that the BRAF mutation is present in a proportion of posterior uveal melanomas and that within these tumours the distribution of this mutation is heterogeneous. This does not appear to carry any prognostic significance but may provide an explanation for the observed upregulation of the MAPK pathway in uveal melanoma.In conclusion, we have shown that the


Among the most prominent metabolic alterations in cancer cells are the increase in glucose consumption and the conversion of glucose to lactic acid via the reduction of pyruvate even in the presence of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, may provide a rationale for therapeutic strategies that inhibit tumour growth by administration of a ketogenic diet with average protein but low in carbohydrates and high in fat enriched with omega-3 fatty acids and medium-chain triglycerides (MCT).ad libitum. Experiments were ended upon attainment of the target tumor volume of 600 mm3 to 700 mm3. The two diets were compared based on tumour growth and survival time .Twenty-four female NMRI nude mice were injected subcutaneously with tumour cells of the gastric adenocarcinoma cell line 23132/87. The animals were then randomly split into two feeding groups and fed either a ketogenic diet  or a standard diet  The ketogenic diet was well accepted by the KD mice. The tumour growth in the KD group was significantly delayed compared to that in the SD group. Tumours in the KD group reached the target tumour volume at 34.2 ± 8.5 days versus only 23.3 ± 3.9 days in the SD group. After day 20, tumours in the KD group grew faster although the differences in mean tumour growth continued significantly. Importantly, they revealed significantly larger necrotic areas than tumours of the SD group and the areas with vital tumour cells appear to have had fewer vessels than tumours of the SD group. Viable tumour cells in the border zone surrounding the necrotic areas of tumours of both groups exhibited a glycolytic phenotype with expression of glucose transporter-1 and transketolase-like 1 enzyme.Application of an unrestricted ketogenic diet enriched with omega-3 fatty acids and MCT delayed tumour growth in a mouse xenograft model. Further studies are needed to address the impact of this diet on other tumour-relevant functions such as invasive growth and metastasis. Cancer is caused by multiple complex processes influencing cellular proliferation, differentiation and death. Genetic alterations favouring growth drive the transformation of normal cells into malignant cells . The relThe conversion of glucose to lactic acid via the reduction of pyruvate, even in the presence of oxygen, is known as aerobic glycolysis or the Warburg effect. Aggressive tumours frequently exhibit this metabolic alteration and reveal an increasing dependency on the glycolytic pathway for ATP generation. Most cells of non-malignant tissues, in contrast, use pyruvate to produce ATP via mitochondrial respiration in the presence of oxygen. Warburg claimed that cancer results from impaired mitochondrial metabolism. The increased glycolysis is thought to be a response to the hypoxic conditions characterising the microenvironment of malignant cells . An upreTherapies designed to target the anaerobic metabolism of tumours may preferentially kill malignant cells exhibiting this metabolic alteration. Promising experimental results in the treatment of certain types of tumours have been obtained with inhibitors of glycolysis  or of thKetogenic diets aim to induce ketosis, a physiological response of the body to limited dietary carbohydrate intake with consequent exhaustion of the glycogen content in liver and skeletal muscle resulting in the body's use of fat for energy. During ketosis, the liver starts to degrade fatty acids and to form acetyl-CoA in fatty acid oxidation. Acetyl-CoA can then be diverted into the ketone bodies acetoacetate and β-hydroxybutyrate (β-OHB), the major ketone body in plasma . Ketone ad libitum feeding. Several groups have focused on the impact of lipid oils on both tumour growth and body weight. Without caloric restriction, an anti-tumour effect was demonstrated for diets rich in omega-3 fatty acids and medium chain (6–12 carbons) triglycerides (MCT), which are not present in lard and scare in soybean oil [in vitro and in vivo [When applied under caloric restriction, different ketogenic diets supplemented with either lard or soybean oil have been shown to have an inhibitory effect on tumour growth -16. Howebean oil -20. Diffbean oil -23. Stilbean oil -27. An a in vivo -30. A re in vivo . Since Pad libitum.To determine the impact of a ketogenic diet on tumours exhibiting aerobic glycolysis, we compared the effects on growth and survival of a nutritionally balanced carbohydrate-restricted diet supplemented with a mixture of vegetable oils and oil extracts possessing elevated levels of polyunsaturated omega-3 fatty acid and MCT with those of a standard diet in a mouse xenograft model. We report a tumour-suppressive effect of this diet with respect to growth, necrosis, and neovascularization of subcutaneously implanted tumours in nude mice, feeding ad libitum. The study was reviewed and approved by the Animal Care Committee of the local government in accordance with the national guidelines for animal care .Twenty-four nude mice  of the NMRI strain obtained from Harlan Winkelmann GmbH  were maintained in groups of six animals per cage in laminar flow hoods in a pathogen-free environment. They were allowed access to food and water We used cells of the human gastric adenocarcinoma cell line 23132/87 .in vitro for their ability to metabolise glucose to lactate in the presence of oxygen. This aerobic glycolytic activity of glucose metabolism may correlate with a unique tumour phenotype characterised by a higher metastatic and invasive potential [Cells of the carcinoma cell line 23132/87 were tested otential . The gluotential . Since nin vivo experiments a freshly thawed tumour cell aliquot was cultured for up to three passages in vitro not more than 3 weeks prior to injection into nude mice. All mice received tumour cells from the same cell passage. The cultured cells (nearly 70–80% confluence) were harvested with trypsin EDTA (PAA) and the viability of the detached cells was routinely checked with the trypan blue exclusion test. All tumour cells prepared for inoculation were highly viable (>90%). They were inoculated subcutaneously in both hind flanks (2.5 × 106 cells per flank). Tumour nodules appeared approximately 8–10 days following cell injection and the larger of the two was selected for analyses. All data presented is based on these tumour nodules. No animal died from tumour growth. The tumour size was measured with calipers and the tumour volume VT (mm3) was calculated using the ellipsoid formula A2 × B × π/6, where A represents the smaller diameter. Endpoint for the experiments was attainment of a tumour volume between 600 and 700 mm3 (target tumour volume), with the interval between subcutaneous tumour cell inoculation and the endpoint defined as the survival time. Tumours reaching the target tumour volume were dissected and the final target volume and wet weight were determined. Subsequently, they were cut through the median, one part was fixed in formalin and embedded in paraffin, the other part was embedded in Tissue-Tek  and snap frozen in liquid nitrogen.For the All mice received a nutritionally balanced diet altromin 1430) provided by the special animal feed manufacturer Altromin GmbH & Co. KG, Lage, Germany, prior to the inoculation of tumour cells. This standard diet was supplied in pellets delivering 12.8 kJ/g gross energy and consisting of 7.0% fat, 23.8% protein, and 36.4% carbohydrates (Table 430 proviBlood glucose and β-OHB levels were measured on the day of tumour cell injection (day 0) and every week thereafter until the last day of the experiments before tumour resection. Measurements were done with a blood glucose and ketone monitoring system  and corresponding test strips  using 2 μl of peripheral blood collected from a snipped tail vein of each animal.2O, the endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol for 10 min. Cryosections (5 μm) were fixed in acetone and subsequently air-dried. All sections were then washed with PBS, blocked for 15 min in 1% goat serum, and incubated with primary monoclonal mouse antibodies for 60 min. The following antibodies, diluted in a commercial antibody diluent , were used: mouse-anti-transketolase like enzyme 1 , final dilution 1:400; mouse anti-pan cytokeratin , final dilution 1:100; anti-Ki-67 antigen , final dilution 1:50; and rabbit anti-glucose transporter type 1 , final dilution 1:100. The slides were washed in PBS, incubated with biotinylated anti-mouse and anti-rabbit immunoglobulins , and treated with streptavidin-peroxidase  according to the manufacturer's protocol. After development in 5% 3,3'-diaminobenzidine (DAKO) and counterstaining with haematoxilin, the sections were dehydrated in graded ethanol and embedded in Vitro Clud .Tumour tissue sections 2-μm thick were deparaffinized with xylene and rinsed in decreasing concentrations of ethanol prior to unmasking by heating for 5 min with 10 mmol/L sodium citrate buffer in a microwave oven at 600 W. After irrigating in distilled HCryosections of dissected tumours were stained with a rat anti-mouse CD34 antibody , final dilution 1:100. Microvessel density was qualitatively assessed by examining the entire vital cellular zone of the tumours with a light microscope at 100× magnification.Sections corresponding to the median line were stained with haematoxyline-eosine and photographed at a magnification of 4× with a digital camera. Images of each whole section were imported into Microsoft PowerPoint and all areas with morphologically well-defined necrosis were circled using the free-hand drawing function of the program at high magnification. The complete area of necrosis per section was quantified using the "analyse particles" option of the public domain Java image processing program ImageJ 1.34 s , Bethesda, MD, USA) and expressed as percentage of the section's total area.GraphPad Prism 4.0 software  was used for statistical analyses. Body weight, tumour growth, plasma glucose, blood ketone levels and necrotic areas were analysed by Mann-Whitney U test to show significant differences between the KD and SD groups after the nonparametric rank order test of Puri and Sen . Probabiin vitro for their ability to metabolise glucose to lactate in the presence of oxygen. First, the glucose consumption was measured with 2-NBDG glucose by flow cytometry. The 2-NBDG glucose uptake, which is time and dose-dependent  determined the end of the experiment and the interval between tumour cell injection and endpoint was defined as the survival time. At the experiments' endpoint, neither tumour volumes  nor tumour weights  differed significantly between the two groups. The results indicate that the determination of the target tumour volume (between 600–700 mm3) is an accurately definable endpoint of experiment.All 24 nude mice receiving a subcutaneous injection of tumour cells of the gastric adenocarcinoma cell line 23132/87 showed growth of solid tumours. On the day of tumour cell injection, animals of the KD group were switched from the standard diet to the ketogenic diet. Tumour progression was followed by measuring tumour volume over time. The difference between the mean tumour volume curves of KD and SD animals is significant  than those of the SD animals. The β-OHB levels of KD and SD animals differed highly significantly after day 6 amino)-2-deoxy-D-glucose; PBS: Phosphate-buffered saline; 23132/87: human gastric adenocarcinoma cell line; PET: positron-emmission tomography; RPMI 1640 medium: Roswell Park Memorial Institute; TKTL1: transketolase like enzyme 1.JFC and RW declare a potential conflict of interest due to the possible application of Tavarlin oil for therapeutic purposes and TKTL1 for therapeutic and diagnostic purposes.CO drafted the manuscript, designed the study, set up the experiments, participated in data collection, analysed and interpreted the results and provided images and figures. UK drafted the manuscript, carried out the histological analysis and analysed the results, and provided images and figures. BI contributed to the conception of the study and acquired funding from the Interdisciplinary Centre for Clinical Research (IZKF) of the University of Würzburg. BM participated in the feeding of the mice, tumour cell injection and data analysis. NP performed the measurements of glucose uptake and lactate production and participated in data analysis. HUV carried out the histological analysis and participated in data interpretation. RW revised the article for intellectual content. AT participated in editorial support and research funding. JFC participated in the study design, experimental concept, and data interpretation. He was responsible for production of the ketogenic diet. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Immunohistochemical analysis of representative tumours of the KD  and SD  groups. The carcinoma cells (pan cytokeratin-positive) located within the viable zone around the necrosis have proliferated (Ki-67 antigen-positive) and exhibit a glycolytic phenotype (TKTL1-positive). The expression of Glut-1 correlated with the strong glucose uptake and lactate production shown in Fig. D animal 6 groups.Click here for fileInfluence of the ketogenic diet on vascularity in tumours of the human gastric adenocarcinoma cell line 23132/87. Representative tumour sections from the KD  and SD  groups are shown. Vessels were stained with the rat anti-mouse CD34 antibody RAM34 as described in Methods (Magnification: ×400).Click here for file


A model system was developed to allow investigation of the frequency at which clastogenic and/or mutagenic events occur in situ in a transplantable murine fibrosarcoma tumour (MC1A-C1) compared with in vitro culture. The marker selected for detecting these events was the X-linked hprt (hypoxanthine-guanine phosphoribosyltransferase) gene. We found that the hprt gene in MC1A-C1 was not suitable for this purpose, most likely because multiple active copies were present. To circumvent the problem, HPRT- [6-thioguanine (6-TG)-resistant] clones were isolated by inactivating all hprt genes with methylnitrosourea. Spontaneous revertants to hypoxanthine/aminopterin/thymidine resistance (HATR) were isolated and found to be approximately 1000 times more sensitive than the parental tumour to induction of 6-TGR mutants by cobalt-60 gamma-rays. This sensitivity is expected for a heterozygous marker, these revertants may therefore possess only one functional hprt locus but two or more active X chromosomes. A clone with a stable hprt gene was identified and a neo gene was introduced. The resulting cell line (MN-11) could be grown as a subcutaneous tumour in syngeneic C57BL/6 animals. The frequency of mutations arising in vivo in the marker hprt gene could be estimated by culturing explanted tumour cells in the presence of 6-TG, using G418 selection to distinguish tumour from host cells. The frequency of mutants in MN-11 cells grown as tumours was found to be 3.4-fold higher than in tissue culture for an equivalent period of time. These data provide the first direct evidence for the existence of mutagenic factors in a tumour environment that might contribute to tumour progression.


Peripheral blood lymphocytes (PBL) were obtained from 13 patients and tumour-intrinsic lymphocytes (TIL) from 20 patients with colorectal cancer. The PBL were separated on a Ficoll-Isopaque gradient and the TIL by digestion of the tumour with collagenase-DNase. Both PBL and TIL were passed through nylon-wool columns and the eluted cells were co-cultured for 2 h with 51Cr-labelled tumour cells from the same patient. If patients in whom spontaneous 51Cr release from the tumour cells was greater than 33% were excluded, PBL showed cytotoxicity for the autoplastic tumour cells in 5/10 cases and TIL in 3/10 cases (NS). In 12 cases the cytotoxicity of the TIL was compared with that for TIL from the same tumour after the lymphocytes had been washed a further 6 times in Medium 199. Three effector: target (E/T) ratios, 5:1, 10:1 and 20:1, were used. The proportion of effector populations showing cytotoxicity was 2/12 for unwashed TIL and 9/12 for washed TIL (P less than 0.006). At the 5:1 E/T ratio the level of cytotoxicity was not significantly greater for washed TIL, but at the 10:1 ratio washed TIL showed significantly more cytotoxicity .


Antibodies directed against the antigen released from viable tumour cells during growth have been raised by cross-immunization of immunocompetent hairy litter-mates with serum from nude mice bearing pancreatic tumour xenografts. Antisera raised against the components released from a primary pancreatic tumour xenograft (WB) or from a tumour cell-line xenograft (GER) showed a titre greater than 1:625 against cultured pancreatic tumour cells by an I125-binding assay. Five out of 14 of the hairy litter-mates immunized with serum from the same tumour (GER) produced antisera that bound more strongly to pancreatic cancer cells than to 13 other non-pancreatic cell lines (binding ratio greater than 2). Absorption of the antisera with pure CEA reduced the level of binding by 11-25% without affecting the specificity for pancreatic tumour cells. The antibody response could be completely removed by absorption with pancreatic tumour cells, whereas 50% and 18% of the activity remained after absorption with normal pancreas homogenate and a mixed non-pancreatic tumour-cell pool, respectively. Immunofluorescent staining of pancreatic tumour sections indicated that the antibody was localized on the membrane of ductular epithelial cells. Challenge of immunocompetent mice using this procedure has produced a polyspecific antiserum with signs of selectivity for the circulating antigens released from pancreatic cancer cells, and may provide a route to the production of antibody against specific tumour components.


Nitro-L-arginine inhibits the production of nitric oxide and can thereby cause vasoconstriction in vivo. One consequence of this is that nitro-L-arginine can increase hypoxia in a range of transplantable and spontaneous murine solid tumours. Bioreductive drugs such as RB6145 are more active under hypoxic conditions, and the combination of RB6145 with nitro-L-arginine in vivo shows greater anti-tumour activity than either agent individually. In mice given nitro-L-arginine at 10 mg kg(-1) i.p. up to 1 h before or after 300 mg kg(-1) i.p. RB6145, survival of KHT tumour cells was reduced by 3-4 logs when assessed by clonogenic assay 24 h after treatment. RB6145 or nitro-L-arginine alone caused no more than 20% cell kill. Similar effects were found in SCCVII tumours. The tumour response to the drug combination was tumour size dependent, with increased tumour cell sensitivity occurring when the tumour volume at the time of treatment was increased. Further, the response of KHT tumours to the combination of RB6145 and nitro-L-arginine was also dependent on the time interval between treatment and on when tumours were excised for determination of survival in vitro. The relative surviving fraction was about 0.3 for intervals less than 4 h but was reduced to 0.01 at 12 h and 0.001 at 24 h. These results were supported by histological examination of tumours, when necrosis at 2 h after treatment was less than 5% but increased to greater than 90% at 24 h. RB6145-induced normal tissue damage, as measured by CFU-A survival, was not altered by combining with nitro-L-arginine. Hence, this drug combination may provide therapeutic benefit. It is likely that the substantial anti-tumour effects are due to enhancement of bioreductive toxicity through increased tumour hypoxia, although additional mechanism(s) may also contribute to the overall response.


Pimonidazole binding occurred at a mean distance of 114 μm from perfused vessels mainly around necrosis. Lipid droplets were principally located in the necrotic tissue. Some smaller droplets were also observed in part of the pimonidazole-binding cells surrounding necrosis. Hence, lipid droplets appeared only in hypoxic cells adjacent to necrosis, at an approximate distance of 181 μm from perfused vessels. In conclusion, our results show that severe hypoxic cells accumulated small lipid droplets. However, a 100% colocalisation of hypoxia and lipid droplets does not exist. Thus, lipid droplets cannot be considered as a surrogate marker of hypoxia, but rather of severe, prenecrotic hypoxia.In C6 rat brain glioma, we have investigated the relation between hypoxia and the presence of lipid droplets in the cytoplasm of viable cells adjacent to necrosis. For this purpose, rats were stereotaxically implanted with C6 cells. Experiments were carried out by the end of the tumour development. A multifluorescence staining protocol combined with digital image analysis was used to quantitatively study the spatial distribution of hypoxic cells (pimonidazole), blood perfusion (Hoechst 33342), total vascular bed (collagen type IV) and lipid droplets (Red Oil) in single frozen sections. All tumours ( A relation between the amount of mobile lipids and the percentage of necrosis was also evidenced in proton spectra of human brain tumour biopsies   supplemented with 2% glutamine and 1% antibiotics) were stereotactically injected 3 mm below the dura in the right caudate nucleus of six female Wistar rats (160–180 g) .A multiple fluorescence staining technique combined with a digital image analysis was used to study quantitatively tumour vascularity, perfusion, hypoxia and lipids in the same section  and a 0.25 ml solution of Hoechst 33342 in PBS . These markers were administrated intravenously via one of the tail veins, 60 and 1 min before killing, respectively.Experiments were carried out by the end of the tumour development (22–25 days after tumour cell implantation). Rats were injected with a 0.25 ml solution of pimonidazole in phosphate-buffered saline  (80 mg kgBrains were quickly removed (less than 3 min after the rat death), frozen in liquid nitrogen then stored at −80°C to prevent the Hoechst from diffusing further into the tissue.μm thick) were cut at mid-tumour (where the tumour was the largest). To analyse the morphology of the tumour, the first section was stained with haematoxylin/eosin (HE). The second section was analysed on the digital image analysis system. This section was first scanned to obtain a composite image with perfused areas (Hoechst binding), followed by fixation in 10% buffered formaldehyde. Next, lipid droplet staining was performed with a specific lipid dye, Red Oil  developed by Olympus . Tumour and necrosis areas were measured and the data were classed according to the necrotic fraction, The perfused areas (Hoechst) were visualised on the fluorescence microscope using a filter with excitation at 365 nm and emission at 420 nm. During each scan, which consisted of a selectable meander pattern from 4 × 4 to 12 × 12 fields, according to the size of the tumour, the previously recorded threshold value was used to isolate the perfused area from the background in a black and white image. After each scan, a composite image of the perfused areas was reconstructed from the recorded and processed black and white images of each field. After immunohistochemical staining for hypoxia and vasculature, sections were scanned on the image analysis system. A filter combination of 450–490 nm excitation and 520 nm emission was used for hypoxia and a filter with excitation at 510–560 nm and emission at 590 nm was used to analyse the vasculature. The sequentially recorded composite images of the tumour sections with detected perfused areas, hypoxia and total tumour vascular bed, respectively, were displayed using pseudocolours for each structure. Lipid droplets were visualised by light microscopy, and the amount of the droplets was qualitatively estimated: low (+), high (++).2 (VD) as well as the perfused vascular density (VDp) were also calculated.A multiparameter analysis of vascularity, perfusion and hypoxia in tumour sections was finally performed. Measured parameters were: the perfused fraction , the relative vascular area , the hypoxic fraction . The number of vascular structures per mmFor each tumour section, the composite images with vascular structures, perfused areas and hypoxic regions were combined, resulting in a new image with the overlapping structures representing the three parameters. In randomly selected regions of interest (approximately 10 ROIs per tumours), each including at least one perfused vessel near a hypoxic region adjoining tissue necrosis, distances from perfused vessels to necrotic areas, crossing hypoxic regions, were measured in manually drawn rectangles. In addition, pimonidazole-binding intensity as a function of distances to perfused vessels was measured using a 12-bit image analysis system . The results are not shown here, since the previous immunohistological protocol did not include the staining for functional and permeable vessels. However, similar results compared to those presented here were observed concerning hypoxia binding and the presence of small lipid droplets in the hypoxic cells adjacent to necrosis.It has been suggested that the presence of lipid droplets in viable cells surrounding necrosis could be related to hypoxia . In the present study, no inverse correlation was found between hypoxic fraction and perfused fraction  . This decreasing gradient in oxygen concentration from vessels has also been reported in in vitro studies (spheroids) (in vivo studies (tumour tissue)  , resulti tissue) , it shouIn their report, et al already showed that lipid droplets were associated with degenerating mitochondria in Ehrlich carcinoma (pO2<10 mmHg) starts to occur (approximately 114 μm according to our results) mitochondrial structures might still allow the activity of the respiratory chain, avoiding the production of lipid droplets. Beyond this critical point, mitochondria might degenerate as the availability of oxygen decreased, leading to a complete disintegration at pO2 below a certain value corresponding to an approximate distance of 181 μm from a perfused vessel, according to our results. Hypoxic cells might thus produce lipid droplets as a temporary  storage compartment for lipids  also induced formation of lipid bodies observable with Red Oil staining. However, in this latter case, the number of lipid bodies appeared fewer than that observed at 0% O2  occurs at a distance of about 100 μm from a perfused vessel, and is mainly detected around the necrotic area. The hypoxia marker binding intensity is considerably variable between tumours and within the different regions of the same tumour. This information on the degree of hypoxia could be useful in tumour treatment. Indeed, a variable dose of irradiation could be applied according to whether the tumour is more or less hypoxic.The present study shows that at late stages of C6 rat brain glioma development, hypoxia (Small-sized lipid droplets are not detected in all hypoxic cells but only in the highly hypoxic, prenecrotic ones. As a consequence, lipid droplets could only be associated with a severe, prenecrotic hypoxia and cannot be considered as a surrogate marker of a moderate hypoxia.


Leucocyte interactions with the cremaster muscle microcirculation in vivo were investigated in response to culture medium conditioned with different cell types in 25 adult male Swiss mice. Animals were divided into five groups. Three groups received ex vivo fluorescently labelled lymphokine activated killer (LAK) cells systemically and had either tumour (murine melanoma K1735)-conditioned medium (TCM), fibroblast (murine 3T3)-conditioned medium (FCM) or fresh culture medium administered topically to the cremaster muscle. In the two remaining groups, the host leucocytes were labelled fluorescently by systemic administration of acridine red, and either TCM or FCM was applied topically to the cremaster muscle. There was an immediate but transient increase in the frequency of rolling and adherent LAK cells, and a subsequent (90-120 min later) increase in rolling and adherent host leucocytes, demonstrating temporal differences in the response to topical administration of TCM. These increases in contact with the vascular endothelium occurred in all vessel types, venules, arterioles and capillaries, with the greatest response observed in the venules. The FCM and normal culture medium did not affect the distribution and localization of either LAK cells or host leucocytes. These data suggest that there are one or more soluble tumour-specific chemoattractants for leucocytes present in the conditioned medium. The mouse cremaster muscle microcirculation is therefore a useful model to investigate the mechanism of leucocyte-endothelium interactions in tumour biology.


The metabolism of 2-[14C] folic acid has been studied in rats with an implanted Walker 256 tumour and in a closely matched group of controls. In animals with tumours, more of the labelled folic acid is converted to 10-formyltetrahydrofolate and 10-formylfolate than in normal animals. No 5-methyltetrahydrofolate could be detected in tumour tissue, or in the livers of tumour-bearing animals. When a mixture of 2-[14C]- and 3',5',9-[3H]-folic acid is given to tumour-bearing rats a similar pattern of metabolites is found. There is apparenly less scission of the folate molecule in tumour-bearing rats than in normal rats.


Correction for:10.1371/journal.pgen.0010011Corbo JC, Cepko CL (2005) A hybrid photoreceptor expressing both rod and cone genes in a mouse model of enhanced S-cone syndrome. PLoS Genet 1(2): e11. doi:Glo1, Hnrpa1, and Dkk3, respectively. This finding led the authors to resynthesize and test all ten probes made in the batch which included these misidentified probes (gene IDs G16-G25). They found that eight of the ten resynthesized probes failed to produce a cone-specific pattern of staining. The probe synthesis protocol used in the manuscript included a PCR amplification step. The authors therefore reason that the original batch of PCR reactions was subject to trace contamination by a strong cone-specific product (possibly deriving from Opn1sw), which resulted in spurious cone-specific staining. In order to remove the possibility of PCR contamination, all ten probes were resynthesized from restriction enzyme-linearized templates. Again, only two probes, Glo1 and 4921511K06Rik (Gene IDs G17 and G24), showed cone-specific staining. Further analysis of other probes used in this study has confirmed that they were free of such contamination. These findings do not alter any of the manuscript's fundamental conclusions.Resequencing has shown that three clones used for making in situ hybridization (ISH) probes for this manuscript were initially misidentified . The correct identities of these clones are as follow:


Of 193 CBA mice kept under prolonged observation after excision of small intradermal transplants of a non-immunogenic tumour (CBA Carcinoma NT), 27 (14%) presented with local recurrence, 19 (10%) with regional lymphnodal metastasis (RNM) and 72 (37%), with pulmonary metastasis +/- other systemic metastases. When mice were exposed to sublethal whole-body irradiation (WBI) before tumour transplantation, the incidence of RNM rose to approximately 80% and the latent period was reduced from approximately 60 days to approximately 40 days after tumour transplantation. This enhancement of RNM by WBI was undiminished when the interval between WBI and tumour transplantation was increased from 1 to 90 days. An explanation for this effect in terms of immunosuppression by the WBI is unlikely for the following reasons: the tumour was non-immunogenic by standard quantitative tests; the effect persisted long after the expected time for recovery of immune reactivity; and i.v. injection of normal marrow and lymphoid cells after WBI failed to reduce the effect. That the effect was systemic was proved by failure of local pre-irradiation of the tumour bed or regional node to enhance RNM. The effect was not observed when WBI was given 4 days after excision of tumours. These and other experiments failed to indicate the mechanism of the effect of WBI, but its long persistence suggests that it may relate to stored lethal radiation damage in migrating cells of slow turnover tissues.


Two techniques for growing colonies of human tumour cells in soft agar have been applied to cell suspensions derived from fresh tumour tissue from 48 patients. Colonies were obtained in 31 cases, with plating efficiencies between 0.01 and 15%. In 11 cases the plating efficiencies were 1% or above. There was evidence that some categories of tumour grew more readily than others under these conditions. The potential applications of the methods to clinical and experimental oncology are discussed.


The aim of the present study was to evaluate the prognostic factors of bilharzial bladder cancer treated by radical cystectomy: good prognosis is defined as a survival of more than one year, free of local recurrence or metastasis. Two groups of 155 patients, one with a good prognosis (GPG) and the other with a bad prognosis (BPG), through the period 1977-1983 at the National Cancer Institute of Cairo were systematically analyzed for 13 variables evaluated at the commencement of the one year follow-up. Nine factors proved to be of high prognostic value: age, tumour stage, size, grade and location in the bladder, lymph node involvement, metastasis, renal insufficiency and type of urinary diversion. Four variables appeared not to have prognostic value viz: sex, type of tumour (multiplicity), histopathology, and presence of ova of schistosoma haematobium in the specimen. Using a discriminant analysis technique to take into account the inter-relationships between the factors, it was found that tumour grade was the most important prognostic factor followed, in order of importance, by tumour stage, renal insufficiency, size of the tumour and lymph node involvement. Moreover, a simplified score for prognosis was determined: X = 10 grade (1 to 3) +5 stage (1 to 4) +6 renal insufficiency (Y/N) +1 diameter of the tumour (cm) +4 lymph node involvement (Y/N). The larger the score, the poorer the prognosis.


Both agents are very toxic, which limits their systemic use. Their applicability may therefore be tested in the isolated limb perfusion setting, by which the tumours can be exposed to high concentrations in the absence of systemic exposure. To study the beneficial effect of the combination in vivo, BN-175 soft tissue sarcoma-bearing rats were perfused with various concentrations of actinomycin D and tumour necrosis factor alpha. When used alone the drugs had only little effect on the tumour. Only when actinomycin D and tumour necrosis factor alpha were combined a tumour response was achieved. However, these responses were accompanied by severe, dose limiting, local toxicity such as destruction of the muscle tissue and massive oedema. Our results show that isolated limb perfusion with actinomycin D in combination with tumour necrosis factor alpha leads to a synergistic anti-tumour response but also to idiosyncratic locoregional toxicity to the normal tissues. Actinomycin D, in combination with tumour necrosis factor alpha, should not be explored in the clinical setting because of this. The standard approach in the clinic remains isolated limb perfusion with tumour necrosis factor alpha in combination with melphalan.Previously we demonstrated that addition of Tumour Necrosis Factor-α to melphalan or doxorubicin in a so-called isolated limb perfusion results in synergistic antitumour responses of sarcomas in both animal models and patients. Yet, 20 to 30% of the treated tumours do not respond. Therefore agents that synergise with tumour necrosis factor alpha must be investigated. Actinomycin D is used in combination with melphalan in isolated limb perfusion in the treatment of patients with melanoma in-transit metastases and is well known to augment tumour cell sensitivity towards tumour necrosis factor alpha British Journal of Cancer (2002) 86, 1174–1179. DOI: 10.1038/sj/bjc/6600169www.bjcancer.comCancer Research UK© 2002  In 1958 In rat osteo- and soft tissue sarcoma ILP models we were able to mimic the clinical ILP with comparable response type and rate  were used for isolated limb perfusions. Rats were fed a standard laboratory diet ad libitum  and were housed under standard conditions. The experimental protocols adhered to the rules outlined in the ‘Dutch Animal Experimentation Act’ (1977) and the published ‘Guidelines of the UKCCCR for the welfare of animals in experimental Neoplasia’ . The pro2×B), where B stands for the largest diameter of the tumour and A the diameter perpendicular to B.The rapidly growing and metastasising BN-175 soft tissue sarcoma, which is transplantable to the BN rat, was used. Fragments of 2–3 mm were implanted subcutaneously in the right hind limb, just above the ankle. Perfusion was performed at a tumour diameter of 13±2 mm, approximately 7 days after implantation. Tumour growth was recorded by calliper measurements and the volume was calculated with the formula 0.4 (A7 U mg−1 as determined in the murine L-M cell assay. Endotoxin levels were <1.25 units (EU) per mg protein. TNF concentrations used were 50 μg in 5 ml perfusate.Recombinant human TNF (rHuTNF) was provided by Boehringer  with a specific activity of 5.8×10−1. Concentrations used were 5 and 10 μg in 5 ml perfusate.Actinomycin D  was diluted in phosphate buffered saline to a concentration of 2 mg ml−1. Concentrations used were 40 μg in 5 ml perfusate.Melphalan  was diluted in phosphate buffered saline to a concentration of 2 mg mlL-glutamine. Media and supplements were obtained from Life Technologies, the Netherlands.Cells isolated from a BN-175 soft tissue sarcoma were maintained in cell culture for a maximum of 20 passages in RPMI supplemented with 10% foetal bovine serum and 4 cells per well and allowed to grow as a monolayer. Actinomycin D and rHuTNF, diluted in RPMI supplement with 10% foetal bovine serum and L-glutamine, were added to the wells and allowed to incubate for 3 days. The range of final drug concentration in the well was 0.05–100 ng ml−1 for actinomycin D and 0–10 000 ng ml−1 for rHuTNF. As a control the TNF sensitive cell line WEHI-164 was used. The cells were incubated in the presence of a concentration of 0.05–100 ng ml−1 actinomycin D and 0–1 ng ml−1 TNF, diluted in RPMI supplement with 10% foetal bovine serum and L-glutamine. The sulphorhodamine B (SRB) protein stain assay was used according to the method of M Tris buffer, pH 9.4 and absorption was measured at 540 nm. Tumour growth was calculated using the formula: percentage tumour growth=(test well/control well) ×100%.BN soft tissue sarcoma cells were added in 100 μl aliquots to 96-well plates at a final concentration of 10−1. A washout with 5 ml oxygenated Haemaccel was performed at the end of the perfusion.The perfusion technique was performed as described previously (2B), where B represents the longest diameter and A the diameter perpendicular to B.Tumour growth was daily recorded by calliper measurement. Tumour volume was calculated as 0.4  means that the rat drags its hind limb without any function; a slightly impaired function (grade 1) means the rat does not use its hind limb in a usual matter, but stands on it when rising; an intact function of the hind limb (grade 2) means a normal walking pattern.Oedema was scored as no formation of oedema (−), or oedema resulting in leg diameter increase of 1.5-fold or more (+).The day after perfusion with actinomycin D alone or in combination with TNF tumour and muscle tissue of the perfused limb was removed fixed for 24 h in 4% formaldehyde and embedded in paraffin. Muscle from the non-perfused limb was also obtained, fixed and embedded. Tissue sections of 4 μm were cut and stained with haematoxylin and eosin, followed by examination using a Leica DM-RXA.U-test was used to compare tumour volumes. Calculations were performed on a personal computer using GraphPad Prism v3.0 and SPSS v8.0 for Windows 98.Mann–Whitney in vitro , TNF perfusions alone (P<0.05) and actinomycin D perfusions alone (P<0.05). The perfusions with a combination of 5 μg actinomycin D and 50 μg TNF resulted in a response rate of 40% (data not shown). When perfusions were performed with a higher concentration of actinomycin D a further increase in tumour response was observed. However due to the severe toxicity with this concentration no further experiments were performed. Perfusions with 40 μg melphalan and 50 μg TNF resulted also in major tumour shrinkage. A significant difference was observed as compared to sham perfusions, TNF perfusions alone and melphalan perfusions alone (P<0.05) (Perfusions with Haemaccel (sham), or with 50 μg TNF or 5 μg actinomycin D alone, resulted in progressive disease in all animals  . Perfusi3. Limb function at this stage was normal and no oedema could be observed. One day after perfusion with actinomycin D in combination with TNF the rats limb was severely swollen with oedema and the remaining tumour was barely visible. Perfusion with buffer or TNF alone had no effect on formation of oedema, whereas perfusion with actinomycin D had only a marginal effect.Shortly after ILP with actinomycin D in combination with TNF severe local toxicity was observed  were examined histopathologically. After sham perfusion some scattered cell necrosis in tumours could be observed, correlating with previous results (Figure 3Aas found . Rats tras found . Histoloas found . One dayas found . Rats tras found . Rats tras found . Treatmeas found .L-NAME to perfusions with melphalan with or without TNF enhanced antitumour effects (in vitro. Treatment with melphalan in combination with actinomycin D has been reported, but these studies showed no significant difference in response between treatment with the single agent melphalan and the combination therapy (in vitro were seen (in vitro studies using melphalan or doxorubicin in combination with TNF on several rat tumour cell lines as colon carcinomas and osteosarcomas (in vivo combination of melphalan as well as doxorubicin with TNF did result in strong synergy both in patients and animal models (Previously we have demonstrated that local treatment of advanced tumours with a combination of melphalan and TNF results in high response rates. We developed a rat tumour model and showed that results comparable to the clinic can be obtained regarding response rate and type of response (In the present study an isolated limb perfusion with TNF, actinomycin D or a combination in BN-175 soft tissue sarcoma bearing rats was performed to investigate the tumour response to these treatments. We demonstrated that the combination of actinomycin D with TNF was much more effective than ILP with the single agents. However, the response rate was less pronounced compared to previously described perfusions with melphalan or doxorubicin in combination with TNF.Application of ILP, with leakage below 5%, allows high local dosages of cytotoxic agents without the systemic side-effects (In conclusion, isolated limb perfusion with actinomycin D and TNF results in a tumour response of 40% which is not better than the standard ILP with melphalan plus TNF. Secondly, this therapy was accompanied by dramatic local toxicity, which increased with higher dosages, whereas response rate improved only marginally. Massive destruction of the muscle tissue was observed and as a result a loss of limb function, indicating that isolated limb perfusion with actinomycin D in combination with TNF should not be explored in the clinical setting.


The transplantable pregnancy-dependent mammary tumour (TPDMT-4), the related hormone-dependent (TPDMT-4EP) and autonomous (T4-0I320 and T4-0I96) subline tumours, and the mammary glands from DDD mice were compared for angiogenic activity on the rabbit cornea by tissue implantation. The TPDMT-4EP tumour was established by serially transplanting TPDMT-4 tumour fragments in oestradiol plus progesterone treated mice. The T4-0I320 and T4-0I96 tumours directly derived from the TPDMT-4 and TPDMT-4EP tumours, respectively. Angiogenic activity was graded by macroscopic and microscopic examinations into 3 classes; negative, partial and complete angiogenesis. These tumours were comparable to mammary glands in activity and induced complete angiogenesis in only 15-23% of the implants. However, when partial and complete responses were combined as positive angiogenesis, TPDMT-4, T4-0I320, TPDMT-4EP and T4-0I96 tumour implants were angiogenic in 25, 29, 42 and 54%, respectively. The T4-0I96 tumour was significantly more angiogenic than the parent tumour but this was not so for the TPDMT-4EP tumour. Spontaneous C3H mouse mammary tumours, human gliomas from nude mice, rat Walker 256 carcinomas and rabbit VX-2 tumours induced complete angiogenesis in 54, 63, 59 and 92% of the implants, respectively. The results suggest that the TPDMT-4 tumour is unique in being weakly angiogenic and able to progress toward greater autonomy with or without augmented angiogenic activity in different conditions.


L -glutamic acid (ZD2767P) and a conjugate of the F(ab')2 anti-CEA antibody A5B7 and the bacterial enzyme carboxypeptidase G2 (CPG2). ZD2767P is converted by antibody-targeted CPG2 into an active bifunctional alkylating drug (ZD2767) at the tumour site. The IC 50 value of the prodrug against the human colorectal tumour LS174T cell line was 55 ± 9 μM following a 1 h exposure. In contrast, co-incubation of ZD2767P with CPG2 resulted in 229-fold increase in activity. Using a modified comet assay, DNA interstrand cross links (ISC) were detected within 1 h of ZD2767P + CPG2 treatment and were repaired by 24 h. A clear dose–response was seen between the level of ISC, growth inhibition and ZD2767 concentration. Administration of a therapeutic dose of ZD2767P 72 h after the F(ab′)2 A5B7 conjugate to mice bearing LS147T xenografts resulted in extensive ISC in the tumour after 1 h; repair was seen at 24 h. Tumour biopsies and peripheral lymphocytes were studied in 5 patients on the ADEPT phase I clinical trial. In 4 patients no ISC were detected. These patients also demonstrated poor localization of conjugate and no tumour response was seen. However a significant level of ISC was detected in one tumour biopsy, which also showed evidence of conjugate localization and clinical response. These studies demonstrate the application of the comet assay in the measurement of ISC in vitro and in clinical material and confirm that activation of ZD2767P results in the formation of DNA crosslinks. © 2001 Cancer Research Campaign http://www.bjcancer.comAn antibody-directed enzyme prodrug therapy (ADEPT) system against CEA-positive tumours is currently in phase I clinical trials. It consists of a prodrug, 4- phenoxycarbonyl


P = 0.03) increase in plasma VEGF level in 29 CLM patients  compared with 19 controls . There were significant correlations between plasma VEGF and tumour vessel count , tumour vessel volume , and CLM volume . A VEGF level in the upper quartile of the plasma VEGF distribution had a 70% sensitivity and 75% specificity in predicting an upper quartile liver metastasis tumour vessel count. No relation was identified between CLM and plasma bFGF levels. Plasma VEGF level predicted CLM vascularity, despite an overlap with normal levels and tumour volume-related variations. © 2000 Cancer Research CampaignThe extent to which plasma levels of angiogenic factors in healthy individuals and tumour volume-related variations in colorectal cancer affect the accuracy of circulating angiogenic factors as predictors of colorectal cancer vascularity is unknown. We used enzyme-linked immunosorbant assay to measure plasma vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels in colorectal liver metastasis (CLM) patients, and ‘no cancer’ controls. CLM volume was determined from computerized tomography scans, and tumour vessel count and vessel volume from anti-endothelial antibody-stained biopsies. There was a significant (


Colchicine injected 5, 9 and 24 h respectively before initiation  led to a significant increase in skin tumour incidence in the --9-h group, and an increase in percentage malignancy in both the --5- and --9-h groups. These times corresponded to the peak of metaphase arrest by the colchicine. The results are discussed in relation in those of Pound and Withers (1963) and others, who found that mitotic stimulation at the time of urethane initiating action raised the ultimate tumour incidence; and the inference is drawn that initiating action in mouse skin may occur during the M phase, rather than during the G1, S, or G2 phases, as suggested by others.


A simple method has been developed which facilitates the detailed cytogenetic analysis of proliferating tumour cells within clusters and colonies arising from clonogenic tumour stem cells in biopsy samples of human cancers. The method uses a simple agar cloning technique for human tumours, which provides marked enhancement in the number of cases with observable mitotic activity and the number of mitotic figures available for detailed karyotypic assessment. The frequency of mitotic figures in cluster and colony samples is much greater than is attainable with standard chromosomal techniques. This novel approach should prove to be a powerful tool for the study of human tumour karyology.


The relationship of serum carcinoembryonic antigen (CEA) levels to tumour size and antigen content was studied in artificially immune-deprived mice bearing human colonic, breast and lung tumour xenografts. Size was measured as in vivo volume and tumour weight at post-mortem. A multiple implant technique combined with early harvest was used to minimize centrilobular tumour necrosis. CEA was extracted from resected tumours with perchloric acid. A radioimmunoassay using chemical precipitation was used to estimate CEA in blood samples. A correlation was found between CEA blood levels and tumour size in half the tumour lines, in contrast to a recent report . The CEA content was found to be constant for one tumour line but not another. The possibility that central necrosis in xenograft tumours may account for the discrepancies is discussed. There may be serious limitations for the use of xenograft tumour models for studying the biology of CEA.


Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-α) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-α was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-α in ILP. © 2000 Cancer Research Campaign


The effect of melphalan on the growth of 4 different lines of human lung-tumour xenografts has been established. The oat-cell carcinoma was the most sensitive, whereas the adenocarcinoma was the most resistant. Two lines of large-cell anaplastic carcinomas were intermediate in sensitivity. The differences in sensitivity were not reflected in the gross uptake of drug into the tumours. There was, with the exception of the adenocarcinoma line, a marked decrease in uptake per g tumour with increasing tumour size. This was partly caused by a decrease in the vascular supply in the same tumours with increasing tumour size. Extravasation of plasma proteins increased with increasing tumour size in all tumours, but was much less pronounced in the adenocarcinoma than in the other tumour lines. The extracellular volume of the various tumour lines did not vary with tumour size.


The effect of the growth of two syngeneic transplanted sarcomata of widely differing biological properties on the number of monocytes in the blood of rats was measured (1) by binding of a specific antimacrophage serum to leucocytes, and (2) by sedimenting in a density gradient rosettes between mononuclear cells and antibody-coated sheep red cells under conditions in which B-cells are not brought down. For the 4 syngeneic sarcomata studied there was a progressive increase in the number of monocytes with tumour growth and the values returned to normal a few days after their surgical removal. The extent of monocytosis was related to the immunogenicity of the tumour and was most pronounced for the HSBPA sarcoma, which is highly immunogenic, has a low rate of spontaneous metastasis and contains many macrophages, and least for the MC-3 sarcoma which is essentially non-immunogenic, invariably gives rise to distant metastases and contains only about 8% macrophages. The growth of sarcomata had previously been found to reduce the number of monocytes which enter inflammatory lesions, both non-specific and due to a delayed hypersensitivity reaction. This "anti-inflammatory" action of sarcomata which is related to their immunogenicity cannot be ascribed to the preferential uptake of monocytes by the tumours and it is concluded that the monocytes in the blood of tumour-bearers, though increased in number, are modified so that they do not enter sites of inflammation.


Subdermal inoculation of the foot of the rat with lethally irradiated (LI) Walker tumour (W256) cells, mixed with viable (V) W256 cells, decreased the latent period for initiation of allogeneic tumour growth without significantly affecting its rate. This Révész effect decreased with increase in the number of inoculated V cells, and with decrease in age of recipient. LI cells of a different (Y-P388) rat tumour exerted a Révész effect, even in recipients which had been immunized with LI (Y-P388) tumour cells. Local pre-irradiation of the site of inoculation of V cells decreased both the latent period and rate of tumour growth. It acted independently of a Révész effect, and the decrease in tumour growth rate was partly due to emigration of V cells from the inoculum, producing metastases. LI, but not heat-killed cells, induced prolonged swelling of the tumour bed in unimmunized and tumour-immunized rats, which, unlike inflammatory swelling, was inhibited by pre-irradiation of the foot. It is postulated that the Révész effect is due to enhancement of survival of V cells by trophic substances which are principally elaborated by LI (AND V) cells, but also by the tumour bed, due to innate growth and trophic reactions of its tissues to the presence of tumour cells.


The relevance of angiogenesis inhibition in the treatment of glioblastoma multiforme (GBM) should be considered in the unique context of malignant brain tumours. Although patients benefit greatly from reduced cerebral oedema and intracranial pressure, this important clinical improvement on its own may not be considered as an anti-tumour effect.GBM can be roughly separated into an angiogenic component, and an invasive or migratory component. Although this latter component seems inert to anti-angiogenic therapy, it is of major importance for disease progression and survival. We reviewed all relevant literature. Published data support that clinical symptoms are tempered by anti-angiogenic treatment, but that tumour invasion continues. Unfortunately, current imaging modalities are affected by anti-angiogenic treatment too, making it even harder to define tumour margins. To illustrate this we present MRI, biopsy and autopsy specimens from bevacizumab-treated patients.Moreover, while treatment of other tumour types may be improved by combining chemotherapy with anti-angiogenic drugs, inhibiting angiogenesis in GBM may antagonise the efficacy of chemotherapeutic drugs by normalising the blood-brain barrier function.Although angiogenesis inhibition is of considerable value for symptom reduction in GBM patients, lack of proof of a true anti-tumour effect raises concerns about the place of this type of therapy in the treatment of GBM. The brain is the highest organised and most complex organ of the body. Some unique features of the brain that have an impact on the biology of brain tumours include extensive three-dimensional structuring with gray and white matter , and hig®). Combined with chemotherapy, this monoclonal antibody against VEGF-A is FDA approved for the treatment of advanced non small-cell lung cancer, advanced breast cancer and advanced colorectal cancer, improving overall survival in these patients compared with chemotherapy alone [Despite attempts to improve treatment results over the last 30 years, GBM remains a highly fatal and devastating disease; a cure is extremely rare, even with aggressive treatment ,7. The cpy alone -13. Indepy alone -17. Due Despite these apparent improvements it should also be realised that the characteristic tumour micro-environment in brain may have a negative impact on the efficacy of such therapies, and may even be responsible for overestimation of efficacy. We outline below how the unique features of GBM may provide this type of tumour with an 'escape mechanism' from anti-angiogenic treatment, and present clinical and histological data that confirm results from preclinical work on orthotopic glioma xenografts. While angiogenesis inhibitors in combination with chemotherapies may effectively inhibit tumour cell proliferation in other tumour types, the brain micro-environment allows furtive invasion and proliferation in case of GBM.One of the aggravating symptoms of high-grade astrocytomas is increased intracranial pressure, a direct result of oedema caused by leaky tumour vasculature. Corticosteroids are used to alleviate these symptoms . AngiogeHowever, the question remains whether angiogenesis inhibitors represent a true anti-GBM regimen. Non-randomised phase II trials in recurrent GBM patients demonstrated high response rates ranging from 20% with single agent TMZ , to 30% Determination of the precise time at which GBM progression occurs is challenging. The often robust and sustained changes on MRI by anti-angiogenic drugs may render subsequent MRI scans difficult to interpret . TherefoA problem with the current enthusiasm for angiogenesis inhibition in GBM is that tumour response evaluations are based on cross sectional contrast enhancing tumour areas . BecauseIn the 1990s, MRI criteria for evaluation of brain tumour size and treatment responses were adjusted after introduction of corticosteroids in GBM treatment . Again, The central hypothesis of angiogenesis inhibition as anti-tumour strategy states that tumour growth and expansion are dependent on the ability of the tumour to generate its own supportive vascularisation -53. The Next to the angiogenic rim, in GBM large areas can be identified where viable and proliferating tumour cells invade the surrounding highly vascularised normal tissue. In the cerebrum, blood vessels and white matter tracts are typically located next to each other and form corridors for invading GBM cells . Close tChemotherapeutic drugs are generally not very effective against GBM, partly due to intrinsic resistance  but alsoThe relevance of angiogenesis inhibition in the treatment of GBM has been placed in the unique context of malignant brain tumours. Patients may benefit from anti-angiogenic therapy by its reduction of peritumoural oedema and intracranial pressure. Several studies also suggest an anti-tumour effect because of improved response rates and prolonged PFS; however, these data are derived from non-randomised trials with PFS as primary endpoint. Available data on survival prolongation are less robust (phase II) and sometimes even conflicting. High costs and side-effects of angiogenesis inhibitors  need to be balanced against advantages related to survival. Therefore, we can only implement angiogenesis inhibitors as standard treatment for patients with GBM when data are available from randomised clinical trials with survival as primary endpoint. Our concerns are not restricted to the limited amount of data on outcome and toxicity, but also arise from preclinical data on the biology of GBM and angiogenesis inhibition.GBM can be roughly separated into an angiogenic component, and an invasive or migratory component. Although this latter component seems inert to anti-angiogenic therapy, it is of major importance for disease progression and survival Figure . Whereas1. GBM can be roughly separated into an angiogenic component, and an invasive or migratory component.2. Although this invasive or migratory component seems inert to anti-angiogenic therapy, it is of major importance for disease progression and survival.3. Whereas symptoms are tempered by anti-angiogenic treatment, furtive invasion of the disease continues, unrecognised by standard imaging modalities.4. Moreover, while treatment of other tumour types is improved by combining chemotherapy with anti-angiogenic drugs, inhibiting angiogenesis in GBM may antagonise the efficacy of chemotherapeutic drugs by normalising the BBB function.5. Although angiogenesis inhibition is of value for symptom reduction in GBM patients, the possible lack of a true anti-tumour effect raises concerns about the place of this type of therapy in the treatment of GBM.The authors declare that they have no competing interests.JJCV, OVT, LJAS, DJR, WPJL and WRVF designed the experiments and the study. JJCV, OVT, AC, WPJL and WRVF collected data for the study. JJCV, OVT, AC, MEVL and WPJL analyzed the data. JJCVV, LJAS, MEVL, DJR and WRVF enrolled patients. JJCV and OVT wrote the first draft of the paper. JJCV, OVT, AC, LJAS, MEVL, DJR, WPJL and WRVF contributed to the writing of the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/444/prepub