Water homeostasis during fetal development is of crucial physiologic importance. It depends upon maternal fetal fluid exchange at the placenta and fetal membranes, and some exchange between fetus and amniotic fluid can occur across the skin before full keratinization. Lungs only grow and develop normally with fluid secretion, and there is evidence that cerebral spinal fluid formation is important in normal brain development. The aquaporins are a growing family of molecular water channels, the ontogeny of which is starting to be explored. One question that is of particular importance is how well does the rodent  fetus serve as a model for long-gestation mammals such as sheep and human? This is particularly important for organs such as the lung and the kidney, whose development before birth is very much less in rodents than in the long-gestation species. There are, at present, eleven known members of the mammalian aquaporin gene family, which encode proteins which function as membrane channels, for water alone , or for water plus small molecules, mostly glycerol and urea AQP 3, 7, 9), or nitrate AQP 6) [ [6]; AQP, 9, or nDuring development there are some unique fluid compartments  and the functions of some organs, such as the kidney, differ from the function in the adult, as discussed below. Although some insights into the developmental roles of aquaporins might be obtained from the study of mice with deletions of various aquaporin genes, this is complicated by the facts that either much of normal organ development occurs postnatally in the rodent, rather than prenatally in the human (e.g.kidney), chick (brain) or the ontogeny of aquaporins differs significantly in the rodent organ (e.g.lung) from that in long-gestation species such the sheep . In addiAmniotic fluid surrounds the developing fetus and is essential for normal morphological development. Inputs into amniotic fluid include the dilute fetal urine and the isotonic lung liquid, and pathways of exit of fluid include fetal swallowing, and transmembrane fluxes ,13. ThusBefore implantation the conceptus develops into a blastocyst, composed of the inner cell mass, and a fluid filled cavity surrounded by trophoectoderm epithelium. In the mouse aquaporins 3, 8, and 9 have been found to be expressed at this time, AQP3 and AQP8 being predominantly in the basolateral membranes of the trophoectoderm, and AQP9 in the apical membrane . The troFrom 45 d gestation (term is ~150 days), AQP3, functioning both as a water and urea channel, and expressed in the trophoblast epithelial cells, is the major AQP, which increases throughout gestation, and is quantitatively the most highly expressed AQP gene in the ovine placenta. The permeability of the ovine placenta to urea increases markedly after ~100 days of gestation, coordinately with a sharp increase of AQP3 expression in the placenta at this time.Similarly, AQP8, which is expressed in the trophoblast epithelial cells and membrane epithelial cell , is alsoIn sheep, the placenta ceases growth close to mid-gestation, despite the dramatic increase in the fetal weight during the last half of gestation . To mainThe fetal metanephric kidney produces a relatively large volume of dilute urine, essential for the maintenance of amniotic and (in some species) allantoic fluid volumes. In the most common animal model (sheep) used for the study of fetal renal function it has been shown that the volume of urine production is 0.3 l/kg/d compared with 0.02 l/kg/d in the adult sheep. This occurs in spite of a glomerular filtration rate which is approximately one third of adult values, and is due to both a decrease in total sodium reabsorption (95 % in the fetus vs 99% in the adult) and to absence of significant concentration of the urine. In the unstressed ovine fetus the urine osmolality is always less than 200 mosmoles.kg water, and may be as low as 60 .In the adult kidney the bulk of the filtrate (81%) is reabsorbed in the proximal tubule and descending limb of the loop of Henle, where AQP 1 is expressed. AQP1 is also expressed in the nonfenestrated descending vasa recta which are thought to be important for the establishment of the hypertonic environment of the medulla. In the mouse with the AQP1 gene deleted there is a lowered capacity to maximally concentrate urine . HoweverMetanephric kidney development varies in different species, being complete before birth in human and sheep, but not until substantially after birth in pigs, mice and rats. The ontogeny of some renal aquaporins has been examined in rats, sheep and humans. In the rat there is very little mRNA for AQP1 detected by Northern blotting or RNase protection, in the kidney, until a few days before birth ,36. HoweAquaporin 2 (AQP2) is low at birth in the rat, but plateaus by 4 weeks post-partum . Later sThere has been one study of AQP3, in fetal kidneys, suggesting it is there by day 18 in the rat . The levThe low level of AQP2 expression, however, seems to be the major factor in allowing the production of a large volume of hypotonic urine to be formed, and this is essential for the maintenance of adequate volumes of amniotic fluid.- in the same direction. It is not known exactly when lung liquid secretion begins, but fluid is present by mid-gestation in fetal sheep and is secreted at 2–4 ml/kg/h between 120 days of gestation and term (~150 d). Fetal lung liquid exits the lungs via the trachea, whereby it is either swallowed (approximately 50%) or passes directly into the amniotic sac, where it contributes to amniotic fluid volume [During fetal life, the future airways of the lung are filled with a liquid that plays a crucial role in the growth and development of the lungs by maintaining them in an expanded state. Lung liquid is secreted across the pulmonary epithelium into the lung lumen due to the osmotic gradient established by the net movement of Cld volume .If the fetal trachea is obstructed, which prevents the outward flow of lung liquid, the fetal lung expands with accumulated liquid. This is a potent stimulus for fetal lung growth and also greatly reduces the proportion of type-II alveolar epithelial cells (AECs). Lung liquid drainage on the other hand, deflates the lung, causes lung growth to cease, but increases the proportion of type II AECs, possibly via type-I to type-II cell differentiation . As a reAt least four AQPs AQP 1, 3, 4 and 5) are expressed in the lungs of various species, including humans, rats, mice and rabbits, although some discrepancies exist in the specific sites of distribution of these proteins. , as well as their respective proteins, are present in the ovine fetal lung well before birth . For AQPIn conclusion, we have shown that the lung of a long-gestation species, such as sheep, expresses both the mRNA and protein of the four typical lung AQPs, beginning well before the expected time of birth. Furthermore, we found that the expression of some, particularly AQP5, is altered by factors known to regulate fetal lung growth and development and parallel changes in fetal lung liquid secretion rates in different animal models. Our findings suggest that gene knock-out studies in mice, in which there is little lung expression of AQPs in fetal life, might not give a realistic picture of the role of AQPs during fetal life in long-gestation species. We predict that these AQPs are also expressed well before birth in the human fetal lung and are also differentially regulated by factors known to influence fetal lung development. As lung liquid is secreted, at least in part, into amniotic fluid, the lung aquaporins are also then implicated in amniotic fluid regulation.The skin of the adult 70 kg man normally contains about 7 l of fluid, about 50% of which is interstitial . The fluIn the human fetus there is a double layer of epidermal cells by 4 weeks; the stratum corneum begins to develop by 24 weeks, and is generally well developed by 34 weeks. . BarrierAquaporin 1 mRNA was found in rat heart ,73. MostCaveolin-3 is a marker for the caveolae – the specialised areas of cell membrane in which a number of receptors cluster . Some ofBased on studies in isolated rabbit hearts, it was concluded that water permeabilitity values were much lower than expected if a functioning aquaporin were present . In a moDuring development AQP1 was found in the endocardium of the sheep fetal heart at a very early stage . Later iUsing RNase protection assay only AQP1  was detected in rat heart , howeverIn a recent study we showed that the small hearts of late gestation growth-retarded ovine fetuses had significantly reduced expression of AQPs 1,3,4 but not AQP8 . It was In conclusion, we demonstrated that the AQP1/3/4/8 are present in the late gestational fetal heart. The low-dose dexamethasone treatment, administered early in gestation, down regulated the expression of AQP1/3/4 in the late gestation fetal heart. In most studies of experimentally induced fetal growth retardation some organs, eg the brain and adrenal gland are 'spared', but others, such as the heart, are reduced in size in proportion to the overall decrease in body size . There aThere is evidence in the literature suggesting that fetuses suffering from severe intrauterine growth retardation (IUGR) show a progressive impairment of cardiac function, as demonstrated by reduced peak velocities at outflow tracts, decreased cardiac output and abnormal venous flow patterns -98. FurtThe exact function for AQP1 in cardiac muscle is unknown. As it is a pure water channel one would suspect that it could regulate the rate at which cells might swell in osmotic stress, such as encountered in myocardial ischemia . Such osIn the adult brain fluid balance is critical, as the inflexible bony skull does not permit big variations in total brain volume without risking severe damage. The extracellular fluid of the brain is specialized as cerebrospinal fluid, with a composition different from that of normal extracellular fluid, as a result of the development of the 'Blood-brain barrier'. There is now increasing evidence that cerebrospinal fluid plays an important part in the correct development of the brain ,104. SpeA number of aquaporins have been found in the central nervous system – AQPs 1,4,5,9 ,112. AQPThe ontogeny of AQP4 in the cerebellum coincides with the development of the blood brain barrier in rat and chick. ,121. In In the ear of the adult rat mRNA for aquaporins 1,2,3,4,5,6 have been found , whereasAQP2 mRNA, at 10% of the levels found in kidney, is found in rat and mouse ear . It is iQuantitatively the most important aquaporin expressed in the ear is AQP4, and it is expressed in Hensen's cells and inner sulcus cells and Claudius cells, which are all supporting cells of the Organ of Corti . In the AQP3 was found by one group  in the sAll these results in rodents are tantalizing, and it will be very interesting to see the ontogeny of brain and sensory organ aquaporins in the primate/human. It is expected that significant expression of these water channels will be seen well before birth, as is the case for the lung in long-gestation species .Much information on the role of various members of the mammalian aquaporin family of water channels has been gained in the relatively short time since Peter Agre and his colleagues described the Channel-forming integral membrane protein of the red blood cell of 28 kD (CHIP28), , and jus


The uptake of myristic (C14:0), palmitic (C16:0), palmitoleic , stearic (C18:0), oleic , linoleic  and arachidonic  acids from plasma free fatty acids (FFA), triglycerides (TGA), phospholipids (PL) and cholesterol esters (CE) was measured in tissue-isolated hepatomas 7288CTC and 7777 in vivo. Adult tumour-bearing Buffalo rats were fed a normal chow diet ad libitum and were subjected to darkness from 1800 to 0600 h. Arterial plasma levels of FFA, TGA, PL and CE were increased during the dark period without change in fatty acid compositions. Arteriovenous difference measurements of tumour lipid uptake were performed between 0600 and 0900 h and included both high (dark) and low (light) arterial blood lipid concentrations. The rate of lipid uptake from each lipid class was directly dependent on the rate of supply of the lipid to the tumour. The efficiency of uptake, however, depended on the type of plasma lipid and the tumour. During one pass of arterial blood, hepatoma 7288CTC (n = 5 to 13) removed 46, 33, 36 and 31%, and hepatoma 7777 (n = 7 to 9) removed 48, 50, 52 and 49% of the fatty acids supplied in FFA, TGA, PL and CE, respectively. Perfusion of tissue-isolated tumours in situ with donor blood containing plasma free (1-14C)palmitic acid showed that 14C-palmitic acid was removed from the arterial blood and was incorporated into tumour lipids and that 14CO2 was released into the tumour venous blood. Uptake of the seven fatty acids over a 24 h period was greatest from PL greater than TGA greater than FFA greater than CE and was estimated to total 18.1 +/- 3.5 mg fatty acids g-1 for hepatoma 7288CTC and 25.9 +/- 3.5 mg fatty acids g-1 for hepatoma 7777. Both hepatoma 7288CTC and 7777 grew at a rate of about 1 g day-1 and contained 13.4 +/- 2.5 and 10.6 +/- 3.9 mg of these 7 fatty acids g-1 tumour wet weight, respectively. We conclude that these two tumours obtain all of the fatty acids needed for daily growth from host arterial blood.


Tumour cords have been measured in 33 cases of squamous cell carcinoma (SCC) of the bronchus and 19 cases of SCC of the uterine cervix. The overall mean cord radius for SCC in both sites was 104 microns, similar to the overall mean for various tumours in rodents. For tumour cells adjacent to blood vessels in cords of SCC, the mean Mitotic Index was 2.1% and from this value a rapid potential doubling time could be inferred (approximately 31 to 66 h). The proportion of dead cells within cords of cervical SCC was higher than in animal tumours. Using measured values for cord radius and published equations that describe the diffusion and consumption of oxygen in metabolising tissue, an attempt was made to calculate the oxygen partial pressure in vessels of cords of these SCC.


Blood pharmacokinetics and tissue distribution of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a boron carrier with postulated melanin-seeking properties for boron neutron capture therapy, were determined in C57/BL mice with subcutaneous pigmented or non-pigmented B16 melanomas. Borocaptate sodium (BSH) was used as a boron compound without melanin-seeking properties in a comparative biodistribution study in the same animal tumour models. Administration of single doses showed that BPTU was retained better in the pigmented B16 tumour than in the non-pigmented variant. BPTU was found in large concentrations in kidney and liver. Brain boron was approximately 10-fold lower than tumour boron. On a molar basis, BPTU demonstrated higher affinity for B16 tumours than BSH. Owing to solubility limits, tumour boron concentrations in this mouse study were too low for effective application of BNCT. However, the high tumour-to-blood and tumour-to-normal tissues ratios indicate that, with appropriate formulation, BPTU could be a promising candidate for clinical BNCT.


In the present studies the effect of bilateral adrenalectomy on the pathophysiologic responses to recombinant human interleukin-1 alpha  was determined in RIF-1 tumour models. Acute vascular injury and haemorrhagic responses were quantitated by the intra-tumour accumulation of 59Fe radiolabelled erythrocytes. In vivo clonogenic tumour cell kill was determined by an excision assay. A single, intraperitoneal rHIL-1 alpha treatment (6.25 x 10(7) D10 units kg-1, 25 micrograms kg-1) resulted in acute tumour haemorrhage and approximately 55% clonogenic tumour cell kill (24 h). Bilateral adrenalectomy, 24 h before rHIL-1 alpha, significantly increased haemorrhagic responses, but haemodynamic toxicity was severe. This toxicity could be ameliorated by giving dexamethasone (5 mg kg-1) before or up to 3 h after rHIL-1 alpha. The effect of dexamethasone on rHIL-1 alpha induced tumour responses in adrenalectomised mice was sequence dependent. Given before rHIL-1 alpha, dexamethasone inhibited tumour haemorrhage. When dexamethasone was given up to 3 h after rHIL-1 alpha, tumour haemorrhage was directly related to sequence interval. Although adrenalectomy and dexamethasone alone had little effect on RIF-1 tumours, adrenalectomy increased rHIL-1 alpha mediated clonogenic tumour cell kill. The surviving fraction 24 h after rHIL-1 alpha (6.25 x 10(7) D10 units kg-1, 25 micrograms kg-1) and dexamethasone  was 1.3 +/- 0.4%. The surviving fraction after this combination in intact mice (36.7 +/- 1.4%) was approximately 30-fold higher than that seen in adrenalectomised mice. The results indicate that adrenal responses secondary to rHIL-1 alpha treatment exert a negative feedback on rHIL-1 alpha mediated responses in solid tumours.


Effects of recombinant tumour necrosis factor (TNF) on functional and structural vascular volumes in solid murine Meth A tumours were investigated by injection of Hoechst 33342 and staining for the vascular basement membrane component laminin, respectively. Systemic injection of 3 x 10(4) U TNF caused an initial increase in functional volume in the tumour, but a strong decrease from 1 to 48 h after treatment. Early effects of intralesional treatment were more moderate. Systemic injection of 10(4) U TNF or 0.3 or 3 micrograms lipid A caused a fall in functional volume at 4 h, but a recovery was seen at 24 h. This recovery did not occur after treatment with a combination of 10(4) U TNF and 0.3 micrograms lipid A. Structural vascular volume was not markedly reduced until 24 h after treatment with the high doses of the separate agents and the combination. All effects appeared generally more prominent in the tumour centre than in the borders. Data suggest that TNF induces initially an active hyperaemia that rapidly converts to passive hyperaemia. A prolonged disturbance of tumour blood supply is probably necessary for therapeutic activity. Breakdown of laminin in the vascular basement membrane may be a cause of loss of vascular integrity.


Previously, we demonstrated that the interaction between leucocytes and endothelial cells in tumour tissues is greatly diminished compared with normal tissues under several induced inflammatory conditions. Radiation has been reported to cause release of inflammatory mediators and to promote neutrophil adhesions to cultured endothelial monolayers. In this study, we tested the hypothesis that radiation would cause increased leucocyte rolling and adhesion in both tumour and normal tissues. We examined these two parameters in response to 6 Gy of gamma-radiation in mammary adenocarcinomas implanted into rat skinfold window chambers as well as normal (i.e. non-tumour-bearing) preparations. Leucocyte rolling and adhesion were measured in terms of flux of rolling leucocytes (F(rolling)) and density of adhering leucocytes (D(adhering)) in microvessels. F(rolling) and D(adhering) were measured in two groups of preparations: irradiated and control. In normal preparations, F(rolling) and D(adhering) were both increased significantly by radiation. In contrast, in adenocarcinoma-bearing preparations, F(rolling) and D(adhering) were either unchanged (in the tumour centre) or reduced  by radiation. Radiation did not cause changes in haemodynamics in these preparations, thus the observed changes in leucocyte rolling and adhesion could not be accounted for by haemodynamic factors. These results indicate that: (1) in normal preparations, radiation could cause inflammation as manifested by increased leucocyte rolling and adhesion; and (2) in tumour-bearing preparations, radiation caused changes in the vascular surface properties such that they became less adhesive to leucocytes. Such differences in radiation response may have important implications for radiation therapy and provide new insights into the unique features of tumours.


The release of components from human kidney tumour xenografts (GYL) and human foetal kidney explants maintained in nude mice has been studied. The GYL tumour released antigens into the serum which could be detected by the generation of antibodies following cross-immunisation of closely related hairy litter mate (HLM) mice. The production of anti-GYL antibody was monitored by an I125 binding assay using viable GYL tumour cells. In 2/16 hairy litter mate mice, cell surface antibody binding by GYL cells was twice that found with 8 other human tumour cell lines (including 2 other kidney cancer cell lines). Absorption of these antisera with 10(7) GYL tumour cells completely abolished this response, where 50%, 38% and 25% of activity remained following absorption with; a normal kidney cell line, a homogenate of normal kidney and a mixed pool of human tumour cells. Six out of 8 GYL tumour bearing nude mice tested had elevated plasma levels of HCG. Absorption of the HLM antisera with an excess of commercial HCG abrogated I125 binding by only 15%, suggesting that antibody production was not directed primarily against ectopic HCG.


Early liver metastases have a predominant portal blood supply. Intraportal (i.port.) vein administration of cytotoxics could theoretically achieve enhanced drug concentrations in tumour cells and be effective as adjuvant therapy after resection of colorectal carcinoma. Pirarubicin (which has a higher hepatic extraction than doxorubicin) was investigated on liver metastases of the VX2 rabbit tumour, which were of less than 2 mm in diameter 7 days after cells injection into the portal vein. To evaluate antitumour activity, 24 rabbits were randomised into three groups 7 days after implantation: (a) control, (b) i.v. pirarubicin, (c) i.port. pirarubicin at doses of 2 mg kg-1 in both groups. Portal infusions led to no hematological or hepatic toxicity. Pharmacokinetic parameters showed a significantly reduced systemic exposure after i.port. administration. Fourteen days after treatment, livers and lungs were analysed. The mean number (+/- s.d.) of tumour foci was (a) 8.62 (+/- 5.4), (b) 4.62 (+/- 3.2), (c) 2.25 (+/- 1.4) (P < 0.05 a vs c). The mean tumour area was (a) 6.31 (+/- 6.1), (b) 1.31 (+/- 2.2), (c) 0.43 (+/- 0.4 cm2) (P < 0.05 a vs c) and the percentage (95% C.I.) of rabbits with lung metastasis was: (a) 87.5% (47-99%), (b) 75% (35-97%), (c) 12.5% (3-52%) (P < 0.02 b vs c). Intraportal pirarubicin seems to be well tolerated and more efficient than i.v. administration, particularly in preventing extrahepatic dissemination.


The ability of pyrazinamide to enhance the in vivo cytotoxicity of cyclophosphamide in Lewis lung and RIF-1 tumours was investigated. Using an in vivo/in vitro excision assay a large single dose of pyrazinamide (500 mg kg-1 i.p.) was shown to enhance the tumour cell killing by cyclophosphamide. This enhancement was greatest when pyrazinamide was administered before the alkylating agent and had a dose-modifying effect on all cyclophosphamide doses tested, giving rise to a mean (+/- 1 s.e.) enhancement ratio (ER) of 1.54 (+/- 0.15) for the Lewis lung and 1.24 (+/- 0.08) for the RIF-1 tumour. Pyrazinamide also increased the cytotoxic action of cyclophosphamide in a normal tissue, namely white blood cell counts. However, the ER was only 1.14 (+/- 0.08), which although not significantly different from the value seen in RIF-1 was significantly less than the ER obtained with Lewis lung, suggesting the possibility of a therapeutic gain. This benzamide analogue did not appear to inhibit recovery from cyclophosphamide-induced potentially lethal damage in tumours, nor did it alter the bioactivation of cyclophosphamide or the subsequent clearance of the cytotoxic species from the plasma, so the mechanism for this chemosensitisation remains unclear.


Analysis of tissue sections from transurethrally resected bladder tumours using anti-CD3 antibody showed the presence of T lymphocytes in intra-epithelial layers in eight of 12 cases investigated. In a larger group of patients, Tumour Infiltrating Lymphocyte (TIL) growth was established from six of 19 cases using Interleukin-2 (IL-2) and conditioned medium (CM) and resulted in the expansion of TILs up to 100-fold. TILs from these individuals were phenotyped with W6/32 , HB55 (anti-DR) and anti-CD3 antibodies using FAC sorter. The mean +/- s.d. frequency of positive staining with these antibodies were 96.7 +/- 4.0%, 87.5 +/- 10.0% and 82.5 +/- 7.8% respectively, indicating the activated nature of these T cells. The cytotoxic activity of these TILs against Daudi  cell line at 25/1 E/T ratios varied from 26.3 +/- 3.2 to 62.8 +/- 5.2%. In one case where TILs and autologous tumour cell line were established, cytotoxicity studies showed low level of cytotoxicity against the autologous tumour cells (15.8 +/- 1.6%) compared with 62.8 +/- 5.2% against Daudi. Staining of tumour sections from these 19 individuals with W6/32 and BBM.1 revealed positive staining in six of six that developed TILs but only six of 13 (46%) cases, whose tumour failed to grow TILs . These results are indicative of the presence of IL-2 passageable T cells in bladder cancer biopsy and demonstrate that the successful expansion of these cells correlates with the normal expression of class I antigens on the tumour cells.


The vascularity of 107 primary cutaneous melanomas has been characterized by morphometric histological analysis. The lesions selected for study were of thickness 0.85-1.25 mm and the aim was to evaluate the prognostic significance of tumour vascularity. Two groups of patients were identified; 86 with no evidence of recurrence after a minimum follow-up period of 5 years and 21 with locoregional recurrence and/or metastasis. The lectin Ulex europaeus type I was used for endothelial cell staining of tissue sections and morphometric analysis was performed to derive the vascular length, surface and volume density from independent measurements of tumour, adjacent dermis and the junctional zone between tumour and underlying tissue. A wide range of values was obtained for each parameter with increased vascularity always found at the tumour base compared with the tumour as a whole. In relation to the adjacent normal dermis, vascularity was generally found to be higher at the tumour base but either higher or lower in the tumour overall. Tumour recurrence could not be predicted by any of the derived vascular parameters either independently or together with other histological and clinical features. This study suggests that tumour vascularity is of no prognostic significance in melanoma of the above thickness range. The highly variable extent of tumour vascularity was not correlated with other clinical or histological parameters, but may have implications for the delivery of pharmaceutical agents used for diagnosis or therapy.


Granular cell tumours (GCT) of the soft tissues are rare benign tumours but some time may be difficult to distinguish from malignant neoplasms. It is important that clinicians are aware of their existence. We present a new case of GCT of the soft tissues followed by a brief review of literature. Granular cell tumours are rare benign tumours and were first described by Weber in 1854  reportedA 47-year-old woman presented to breast clinic with a swelling over sternal region that was causing discomfort. On physical examination, there was a firm, mildly tender, 5 × 4 cm palpable lump in the midline, anterior to sternum. Overlying skin was normal and mobile. Fine needle aspiration of the lesion revealed only blood. Ultrasound guided core biopsy of the lesion showed sheets of cells with abundant granular cytoplasm and small unremarkable bland nuclei. A diagnosis of granular cell tumour was made. No evidence of malignancy was noted on the core biopsy. The patient underwent a wide surgical excision of the tumour and had an unremarkable post-operative recovery. She was discharged the next day.The specimen submitted to pathology contained an irregular piece of soft tissue measuring 5 × 4.5 × 3 cm. The specimen weighed 27 grams. On sectioning, there was a firm nodule, which measured 2.5 cm in diameter and was whitish in colour. The nodule appeared very close to the posterior wall of the specimen. Immunohistoichemistry showed S100 to be diffusely positive, while NSE faintly positive. All these features were suggestive of granular cell tumour. Histology confirmed that excision margins were clear of the tumour.  declare that they have no competing interest.


The in vivo characteristics of four analogues of benzoporphyrin derivative (BPD) have been investigated. Biodistribution data obtained in DBA/2J mice with BPD-MA  which had been tritiated or internally labelled with 14C showed that both labelled materials acted in an essentially identical manner during the period of study. Biodistribution and clearance studies showed that relative distribution in a variety of mouse tissues was similar for all BPD analogues. M1 tumour cells (rhabdomyosarcoma in DBA/2J mice) taken from tumours excised from animals treated 3 h earlier with BPD, and tested in vitro for photosensitivity provided evidence that significant levels of photosensitiser detected in tumour was both active and associated with tumour cells. The monoacid forms of BPD were found to be much more photodynamically active in this test than were the diacid analogues. The ability of the analogues to ablate tumours in mice by photodynamic therapy was also tested. Again, BPD-MA and BPD-MB proved to be measurably better than the diacid analogues. These findings are discussed in reference to structural and physical differences between the analogues.


Flavone Acetic Acid (FAA) exerts much of its effect by reducing tumour blood flow. Previous studies on FAA-induced changes in blood flow have used established tumours with a functional microvasculature. Using radioactive Xenon(133Xe) clearance to monitor local blood flow we show that the effects of FAA are dependent on the presence of this functional microvasculature with no evidence that FAA inhibits the actual development of tumour microcirculation. Thus, administration of multiple doses of FAA around the time of tumour cell injection failed to diminish t1/2 values of 133Xe  or to affect tumour volumes . In marked contrast a single dose of FAA (200 mg kg-1 body weight) 2 weeks after tumour cell injection dramatically extended t1/2 times  and significantly reduced tumour burden. This effect is specific for tumour microvasculature and is not directed simply at new vessels since a similar treatment of animals with implanted-sponge-induced granulation tissue had no effect on t1/2 times (6.8 +/- 1.1 min for FAA at 200 mg kg-1 vs 7.2 +/- 1.0 min for saline-treated controls.


Pentoxifylline (PTX) has been shown to increase radiation damage to tumours and to decrease late radiation-induced injury to normal tissues. This tumour radiation sensitisation results from increased oxygen supply via improved tumour perfusion. We propose that the improved perfusion results from decreased viscous resistance and/or geometric resistance. The decreased flow resistance may be accompanied by a reduction in microvascular pressure (MVP). Since MVP is approximately equal to the interstitial fluid pressure (IFP), PTX should lead to a decrease in IFP. To test this hypothesis, we measured PO2, laser Doppler flow (RBC flux) and IFP in FSaII murine tumours at two doses (PTX at 25 and 100 mg per kg body weight) which sensitise this tumour to X-irradiation. We found that 25 mg kg-1 PTX was ineffective, but 100 mg kg-1 PTX was effective in increasing the PO2 of this tumour. PTX at 100 mg kg-1 (i.p.) increased median PO2 from 5 to 7 mmHg (P < 0.05) within 2 h, and decreased the fraction of PO2 values < 5 mmHg from 65% to 45% (P < 0.05). In support of our hypothesis, we found that with this dose of PTX, RBC flux in the tumour centre increased significantly  prior to an approximately 40% decrease  in tumour interstitial fluid pressure (TIFP), without changes in mean arterial blood pressure (MABP). In conclusion, a single i.p. administration of PTX at 100 mg kg-1 can increase oxygen availability in the tumour due to ameliorate hypoxia in tumour microregions. Second, PTX can lower the elevated TIFP without lowering the MABP.


The effect of the synthetic progesterone, megestrol acetate, on weight loss induced by both tumour necrosis factor alpha (TNF) as a model for the cachexia accompanying the acquired immunodeficiency syndrome and by a cachexia-inducing tumour (MAC16) has been studied in NMRI mice. Megestrol acetate was effective in preventing weight loss in both model systems with treated animals having an increase in intake of both food and water. Megestrol acetate was unable to prevent loss of body weight in animals pair-fed with TNF treated animals, suggesting that the increase in food and water intake was responsible for the increase in body weight. Analysis of body composition showed that the major contribution to the increase in body weight in animals treated with megestrol acetate was an increase in water content, although there was also an increase in carcass fat in animals bearing the MAC16 tumour given the high dose of megestrol acetate. Animals bearing the MAC16 tumour had a significant increase in tumour weight after treatment with megestrol acetate, possibly owing to the increased plasma glucose levels. These results suggest that an increase in appetite and weight gain alone are not sufficient to justify the anticachectic effect of a particular agent and that body composition analysis and tumour growth rate are very important parameters.


The ability of cancer cells to survive at a distance from blood vessels should be dependent on the local supply of nutrients to each vessel. The corded growth of tumour cells around blood vessels within regions of necrosis in the RH carcinoma in the mouse allows the limit to which cells can be supported by individual vessels to be observed. The thickness of individual tumour cords was measured in conventionally stained tumour sections using a scanning technique to determine the distance between the blood vessel wall and the most distant viable cell adjacent to necrosis. Cord radius was found to vary with the oxygen supply conditions. Control animals had a mean radius of 105 +/- 2 microns while animals that had breathed 10% oxygen had significantly narrower cords (93 +/- 3 microns after 48 h) and animals breathing 100% oxygen had significantly wider cords (117 +/- 3 microns after 24 h). Mice made anaemic (mean hct. 28%) by phlebotomy and plasma transfusion had cord radii that were not significantly different from controls at any time up to 48 h. We conclude that this relatively slow growing mouse tumour is capable of rapid morphological adaptation (less than 3 h) to changes in nutrient availability and that oxygen is probably the limiting substrate.


Vasoactive drugs have a variety of effects upon splanchnic and hepatic haemodynamics which may alter tumour blood flow and potentiate the delivery of a chemotherapeutic drug to hepatic tumour. We have investigated the effects of vasopressin infusion on hepatic tumour blood flow in an experimental model of liver tumour. Hepatic tumour was induced by the intraportal inoculation of HSN sarcoma cells. Hepatic and splanchnic blood flow was determined using a dual reference microsphere technique before and after an intravenous infusion of vasopressin at a dose of 0.1 mU kg-1 min-1 for 10 min. There was a significant increase in systemic arterial blood pressure associated with a rise in portal venous inflow  and a significant fall in hepatic arterial flow (P less than 0.05). The tumour: liver blood flow ratio was significantly increased by vasopressin infusion (P less than 0.02). Vasopressin infusion decreases hepatic arterial flow and increases tumour blood flow which may potentiate the delivery of a regionally delivered chemotherapeutic drug to hepatic tumour.


Histopathology50, 591–596 Erythropoietin/erythropoietin-receptor system is involved in angiogenesis in human hepatocellular carcinomaRibatti D, Marzullo A, Gentile A, Longo V, Nico B, Vacca A & Dammacco F (2007) To correlate microvascular density and erythropoietin (Epo)/Epo-receptor (EpoR) expression in endothelial and tumour cells with histopathological type in hepatocellular carcinoma (HCC).Specimens of primary HCC obtained from 50 patients who had undergone curative hepatectomy were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. Poorly differentiated HCC had a higher degree of vascularization than other stages and Epo/EpoR expression in both tumour and endothelial cells increased in parallel with grade of malignancy and was highly correlated with the extent of angiogenesis.Epo/EpoR levels correlate with angiogenesis and progression of patients with HCC and these findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. It is thus suggested that Epo is an important factor in hepatic tumour angiogenesis. Understanding the mechanisms of HCC angiogenesis provides a basis for a rational approach to the development of antiangiogenic therapy in patients with hepatic cancer. Erythropoietin (Epo) is a low-molecular-weight glycoprotein hormone stimulator of erythropoiesis produced in the fetal liver and subsequently in the adult kidney.2in vitro.et al.Epo is a pleiotropic cytokine that exerts diverse biological effects in many non-haematopoietic tissues. There is increasing evidence suggesting a wider biological role for Epo/EpoR unrelated to erythropoiesis. Angiogenesis, the process by which new blood vessels arise from pre-existing ones, has been shown to be one of the extrahaematopoietic functions of Epo.The potential role of Epo in angiogenesis may be considered as a subsidiary of its possible function in improving overall tissue oxygenation and of its antiapoptotic role. The expression of EpoR in tumour vascular endothelium suggests that Epo may affect the tumour microenvironment, perhaps by stimulating tumour angiogenesis.3Experimental and clinical data indicate that tumour progression in human hepatocellular carcinoma (HCC) is associated with angiogenesis and that an increase in microvascular density is associated with a poor prognosis.11In this study, we investigated immunohistochemical expression of Epo/EpoR in HCC and correlated this with CD31 expression in vascular endothelial cells with the aim of establishing a potential role for Epo in angiogenesis in HCC.Fifty patients  with a singular nodule of HCC who had undergone curative hepatectomy from January 1996 to December 2002 at the Hepato-Biliary-Pancreatic Surgery Division, University of Bari Medical School, were included in the study. Patients who had had a previous hepatectomy or hepatic arterial chemoembolization were excluded. All patients had liver cirrhosis, they were not alcoholic and tested negative for hepatitis B surface antigen (HBsAg) and for antibodies to hepatitis C (anti-HCV). HCC tissues and surrounding cirrhotic liver tissues were examined. Liver biopsy samples from 10 patients with minimal reactive changes  or with histological changes of non-immune-mediated cholestasis  were also studied. Biopsies were obtained during abdominal surgery for uncomplicated cholelithiasis and all these patients were negative for HBsAg, anti-HCV and autoantibodies. Tumours were graded as well differentiated (tumour size maximum diameter = 30.0 ± 15.4 mm), moderately differentiated (tumour size maximum diameter = 51.5 ± 24.6 mm) or poorly differentiated (tumour size maximum diameter = 82.4 ± 42.3 mm). HCC develops and progresses from a small-sized, well-differentiated histotype with no developed blood vessels to a larger and moderately differentiated or poorly differentiated one with characteristic hypervascularity during the differentiation process.A murine monoclonal antibody (MAb) against the endothelial cell marker CD31, a more sensitive marker for endothelial cells than factor VIII antigen2) inserted in the eyepiece. Care was taken to select microvessels, i.e. capillaries and small venules, from all the CD31-stained vessels. They were identified as transversely sectioned tubes with a single layer of endothelial cells, with or without a thin basement membrane. Each assessment was agreed upon in turn. Microvessels were counted with a planimetric point-count method with slight modifications to restrict counting to transversely cut microvessels occupying the reticulum intersection points.2 test or logistic regression analysis. A cut-off value of 10 marked cells was assigned to microvessel density and to EpoR expression in endothelial cells and of 30 marked cells to EpoR expression in tumour cells. Statistical significance was defined as P < 0.05.These were simultaneously assessed without knowledge of the final pathological diagnosis by two investigators with a double-headed light microscope . Four to six 200× fields covering almost the whole of each of three sections per sample were examined with a 144-intersection point square reticulum . Regarding differences in microvessel, Epo and EpoR expression in both endothelial and tumour cells, the within-group comparison showed that both counts were always significantly correlated. There was a trend for these three parameters to increase with histological grade. The counts of patients with poorly differentiated HCC were significantly higher (P < 0.001) than those of patients with moderately and well-differentiated HCC, respectively.The results of this study have shown that ‘poorly differentiated’ HCC has a higher degree of vascularization than ‘moderately’ and ‘well-differentiated’ HCC, respectively, and that Epo expression in tumour cells and EpoR expression in both tumour and endothelial cells increases in parallel with the grade of malignancy and is highly correlated with the extent of angiogenesis. Tumour angiogenesis does not depend on a single molecule, since many angiogenic inducers and inhibitors are simultaneously expressed. Secretion by HCC cells, infiltrating inflammatory cells and hepatic stellate cells of several angiogenic cytokines, such as VEGF, FGF-2, angiogenin, angiopoietins and Epo promotes the sprouting of new vessels from pre-existing ones.The presence of an autocrine-paracrine Epo–EpoR system in tumours and the possible effects of Epo on the tumour microenvironment and angiogenesis are consistent with a complex biology for Epo–EpoR signalling in cancer.et al.Kayser and GabiusThere is evidence that HCC produces marked erythrocytosis and is associated with increased levels of serum Epo or the production of Epo-like activity in cell cultures.et al.et al.Sugimachi These findings suggest the presence of a loop in the Epo–EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. Thus, it is suggested that Epo is an important factor in hepatic tumour angiogenesis.et al.Understanding the mechanisms of HCC angiogenesis provides the basis for a rational approach to the development of antiangiogenic therapy in patients with HCC. Inhibition of Epo signalling, by the injection of an anti-Epo monoclonal antibody or a soluble form of EpoR, results in delay of tumour growth in ovarian and uterine cancers.These results may have implications for the treatment of HCC by inhibition of the Epo–EpoR system.


The effects of different doses of hydralazine and prostacyclin on the 31P magnetic resonance spectra of the LBDS1 fibrosarcoma were investigated and related to their effects on mean arterial blood pressure (MABP) and heart rate. The effect of reducing MABP by bleeding the animals, via the tail artery, was also investigated. Tumour spectral changes following high dose drug treatment  were consistent with nutrient deprivation. These changes were dose dependent. Changes in MABP and heart rate were consistent with vasodilatation in normal tissues. However, for the same fall in MABP, hydralazine produced a greater rise in tumour inorganic phosphate (Pi) and a greater fall in tumour pH than did prostacyclin. Controlled bleeding was effective in reducing MABP. It also reduced tumour pH but had no significant effect on tumour Pi. The clinical application of the two drugs for reducing tumour blood flow and pH for therapy is likely to be limited by the large degree of hypotension necessary to produce an effect. The differential effect of the two drugs for the same fall in MABP may be related to different degrees of direct tumour vasodilatation or to a direct effect of hydralazine on tumour energy metabolism. The observation that controlled bleeding does not change tumour Pi is further evidence indicating that the degree of arterial hypotension is not the sole factor in determining tumour energy status.


The presence of soluble laminin fragments in urine of healthy subjects, patients with diabetes, and patients with tumours was studied using sandwich immunoenzymometric assay technique. The form of urinary laminin (ULN) fragments was dramatically different from that of intact laminin, so ULN could be detected only by using monoclonal antibodies. Mean levels of ULN in lung tumour were significantly higher (171 micrograms gram-1 creatinine) than those in healthy subjects, patients, with diabetes, patients with stomach tumour, and patients with colon tumour . Immunopurified ULN fragments showed an apparent molecular mass of 42 KD on electrophoresis. This fragment was recognised as being derived from the N-terminal region of laminin B2 chain, because the N-terminal residues of ULN were found to be completely homologous to B2 chain. These data suggested that ULN was almost all fragmented, consisted mainly of N-terminal domain of the B2 chain, and was suspected of a tumour-associated protein fragments probably derived from basement membrane degraded proteolytically by tumour cells. ULN, increased in tumour patients, could be a potential clinical marker for monitoring the turnover of basement membrane in tumours.


Decay-accelerating-factor , a phosphatidyl-inositol anchored glycoprotein, is a member of the cell membrane bound complement regulatory proteins that inhibit autologous complement cascade activation. DAF was found expressed on cells that are in close contact with serum complement proteins, but also on cells outside the vascular space and on tumour cells. Using CD55(BRIC110) and CD55(143-30) we show here that DAF(CD55) is only sporadically expressed on the luminal surface of normal colonic epithelium. However, 5/20 adenomas expressed DAF(CD55) on the cell surface of all tumour cells, 5/20 adenomas were completely negative, 10/20 adenomas expressed DAF(CD55) in various amounts. DAF(CD55) was expressed in various intensities on almost all tumour cells of the colon carcinoma cell line HT29. In 5/88 colorectal carcinomas DAF(CD55) was localised on the apical cell surface of all tumour cells, 31/88 were completely negative, 52/88 expressed DAF(CD55) in parts of their neoplastic populations. There was no correlation between the tumour grading, staging and location and the mode of DAF(CD55) expression, but DAF(CD55) was found more often in mucinous carcinomas (P = 0.007). Although the mode of DAF(CD55) expression is not correlated with tumour prognostic parameters, the upregulation of DAF(CD55) in a subset of adenomas and carcinomas needs further investigation concerning protection of tumour cells against complement cytotoxicity.


Human tumour lines established in athymic nude mice were grafted under the renal capsule of immunocompetent mice. Grafts from 27 human tumour lines comprising 9 malignant melanomas, 10 sarcomas, 2 colon carcinomas, 4 lung carcinomas and 2 mammary carcinomas, grew well under the renal capsule of the immunocompetent mice and retained morphological and functional characteristics of the parent tumours, as judged by light and electron microscopy and immunohistochemical examinations. Numerous mitoses were detected. Granulation tissue and necrosis were not predominant features. After Day 4, the grafts became infiltrated from the periphery by mouse inflammatory cells. The infiltration could be prevented by pretreatment of the animals with cyclophosphamide. Anti-human antibodies were detected after Day 3. Single cell suspensions from the subrenal grafts were able to form colonies in soft agar. and upon reimplantation in nude mice, subcutaneous tumours were formed showing that the grafted tumour tissue had also retained its malignant character. Altogether the results support the view that human tumour xenografts grow well under the renal capsule of immunocompetent mice and that the grafts retain important characteristics of the original tumour.


The cellular distribution of 4-borono-2-[18F]fluoro-L-phenylalanine , a potential agent for boron neutron capture therapy (BNCT), and [6-3H]thymidine  in murine two B16 melanoma sublines and FM3A mammary carcinoma was studied in vivo using double-tracer microautoradiography. Tumour volume, tumour age, cell density in the tissues and the proportion of S phase cells in the cell cycle were the same in the three tumour models. Volume doubling time, which represents tumour growth rate, was fastest in B16F10, followed by B16F1 (P < 0.05), the slowest being in FM3A (P < 0.001). The rate of DNA synthesis in S phase cells corresponded to the volume doubling time. The greatest amount of [18F]FBPA was observed in S phase melanocytes and the lowest amount was found in non-S phase non-melanocytes. The [18F]FBPA accumulation was primarily related to the activity of DNA synthesis and, secondarily, to the degree of pigmentation in melanocytes. The therapeutic efficacy of BNCT with p-boronophenylalanine may be greater in melanoma that exhibits greater DNA synthesis activity and higher melanin content.


Immunohistochemical expression of the tumour associated mucin-type glycoprotein A-80 was investigated in a series of 173 breast cancer patients with a clinical follow-up between 13 and 19 years. A routine immunoperoxidase technique was used in formalin-fixed, paraffin-embedded surgical tumour specimens. One hundred and fifty of 173 tumours (87%) immunostained with MAb A-80. The degree of A-80 immunoreactivity was related to the tumour grade but not to lymph node status, tumour size, or nuclear DNA distribution pattern. In univariate analysis the degree of A-80 expression was found to be of significant prognostic value both in node negative and in node positive breast cancer patients (P = 0.03). Patients with non-A-80 immunoreactive tumours had significant longer distant metastases-free survival times and fewer relapses than women with carcinomas composed of A-80 immunoreactive tumour cells. This prognostic value was reduced in a multivariate analysis, including lymph node status, tumour size, and nuclear DNA distribution pattern, but retained borderline significance (P = 0.08). In conclusion, the findings of this study indicate that expression of the mucin-type glycoprotein A-80 as determined by immunohistochemistry seems to be related to clinical outcome in breast cancer patients.


Non-invasive predictive assays which can confirm the presence or absence of hypoxic cells in human tumours show promise for understanding the natural history of tumour oxygenation, and improving the selection of patient subsets for novel radiotherapeutic strategies. Sensitiser adducts have been proposed as markers for hypoxic cells. Misonidazole analogues radiolabelled with iodine-123 have been developed for the detection of tumour hypoxia using conventional nuclear medicine techniques. In this pilot study, we have investigated one such potential marker, 123I-iodoazomycin arabinoside (123I-IAZA). Patients with advanced malignancies have undergone planar and single-photon emission computed tomographic (SPECT) imaging after intravenous administration of 123I-IAZA. We have observed radiotracer avidity in three out of ten tumours studied to date. Normal tissue activity of variable extent was also seen in the thyroid and salivary glands, upper aerodigestive tract, liver, intestine, and urinary bladder. Quantitative analysis of those images showing radiotracer avidity revealed tumour/normal tissue (T/N) ratios of 2.3 , 1.9  and 3.2  at 18-24 h post injection. These preliminary data suggest that the use of gamma-emitter labelled 2-nitroimidazoles as diagnostic radiopharmaceuticals is feasible and safe, and that metabolic binding of 123I-IAZA is observed in some, but not all tumours. The inference that tumour 123I-IAZA avidity could be a non-invasive measure of tumour hypoxia deserves independent confirmation with needle oximetry.


A 78-year-old woman was diagnosed with a proximal gastric adenocarcinoma and underwent an elective D2 total gastrectomy with splenectomy. Subsequent histopathology revealed the presence of another tumour at the gastric antrum. This was a small benign gastrointestinal stromal tumour (GIST) mixed with gastric adenocarcinoma cells similar to those of the main gastric tumour i.e. a collision tumour. The literature has only few previous reports of this very rare association. It is not known whether this synchronicity is incidental or there is a causative factor inducing the development of tumours of different histotypes in the same organ. Pathologists, oncologists and surgeons should be aware of this interesting condition. Adenocarcinoma is the most common histological type of gastric tumour. It may coexist with another synchronous tumour of different histological type in a different part of the stomach. Gastric adenocarcinoma may coexist most commonly with lymphoma and less commonly with carcinoid and gastrointestinal stromal tumour (GIST). Rarely, cells of different histological types may intermix and form a collision tumour in the stomach. We present here the very rare combination of a synchronous proximal gastric adenocarcinoma and a distal gastric collision tumour consisting of GIST and adenocarcinoma cells similar to those of the main gastric tumour.A 78-year-old woman presented with a 6-month history of dyspeptic symptoms, epigastric pain and weight loss. Gastroscopy showed mucosal nodularity and ulceration at the proximal gastric body with an "hour glass" deformity, an appearance suggestive of malignancy. Multiple mucosal biopsies were obtained and histopathology revealed a poorly differentiated adenocarcinoma and chronic gastritis. Computed tomography of the abdomen showed diffuse thickening of the gastric wall and a few enlarged lymph nodes in the lesser sac.The patient underwent an elective D2 total gastrectomy with splenectomy as the bulky gastric tumour was extending into the splenic hilus and a Roux-en-Y reconstruction was performed.The histopathological examination of the specimen macroscopically showed firm texture of the proximal stomach and vague nodular appearance of the mucosa over a 100 by 80 mm area. Microscopically the proximal stomach showed transmural infiltration by poorly differentiated diffuse adenocarcinoma, which had reached the serosal surface. There was vascular invasion, infiltration of 10 out of 34 nodes and numerous extranodal tumour deposits. A nodule of firm white tissue, 9mm diameter, was present on the external surface of the gastric antrum. Histological examination revealed this nodule to be a benign gastrointestinal stromal tumour (GIST), which had arisen from the muscularis propria. It was composed of interwoven cytologically bland spindle shaped cells that were demonstrated by immunohistochemistry to be uniformly positive for CD117. Scattered spindle cells of the GIST also expressed smooth muscle actin, desmin and S-100 Fig. , 3. CytoThere are only a few previous reports of simultaneous adenocarcinoma and GIST in the stomach -3. In thGISTs are usually sessile, big, soft tumours and can develop necrosis or ulceration of the overlying mucosa. However, when the GIST is submucosal or subserosal the gastric mucosa may not be invaded and the endoscopic biopsies can be normal. In most of the reported cases of synchronous gastric adenocarcinoma and GIST, the preoperative biopsy fragments showed only adenocarcinoma and the GIST were detected only following laparotomy and examination of the resected stomachs. In our case the total gastrectomy was performed for the proximal gastric adenocarcinoma and a small GIST was found incidentally with the histopathological examination of the specimen. The coexistence of primary gastric adenocarcinoma and GIST has often been detected incidentally on gastric mucosa or serosa, or occasionally intramurally, at surgery or gastroscopy for other reasons.Gastric collision tumours are uncommon and most are composed of adenocarcinoma intermixed with gastric lymphoma -6 or witVarious hypotheses have been proposed regarding the simultaneous development of GIST and adenocarcinoma. It is not known whether or not this association is a simple incidental coexistence or whether the two lesions are connected by a causal relationship. It has been suggested that gene mutations or a single carcinogenic agent might interact with two neighbouring tissues resulting in the development of tumours of different histotypes in the same organ . In the The authors declare that they have no competing interests.IEK conceived the idea, contributed to the literature search and wrotethe final manuscript.MB performed the literature search and wrote the case presentation.PIR contributed to the writing of the case presentation and provided the figures.JIL revised the manuscript.All authors read and approved the final manuscript.


It has been established that loss of tumour suppressor genes is crucial in carcinogenesis. There has been no reported study on searching for tumour suppressor genes in cholangiocarcinomas as yet. In order to investigate the loss of heterozygosity (LOH), which may represent such gene loss, in cholangiocarcinoma, we studied 14 patients with this tumour using restriction fragment length polymorphism analysis. Twenty-two probes assigned to chromosomes 1, 5, 7, 9, 11, 12, 13, 14, 16, 17 and 18 were used. Allelic losses were found in chromosomal regions 5q35-qter and 17p13. Loss of genetic material in these regions in cholangiocarcinoma was shared with hepatocellular carcinoma. Probes for other chromosomes have as yet shown no consistent LOH. In conclusion, this study for the first time showed LOH on chromosomes 5 and 17 in cholangiocarcinoma.


Resistance to cytotoxics precludes the successful treatment of many solid tumours. Inhibition of DNA synthesis in normal tissues with antimetabolites such as hydroxyurea (HU) may be a useful means of improving the selective uptake of toxic thymidine analogues by the relatively resistant tumour cells. HU also inhibits DNA repair by the critical depletion of intracellular deoxyribonucleotides. Twenty-five patients with various malignancies received 5-iodo-2-deoxyuridine (IUdR) 100 mg m-2 as a 20 min i.v. infusion and the uptake of IUdR was determined 1 h later immunocytochemically. Of these patients, 14 received IUdR 23 h from the start of a continuous i.v. infusion of HU (36 g over 36 h). Uptake of IUdR was equally suppressed in bone marrow and tumour aspirates, 0.1% (+/- 0.2%) of marrow precursor cells and 0.5% (+/- 0.4%) of tumour cells respectively, in patients who received HU compared to the uptake of IUdR in 11 patients who were not given HU 6.8% (+/- 1.1%) and 12.2% (+/- 1.8%) respectively. Mean HU plasma concentrations at the time of IUdR administration was 1.7 +/- 0.2 mM. The growth fraction of tumour cells (using Ki67 labelling) was not changed after treatment with HU. It is concluded that (1) since DNA synthesis is effectively inhibited by HU in tumour cells, differential uptake of radiolabelled IUdR by those cells will not be feasible using the current schedule of HU administration, (2) HU may be used as an inhibitor of DNA repair in vivo since the degree of inhibition correlates with that required to inhibit repair experimentally and that (3) Ki67 labelling index is not useful in studying cell kinetics in patients treated with HU.


Antimetabolites are S-phase specific anticancer drugs. Administration of bromodeoxyuridine (BrdUrd) to tumour bearing mice was followed by treatment with cytosine arabinoside or hydroxyurea. Anti-BrdUrd immunocytochemistry visualised susceptible tumour and intestinal crypt cells at electron microscope level, showing unequivocally that cells that were in S-phase at the time of administration of the drugs subsequently died by apoptosis.


The effect of acute carbon monoxide (CO) breathing on blood oxygenation and tumour hypoxia was related to the radiation response of the C3H/Tif mammary carcinoma. Blood gas analysis showed that CO breathing caused a time- and dose-dependent formation of carboxyhaemoglobin (HbCO), a significant left shift of the oxygen dissociation curve and a reduction in tumour blood perfusion. These factors all contributed to a marked drop in tumour oxygen supply. In agreement with this, tumour hypoxia was found to be significantly increased: Microelectrode PO2 measurements showed a clear relationship between CO concentration and the proportion of low PO2 measurements (< or = 5 mmHg). The fraction of clonogenic hypoxic cells increased from 8% in air-breathing animals to 13%, 18% and 54% with 75,220 and 660 p.p.m. CO respectively. The tumour hypoxia resulted in significant radiation modification. The local tumour control after single-dose and fractionated irradiation gave TCD50 enhancement ratios (relative to air-breathing controls) of 0.90, 0.85 and 0.89 for single dose and five or ten fractions given in 5 days . For 15 fractions in 5 days with 6- 6- and 12 h intervals, the TCD50 was similar in CO- and air-breathing mice, presumably as a consequence of insufficient reoxygenation during the short inter-fraction intervals. It is concluded that elevated HbCO levels to increased tumour hypoxia and that the induced hypoxia has a significant impact on the local tumour control also after fractionated irradiation.


Bile acids enhance colorectal carcinogenesis in animals and man, perhaps after degradation by faecal anaerobes. The promotional effect of sodium deoxycholate (SDC) and its relationship to bacteria was examined in male Sprague-Dawley rats (n = 115) which had received a 6-week course of azoxymethane  Two groups received 3 X weekly intrarectal (i.r.) instillations of N saline or 0.12 M SDC for 18 weeks. Another group received SDC i.r. plus metronidazole (22.5 mg kg-1) daily in the drinking water. Controls had no instillations or metronidazole alone. By 28 weeks SDC had increased mean colonic crypt depth by 9% (P less than 0.001), and had almost trebled colorectal tumour yields from 2.4 +/- 0.4 per rat (mean +/- s.e.) in controls to 6.4 +/- 0.5 (P less than 0.001). Tumour yields after SDC + metronidazole (4.2 +/- 0.5) remained 75% higher than in controls (P less than 0.01) but were 33% less than after SDC alone (P less than 0.01), and the increase in crypt depth was maintained at 7% (P less than 0.001). Neither metronidazole alone nor saline i.r. had any effect on tumour yield, but metronidazole alone reduced crypt depth by 9% (P less than 0.001). Deoxycholate is a potent cocarcinogen and also stimulates mucosal hyperplasia. Metronidazole reduces its tumour-promoting effect, suggesting that faecal anaerobes are important in bile acid cocarcinogenesis.


A human tumour cell line, designated RMS-YM, was established from a childhood rhabdomyosarcoma. The monolayer cells were polygonal, round or spindle-shaped. The cells became multilayered and formed many focal piles when confluent. RMS-YM became stable with a doubling time of about 30 h and has been maintained for 104 passages to date. Tumourigenicity of the cells was confirmed by heterotransplantation into nude mice. Morphological features were similar to those of the primary tumour, and myofibrils were found by electron microscopy. The expression of desmin and human myoglobin, and high levels of striated muscle system specific enzymes were recognised. Chromosomal analysis revealed possible gene amplification in the form of homogeneously staining regions. Oncogene analysis was performed on the primary tumour and the cell line, but neither N-myc nor N-ras genes were amplified, nor were Ki-ras, Ha-ras or N-ras genes mutated at the 12th, 13th and 61st codons. The RMS-YM cell line may provide a system to identify novel genes which are amplified in rhabdomyosarcoma.


Overt liver tumour was induced in Fisher rats by intraportal administration of 1.6 x 10(7) Walker carcinosarcoma cells. Control groups of rats received similar volumes of dead cells or saline intraportally. All animals were studied at 3 weeks when overt tumour was present. The Hepatic Perfusion Index (HPI) was significantly raised in rats with overt tumour compared to both groups of control animals. Portal flow and portal venous inflow were significantly reduced in the presence of overt tumour but hepatic arterial flow did not alter. These observations suggest that the alteration in the HPI in the presence of overt tumour results from an alteration in portal venous flow and inflow even though the blood supply to the tumour is principally derived from the hepatic artery. The changes in hepatic haemodynamics in the presence of tumour were accompanied by a reduction in portal pressure, an increase in splanchnic vascular resistance and an increase in the degree of arteriovenous shunting through the liver. Portal vascular resistance was unchanged. These findings indicate that the presence of overt hepatic tumour results in gross derangements of hepatic blood flow. These changes must be taken into consideration when attempting to potentiate the delivery of cytotoxic drugs to hepatic tumour by manipulation of hepatic haemodynamics.


Synthetic peptides derived from the conserved p15E sequence were found to suppress some aspects of the immune response implicated in anti-tumour activity. These data suggest that a p15E-related material with immunomodulatory properties is elaborated within human tumours, either by the tumour itself or as a normal component of the endogenous anti-tumour reaction.


The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane proteins (matrigel). Tumour growth is promoted and the latency period is low (2 to 4 weeks). In the absence of matrigel, the simultaneous inoculation of fibroblasts and MCF7 cells also resulted in the development of tumours, but with a longer latency period (about 2 months). The tumorigenic synergy between matrigel and fibroblasts was evidenced by co-inoculating MCF7 cells MDA-MB 231 cells with fibroblasts and matrigel. This co-inoculation decreased the delay of appearance of the tumours and/or accelerated the tumour growth, depending upon the number of fibroblasts injected. Repeated injections of fibroblasts conditioned medium, at the site of inoculum of tumour cells also enhanced tumour growth, suggesting the involvement of soluble factors secreted by fibroblasts. Histologically, tumours induced by co-inoculation of tumour cells and fibroblasts contained more stromal structures including vimentin-positive cells, fibronectin and interstitial collagens. These data suggest that human tumours may be reconstituted and grown in athymic nude mice using basement membrane components and fibroblasts as inductors.


Mast cells were shown to accumulate around the periphery of the invasive and metastatic rat mammary adenocarcinoma (MTLn3), and histological evidence of mast cell degranulation was observed during the later stages of this model. To assess the physiological role of mast cells in vivo we have used the mast cell-stabilising compound FPL 55618 applied i.p. daily at 1 mg kg-1 for 23 days. Using groups of 12 rats we have found that this compound inhibited tumour growth at the primary site by as much as 70% in most of the treated animals compared with the control group which received equivalent volumes of saline. When the drug treatment was stopped after 23 days, tumour growth of the test group accelerated over the next 7 days and reached a similar tumour size to that of control animals. Histological studies of the tumour and contiguous host tissue at day 24 of the experiment revealed numerous extra-tumoural mast cells often showing signs of degranulation at several sites around the tumour periphery in the control animals. Such observations were not seen in those animals receiving FPL 55618 where, in contrast to controls, numerous intact mast cells were often seen within the tumour mass. Following cessation of the MC-stabilising treatment progressive mast cell activation was evident within 2-4 days, primarily at the tumour periphery. In vitro studies have shown that drug concentrations equivalent to five times the in vivo dose had no effect on the proliferative rate or viability of the MTLn3 cells. Moreover, the proliferative rate of these cells in culture was significantly increased when exposed to soluble mast cell products. Thus our data indicate that a mast cell-stabilising compound has significant benefits in reducing tumour growth in vivo, an observation which supports the concept that mast cell:tumour cell interactions are important for the growth and invasive properties demonstrated by this model of breast carcinoma.


Detailed studies of the effects of misonidazole (MISO) on the pharmacokinetics of CCNU in the KHT tumour, bone marrow and the gut have been carried out in order to elucidate the mechanism of chemosensitisation by MISO, and the therapeutic gain often obtained due to the preferential enhancement of tumour toxicity. In experiments where CCNU concentration and growth delay were both measured in the same transplant group of tumours, we found that tumour response is well correlated with tumour peak CCNU concentration. Further, with MISO treatment the tumour peak CCNU concentration was increased such that the enhancement of tumour response can be entirely accounted for by this increase. The effects of MISO on the CCNU pharmacokinetics in bone marrow and in the gut were different from the tumour in that peak CCNU concentration was not increased. We suggest that this is the explanation for the therapeutic gain.


The effect of cryosurgery on normal liver and liver tumour was investigated using 60 adult male rats. Animals were divided into four groups Group A implanted tumour/cryosurgery (n = 19), Group B normal liver/cryosurgery (n = 17), Group C normal liver/sham cryosurgery (n = 10) and Group D implanted tumour/sham cryosurgery (n = 14). At laparotomy animals were injected into the left lateral lobe of the liver with 10(5) HSN fibrosarcoma cells or vehicle. Two weeks after implantation red cell flux was recorded in all animals, the appropriate groups treated with cryosurgery and after thawing red cell flux was monitored over the tumour and at the edge of the cryolesion and over the corresponding normal area in controls. In certain animals red cell flux was measured at hourly intervals for 8 h, and in further groups at 24 h and at 2 weeks after cryosurgery. Results demonstrated that cryosurgery significantly reduced (P < 0.01) red cell flux in both normal and tumour liver, immediately after treatment. Red cell flux remained significantly reduced (P < 0.005) at 8 h after treatment but by 24 h had returned to preoperative levels which was maintained at 2 weeks. The results suggest that microcirculatory shutdown may be a contributing factor to the tumour necrosis occurring after cryosurgery.


The pharmacokinetics of Zn-phthalocyanine (Zn-Pc) in mice bearing a transplanted MS-2 fibrosarcoma has been studied using dipalmitoyl-phosphatidylcholine (DPPC) liposomes and low density lipoproteins (LDL) as drug delivery systems. LDL induce a higher Zn-Pc uptake by the tumour and improve the selectivity of tumour targeting as compared to DPPC liposomes. Experimental photodynamic therapy (PDT) of the MS-2 fibrosarcoma has been performed using liposome-delivered Zn-Pc and the efficiency of tumour necrosis has been measured following four different irradiation protocols. We found that Zn-Pc doses as low as 0.07-0.35 mg kg-1 are sufficient for inducing an efficient tumour response that is linearly dependent on the injected dose. The amount of Zn-Pc in the tumour decreases very slowly as a function of time, hence PDT gives satisfactory results even if performed at relatively long time intervals after administration.


In the early period following radical hepatectomy for hepatoma, recurrences in the remaining liver are frequently found. In regenerating liver, implantation and growth of tumour cells released into the portal system during surgical treatment might be promoted. We examined the relationship between liver regeneration and the formation of metastases following hepatic resection. Intraportal injections of rat ascites containing hepatoma AH130 cells at a concentration of 1 x 10(5) cells 0.2 ml-1 were made at various periods following two thirds liver resection in rats. Tumour cell injections immediately at 24 h after surgery resulted in an increased number of hepatic metastases compared with control animals. Tumour cell injections 2 weeks after hepatectomy, however, had no significant difference in effect compared with control rats. In contrast, tumour cells injected immediately after removal of half of the caudate lobe resulted in the same number of metastases as control animals. These results demonstrate that the number of artificially induced hepatic metastases was increased during an initial period of active liver regeneration and was proportional to the volume of hepatectomy. The effect of 5-fluorouracil (5FU) or mitomycin C (MMC) as inhibitors of hepatic regeneration on liver metastasis after hepatectomy was studied. The administration of 5FU (20 mg kg-1) or MMC (0.2 mg kg-1) immediately, 24 and 48 h after hepatectomy resulted in a marked reduction in metastatic lesions. The administration of 5FU caused delays in weight gain and decreases in the wet weight of remaining liver, while MMC had no effect on either. Accordingly, results of 5FU administration may be due to inhibitory effects on liver regeneration whilst that of MMC administration may be due to cytocidal antitumour effect. The effect of OK-432 as an immunoactivator on the implantation and growth of tumour cells in regenerating liver was also studied. Pretreatment with OK-432, 0.5 mg intraperitoneally on 7 consecutive days, had no effect on hepatic metastases. The pathophysiology of liver regeneration may enhance hematogenous hepatic metastasis and release of tumour cells during surgical manipulation may represent an important cause of recurrence following hepatic resection.


The means by which oncogenes and their products activate malignant transformation are currently under intense investigation. However, published papers on experiments using human tumour material do not always report in detail their methods of collection or storage of the specimens. In order to assess the stability of oncogene encoded proteins following collection or storage of human tumour biopsies, we have examined the rate of decay of the c-myc, neu and EGF-receptor proteins. Solid tumours, containing amplified copies of each oncogene, were established in nude mice and the stability of the oncogene protein in portions of each tumour, left in phosphate buffered saline at room temperature for varying time intervals, was examined by immunoblotting. Intact EGF-receptor and neu oncoproteins were present even after 24 h under these conditions while the c-myc protein was apparently rapidly degraded after 20 min. These data demonstrate that oncogene products decay at different rates after tumour resection and that collection of human biopsies should take this into account in order to provide the basis for consistent measurements of protein expression.


Experiments on s.c. rat tumours (DS sarcoma) were performed to determine whether chronic or acute changes in tumour perfusion necessarily lead to changes in tissue oxygenation and bioenergetic status since, as a rule, blood flow is thought to be the ultimate determinant of the tumour bioenergetic status. Based on this study, there is clear experimental evidence that growth-related or acute  decreases in tumour blood flow are accompanied by parallel alterations in tissue oxygenation. In contrast, tumour energy status remains stable as long as flow values do not fall below 0.4-0.5 ml g-1 min-1, and provided that glucose as the main substrate can be recruited from the enlarged interstitial compartment. Perfusion rate seems to play a paramount role in determining energy status only in low-flow tumours or low-flow tissue areas.


Between October 1977 and December 1983, 379 consecutive patients have been treated for unilateral, non-metastatic breast cancer, either with conservative (n = 205) or radical surgery (n = 174), with axillary dissection in all the cases. None of them had histologically proved lymph node involvement. Oestrogen receptor (ER) and progesterone receptor (PR) levels were measured on each tumour. Levels greater than 5 fmol mg-1 cytosolic protein were considered as positive for both ER and PR. At 5 years, overall survival (OS) and disease-free survival (DFS) are respectively 88% and 78%. Unifactorial analysis using Kaplan and Meier estimates and the log rank test revealed that OS was significantly related to age (P less than 0.05), tumour size (P less than 0.001), histological grading (SBR) (P less than 0.01), ER (P less than 0.001) and PR (P less than 0.001). DFS was significantly related to the same factors. Menopausal status, number of breast tumour foci and previous familial history of breast cancer were not significant. Multifactorial analysis revealed that DFS was significantly related to age (bad prognosis (b.p.) less than or equal to 37 years old), tumour size and histological grading (b.p. SBR = 3), and that OS was significantly related to tumour size and PR . A prognostic score has been constructed for both DFS and OS. These scores divide our patients into three significantly different (P less than 0.0001) groups with good, intermediate and bad prognosis.


When human melanoma cells are injected into nude mice they usually give rise to tumours that grow progressively and do not elicit a prominent host response. We have recently developed a melanoma cell line, UCT-Mel 7, that did not show these characteristics. In the first place UCT-Mel 7 showed a consistently unusual, phasic growth pattern. After a short initial period of limited growth (phase 1), the tumour ceased growing and remained static for 2-3 months (phase 2). The tumour then regressed (phase 3) to enter a second period of quiescence (phase 4) which was eventually broken by the emergence of a rapidly growing lethal tumour (phase 5). Of particular interest was the fact that the rate at which the tumours grew correlated closely with their collagen content. During the prolonged, phase 2 plateau, the tumours were intensely desmoplastic; rapidly growing phase 5 tumours, that had escaped from dormancy, contained very little collagen and virtually no reticulin. This cell line helps to fill an important need for an experimental system for the study of desmoplasia, dormancy and progression.


The effect of the 2-nitroimidazole Ro 03-8799 (8799) on the activity of 11 chemotherapeutic agents against the anaplastic MT tumour in mice has been determined by soft agar cloning. The 8799, whilst producing little cytotoxicity by itself, potentiated the cytotoxic actions of the alkylating agents melphalan and cyclophosphamide, and the nitrosoureas BCNU, CCNU and MeCCNU. This potentiation was influenced by the time interval between the administration of 8799 and the chemotherapeutic agents, and also by the site of tumour implantation. However, 8799 did not potentiate the cytotoxicities of the compounds CBDCA, cisplatin, adriamycin, vincristine, 5-fluorouracil and bleomycin. A review is included of the reported in vivo effects of nitroimidazoles on the chemotherapeutic agents investigated here.


We have examined the effectiveness of photodynamic therapy against R3230AC rat mammary adenocarcinoma and human mesothelioma as xenografts in the same host. The results demonstrate that the xenografted human tumour is significantly more responsive to photodynamic treatment than the rodent mammary tumour. Studies also showed that the mesothelioma xenograft was fluence rate- and fluence-dependent while the rat tumour exposed to the same conditions demonstrated neither of these dependencies. This disparity in response was not attributable to a difference in either whole-tumour uptake or subcellular distribution of the porphyrin photosensitiser. Analysis of the effects of visible irradiation on cytochrome c oxidase activity, measured in mitochondria prepared from tumours borne on hosts injected with photosensitiser, demonstrated that photoradiation-induced enzyme inhibition was significantly greater in mesothelioma than in R3230AC mammary tumour preparations. However, in parallel studies conducted in vitro, when photosensitiser and light were delivered to previously unperturbed mitochondria, rates of enzyme inhibition were not significantly different. Both tumours were established in long-term cell culture. While the uptake of porphyrin photosensitiser was equivalent in both cell lines, the R3230AC cells displayed a significantly greater photosensitivity than the mesothelioma cells. The data presented here demonstrate that the mechanisms that govern response to photodynamic therapy are complex, but in the case of these two xenografted tumours host response to therapy is not likely to play a significant role.


The protein and glycoprotein compositions of a subcutaneous lymphosarcoma (1 degree) and its metastatic deposits in the liver (2 degrees) have been investigated in Triton X-100 extracts obtained from tissue, single cells and membrane preparations. No consistent differences in the electrophoretic patterns for 1 degree and 2 degrees tissue or cells were observed for separations visualized with the protein stain Coomassie Blue. Substantial and consistent reductions in the glycoprotein content of extracts from 2 degrees tissue or cells were observed if the separated proteins were treated with the radioiodinated lectin, Wheat Germ Agglutinin (WGA). Densitometric scans of autoradiographs indicated that WGA binding occurred in 4 major areas; the approximate mol. wts of these were 180,000, 102,000, 84,000 and 23,000 daltons. All these components except the 23,000 component were shown to be located in the cell membrane and to be reduced in 2 degrees preparations. Possible sources of host contamination were also investigated, but these did not show WGA binding patterns that were similar to that obtained for the tumour. If 2 degrees tumour was transplanted into the 1 degree site, the resultant growth exhibited a WGA-binding pattern normally shown by a 1 degree tumour growing in this site. Conversely, if 1 degree tumour was transplanted into the liver, the WGA binding of the resultant growth was substantially reduced. The results suggest that local and metastatic tumours do contain cells that express different glycoproteins on their surfaces but that the site of tumour growth is a very important factor in determining this difference in surface expression.


In this study the localisation of porphyrinoid photosensitizers in tumours was investigated. To determine if tumour selectivity results from a preferential uptake or prolonged retention of photosensitizers, intravital fluorescence microscopy and chemical extraction were used. Amelanotic melanoma (A-Mel-3) were implanted in a skin fold chamber in Syrian Golden hamsters. Distribution of the porphyrin mixture Photofrin and three porphycenes, pure porphyrinoid model compounds, was studied quantitatively by intravital fluorescence microscopy. Extraction of tissue and blood samples was performed to verify and supplement intravital microscopic results. Photofrin accumulated in melanomas reaching a maximum tumour:skin tissue ratio of 1.7:1. Localisation of the different porphycenes was found to be highly tumour selective (3.2:1), anti-tumour selective (0.2:1), and non-selective (1:1) with increasing polarity of the porphycenes. The two non-tumour selective porphycenes had distinctly accelerated serum and tissue kinetics; serum halflife times being as short as 1 min. The specific localisation of the slowly distributed, tumour selective photosensitizers, occurred exclusively during the distribution from serum and uptake into tissues. For the most selective porphycene, the tumour selection process had a halflife of 260 +/- 150 min and led to a strongly fluorescent tumour edge edema. Accumulation of porphyrines by the amelanotic melanoma (A-Mel-3) can be attributed to an enhanced uptake rate for lipophilic molecules in this subcutaneously growing neoplasm. The slow distribution of the two tumour specific photosensitizers and the strong fluorescence of these hydrophobic molecules in the tumour compartment with a high water content indicate a carrier role of serum proteins in the selection process. Enhanced permeability of the tumour vasculature to macromolecules appears to be the most probable reason for the tumour selectivity of these two sensitisers.


The relationship between the size of the primary tumour upon initial treatment and the incidence of distant metastasis during the course of the disease was investigated using data from 2648 breast cancers treated at the Institut Gustave Roussy between 1954 and 1972. This analysis suggests the existence for each tumour of a critical volume (threshold) at which the first remote metastasis is initiated. The correlation between the size of the primary tumour and the probability of metastatic dissemination was assessed as well as the influence on this correlation of two prognostic indicators: histological grade and number of involved lymph nodes. It was found that the threshold volume is strongly correlated with the number of involved lymph nodes and the histological grading.


Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.


On the outside, humans and other vertebrates seem to be bilaterally symmetrical. Draw a line down your body from the top of your head to your feet, and what is to the left of the line is pretty much the mirror image of what is to the right. But think about your internal organs. Your heart is on the left of your body, while your liver is over to the right. This left-right asymmetry, like the other asymmetries of vertebrate bodies, is established early in embryonic life. The symmetrical ball of cells formed after fertilization of the egg quickly develops a head and tail end (an anteroposterior axis) and a front and back . Once these identities have been established, the embryo then specifies its left and right sides—an event called left-right symmetry breaking.Work in early mouse embryos suggests that left-right symmetry breaking arises from the leftward flow of extra-embryonic fluid. This flow is somehow generated by beating cilia, rod-like unicellular organisms through water. During evolution, however, multicellular organisms retained cilia on cells that move extracellular fluid; for example, respiratory tract cells use cilia to flush away bacteria and other debris. And in the embryo, cilia on cells in a region called the node produce the symmetry-breaking leftward flow, or “nodal flow.” As a result, in mouse embryos with mutant cilia that fail to beat, nodal flow is not established, and some mice develop with their internal organs on the wrong side. But it's not clear how beating nodal cilia actually produce a leftward fluid flow. Unlike most cilia, which beat in a whip-like back-and-forth motion, nodal cilia beat by twirling in a circle; cilia twirling in fluid should act like a propeller and create a vortex (a circular flow of water). But nodal cilia don't produce a vortex; they produce a leftward flow. Models of fluid dynamics suggest that the nodal cilia might be able to do this if they are tilted. And as Hiroshi Hamada and colleagues now report, this is indeed the case; nodal cell cilia are tilted toward the embryo's tail end, rather than sticking straight up out of the plane of the node.To investigate whether the cilia in mouse embryonic nodes are tilted, the researchers first traced their trajectories using a high-speed camera. Tracing the path of the tip of a straight-up cilium as it beats should yield a circular trace, but the researchers actually recorded elliptical and D-shaped traces. The authors explain that elliptical traces are representative of beating cilia tips viewed at an angle, while the D-shaped traces result when the cilia are tilted so much that they slam into the “floor”—the embryonic cell surface—during their circuit. Scanning electron microscopy then revealed that all the cilia in the node tilted toward the posterior of the embryo and that each cilium was located toward the back of its node cell. Finally, the researchers built a 1,000× scale model of an embryonic node using wires to represent cilia and thick silicone fluid to represent the extra-embryonic fluid. As predicted by theoretical fluid dynamics, the liquid in this model always flowed leftward when the tilted wires were rotated clockwise.Hamada and colleagues propose that the position of the cilia and their tilt is determined by the pre-existing axis asymmetries  in the embryo. The tendency of the cilia to rotate clockwise then produces the leftward fluid flow across the node that breaks embryonic left-right symmetry. Whether a similar mechanism occurs in vertebrates other than mice, and how exactly leftward flow contributes to left-right axis establishment remains to be determined, but at least the mystery of how rotating cilia can produce a linear flow has now been solved.


Low-level direct electric current has been shown to be capable of destroying tumour tissue. Using an early-passage subcutaneous murine mammary carcinoma, the relationships between the volume of tumour destruction, charge and polarity have been examined. The results revealed a direct correlation between charge passed and absolute volume regression when the intratumoral electrode was made either an anode or a cathode. Tumour destruction for a given charge was significantly greater following anodic than cathodic treatment. A direct correlation was also observed between the percentage volume of prompt treatment-induced regression and the in situ end point of tumour growth delay. During the course of these experiments, a highly reproducible toxic effect was discovered, which has not been previously reported for this modality. An anodic charge greater than 10.6 coulombs or a cathodic charge greater than 21.6 coulombs resulted in 100% mortality at 24-72 h, while lower charges had no influence on mortality. Quantitative assays of a number of blood parameters showed that mortality was associated with serum electrolyte imbalances and appeared to be the result of the metabolic load of tumour breakdown products. These effects are similar to the tumour lysis or surgical crush syndromes and should not constitute a significant problem in clinical practice, where the tumour mass to total body mass ratio will normally be much smaller.


Recombinant interleukin-2 (rIL-2) therapy has been shown to be of value in the treatment of some cases of melanoma and renal cell carcinoma. However its use can be limited by severe systemic toxicity. Targeting rIL-2 to the tumour should improve the anti-tumour immune response and decrease the systemic toxicity. With this aim we have employed recombinant DNA techniques to construct a single chain antibody interleukin-2 fusion protein (SCA-IL-2). The protein used in this model system comprises the variable domains of the anti-lysozyme antibody D1.3 fused to human IL-2. It has been expressed by secretion from Escherichia coli and the purified product possesses antigen binding specificity and retains the immunostimulatory activities of rIL-2. This approach can be taken to generate SCA-IL-2 proteins that bind to appropriate cellular antigens. In vivo administration of a tumour binding SCA-IL-2 should result in a localised high concentration of IL-2 in tumour tissues, maximising the anti-tumour immune response, whilst keeping systemic side effects to a minimum.


Oestrone sulphatase may be an important means of production of intra-tumoural oestrogens in breast cancer cells. The N-nitrosomethylurea induced rat mammary tumours, which is a good model of human breast carcinoma, was utilised to examine the significance of intra-tumoural oestrone sulphatase levels. The particular fraction  was prepared from both the liver and the tumour of NMU treated rats and assayed for sulphatase activity. The tumour enzyme had an optimum pH of 7.2, Km value of 14.8 microM and Vmax of 0.90 nmoles min-1 mg-1, while the hepatic enzyme was optimal at pH 7.4, Km of 10.8 microM and Vmax of 3.71 nmoles min-1 mg-1. The relationship of intra-tumoural sulphatase levels with tumour regression and progression in endocrine responsive tumours was investigated. Tumour regression as a result of oophorectomy was associated with a significantly decreased intra-tumoural sulphatase level (mean level = 0.165 nmoles min-1 mg-1) in comparison to a control group  in which the tumours remained stable. This significant difference was not observed in the corresponding hepatic samples suggesting that it is the intra-tumoural rather than the peripheral production of oestrogens by oestrone sulphatase that is important in supporting growth of endocrine responsive tumours.


Flavone acetic acid (FAA) showed impressive effects against murine solid tumours but no activity in clinical studies. The mechanism of action in mice may involve damage to tumour vasculature or immunomodulation, and these effects may be species-specific. Alternatively, concentrations of FAA achieved in mouse tumours may be higher than in human tumours. It is important to resolve this issue since it raises important questions about the relevance of in vitro versus in vivo tumour screens and the development of FAA analogues. As part of a Cancer Research Campaign Phase II study of metastatic melanoma in which 8.4 g m-2 FAA was given as a 6 h infusion, six tumour biopsies were obtained from four patients. FAA tumour concentrations were determined by HPLC and compared with subcutaneous murine solid tumours within the same analytical laboratory. Tumour/plasma percentages  were similar to those in mice, as was the area under the curve (AUC) extrapolated to infinity and the AUC above the putative activity threshold of 100 micrograms ml-1. We conclude that the exposure of drug-refractory human melanoma tissue to FAA was comparable to that of sensitive mouse tumours. This suggests that reduced penetration of FAA into human tumours is unlikely to explain the lack of antitumour activity observed in clinical studies and that differences in mechanism of action are predominant.


Eighteen consecutive patients with measurable locally advanced or metastatic pancreatic adenocarcinoma were treated with goserelin (Zoladex) 3.6 mg subcutaneously every 4 weeks. Hydrocortisone 20 milligrams twice daily was commenced with the second injection of goserelin. Objective tumour response was monitored by computerised tomography of the abdomen. There was no objective remission in disease sites. Serial measurements of serum tumour markers showed no reduction in serum CA 19-9 and CA 195 concentrations. The median duration of survival of all cases was 5 months. Administration of goserelin resulted in significant reductions in oestradiol, testosterone, androstenedione in males and reductions in FSH and LH in both males and females. The addition of hydrocortisone resulted in further reductions of androstenedione and testosterone levels in males. Thus goserelin showed no anti-tumour effect, but concentrations required for direct inhibitory effects may be higher than those required to produce effects on hormone suppression.


In this review we shall discuss the biological rationale and the clinical findings obtained using Interleukin 2 (IL2)-based immunotherapy in the management of cancer patients. Objective and long-lived clinical responses have been documented in a proportion of cases, particularly renal cell carcinoma, melanoma and acute myeloid leukaemia. Though encouraging, the clinical use of IL2 has so far been limited by toxicity, as well as by the heterogeneous and unpredictable responses and by the lack of specific anti-tumour effect. These considerations have led to the belief that more sophisticated technologies aimed at introducing the IL2 gene into the neoplastic cells may potentially overcome some of the limitations coupled to the in vivo infusion of high doses of IL2. The data accumulated in animal models and, more recently, also with human tumour cells indicate that the IL2 gene may be successfully inserted into neoplastic cells. The constitutive secretion of IL2 by the tumour cells leads to a reduced or abrogated tumorigenicity in several different tumour models. The evidence that in some experimental tumours the transduction of the IL2 gene into the neoplastic cells may elicit a specific cytotoxic response and confer anti-tumour memory, suggests that vaccination protocols based on this innovative strategy may represent a potential new tool in the management of cancer patients.