E1B-55 kDa-gene-deleted adenovirus ONYX-015  in subcutaneous human malignant glioma xenografts deriving from primary tumours. Here, we show the combined efficacy of this oncolytic therapy with radiation therapy. Total body irradiation (5 Gy) of athymic nude mice prior to intratumoral injections of ONYX-015 1 × 108 PFU daily for 5 consecutive days yielded additive tumour growth delays in the p53 mutant xenograft IGRG88. Radiation therapy was potentiated in the p53 functional tumour IGRG121 with a ‘subtherapeutic’ dose of 1 × 107 PFU daily for 5 consecutive days, inducing significant tumour growth delay, 90% tumour regression and 50% tumour-free survivors 4 months after treatment. These potentiating effects were not due to increased adenoviral infectivity or replication. Furthermore, cell lysis and induction of apoptosis, the major mechanisms for adenoviral antitumour activity, did not play a major role in the combined treatment strategy. Interestingly, the oncolytic adenovirus seemed to accelerate radiation-induced tumour fibrosis. Potentiating antitumour activity suggests the development of this combined treatment for these highly malignant tumours.In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the  These aIGRG88 (p53 mutant) and glioblastoma xenograft IGRG121 (p53 wild-type) derived from primary tumours, previously described =width2 (mm2) × length (mm)/2. The experiment was stopped after 120 days when there were tumour-free survivors. Statistical significance between treatment groups and controls was estimated by the two-tailed nonparametric Kruskal–Wallis test. Tumour regression was described using standard terminology. ‘Additive’ antitumour activity was defined as an antitumour effect of the sum of both agents alone, ‘synergy’ as effects greater than the sum of both agents alone, and ‘potentiation’ as the effect of an itself nontoxic agent to increase the efficacy of the combined agent.Antitumour activity was evaluated against advanced stage human malignant glioma 8 PFU ONYX-015 in 50 μl PBS for 5 consecutive days. Animals bearing IGRG121 xenografts, received TBI and a dose of 1 × 107 PFU ONYX-015 for 5 days. The first virus injection was performed 6–7 h following irradiation in anaesthetized animals. Different sites of the tumour were chosen for each injection. Simultaneously, groups of animals were either irradiated with 5 Gy and injected with vehicle PBS without glycerol  for 5 days, or were enclosed in a radiation chamber without radiation and received injections of 1 × 108 PFU ONYX-015 or 1 × 107 PFU ONYX-015, respectively, for 5 days, or control animals were enclosed in the radiation chamber without radiation and were injected with PBS in an equivalent volume and schedule. Experiments were carried out with ethical committee approval and under the conditions established by the European Community (Directive 86/609/CCE) and in accordance with the UKCCCR guidelines . Following irradiation, animals were placed in a system with constant air renewal allowing to perform experiments under L2 conditions. For combined treatment studies, animals bearing IGRG88 xenografts received 5 Gy TBI and intratumoral injections of 1 × 104/well in a 96-well plate) in triplicate were irradiated with 0, 2, and 5 Gy and incubated 48 h later with AdCMVLuc at 10 or 100 MOI. Luciferase activity in the cells was assayed 48 h after infection using the Luciferase Assay System  and a Lumat LB 9507 luminometer . Infectivity of glioma cells was defined by the amount of luciferase activity measured after adenoviral-mediated gene transfer in the tumour cells.Short-term cultures of IGRG88 and IGRG121 glioma cells  without further enzymatic cell-separation step. Cells were stained with propidium iodide buffer  for 1 h. The DNA content of individual cells was measured using a FACScalibat (Becton Dickinson).Subcutaneous IGRG88 and IGRG121 tumours were harvested at baseline and 7, 24, and 48 h after 5 Gy TBI irradiation treatment and were frozen at −80°C. Tumour cells were aspirated from frozen tumours with a 25 Gauge needle and resuspended to approximately 3 × 10in vivo once with 107 PFU ONYX-015 or 108 PFU ONYX-015, or 107 PFU ONYX-015, 7 h after 5 Gy TBI. Experiments were carried out with ethical committee approval and under the conditions established by the European Community (Directive 86/609/CCE) and in accordance with the UKCCCR guidelines  in 96-well plates and infected after 24 h with the virus solution at end point dilutions according to standard procedures. Plates were incubated in humidified conditions at 37°C and monitored daily for cytopathic effect during 14 days. Each sample was done in triplicate. Uninfected wells were used as a control for cell survival.IGRG121 tumour xenografts were injected μm thick sections, and rehydrated, were incubated for 1 h at 35°C with the polyclonal antibody AB 1056  diluted 1 : 300. Detection was performed by a biotinylated rabbit secondary antibody to goat immunoglobulin streptavidin–horseradish peroxidase conjugate , and the chromogen diaminobenzidine. Slides were counterstained with haematoxylin. Haematoxylin–eosin–safranin staining was performed for morphology; Masson's trichrome staining for collagen type I, was performed according to the manufacturer's instructions and using aniline blue .Formalin-fixed paraffin-embedded tumours, cut into 4-In situ Cell Death Detection Kit, AP  according to the manufacturer's instructions. Tissue sections were deparaffinised, rehydrated, and received microwave pretreatment in citric acid buffer for antigen retrieval. DNA strand breaks were labelled by polymerisation of fluorescein-dUTP to the 3′-OH sites, catalysed by terminal deoxynucleotidyl transferase (TdT), revealed by antifluorescein Fab fragments conjugated with alkaline phosphatase (Converter-AP), and visualised using Fast Red . TdT and dUTP were omitted for negative controls. TUNEL-positive cells, displaying compaction or segregation of the nuclear chromatin, or breaking up of the nucleus into discrete fragments, were counted per view at × 100 magnification. Five representative fields were chosen for counting; necrotic fields were excluded.Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) was performed to quantify apoptotic cell death using the in vivo was evaluated in the moderately radiosensitive human malignant glioma xenografts IGRG121 and IGRG88 deriving from primary tumours. To prevent potential mutagenesis of adenoviral DNA, irradiation was delivered prior to intratumoral injections of ONYX-015. Intratumoral administration of ONYX-015 at 1 × 108 PFU/injection on 5 consecutive days to mice bearing advanced stage subcutaneous IGRG88 tumour xenografts resulted in significant tumour growth delay of 10 days compared to controls (P<0.05) and one and two out of 10 animals experienced complete and partial tumour regression, respectively (Table 1P< 0.01). Moreover, combined treatment of irradiation given 6–7 h prior to the first intratumoral ONYX-015 treatment yielded an additive antitumour activity with 73% partial tumour regressions and TGD of 30 days  (Antitumour activity of ONYX-015 in combination with radiation therapy ontrols) .IGRG121 xenografts, intratumoral treatment with ONYX-015 at 1 × 108 PFU times 5 has previously been proven highly effective, inducing 73% tumour-free survivors at 4 months . Most importantly, the ‘subtherapeutic’ dose of ONYX-015 potentiated antitumour activity of total body irradiation and induced a significant tumour growth delay of 75 days compared to untreated controls (P<0.001) and 40% partial and 50% complete tumour regressions with five of 10 animals surviving tumour-free 120 days after treatment . In addition, we examined whether irradiation influences viral infectivity of glioma tumour cells using an adenovirus vector expressing luciferase under the CMV promoter. Transgene expression of the IGRG88 and IGRG121 glioma cells in vitro, infected at 10 and 100 MOI, was not increased 48 h after irradiation with 2 or 5 Gy compared to untreated controls  plays a crucial role in the viral infection of cells, with P53 mutant IGRG88 cells arrested only in G2 M and not in G0/1. However, there was only a minor increase in tumour cells in S phase of 3% (IGRG121) and 5% (IGRG88) after 7 h, with a subsequent drop to 50% of the initial S fraction. These minor and transient changes might not be considered significant enough to explain the potentiating effect of combination treatment in vivo.Cellular S-phase fraction has been suggested to determine sensitivity of cells to viral infection . To inveFigure 3in vivo. Viral titres of ONYX-015 in tumours injected once with 107 or 108 PFU ONYX-015 were increased by 5 and 7 log, respectively, at days 3 and 6 compared to day 1 after injection, demonstrating significant intratumoral adenoviral replication in vivo  in human malignant glioma models in vivo  is currently ongoing. Nevertheless, it is unlikely that adenoviral therapy applied as a monotherapy will cure glioblastoma. Data from preclinical studies and clinical trials have suggested that ONYX-015 sensitises tumour cells to killing by chemotherapy  showed major post-therapeutic changes, including apoptosis, single cell necrosis and stromal fibrosis. However, these changes were not seen in tumours treated with radiation alone or with radiation and ONYX-015 at the lower dose. In the latter case, antitumoral effects were primarily associated with infiltrations of macrophages and lymphocytes, and predominant, early appearing tumour fibrosis. We therefore suggest, that the potentiating antitumour activity of radiation therapy and ONYX-015 is related to an induction of inflammatory processes through adenoviral infection and subsequent acceleration of radiation-induced processes of tissue fibrosis.IGRG121 tumours treated with the high dose of ONYX-015 (5 × 10E1B-deleted, replication-competent adenovirus ONYX-015 in combination with radiation therapy in human glioblastoma xenografts. The combination treatment was particularly effective on the p53 functional IGRG121 xenografts. Although it remains to be determined whether ONYX-015 exerts any toxicity to normal brain tissues, combination therapy with replication-selective oncolytic viruses and radiation therapy holds promise as a new treatment paradigm for glioma.In summary, our investigations demonstrate the potentiating antitumour efficiency of local treatment with the


Xenografted artificial heterogeneous tumours (AHTs) were created by admixing, in a ratio of 9:1 or 1:9, two clonal subpopulations (designated as clones A and D) obtained from a heterogeneous human colon adenocarcinoma. In unperturbed AHTs these percentages remain constant with increasing tumour size. At average volumes of 250 mm3, AHTs were X-irradiated (15 Gy) and changes in growth rate and composition assayed. A and D cells exhibited equivalent levels of survival after in vivo irradiation as determined by excision assay procedures. At about 2-3 weeks post-irradiation AHTs exhibited a significant enrichment of the majority population in both the 1:9 or 9:1 A:D AHTs. Additional studies were concomitantly performed to determine whether these changes were mostly a function of normal tissue damage or of parenchymal tumour cell killing. In these studies, the normal tissue only was irradiated, tumour cells were implanted one day after irradiation, and the composition of AHTs assayed as a function of time post-irradiation. In these studies, similar shifts in composition with similar kinetics to that seen in the in situ irradiations were found. We therefore propose that these compositional shifts are mainly a reflection of radiation damage to the stromal microenvironment, which is consequently unable to support tumour growth adequately leading to competitive exclusion of the minority subpopulation.


A significant correlation (r = -0.48) was found between the logarithm of the S-phase fraction of the primary tumour (SPF) and the logarithm of the doubling time of lung metastases (T2). The estimated median cell loss factor was 88% (range 35-99%).


The efficacy of photodynamic therapy (PDT) using delta-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) sensitisation and laser light at 635 nm was investigated in the treatment of experimental hepatic tumours. The model of liver tumours was induced either by local inoculation or by administration of tumour cells through the portal vein in rats. ALA at a dose of 60 mg kg(-1) b.w. was intravenously administered 60 min before PDT. PpIX accumulation in tumour, normal liver and abdominal wall muscle was detected by means of laser-induced fluorescence (LIF). Laser Doppler imaging (LDI) was used to determine changes in the superficial blood flow in connection with PDT. Histopathological examinations were performed to evaluate the PDT effects on the tumour and the surrounding liver tissue, including pathological features in the microvascular system. The accumulation of PpIX, as monitored by LIF, showed high fluorescence intensities at about 635 nm in both the hepatic tumour tissue and normal liver and low values in the abdominal wall. LDI demonstrated that the blood flow in the treated tumour and its surrounding normal liver tissue decreased immediately after the PDT, indicating an effect on the vascular system. A large number of thrombi in the irradiated tumour were found microscopically 3 h after the PDT. The tumour growth rate showed a marked decrease when evaluated 3 and 6 days after the treatment. These results show that the ALA-PDT is effective in the inhibition of growth of experimental hepatic tumours.


Five high-grade (3 grade III and 2 grade IV) astrocytoma tumour cell populations were treated with a preparation of Human Leukocyte Interferon either in monolayer cell culture or as multicellular spheroids in vitro or as xenografts growing in immune-deprived mice in vivo. A moderate and transient sensitivity was seen in one grade III tumour when tested in both of the in vitro assays, but no inhibition of growth was seen in vivo. Two tumours which were apparently resistant to Interferon treatment responded to orthodox chemotherapy. When used in conjunction with BCNU, Interferon was not effective in prolonging delay in tumour growth. It is concluded that Interferon is unlikely to be an effective agent in the treatment of malignant brain tumours.


Sir,A six-year-old girl was posted for a posterior pharyngeal wall tumour excision. The patient was submitted for preanaesthetic review with chest X-ray as well as X-ray of head and neck. There was no gross abnormality in either and no history suggestive of sleep apnoea. However,As the surgery was underway, there was difficulty in ventilating the child and it was found that the tube had come off as the tumour was being resected, giving a big passage larger than the existing endotracheal tube size (4.0 uncuffed). Since the field was bloody and the saturation kept falling, a cricothyroid puncture was made to oxygenate and later intubation with a 6.0 cuffed tube after clearing the bloody field.Changing the tube with a different size, as the tumour was resected, was not spontaneous, though it was anticipated and planned for a difficult airway management. This may be a good lesson for the beginners who involve in oropharyngeal wall tumour surgeries.


NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients. © 2000 Cancer Research Campaign


During the terminal stages of tumour growth (6-8 weeks) in Wistar rats bearing a syngeneic squamous cell carcinoma (Sp1), their sera can block in vitro anti-tumour cytotoxicity by immune splenic T lymphocytes. At an earlier stage of tumour growth (4-6 weeks) the sera do not block this cytotoxicity, but can induce anti-tumour cytotoxicity by non-immune spleen cells in the absence of complement. Sera taken at these 2 stages of tumour growth have been fractionated by ion-exchange chromatography, using DEAE-cellulose. The fractions have been examined by immunoelectrophoresis and tested for anti-tumour reactivity. Blocking activity was found in the Week-8 serum fraction eluted with 0-005M phosphate buffer, pH 7-4, whilst the "cytotoxic" activity of Week-4 serum was eluted with 0-02M phosphate buffer, pH 6-2. It is suggested that different IgG sub-classes are responsible for the 2 activities.


Many studies underline the importance and complexity of the initial arrest of the circulating tumour cells in the target organ, a key stage in metastasis occurrence. In our study, we evaluated by visual means the metastasis formation using an in vivo microscopy system in a murine model. Moreover, we investigated the involvement of P-selectin in these processes using immunohistochemistry and P-selectin knockout mice. The present study offers direct evidence of distinct pathways for tumour metastasis formation by a lymphoma cell – EL-4 and a solid tumour cell – C26. Off-line analysis of the images and histological data confirmed that mechanical entrapment of the solid tumour cell, which had a bigger diameter than that of the liver sinusoids, promoted metastasis without any detectable involvement of adhesion molecules. On the other hand, we observed that lymphoma cells, in spite of their smaller diameter as compared to the sinusoids, promoted liver metastasis as well, but with the essential participation in their arrest of P-selectin, indicating an adhesion molecule-mediated pathway. © 2001 Cancer Research Campaign


Fluids are often given liberally after the return of spontaneous circulation. However, the optimal fluid regimen in survivors of cardiac arrest is unknown. Recent studies indicate an increased fluid requirement in post-cardiac arrest patients. During hypothermia, animal studies report extravasation in several organs, including the brain. We investigated two fluid strategies to determine whether the choice of fluid would influence fluid requirements, capillary leakage and oedema formation.19 survivors with witnessed cardiac arrest of primary cardiac origin were allocated to either 7.2% hypertonic saline with 6% poly (O-2-hydroxyethyl) starch solution (HH) or standard fluid therapy  (control). The patients were treated with the randomised fluid immediately after admission and continued for 24 hours of therapeutic hypothermia.During the first 24 hours, the HH patients required significantly less i.v. fluid than the control patients  with comparable use of vasopressors. Systemic vascular resistance was significantly reduced from 0 to 24 hours (p = 0.014), with no difference between the groups. Colloid osmotic pressure (COP) in serum and interstitial fluid (p < 0.001 and p = 0.014 respectively) decreased as a function of time in both groups, with a more pronounced reduction in interstitial COP in the crystalloid group. Magnetic resonance imaging of the brain did not reveal vasogenic oedema.Post-cardiac arrest patients have high fluid requirements during therapeutic hypothermia, probably due to increased extravasation. The use of HH reduced the fluid requirement significantly. However, the lack of brain oedema in both groups suggests no superior fluid regimen. Cardiac index was significantly improved in the group treated with crystalloids. Although we do not associate HH with the renal failures that developed, caution should be taken when using hypertonic starch solutions in these patients.NCT00347477. Few studies have described fluid requirements in cardiac arrest patients -3, but fThe application of a hypertonic colloid during cardiopulmonary bypass has been shown to reduce fluid overload ,12. CollThe aim of the study was to determine whether a capillary leakage was present in OHCA survivors during therapeutic hypothermia. We compared two fluid regimens and studied the impact on capillary leakage. The intervention group received an additional 500 ml of 7.2% hypertonic saline with 6% poly (O-2-hydroxyethyl) starch solution during the first 24 hours, and was compared with standard therapy. The primary endpoint was the amount of fluid administered during the first 24 hours. The secondary endpoint was the magnitude of capillary leakage as a surrogate marker for oedema formation.The study was approved by the Regional Committees for Medical Research Ethics, the Data Inspectorate, the Directorate for Health and Social Affairs and the Norwegian Medicines Agency. Deferred consent was used, and the patients' families were entitled to withdraw the patients at any time. All patients included were informed about the study when they were able to receive the information and signed a written informed consent form.The study was performed on 19 patients with witnessed out-of-hospital cardiac arrest (OHCA) and carried out between September 2005 and March 2007 at Haukeland University Hospital , an 1,100-bed hospital serving 600,000 people. All inclusion/exclusion criteria are presented in Table ® Fresenius Kabi, Germany) (HH). Fluid was administered to achieve the treatment goals listed in Table On admission, the patients were allocated by means of stratified randomisation to one of two fluid regimens administered via infusion pumps: Ringer's Acetate and saline 9 mg/ml (control), or hypertonic colloid,7.2% NaCl with 6% Hydroxyethyl starch 200/0.5 .Before admission to the MICU, after cardiac intervention and if the patient did not have an intra-aortic-balloon pump (IABP), magnetic resonance imaging (MRI) of the brain was planned . Repeated MRI was scheduled after 24 and 96 hours.®, Pulsion Medical System AG, Germany). Fluid balance was measured as the total amount of fluid administered intravenously and enterally in relation to output measured by hourly diuresis and 24-hour faecal loss. Systemic vascular resistance (SVR) was calculated 0, 8, 16 and 24 hours after admission to the MICU. Vasopressors  were administered if the mean arterial blood pressure was <60 mmHg and the fluid load proved ineffective, guided by PiCCO measurements. Dopamine was replaced with noradrenaline if tachycardia occurred (>100 beats min-1) or if dopamine failed to achieve the required blood pressure. Sedatives  were administered to achieve a motor activity assessment score of 0 (MAAS). If necessary, vecuronium was administered to prevent shivering. Ventilation was provided by Evita XL , using a bi-positive airway pressure mode. Cooling was initiated outside the hospital for all patients who had return of spontaneous circulation (ROSC) and remained unconscious. At the scene, cooling was performed using icepacks placed on the neck, armpits and groin. A Coolgard catheter  was installed in the right femoral vein in the PCI lab and activated in the MICU, cooling the patient at a rate of 1°C per hour. The target temperature was set at 33°C and measured in the urine bladder. After 24 hours of cooling, rewarming at a rate of 0.5°C per hour was stopped at 35.0°C.Cardiac arrest data were recorded according to the Utstein style . In the Blood samples were taken from the artery line after 0, 8, 16 and 24 hours, and analysed at the Laboratory of Clinical Biochemistry at Haukeland University Hospital. Colloid osmotic pressure (COP) was measured at 0, 8, 16 and 24 hours in serum and in interstitial fluid that was sampled using the wick method, installed for 60 minutes -20. A stThe randomisation was stratified with respect to initial heart rhythm. Numbered envelopes were distributed from the MICU and opened when the physician in the emergency room enrolled a patient, filling in the inclusion criteria. The allocation was generated by the authors. The sample size was determined by power calculations on the basis of a required volume load of 8000 ml crystalloids during the first 24 hours and a standard deviation of 500 ml. A power of 80% and a significance level of 0.05 for a two sample t-test suggested that it would be sufficient to have three patients in each group if HH reduced the required volume by 30% to 5600 ml. Due to lower power in non-parametric tests, a higher number was chosen. The unconscious patients, as well as the neuroradiologist, were blinded to the treatment. The two treatment groups were descriptively compared at baseline. Fluid load, urine output and fluid balance were compared using an exact Mann-Whitney test. Mixed effects models were used for group comparisons of repeated measurements of variables . Time frTwenty-four patients were randomised. Five were excluded due to lack of witnessed arrest, inclusion in another study, probable respiratory cause of the cardiac arrest, and age >80 years Fig . Ten patDuring the first 24 hours in the hospital, the HH group required significantly less fluid than the control group to meet the treatment goals. Fluid calculations are presented in Table COP in plasma showed a significant decline in both groups Fig. . The red2 (0.2) on admission to the MICU. At 24 hours, before rewarming, the CI was higher in both groups and significantly higher in the control group . Both groups had a significant drop in SVR, and demonstrated increased extravasation through the drop in COP. The extravasation did not show as vasogenic brain oedema.The strength of the study lies in its design and the multiple determination of leakage. The weakness of our design is that the treating physicians were not blinded. This could have caused a tendency to replace fluid with vasopressors. However, there were no differences between the groups regarding the use of these drugs. Furthermore, as sedation can cause vasodilatation, the use of sedation may influence the use of fluid and vasopressors. The lowest doses of sedation were used in all patients to achieve MAAS 0-1. The number of patients in our study is not sufficient to determine whether fluid load can affect neurological outcome/survival.A large cohort study recently reported on the challenging aspects of therapeutic hypothermia . In spitTo our knowledge, there are no papers describing repetitive MRI in the initial treatment of OHCA patients. Järnum and colleagues performed MRI on 20 cardiac arrest patients who remained unconscious 72 hours after normothermia . They foWe found reduced fluid leakage to the interstitial space in the HH patients compared with controls. Maintenance of intravascular COP is one important factor in determining fluid flux across the capillary membrane. The decline in COP in plasma was probably due to hemodilution, which is also reflected in a reduction in haemoglobin and erythrocyte volume fraction.The reduction in COP in interstitial fluid is probably caused by the escape of fluid with a lower COP through the capillaries. Since we observed a simultaneous reduction in COP both in plasma and interstitial fluid, the increased extravasation cannot be explained by the differences in the COP gradient between the groups. However, the change may be attributed instead to capillary leakage, which has also been demonstrated in several animal studies ,27,28. TBoth groups demonstrated a comparable and significant reduction in SVR, suggesting a similarity between septic and post-cardiac arrest patients . As hypoSmall volume resuscitation with hypertonic saline during CPR is described as feasible and safe , and, inDespite lower body temperature, CI was higher at 24 hours than on admission to the MICU. The increase was significant in the control group. Laurent and collaborators made the same observation when they monitored more than 160 OHCA patients with pulmonary artery catheter .The clinical implication of the present study is that post-cardiac arrest patients can be liberally infused with crystalloids during the first 24 hours without cerebral oedema resulting. They also have a high fluid requirement, which is partly because of increased extravasation, measured by means of colloid osmotic pressures, systemic vascular resistance and fluid calculations. Both fluid regimens stabilise hemodynamics. The reduced fluid load achieved by the application of HH should be further investigated in cardiac arrest caused by asphyxia, where a disrupted blood-brain barrier is more likely. The lack of vasogenic brain oedema in these patients is encouraging. This supports a liberal use of crystalloids, especially due to an increased need for intravascular volume and the possible side effects of colloids. Furthermore, the impact on neurological outcome and survival should be examined.Post-cardiac arrest patients have high fluid requirements during therapeutic hypothermia, probably due to increased extravasation. The use of HH reduced the fluid requirement significantly. However, the lack of brain oedema in both groups suggests no superior fluid regimen. Cardiac index was significantly improved in the group treated with crystalloids. Although we do not associate HH with the renal failures that developed, caution should be taken when using hypertonic starch solutions in these patients.The authors declare that they have no competing interests.BEH participated in the design of the study, the application for official approvals and the collection and interpretation of data. JKH, ABG participated in the design of the study and in collection and interpretation of data. JL, RF participated in the design of the study and collection of data. SMH, EML participated in the collection and interpretation of data. TWL participated in the design of the study and statistical analysis of the data. All authors read and approved the final manuscript.


We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells  from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity  at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy.


A significant positive correlation was found between AI and the change in tumour oxygenation  following radiotherapy . The lack of correlation between apoptosis and hypoxia may occur because the Eppendorf measures both acute and chronic hypoxia, and the relative ability of acute hypoxia to induce apoptosis is unknown. These results indicate that cell death via apoptosis may be a mechanism of tumour reoxygenation during radiotherapy. © 2000 Cancer Research CampaignA relationship between hypoxia and apoptosis has been identified in vitro and in experimental tumours. The aim of this study was to investigate the relationship between apoptosis, hypoxia and the change in oxygenation during radiotherapy in human squamous cell carcinoma of the cervix. Forty-two patients with locally advanced disease underwent pretreatment evaluation of tumour oxygenation using an Eppendorf computerized microneedle electrode. Twenty-two of these patients also had a second evaluation of tumour oxygenation after receiving 40–45 Gy external beam radiotherapy. Paraffin-embedded histological sections were obtained from random pretreatment biopsies for all 42 patients. Apoptotic index (AI) was quantified by morphology on TUNEL stained sections. No correlation was found between pretreatment measures of AI and either the median pO


Herein, in vivo efficacy and mechanism of action studies have been completed for the lead agent, ADMP06.Photodynamic therapy (PDT) is a treatment modality for a range of diseases including cancer. The BFA multi-modality imaging approach was employed to assess efficacy of treatment, as well as probe the mechanism of action of ADPM06-mediated PDT.−1 of ADPM06 followed immediately by light irradiation with 150 J cm−2. The inherent fluorescence of ADPM06 was utilised to monitor organ biodistribution patterns, with fluorescence reaching baseline levels in all organs within 24 h. Mechanism of action studies were carried out using dynamic positron emission tomography and magnetic resonance imaging techniques, which, when taken together, indicated a decrease in tumour vascular perfusion and concomitant reduction in tumour metabolism over time after treatment.Tumour ablation in 71% of animals bearing mammary tumours was achieved after delivery of 2 mg kgin vivo and vascular-targeting mechanism of action continue to indicate therapeutic benefit for this new class of photosensitiser.The encouraging treatment responses  Photodynamic therapy (PDT) is an emerging treatment modality whereby a photosensitiser is administered systemically or locally and subsequently activated by illumination with visible light, leading to the generation of cytotoxic reactive oxygen species . The intvs direct tumour toxicity depend on the sensitiser and on the interval between sensitiser administration and illumination. For short intervals , when there is a high drug concentration in circulating plasma, the vascular damage is maximised. For longer intervals, when the drug has been distributed to tumour tissue, direct cell toxicity becomes more important , and have shown in vitro that their photophysical and biological characteristics have significant potential for the development as new anti-cancer therapeutics  and magnetic resonance imaging (MRI), we have also shown that our current ADPM06 treatment regimen elicits a vascular-targeting effect.In this study, we have used a short drug–light interval protocol with lead compound ADPM06 to show tolerance and efficacy in several animal models of cancer. We have also exploited the inherent fluorescent properties of a candidate PDT agent to directly assess The synthesis and formulation of the ADPM family of compounds in a mixture of Cremophor EL (CrEL)/1,2-propanediol (10 : 3 v/v) has been described previously  sodium pyruvate, and 1% (v/v) L-glutamine. All cells were maintained in 5% CO2 (v/v) and 21% O2 (v/v) at 37°C. MDA-MB-231 luciferase-expressing cell line (MDA-MB-231-luc) was obtained from Caliper LS  and maintained as above. Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics  and maintained in endothelial cell growth medium , containing 12 μg ml−1 bovine brain extract, 10 ng ml−1 EGF, 1 μg ml−1 hydrocortisone, 50 μg ml−1 gentamicin, 50 ng ml−1 amphotericin B, and 2% foetal bovine serum (Clonetics).MDA-MB-231 tumour cells were obtained from the American Type Culture Collection (ATCC) and cultured in minimum essential medium supplemented with 10% (v/v) foetal calf serum, 1% (v/v) non-essential amino acids, 50 U mlμm filter. Viral supernatant was placed directly on subconfluent MDA-MB-231 cells, which were then incubated overnight before the cells were washed gently with sterile PBS and the media was refreshed.A stable MDA-MB-231 breast cancer cell line capable of constituitive, enhanced GFP expression was generated through lentiviral transduction. For this, viral particles were produced by transient calcium phosphate transfection of HEK 293t cells with the GFP-encoding lentivector, pLVTHm, the envelope vector, pMD2G, and the packaging vector, psPAX2 . Transfected cells were incubated for 48 h, after which medium containing the lentiviral particles was harvested and passed through a 0.45-in vitro Angiogenesis Assay Kit . Briefly, solid gels were prepared according to the manufacturer's instructions on a 96-well tissue culture plate. HUVECs seeded into 25-cm2 flasks and grown to confluency of 70–80%, were irradiated 3 h after ADPM06 administration (150 nM). Cells were then trypsinised and seeded at a density of 5 × 104 cells per well in EGM onto the surface of the solid endothelial cell gel matrix. The cells were incubated for 7 h at 37°C in a CO2 incubator. Tube formation was observed under an inverted light microscope at × 20 magnification. Microscopic fields were photographed with a digital camera. The number of branch-points per field-of-view was counted, and the length of tubules was quantified using ImageJ 1.41 . Mann–Whitney U-test was used to compare control and treated samples.Tube formation assays were performed using an −1) to establish a basic toxicity profile for the novel photosensitiser. There was no evidence of either acute or chronic (6-month follow-up) toxicity. No significant histopathological changes were observed on veterinary pathologist's examination of the liver, kidney, heart, or spleen. Specifically, no necrotic, degenerative, inflammatory, or hyperplastic processes were observed (data not shown). For light+drug toxicology studies (as well as drug efficacy studies), C57/Bl mice were shaved on the right hind limb and depilated with Nair . Mice were anesthetised with an i.p. injection of ketamine/xylazine cocktail (90 mg kg−1 ketamine and 10 mg kg−1 xylazine). One million LLC/1 lung carcinoma cells were injected subcutaneously in one mid-thigh area suspended in 100 μl PBS. Tumours grew predictably in all the mice and reached a size of 5- to 6-mm diameter 5–7 days after injection, at which time they were used for PDT. Female Balb C nu/nu mice were similarly anesthetised and five million MDA-MB-231-GFP or MDA-MB-231-luc cells were injected subcutaneously in 100 μl PBS/Matrigel (50 : 50) into the right forelimb. Both cell lines similarly reached 5–6 mm in diameter within 7–10 days and were subjected to PDT as described below. For dynamic PET and MRI studies, MAT III B tumour cells (obtained from ATCC) were inoculated intradermally (5 × 106 cells in PBS) into both axillary areas on rats 6–8 days before the experiment. Rats were fasted 12 h before PDT treatment.All animal experiments were licensed by the Department of Health and Children, Ireland and specific protocols reviewed by the Animal Research Sub Committee (ARSC) at University College Dublin. Imaging protocols were conducted in accordance with the recommendations of the Canadian Council on Animal Care and of the in-house Ethics Committee for Animal Experiments at Université de Sherbrooke. All studies were in full compliance with EU guidelines. Female C57/Bl or Balb C nu/nu mice were received to the SPF-grade Conway Institute Biotechnical Services (CIBS) Xenograft Facility at 4–6 weeks old . Initially, normal C57/Bl mice were parenterally treated (i.v.) with ADPM06  emitting light at 690 nm (±25 nm). The illuminating spot had a diameter of 11 mm and was positioned so that the entire tumour and a surrounding 2–3 mm area of normal tissue were exposed to light with no evident temperature increase at the site of irradiation. Mice were anesthetised as described above and the tumour-bearing limb positioned under the spot. Total fluences between 50 and 200 J cm−2 were delivered at a fluence rate of 717 mW cm−2. Some mice had LLC/1, MDA-MB-231-luc, or MDA-MB-231-GFP tumours illuminated without having received an earlier injection of ADPM06. In addition, another group of animals were treated with ADPM06 i.v. in the absence of light. At the completion of illumination, mice were allowed to recover in an animal warmer until they resumed normal activity. A positive tumour response was assigned to tumours that appeared macroscopically as flat blackened eschars and necrotic tissue within a few days after PDT. Animals were considered cured after complete tumour regression; this was defined as the absence of a palpable tumour.ADPM06 in PBS/Cremophor solvent was injected at a dose range of 1–10 mg kg−1 of the photosensitiser was administrated to rats lying in the scanner through a second cannula 30 min after the commencement of 18F-FDG infusion. Illumination was provided immediately after the photosensitiser injection (less than 5 min between photosensitiser injection and tumour illumination). One tumour was illuminated with a 670-nm light beam delivered through a fibre optic by a diode laser  and the other tumour was masked and served as control. The light beam was spread uniformly over the whole tumour area and maintained for 215 s at a fluence rate of 700 mW cm−2 for a total fluence of 150 J cm−2, as before. For PET and MRI imaging studies at specific time points before or after PDT, the photosensitiser administration and tumour illumination were performed off the scanner after the same treatment protocol as above.Rats bearing 13762 MAT B III tumours were anesthetised and two cannulas were inserted in the caudal veins, one for radiotracer infusion or contrast agent injection (MRI) and the other for photosensitiser administration. The animals were then placed on the scanner bed equipped with a heat pad and instrumented for life sign monitoring. For the dynamic PET studies, 0.8 mg kgin vivo imaging system , having both bioluminescence and fluorescence capabilities. Images and measurements of fluorescent signals were acquired and analysed using Living Image Software v3.0 . Excitation and emission wavelengths of 475 and 540 nm, respectively, were used for acquiring in vivo GFP fluorescent images, whereas excitation and emission wavelengths of 640 and 720 nm, respectively, were used for acquiring in vivo ADPM06 fluorescent images. All images were attained using a 1-s exposure time and an f/stop of 1, with a sampling of multiple angles with animal remaining sedated. For biodistribution studies, mice were injected with 2 mg kg−1 ADPM06 through the tail vein, anesthetised before being imaged at ADPM06 excitation and emission wavelengths at various time points. Mice were then euthanized, the tumour, liver, lungs, heart, spleen and kidneys were extracted, and ex vivo fluorescence images were acquired. MDA-MB-231-luc tumours were imaged following i.p. administration of luciferin substrate (150 mg kg−1).Optical imaging was performed with an IVIS Spectrum small-animal μl volumetric resolution). The scanner is made of four detector rings with a total of 512 pixel detectors defining an axial field-of-view of 2.5 cm with a diameter up to 10 cm. The massively parallel processing of pixel detectors makes it possible to image over a large range of radioactivity with negligible dead time losses. Therefore, dynamic PET imaging of tumour 18F-FDG uptake during PDT treatment could be performed with radiotracer infusion for up to 4 h, starting at the same time as the dynamic image acquisition. Rats were typically infused at 0.008 ml min−1 with an initial 18F-FDG radiotracer concentration of ∼1.5 mCi ml−1. Tumours were positioned at the centre of the scanner axial field-of-view in such a way to allow illumination of one tumour with the laser light during the scan, whereas the other one (used as control) was masked. Rats were anesthetised using a mix of 2% isofluorane in oxygen with the flow set at no more than 2 ml min−1 during these extended scans and the animal vital signs were monitored and recorded throughout the entire scan time to ensure a stable physiological status at all times and for later reference. Scanning was stopped if vital signs were not at physiological status. The list mode PET data were sorted out into 1-min frames and PET images were reconstructed using 10 iterations of a maximum likelihood expectation maximisation (MLEM) algorithm that models detector response. Image series consisted of up to 240 1-min frames. These PET measurements were performed with PDT treatment (n=5), with ADPM06 alone (n=1) and light alone (n=1). Regions of interest (ROIs) were traced over the treated and the control tumours using the last frame of the dynamic image series and applied to all frames. Time-activity curves corrected for radioactive decay were generated from the maximum counts averaged over 4 pixels in the ROIs. Taking the control tumour as reference, the relative metabolic activity of the treated and control tumours was estimated as the ratio of the slope of these two curves. Mean group values were tested for statistical significance using a one-way analysis of variance. The PET images of the tumours after a bolus injection of 1–1.3 mCi 18F-FDG (in <1 min) were also obtained at specific time points before PDT and at 30 min, 24 and 48 h after PDT treatment to monitor tumour metabolism over a more extended period of time by simply comparing 18F-FDG uptake values at equilibrium in tumour ROIs. A static 1-min image frame at 30 min after the bolus injection was extracted from the list mode data and reconstructed using 10 MLEM iterations. The relative metabolic activity of the treated and control tumours were also estimated by ROI analysis as described above.PET imaging was performed with the Sherbrooke avalanche photodiode PET scanner  . Sets of images were acquired continuously. A bolus of 0.6 ml gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA)  was injected i.v. 5 min after the start of the scan (with an infusion rate of 600 μl min−1) through the tail vein after the second set of images.The MRI was performed with a Varian 7T small animal scanner  using a 63-mm volume coil. Rats were anesthetised using a mix of 2% isofluorane in oxygen with the flow set at 2 ml minThe capacity of HUVECs to form new tubules in Matrigel  was directly inhibited after PDT with ADPM06 , indicat−2) provided early evidence of tumour response  in the immediately adjacent, non-illuminated, normal tissue was invariably observed. In contrast, tumour healing took approximately 20–30 days. The skin covering the tumour was initially damaged, but underwent remodelling during the healing process.LLC/1 tumour-bearing mice were used to determine drug–light combination tolerance. Initial studies comparing toxicity profiles of ADPM06 in combination with light  is shown in For xenograft studies, MDA-MB-231-GFP cells inoculated subcutaneously into nude mice developed tumours. Within 7–10 days, when the tumours reached a diameter of about 7–9 mm, PDT treatment was performed, using different light–drug concentrations. In all cases, tumours were irradiated immediately after i.v. drug delivery. The illumination of the tumour always included 2–3 mm of surrounding normal tissue. Tumour ablation over time, as shown by reduction in GFP fluorescence, is shown in Ex vivo imaging and fluorescence quantification from excised organs was performed to establish drug biodistribution patterns (patterns . A peak patterns . Fluorespatterns . Criticain vivo (18F-FDG uptake rate in the treated tumour relative to control tumour after light treatment. Photosensitiser and light controls showed no significant modification of the 18F-FDG uptake rate relative to the control (untreated) tumour in PDT-treated rats, confirming the absence of any detectable metabolic effects due to ADPM06 or light alone. Therefore, transient changes in the 18F-FDG uptake rate, which occurred from 10 to 30 min after the commencement of illumination can clearly be related to the effect of PDT. The subsequent slope change of the 18F-FDG uptake curve observed >30 min after PDT is likely the result of the time-confined, short PDT insult causing a partial, but permanent, drop in tumour metabolic activity, specifically 50%  from the slope value (counts per pixel per minute) of the 18F-FDG uptake curves after PDT. Such behaviour does not suggest extensive cellular damage during this observation time window and is consistent with a vascular-targeting response depriving tumour cells of blood supply, when light irradiation is performed immediately after photosensitiser delivery. Bolus tracer injection confirmed reduced tumour metabolism at 30 min post PDT and almost complete suppression of tumour metabolic activity at 24 and 48 h post PDT  as described above, 0.6 ml of MRI contrast agent (Gd-DTPA) was injected i.v. at 0.6 ml minnsitiser .in vivo, when a short drug–light interval protocol was used. Moreover, we have also shown a classical PDT-associated light and photosensitiser dose-dependent response in terms of efficacy, as well as evidence of anti-angiogenic effects in vitro. The observed cure rates (>70%) in a xenograft model of breast cancer are encouraging and comparable with the leading vascular-targeting PDT agents currently in clinical development (Our previous studies have shown the ADPM family of photosensitisers to be an exciting new class of agents having significant potential for preclinical development (elopment . SimilarWe have also shown that ADPM06 has an organ biodistribution/clearance pattern consistent with that of an ideal photosensitiser (in vivo mechanism of action (in vivo setting and, most importantly, to determine vascular-targeting effects after treatment (Dynamic PET has been previously used to investigate photosensitiser f action . AccordiWhen compared with the historical gold-standard, that is, clinical PDT protocols that allow tumour cell accumulation of photosensitiser before light activation, it has been proposed that the anti-vascular strategy comprises the following benefits: First, blood oxygen levels are known to be the highest at the vascular site in comparison to surrounding tissues, and sensitiser levels in the blood are maximal shortly after injection – an optimal situation for successful PDT. Second, the vascular endothelial cells are directly accessible to the blood-borne sensitiser molecules. Third, it is likely that vascular rupture and haemorrhage formation facilitate rapid discharge of the sensitiser molecules into the tumour site more efficiently than expected by passive diffusion across capillary walls (in vivo. The drug quickly clears both from the skin and internal organs as shown by optical imaging. Application of a combined PET/MRI approach provided evidence for a vascular-targeting response to therapy with this agent. Both imaging modalities may be further used during clinical evaluation as useful biomarkers of therapeutic response. Taken together, the preclinical data set presented here clearly establishes lead BF2-azadipyrromethene ADMP06 as an exciting new photosensitiser for potential application in a Phase 1 clinical trial.In summary, we have shown that our lead novel photosensitiser, ADPM06, elicits an impressive therapeutic response in tumour models and is well tolerated


The effect of methotrexate on solid rodent tumours has been investigated using the spontaneous mammary adenocarcinoma of the female C3H/Bts mouse. As these tumours exhibit a wide range of volume doubling times, calculations of tumour response to methotrexate must be related to doubling time. When methotrexate is injected into the tumour a dose-dependent tumour response is obtained. Systemic citrovorum "rescue" prevents methotrexate lethalities but reduces tumour response by a factor of 2. Methotrexate-treated tumours have an increased volume doubling time post treatment.


The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects.vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients.Herein, we evaluated the anti-tumour efficacy of metronomic Ktrans values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1α, IL-8, ICAM-1, and VCAM-1.Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment. Myelosuppression is the dose-limiting toxicity. This pulsatile maximum tolerated dose (MTD)-driven gemcitabine regimen significantly improves clinical benefit response but only provides modest survival benefits . When the subcutaneous tumours reached 600–800 mm3, tumour-bearing mice were randomised into three groups (n=5 each) and treated with vehicle control , one cycle of MTD gemcitabine , or metronomic gemcitabine  for 4 weeks. The dose and schedule used in the MTD gemcitabine regimen were chosen to emulate clinical treatment protocols. When administered on a weekly basis, 240 mg kg−1 (∼724 mg m−2 for a 20-g mouse) was previously reported to be the MTD of gemcitabine in mice  was purchased from the British Columbia Cancer Agency pharmacy and dissolved in 0.9% saline . When the orthotopic xenografts reached a palpable size of ∼200–400 mm−1. Serial images were acquired to monitor changes in the concentration of the contrast agent in each pixel during the initial uptake and subsequent washout in the tumour. Magnetic resonance imaging scans followed the technique previously described by 1-weighted scans were acquired, with each scan lasting 64 s . In-plane spatial resolution was 312 μm with 10–14 slices at a thickness of 1 mm or 1.5 mm, depending on the size of the tumour. The Gd-DTPA concentration–time curve for each pixel was fitted to a two-compartment Kety model , with a quadrature birdcage coil for transmission and a 1.7 × 1.4 cm rectangular surface coil for reception. At the end of the treatment cycle (day 29), the three groups of tumour-bearing mice were anaesthetised with isoflurane  and administered an i.v. bolus injection of the magnetic resonance contrast agent gadolinium (Gd)-DTPA  at 0.3 mmol kg−1 i.v. and with proliferation marker BrdU  at 1500 mg kg−1 i.p. at 3 h and 2 h before euthanasia, respectively. The orthotopic tumours were excised immediately after euthanasia. Perpendicular tumour diameters were measured with a caliper, and tumour volumes were calculated using the formula π/6 × a × b2, where a is the longest dimension of the tumour and b is the width. Tumours were then snap frozen in OCT  in liquid nitrogen. Cryosections of 10 μm were cut from each tumour and fixed in 50% (v/v) acetone/methanol for 10 min at room temperature. Endothelial cells were stained with a monoclonal rat anti-mouse CD31/PECAM-1 antibody , followed by an Alexa 647-conjugated goat anti-rat secondary antibody . Reduced EF5 adducts in viable hypoxic cells were stained on the same section using a Cy3-conjugated monoclonal antibody to ELK3-51 (1 : 200) (http://rsb.info.nih.gov/ij/) running on a G5 Macintosh computer . All parameters stained on the same section were imaged separately, and subsequently overlaid and aligned with the removal of imaging artefacts. Tumour hypoxia and cell proliferation were visualised by EF5+ and BrdU+ staining, respectively. False colour images were generated using Adobe Photoshop CS3. Using NIH software applications and user-supplied algorithms, images were cropped to tumour tissue boundaries with necrosis removed; fluorescent images were inverted and a threshold was applied to the CD31 image. Neighbouring CD31+ pixels were grouped as CD31+ objects . All image pixels were sorted to determine their distance from the nearest vessels and the average distance between vessels, which reflects microvessel density, was reported. A short average distance between vessels indicates high microvessel density. The extent of tumour hypoxia or cell proliferation was reflected by the percentage of EF5+ or BrdU+ pixels, respectively, which was calculated as the number of EF5+ or BrdU+ pixels divided by the total number of tumour tissue pixels, and multiplied by 100%.At the end of the treatment cycle (day 29), the three groups of tumour-bearing mice were injected with hypoxia marker EF5  at 30 mg kgμm were cut, air dried, and fixed in 50% (v/v) acetone/methanol for 10 min at room temperature. Sections were stained with H&E for histological examination. To detect apoptosis in cancer-associated fibroblasts, sections were double stained with a FITC-conjugated monoclonal anti-αSMA antibody  and an anti-cleaved-caspase 3 antibody , followed by an Alexa 546-conjugated secondary antibody . To determine apoptosis in endothelial cells, sections were incubated with anti-CD31/PECAM-1 antibody (1 : 100), followed by a FITC-conjugated secondary antibody , and with anti-cleaved-caspase 3 antibody, followed by the Alexa 546-conjugated secondary antibody (1 : 200). Images of whole tumour sections were captured ( × 20 objective) using a Leica fluorescence microscopic imaging system  equipped with both cooled 350FX monochrome and 300FX colour cameras for fluorescence and bright field imaging, respectively, an automated scanning stage (DM6000B), and Surveyor software . After removal of artefacts, composite tumour images for αSMA, CD31, and cleaved-caspase 3 staining were overlaid, aligned, thresholded, and analysed using in-house software programs developed in MATLAB . The percentage of apoptotic cancer-associated fibroblasts (CAFs) or endothelial cells was reported as the number of αSMA+cleaved-caspase 3+ or CD31+cleaved-caspase 3+ pixels divided by the total number of αSMA+ or CD31+ pixels, respectively, and multiplied by 100%.Cryosections of 10 M HEPES, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 100 mM NaF, 10 mM Na4P2O7, 100 μg ml−1 PMSF, 5 μg ml−1 leupeptin, and 5 μg ml−1 aprotinin) using a Polytron PT10-35 homogeniser  and stored at −80°C. Total protein concentration in each tumour lysate was determined using a Micro BCA proteins assay kit . The protein levels of 89 human antigens that include numerous growth factors and cytokines were quantified in the tumour lysates using HumanMAP Antigen version 1.6 .Harvested tumours from all treatment groups were homogenised in lysis buffer  compared with the vehicle control group . Many of the downregulated proteins are pro-angiogenic growth factors and cytokines. In general, the decreases in the levels of these proteins caused by metronomic gemcitabine were more pronounced than those induced by MTD gemcitabine .Metronomic gemcitabine induced significant changes in the levels of 37 and 14 out of 89 proteins in the PaCa8 and PaCa13 tumours, respectively, compared with vehicle control . The provs 720 mg kg−1, respectively). Metronomic gemcitabine did not induce significant weight loss in tumour-bearing mice, indicating a lack of toxicity. These findings suggest that the use of metronomic gemcitabine in the clinic would be advantageous because such a regimen is more effective and better tolerated in terms of total drug dose than MTD weekly gemcitabine.This study demonstrated that metronomic dosing of gemcitabine is active against pancreatic ductal adenocarcinoma. It is noteworthy that, although both metronomic and MTD gemcitabine induced significant tumour volume reductions in PaCa8 and PaCa13 primary xenografts, the total dose of gemcitabine administered over 4 weeks in the metronomic group was less than half of that given in the MTD group . Pancreatic ductal adenocarcinoma is characterised by a dense fibrovascular stroma consisting of different cell types   . These a


For designing the experiments, human neuroblastoma xenografts growing subcutaneously in nude rats were analysed in vivo with 1H MRS and magnetic resonance imaging at 4.7 T. The effects of spontaneous tumour growth and antiangiogenesis treatment, respectively, on spectral characteristics were evaluated. The spectroscopic findings were compared to tumour morphology, proliferation and viable tumour tissue fraction. The results showed that signals from choline (Cho)-containing compounds and mobile lipids (MLs) dominated the spectra. The individual ML/Cho ratios for both treated and untreated tumours were positively correlated with tumour volume (P<0.05). There was an inverse correlation between the ML/Cho ratio and the viable tumour fraction . Higher ML/Cho ratios concomitant with pronounced histological changes were seen in spectra from tumours treated with the antiangiogenic drug TNP-470, compared to untreated control tumours (P<0.05). In conclusion, the ML/Cho ratio obtained in vivo by 1H MRS enabled accurate assessment of the viable tumour fraction in a human neuroblastoma xenograft model. 1H MRS also revealed early metabolic effects of antiangiogenesis treatment. 1H MRS could prove useful as a tool to monitor experimental therapy in preclinical models of neuroblastoma, and possibly also in children.The aim of the study was to evaluate proton magnetic resonance spectroscopy ( Treatment with cytotoxic and cytostatic agents in addition to surgery is the cornerstone of clinical therapy for childhood solid tumours. Alteration in tumour size, assessed by magnetic resonance imaging (MRI), computed tomography (CT) or ultrasound, is currently the basis for the evaluation of treatment response . This evThe development of sensitive methods for repeated and noninvasive measurement of tumour biological activity during treatment needs to be attempted in order to optimise and individualise the treatment of childhood solid tumours. MRI is currently the method of choice for radiological evaluation of many childhood tumours and is increasingly used in clinical routine (1H MRS) can complement MRI in useful ways. 1H MRS allows sampling of detailed biochemical information from normal tissues or pathological processes and has proved valuable for the characterisation of a wide range of malignant disorders . A molecular configuration that enables a high level of proton mobility is a prerequisite for detection with 1H MRS. Accessible metabolites include a large number of intracellular compounds such as phospholipids and polypeptides.The use of proton magnetic resonance spectroscopy (isorders . 1H MRS  reviews ). Using 1H MRS spectra of human tumours (2) and methyl (CH3) groups, and are commonly designated mobile lipids (MLs). Several reports indicate a possible correlation between the amount of MLs and tumour cell death  to rapid tumour dissemination and a dismal prognosis in the majority of older children. Metastases to bone or bone marrow or the finding of certain specific genetic aberrations  . The medium was changed twice weekly and confluent cultures were subcultivated after treatment with 0.5 g l−1 trypsin and 0.2 g l−1 EDTA (Gibco BRL). Cultures were free from mycoplasma as verified by DNA staining. For xenotransplantation, a single-cell suspension, approximately 108 cells ml−1, was prepared in culture medium supplemented with L-glutamine (2 mM). The viability and exact cell concentration were assessed by trypan blue dye exclusion using a haematocytometer.The human neuroblastoma cell line SH-SY5Y  (7 cells suspended in 0.2 ml medium were injected into each hind limb. A 23-gauge cannula was used to deposit the suspension subcutaneously (s.c.). Attention was paid so as not to pierce the muscle fascia or lose cells by leakage from the injection site.A total of 20 male nude rats  at the age of 5–6 weeks were used for the establishment of xenografts as previously described  with final tumour volumes of 3.4±2.9 ml (mean±s.d.), n=9, range 0.4–8.6 ml. In this group, the two largest tumours were followed up to a size of approximately 25 mm, which represented days 19 and 21 from tumour take, respectively.Tumour take was defined as the observation of a palpable and measurable nodule in the area of cell injection. Tumours were measured with a digital calliper every other day and tumour volume was calculated by length×widthn=10) or treatment group (n=8). Animals in the treatment group received the antiangiogenic compound TNP-470  10 mg kg−1 s.c. every other day for 10 days . Tumours were investigated using a circular surface coil with a planar circular detection area of 3 cm. The surface coil was fitted into a customised Plexiglas rig placed in the centre of the gradient system. In order to facilitate the positioning, the rat was placed on a separate rig in side position. Animals were anaesthetised with isofluorane and respiratory activity was monitored continuously. Body temperature was registered and maintained at 37±0.5°C with a heated air stream. The manufacturer's implementation of RARE sequence was used for anatomical imaging, with the following parameters: T1-weighted images: repetition time (TR)=535 ms, echo time (TE)=10 ms; T2-weighted images: TR=2500 ms, TE=115 ms; proton density-weighted images: TR=2500 ms, TE=35 ms.MRI examinations were performed using a 4.7 T magnet with horizontal bore . The magnet was equipped with a 12 cm inner diameter self-shielded gradient system  and additional spoiling gradients. VOIs were carefully inscribed within the xenografts to avoid spectral contamination from adjacent muscle or fatty tissue. The VOI was chosen to include a maximal volume of the tumour, and thus varied with tumour size between approximately 0.2 and 6 cmAfter suppression of the water signal, acquisitions were averaged (64–128) until a signal-to-noise ratio sufficient for spectral evaluation was reached. A long TR (6 s) was employed in order to achieve full relaxation between scans and to avoid different degrees of saturation because of differences in T1 and spatial variation of the excitation flip angle. The TE was varied to differentiate between the trimethyl singlet of Cho-containing compounds , multiple of macromolecules/lipid resonances (ML) and weakly coupled lactate . The first spectrum was recorded at 135 ms, where the slowly relaxing Cho is preserved and Lac is inverted. If a residual signal of fast relaxing lipids was present, the measurement was repeated at TE=270 ms to improve the relaxation differentiation. Finally, a spectrum at short TE=20 ms was recorded showing all metabolites with negligible T2 weighting. In addition, spectra from skeletal muscle and subcutaneous fat were recorded to identify corresponding contamination. This was found to be negligible. The metabolic levels were estimated by peak area integration after appropriate baseline correction. For calculation of the ML/Cho ratio, the methylene lipid peak appearing at 1.2–1.4 ppm (TE=20) was employed.In the control group, MRI/MRS was performed at time points between days 4 and 21 from tumour take in order to study the influence of tumour volume on spectroscopic characteristics. In the treatment group MRI/MRS was performed after completion of therapy (day 10±1). In both groups, there was a sufficient variation of tumour size such that spectroscopic measurements could be correlated to the volumes. On the day of MR-examination, the tumour volume was measured as described.μm) were obtained from different parts of each tumour and processed for histology. Tumour cell proliferation was assessed using a rabbit monoclonal antibody raised against the tumour proliferation marker Ki-67 . A Ki-67 labelling index (LI) was calculated from 1500 nuclei at a magnification of ×400 in three randomly chosen viable areas. The terminal deoxynucleotidyl transferase (TUNEL) reactivity assay  was employed for quantification of apoptosis .Statistical analysis was performed using appropriate software . Parameters were correlated to tumour volume or viable tumour fraction in univariate regression models and the Pearson correlation factors were calculated. For comparison between different groups, Tumours <1.5 ml generally appeared homogeneous but occasionally contained foci with low signal intensity on T2 images. Such low signal areas were seen more often in larger tumours. All tumours >2 ml had a heterogeneous appearance, containing both hyperintense and hypointense areas  were dominated by signals from Cho at 3.2 ppm and ML signals, the latter contributing to resonances at 0.7–0.9 ppm and 1.2–1.5 ppm, respectively  typically had intense Cho signals together with moderate ML levels (N-acetyl-aspartate) (V>2 ml) generally displayed a ML peak of high intensity at 1.2–1.4 ppm, a variable lipid peak at 0.9 ppm and a less prominent Cho signal  . Spectrao signal . ControlV<1.5 ml) generally showed a dense and mostly viable appearance on macroscopic examination while larger tumours were mainly necrotic and haemorrhagic. These macroscopic findings were corroborated to microscopic analyses  (P<0.05). Viable tumour fraction was negatively correlated to tumour volume in untreated animals  (P=0.058). Comparison with histological findings revealed a significant inverse correlation between the ML/Cho ratio and the viable tumour cell fraction   . In the P<0.001) . In treain vivo by 1H MRS in a xenograft model of human neuroblastoma. We found that the ML (methylene)/Cho ratio constitutes a noninvasive measure of the viable tumour fraction in this tumour model.Noninvasive methods that allow repeated evaluation of tumour status and response to therapy could improve efforts to individualise treatment of cancer patients. Proton magnetic resonance spectroscopy is emerging as a highly useful tool for this purpose . In the 1H MRS in neuroblastoma have been reported. Intense creatine and Cho signals were found in 1H MRS spectra from perchloric extracts of neuroblastoma cells.  lipid resonance (at 1.3 ppm) to the methyl (CH3) resonance (at 0.9 ppm) is correlated to the percentage of apoptotic lymphoblasts after doxorubicin treatment . This observation is in line with the finding of ML droplets inside growth-compromised, but viable, glioma cells  is one of the major contributors to the Cho resonance observed in  spectra  and has 1H MRS. After 10 days of treatment, there was no significant difference in tumour volume between the two groups. We could, however, detect higher ML/Cho ratios in relation to tumour volume in treated tumours . In the present paper, we provide evidence that the ML/Cho ratio inversely correlates to tumour viability. Hence, we suggest that 1H MRS could be a tool for monitoring the treatment of neuroblastoma in children by repeated examinations during the course of therapy.The SH-SY5Y xenograft model employed in this study is a well-characterized and highly reproducible in vivo with 1H MRS in a model of human neuroblastoma. Our findings also suggest that the ML/Cho ratio may be valuable as a noninvasive surrogate marker to detect response to experimental therapy in neuroblastoma.In conclusion, we have shown that the viable tumour fraction can be accurately estimated


Heterotransplantation of a human colonic neoplasm with classical morphologic characteristics of a carcinoid was sucessful in the cheek pouches of unconditioned, adult golden hamsters after a short sojourn in cell-impermeable chambers in rats. Although no mucin-secreting cells were detected in the donor carcinoid, the cheek pouch transplants exclusively exhibited mucinsecreting tumour cells of signet-ring type consistent with adenocarcinoma. This transplantable tumour, designated GW-77, has retained this appearance as well as expansive growth characteristics in xenogeneic hosts for a period of 4 years.These findings represent strong biological evidence consonant with views, based upon morphological findings, advocating a histogenetic relationship between colonic carcinoid and adenocarcinoma. It is believed that colonic adenocarcinoma has a selective advantage over carcinoid for serial propagation in an alien environment, indicating the less differentiated nature of its cellular components. Since the donor carcinoid cells failed to exhibit argentaffin reactions, these conclusions may be limited only to the nonreactive forms of carcinoid.


To the Editor: Varicella-zoster virus (VZV), a human herpesvirus, is the causative agent of varicella (chickenpox) and herpes zoster (shingles). Worldwide, children are routinely vaccinated with a live attenuated varicella vaccine containing the Oka vaccine (vOka) strain of VZV, originally developed in Japan . She received oral acyclovir treatment and fully recovered by day 19 after herpes zoster onset. On the same day that the girl recovered, her immunocompetent 2-year-old brother was found to have fever and a rash consisting of 10–20 papulovesicles; mild varicella was diagnosed. The boy had no known history of contact with persons infected with varicella or with persons who administered the varicella vaccine. After receiving oral acyclovir treatment, the boy recovered without systemic complications.On day 19 after the girl’s onset of herpes zoster, an enzyme immunoassay  confirmed the presence of VZV-specific immunoglobulin (Ig) G  but not IgM. The boy showed seroconversion of VZV-specific IgG from a titer of <2.0 on day 3 after his disease onset to 19.3 on day 30. Although vesicular fluid or crust specimens were obtained from both children, only the specimens from the boy contained detectable amounts of VZV DNA.PstI and BgII , and GlaxoSmithKline Biologicals  possess similar immunogenicity and safety characteristics . Thus, the case reported here is considered to be rare in that vOka was transmitted to a healthy susceptible person through close contact with a vaccinee with herpes zoster.vOka is composed of a mixture of genotypically distinct virus strains that have 15 base substitutions in gene 62 compared with the parental Oka strain. Gomi et al. have suggested that the amino acid alterations in the gene 62 products of vOka are associated with the characteristics of vOka, i.e., slower growth and less efficient cell-to-cell spreading in vitro compared with parental Oka (


Sir,The most common site of metastatic disease in melanoma is regional lymph nodes. Several well-documented factors predictive of regional nodal metastasis particularly include tumour ulceration and thickness (


In large families of closely related target sequences, the high degree of similarity makes it impossible to find a unique probe for every target. We studied how to select a minimum set of non-unique probes to identify the presence of at most d-disjunct matrices. Our non-unique probes selection can also identify up to d targets in a sample with at most k experimental errors. The decoding complexity of our algorithms is as simple as O(n). The experimental results show that the decoding time is much faster than that of the methods using d-separable matrices while running time and solution size are comparable.We proposed efficient algorithms based on Integer Linear Programming to select a minimum number of non-unique probes using d targets with error tolerance and the decoding complexity is O(n).Since finding unique probes is often not easy, we make use of non-unique probes. Minimizing the number of non-unique probes will result in a smaller DNA microarry design which leads to a smaller chip and considerable reduction of cost. While minimizing the probe set, the decoding ability should not be diminished. Our non-unique probes selection algorithms can identify up to  One of recent important developments in biology is the success of Human Genome Project. This project was done with a great deal of help from computer technology. The technique for obtaining sequenced genome data is getting mature. More and more sequenced genome data are available to scientific research community. Based on those data, the study of pooling designs has become a very important research direction. Doing research in this direction requires high-quality DNA libraries. A DNA library is a collection of cloned DNA segments, usually from a specific organism. Those cloned DNA segments are called clones. Given a DNA library, the problem is to identify each clone whether it contains a probe from a given set of probes. A probe is a piece of DNA, labeled with radioisotope or fluorescence to identify specific DNA sequences by hybridization. A clone is said to be positive if it contains a given probe, and negative, otherwise. A pool is positive if it contains a positive clone, and negative, otherwise. In a pooling design algorithm, a clone may appear in two or more pools. Hybridization is one of techniques to reproduce clones or to do DNA cloning.DNA microarray is a solid surface where sequences from thousands of different genes are immobilized, or attached, at fixed locations. DNA microarray is also a tool for performing large numbers of hybridization experiments in parallel. GeneChip  is one oDNA microarray technology falls into two applications. We can determine the expression levels of gene-specific probes by observing hybridizations of mRNA to different probes on a microarray. This can be used for drug development, drug response, and therapy development. We can also identify the presence or absence of gene sequences by observing appropriate hybridization reactions. Many domains benefit from this application, such as medicine, environmental science, and industrial quality control.A biological sample is a biological object such as blood. Targets are for example viruses or bacteria. In order to identify the target sequences in a biological sample, unique probes are preferred since each unique probe only hybridizes to a specific target. In terms of identification, it has strong separability of target sequences. Thus establishing the presence of targets in a sample by using unique probes is obvious and straightforward. Although probes selection is of great importance in many applications, only a few works have been done on this problem. Other than using random probes, some methods choose probes based on their frequencies in the clones . Severald probes. The slave ILP guarantees the separation between pairs of small target groups. A cutting plane approach has been proposed to handle the master ILP and the slave ILP. Whenever there is a feasible solution to the master ILP, the slave ILP is applied to check for violated group inequalities which are added to the master ILP to solve it again. This process is iterated until no further violated inequalities are found. The ILP algorithm studies a 1-separable matrix with k errors. Therefore, there is no improvement in terms of decoding complexity.In reality, finding unique probes for every target is a difficult task because of the strong similarity of closely related targets, for example, virus subtypes, though temperature and salt concentration are helpful experimental conditions for a probe to uniquely hybridize to its intended target. We need to make use of non-unique probes which hybridize to more than one target in a sample. In terms of non-unique probes, only two works have addressed this problem in literature to the best of our knowledge. A greedy heuristic algorithm has been proposed in  to identposed in  to reducM . ijM=1 if probe ip hybridize to target jt; otherwise, ijM=0. V is a test outcome vector. iV=1 if probe ip hybridizes to at least one target in the sample; otherwise, iV=0. In Figure V is given when two targets t1 and t3 are present in a given sample. A column is called an isolated column iff there is a row containing only one 1-entry at the intersection with that column. In the non-unique probes selection problem, a column (target) jt is isolated if there is a probe ip that only hybridizes to one target jt, that means ip is a unique probe. Since we only consider non-unique probes, we can assume that the given matrix M does not contain any isolated column. Our study focuses on the second step which is called non-unique probes selection. It is concerned about selecting a minimum set of probes while maintaining good decoding ability. Using all probes in M always gives the best separation properties of the design. However, some probes have overlapped decoding ability and several probes might hybridize to the same target set so that keeping all of them in the design is not necessary. The number of selected probes is exactly the number of hybridization experiments needed. By selecting a minimum set of probes, we can reduce the number of hybridization experiments, which results in reducing the experimental cost and effort. And also we can have a smaller design which leads to a smaller chip and significant reduction of cost.The identification problem of determining the presence of targets in a biological sample using non-unique probes can be solved in three steps suggested by Schliep and Torney : 1) PreM ,11. (2)  PreM ,11d-separable matrix iff the unions of any d columns are distinct. The union of d columns is the boolean sum of these d columns. A matrix is defined as a d columns are different. A matrix is a d-disjunct matrix iff the union of anyd columns does not contain any other column. For example, the submatrix H in Figure d-disjunct matrices have a decoding complexity of O(n), which is much lower than that of d-separable matrices, which is O(dn). We choose to construct a d-disjunct matrix H instead of a d-separable matrix considering the computational complexity of decoding. In this study, we propose efficient non-unique probes selection algorithms using d-disjunct matrices to identify the presence of up to d targets in a given sample. We also consider the error tolerance case where there are at most k experimental errors. The experimental results show that the decoding time is much faster than that of the methods using d-separable matrices while running time and solution size are comparable.While minimizing the number of probes, the decoding ability should not be compromised. The following definitions need to be introduced first. A matrix is a In this section, we study the following problem:Problem 1: MIN-d-DS (Minimum d-Disjunct Submatrix): Given m non-unique probe candidates and a m × n probe-target incidence matrix M, select a minimum set of the probe candidates such that the h × n submatrix H is d-disjunct, where h≤n.d-DS is NP-hard [d=1. Recently, Thai and Znati have shown that MIN-d-DS is NP-hard for any fixed d≥1[d-DS problem.In , Du and  NP-hard  when d=1fixed d≥1,15. Due d-disjunct matrix:The following is another definition for a Definition 1: H is a d-disjunct matrix iff for any (d+1) columns, there exist a row such that an entry at one of those (d+1) columns is 1 and entries at the remaining d columns are 0.M does not contain any isolated columns since we only consider non-unique probes. The submatrix H certainly does not contain any isolated columns either. We have the following definition and lemma:We assume that a given matrix Definition 2: H is called -disjunct if for any column jt,jt must have at least k+11-entries not contained in the union of other d columns.Lemma 1: Every (d+k)-disjunct matrix without isolated column is a -disjunct matrix. [ matrix. n-d+1 targets since they do not contribute to d-disjunctness. Then based on Lemma 1, we use ILP1 to construct a -disjunct matrix which facilitate the construction of a d-disjunct matrix later. ILP2 is applied to find violations of d-disjunctness and those violations are addressed one by one. Finally, the resulting submatrix H is d-disjunct.We propose an ILPEF  algorithm which consists of two ILP formulations (ILP1 and ILP2) to deal with Problem 1. According to Definition 1, we eliminate those probes that hybridize to at least ILPEF Algorithm:n-d-1 targets1. eliminate all the probes that hybridize to more than P to construct a -disjunct matrix using ILP12. select the minimum number of probes (ILP2 finds target sets R and S which violate d-disjunctness)3. while p that hybridizes to the single column in S and does not hybridize to any columns in set Rif there is a probe elsereturn no solution;end ifend whileM is an m × n binary matrix with rows representing a set of non-unique probes ip, jt, ip hybridize to target jt; otherwise, ijM=0. ix is a set of binary variables with ix=1 if probe ip is chosen in the submatrix H and 0 otherwise. The details of the ILP1 are as follows.Our formulation of the ILP1 is based on the following definitions. ILP1 Formulation:M has at least d 1-components not contained in any other column.The first constraint simply guarantees that any column in M, we have a submatrix which is a -disjunct matrix. In order to construct a d-disjunct matrix H, we need to find any target sets R and S(S is a singleton set) that violate d-disjunctness and add an appropriate probe to cover R and S until no violation can be found. A probe is said to cover R and S if this probe hybridizes to the single column in S and does not hybridize to any columns in set R. The followings are definitions for the formulation of the ILP 2. Rw is the vector that results from the union of columns in target group R. X is the index set of the currently chosen probes. x* is a solution vector of the ILP 1. We define r and s are variable vectors of target groups R and S. We define jr=1 if jt is in group R and js=1 if jt is in group S. The formulation of the ILP 2 is as follows.After applying the ILP1 on ILP2 Formulation:s.t.S is a singleton set and constraint (5) makes sure that R is a set of d targets. Constraint (6) keeps R and S disjoint and the rest constraints are trivial. The ILP2 is repeated until the minimum value found is at least 1, which means no violation of d-disjunctness is found.The first two constraints defines two conditions that d-disjunctness, an appropriate probe needs to be added to the design. If there is more than one probe that hybridizes to the single column in S and does not hybridize to any columns in set R, p is selected using a greedy heuristic. We pick the one that covers the most number of  pairs. A probe covers a pair  when it hybridize toS but does not hybridize to any target in R. For a probe candidate that hybridizes to k targets, the number of covered pairs is When there is a violation of H is d-disjunct. The decoding algorithm for a d-disjunct matrix is simply based on the following lemma:After applying our ILPEF algorithm, the resulting submatrix Lemma 2: For testing based on a d-disjunct matrix, the number of targets not appearing in any negative results is no more than d. [ than d. hn), where n is the number of targets in M and h is the number of selected probes inH.In the decoding algorithm, we just need to remove all columns that appear in the negative results. The remaining columns are the targets that need to be identified in the sample. The time complexity of this decoding algorithm is O-disjunct matrix [The presence of errors due to the noise of hybridizations complicates the non-unique probes selection problem. Test outcomes may consist of two kinds of experimental errors: false positives and false negatives. We can easily add error tolerance in order to identify at most t matrix  and studProblem 2: MIN--DS  Disjunct Submatrix): Given m non-unique probe candidates and a m × n probe-target incidence matrix M, select a minimum set of the probe candidates such that the h × n submatrix H is  disjunct, where h<n.n-d+1 targets since they do not contribute to d-disjunctness. Then ILP3 is used to select the minimum number of probes P to construct a  disjunct matrix. The ILP2 is then applied to the constructed matrix to find any violations of  disjunctness. The violations will be addressed and the ILP2 is applied again until all the violations have been addressed. The resulting matrix is a  matrix. The detailed algorithm is given as follows.We propose an ILPET , which also consists of two ILP formulations (ILP2 and ILP3) to find a solution for problem 2. Same as in the ILPEF algorithm for problem 1, we eliminate those probes that hybridize to at least ILPET Algorithm:n-d-1 targets1. eliminate all the probes that hybridize to more than P to construct a -disjunct matrix using ILP32. select the minimum number of probes ILP2 finds target sets R and S which violate -disjunctness, i.e., S only contains v (v<2k+1) entries not contained in the union of all the columns in R)3. while  disjunct matrix. Du and Hwang have proven the following lemma:The resulting matrix of the ILPET algorithm is a -disjunct matrix is a k-error-correcting d-disjunct matrix. According to lemma 3, our ILPET algorithm can correct at most k errors. The decoding is made very simple by the following lemma from [Lemma 3  Every  – disjunct matrixH with at most k errors, a target is present iff it appears in at most k negative results.Lemma 4  Suppose O(hn), h is the number of probes in the matrix. For each target, we just count the number of negative results containing it. If this number is less than k, then this target must be present.Based on lemma 4, the decoding can be done with time complexity of Our experimental study is carried out on Sun Fire 280R, with Solaris 8 operating system. The ILP software package we utilize is ILOG INC's CPLEX 8.1 . We use d–1) matrices from the ILP1 are already d-disjunct matrices, which saves the effort to run the ILP2 iteratively. Moreover, our ILPEF approach has much faster decoding time. As shown in Figure We tested our ILPEF algorithm on three randomly generated datasets, with 3000, 6000, 15000 probe candidates and 256, 400, 679 targets respectively. The result is shown in Figure d-disjunct matrix using our ILPEF approach for the simplicity of decoding. Our experimental study shows that our ILPEF approach can greatly reduce the decoding time while maintain comparable running time and solution size compared with previous approaches, which achieves our design goal.In summary, we chose to generate a A minimization problem arises from the study of non-unique probes selection. However, it is misleading to minimizing the number of probes regardless of the decoding ability of the corresponding design. Efficient non-unique probes selection algorithms for DNA microarray design should reduce the number of probes, which results in the reduction of experimental cost and time, while maintain good decoding ability. In addition, the designed algorithms should be error tolerant since errors are very common in biology experiment.d targets in a given sample. Our non-unique probes selection can also identify the presence of at most d targets in a case of at most k experimental errors. In addition, we have also analyzed the running time of how to decode the test results obtained from the microarrays experiments. The time complexity of our decoding algorithms is O(hn) where h is the number of selected probes. Experimental study has been carried out for the ILPEF algorithm and the results have shown that our ILPEF algorithm can greatly reduce the decoding time of existing approaches while maintain comparable running time and solution size.We have presented solutions for two cases, error free and error tolerance. For each case, we have shown efficient algorithms to select a minimum number of probes as well as to decode the results. These algorithms are able to identify at most d-disjunct matrices have a stronger property than that of d d-disjunct matrix should have more rows (probes) than that of d-disjunct matrix which results in a very simple decoding algorithm.Note that Since the number of selected non-unique probes is equal to the number of hybridization experiments, we need to find a minimum number of selected probes in order to reduce the experimental cost and time. Furthermore, the selected probes must result in an efficient decoding algorithm, which is used to infer the test outcomes. Finally, the designed algorithms must be robust in order to handle the experimental errors due to noises.Integer linear programming involves the optimization of a linear objective function, subject to a number of linear equality or inequality constraints. All the variables in ILP can only take integer values. ILP has been widely used for optimization problems.d-disjunct matrix using one ILP. Then we can formulate another ILP to find out violations of d-disjunctness. The violations are addressed and the other ILP is formulated to find out further violations. This goes on until all the violations have been addressed. Then we can obtain a d-disjunct matrix. Similar ideas apply to construct a -disjunct matrix.Our design is to build a matrix that is close to a d and n –d. d is set to be 5, which is the same as in [d targets are randomly picked and the union of those columns is the result vector that can be used to verify the correctness of the decoding process.The datasets used for the experimental study are randomly generated. We generate three different datasets, with 3000, 6000, 15000 probe candidates and 256, 400, 679 targets respectively. The values are picked to facilitate comparison with previous approaches and future comparison with real biological datasets. Each candidate hybridizes to a random number of targets. This random number is set to be between The authors declare that they have no competing interests.All authors contributed to the design and the writing of the manuscript. All authors read and approved the final manuscript.


The in vitro thymidine labelling indices (TLI) of 58 human lung tumours were assessed using autoradiography. The labelling technique involved incubation of 1 mm3 tumour fragments with 3H-thymidine (5 muCi ml-1) under conditions of hyperbaric oxygenation at a pressure of 3 atmospheres. Only a rim of labelling was achieved along the edges of fragments and the depth of this rim varied from tumour to tumour. A technique for counting TLIs was therefore devised to take this into account. In general, those tumours showing low TLI values of less than 5.0% showed a greater depth of labelling. The common malignant tumours of the bronchus showed a wide range of values (2.2-30.4%) though the adenocarcinomata had a lower average value than the other groups. With the squamous carcinomata a relationship with differentiation was shown. The mean value for small cell carcinomata (16.9%)--a highly aggressive tumour--was no higher than for the other groups. The low grade malignant tumours showed TLIs of less than 3.0% and these values correlate with their less aggressive clinical behaviour. Labelling of stromal cells and inflammatory cells varied greatly from tumour to tumour; however, no correlation was found with the TLIs of tumour cells.