Microarray diagnostics of tumour samples is based on measurement of prognostic and/or predictive gene expression profiles. Typically, diagnostic profiles have been developed using bulk tumour samples with a sufficient amount of tumour cells . Consequentially, a diagnostic results depends on the minimal percentage of tumour cells within a sample. Currently, tumour cell percentage is assessed by conventional histopathological review. However, even for experienced pathologists, such scoring remains subjective and time consuming and can lead to ambiguous results.In this study we investigated whether we could use transcriptional activity of a specific set of genes instead of histopathological review to identify samples with sufficient tumour cell content. Genome-wide gene expression measurements were used to develop a transcriptional gene profile that could accurately assess a sample's tumour cell percentage.Supervised analysis across 165 breast tumour samples resulted in the identification of a set of 13 genes which expression correlated with presence of tumour cells. The developed gene profile showed a high performance (AUC 0.92) for identification of samples that are suitable for microarray diagnostics. Validation on 238 additional breast tumour samples indicated a robust performance for correct classification with an overall accuracy of 91 percent and a kappa score of 0.63 (95%CI 0.47–0.73).The developed 13-gene profile provides an objective tool for assessment whether a breast cancer sample contains sufficient tumour cells for microarray diagnostics. It will improve the efficiency and throughput for diagnostic gene expression profiling as it no longer requires histopathological analysis for initial tumour percentage scoring. Such profile will also be very use useful for assessment of tumour cell percentage in biopsies where conventional histopathology is difficult, such as fine needle aspirates. Microarray diagnostics of tumour specimens is based on gene expression measurement of a specific set of predictive or prognostic genes. Bulk tumour samples that consist of tumour cells admixed with surrounding stromal tissue are commonly used for microarray analysis. Although tumour stroma likely plays an important role in tumour development and metastasis -3, gene Currently, tumour cell percentage assessment is assessed using heamatoxilin-eosine (H/E) stained specimen for histopathological review. However, even for experienced pathologists, histopathological tumour scoring remains subjective and time consuming and can lead to inconclusive results -8 and vaHerein we report the development of a molecular profile that can accurately identify breast cancer samples with sufficient tumour cell content for diagnostic purpose. This assessment is based on transcription levels and is able to reduce the time that is needed for a microarray experiment as no initial pathological tumour cell percentage (TCP) scoring will be required before sample processing. Moreover, the identified tumour percentage profile would facilitate microarray diagnostics of small specimens for which tumour sections can not be generated for pathological scoring.Four hundred and three frozen tumour samples or tumour samples preserved in RNALater from breast cancer patients were used. At the time of initial diagnosis, all patients had provided consent in the sense that their tumour samples could be used for investigational purposes. The study was carried out in accordance with the ethical standards of the Helsinki Declaration and was approved by the Medical ethical Board of the participating medical centers and hospitals. All samples were de-identified, analyzed anonymously and were part of research implementation studies for MammaPrint.Histopathological tumour cell percentage assessment was done based on (H/E) coloured tumour sections. Gene expression analysis of breast tumour samples was performed using custom-made Agilent 44K high-density microarrays according to manufacturer's protocols. Ninety-five selected samples were hybridised against the MammaPrint reference pool (MRP)  and inclA 3-fold cross validation (CV) method was used to identify a gene expression profile (nearest-mean classifier) that showed a strong association with pathological TCP. Within each CV loop, two-thirds of the samples were randomly selected as training samples and their TCP was adjusted randomly by either -10, 0 or +10 percent to represent pathological variation, as indicated above. For all genes the association between transcriptional levels and continuous pathological TCP was determined (Pearson correlation) and the top 200 genes (100 with positive association and 100 with negative association) were selected for inclusion in the classifier model. This procedure was repeated five-hundred times (multiple sampling approach) and selected genes were ranked according to their CV performance. To identify the optimal number of genes to be used in the classifier model, the top-ranked gene set was gradually expanded and the classifier performance was determined for each gene set size, using leave-one-out CV and measuring the area under ROC curve (AUC). Next, within the set of 35 top-ranked genes, genes were removed that showed a large variation across an additional set of 70 tumour samples with high (≥50%) TCP. Twenty-two genes that showed a variation in gene expression greater than 0.4 (stdev across 70 samples) were excluded from further analysis. This stringent threshold was used to ensure a stable profile in tumours with a relative high tumour cell percentage. The remaining set of 13 genes . Full genome gene expression measurement of all samples analysed in this study is publicly available at the Gene Expression Omnibus (GEO) with accession number GSE16201.To determine the variation in histopathological tumour cell percentage scoring, H/E stained tumour sections were repeatably analysed in a period of six months by five different pathologists Figure . AlthougUsing 95 breast tumour samples that ranged in tumour cell content from zero to 85 percent, based on analysis of HE slides, a tumour percentage gene expression profile was identified. A 3-fold cross validation (CV) method was used to identify a gene expression profile (nearest-mean classifier) that showed a strong association with pathological TCP. This procedure was repeated five-hundred times (multiple sampling approach) and genes were ranked according to their CV performance. The multiple sampling CV performance indicated a strong association of the selected nearest-mean classifier model with pathological TCP . To identify the optimal/minimal number of genes to be used in the classifier model, the top-ranked gene set was gradually expanded and the classifier performance  was determined for each gene set size.The classifier showed a strong performance (AUC >0.88) when at least ten of the identified TCP associated genes were included in the classifier model Figure . The higAlthough optimal performance was reached with the complete set of 35 genes, a substantial number of genes from the 35-gene set showed a significant single-gene performance . A minimal combination of just two random genes from within the 35-gene set resulted in an AUC of greater than 0.80. This performance increased to >0.85 for random set of least 5 genes (data not shown).In a next step to further optimise the classifier, an additional set of 70 tumour samples was used to select from the 35 gene set those genes that showed stable expression in samples with a high tumour percentage. As expected, genes with a higher expression for high TCP Figure , which wMGAT3, AQP1, TPM3, MYLK, SFRP1, NTRK2: p = 1.7e-3), cell-to-cell signalling , cellular movement and invasion  and cellular survival and apoptosis .Biological function analysis indicated that the 35 TCP associated gene set was enriched for genes associated with cancer related processes such as cellular morphology . The TCP distribution across this validation cohort was representative of typical diagnostic samples. The 13-gene molecular TCP profile correctly assigned 78, 71 and 95 percent of the low, medium and high TCP samples as assessed by pathological scoring Figure . The pro2 = 0.48, Wilcoxon P < 0.001, AUC 0.90) and indicated that the profile might also be useful for indication of accurate tumour cell percentage instead of a low-, medium- or high-TCP classification. Statistical analysis of the profile indexes indicated a significant difference in outcome between samples with a low and medium TCP  and between samples with a low and high TCP (P < 0.0001)  Figure . Althouget al. indicated that formalin fixation of slides can result in tumour cell shrinkage [This study reports the development of a tumour cell percentage (TCP) assessment method for breast cancer samples based on transcriptional analysis of 13 genes. The 13-gene molecular profile has been validated on an independent cohort of 238 samples and can accurately identify breast tumour samples with a sufficient number of tumour cells for microarray diagnostics. Tumour percentage scoring based on the molecular profile is identical to the pathologist scoring for more than 90 percent of all analysed tumour samples. Although the variation in pathologist TCP scoring is in agreement with the number of discrepancies between pathological and molecular profile classification, this inconsistency is likely caused by the difference between the number of tumour cells present in a tumour sample and the actual tumour cell specific mRNA levels. In addition to the known variation and inconsistency in pathological scoring of tumour slides  pathologist. The profile, on the other hand, is able to distinguish samples with low-, medium- or high-tumour cell content. Although the 13-gene profile provides a more qualitative measurement compared to the quantitative pathological assessment, it is a value tool for an objective TCP scoring based on transcriptional levels that can quickly identify samples suitable for diagnostics.Since the gene profile was developed in such a way to mimic a pathological scoring which has been described as inconsistent and subjective, one might argue that the TCP profile also suffers from these factors. The main goal of this study, however, was not to develop a more accurate tool for TCP assessment but an objective method that can be performed independent of pathological expertise and which is based on transcriptional gene levels instead of the number of tumour cells. The use of a robust cross validation procedure that included a mimicked pathological variation was therefore included in the selection model that should, in principle, select a set of genes that are robust to this variation. Nevertheless, the 10 percent misclassification between the gene profile and pathological scoring might partly be attributed to this phenomenon.Future application of the developed TCP profile could significantly improve the throughput of microarray diagnostics. Currently, after sample arrival, RNA processing and expression analysis cannot proceed until a histopathological TCP analysis confirms that the tumour sample contains sufficient tumour cells for analysis. As indicated above, pathological analysis will remain necessary for detection of ductal carcinoma in situ, necrosis and a detailed assessment about the percentage of tumour cells to define the suitability of the specimen. However, replacement of initial pathologist TCP scoring by a faster transcription based analysis that is able to identify whether a sample likely contains sufficient tumour tissue will shorten the processing time for microarray diagnostics. More importantly, the 13-gene molecular profile for tumour cell percentage (TCP) enables gene expression diagnostics for small samples or those on which no H/E evaluation is possible.While gene expression profiles tend to be more robust with inclusion of a larger set of genes , we deciWhether one uses a qPCR assay for specimen selection or uses parallel microarray readout for final approval, the developed 13-gene TCP profile will provide an additional tool for objective assessment of sufficient tumour cell content in breast cancer tissue. It will improve the efficiency and throughput for diagnostic gene expression profiling as it eliminates the need for histopathological analysis in initial tumour percentage scoring. Moreover, it will also allows likely qualify small clinical samples, such as fine-needle aspirates, for transcriptional diagnostics.All authors are employed by Agendia BV. PR and AMG have filed a patent application for the use of the tumour cell percentage profile.PR, ANF and AMG were involved in development of the study design. AS, LJMJD and ATW performed the microarray gene expression analysis. PR performed the data analysis and PR, ANF and AMG were involved in interpretation of the results. PR and AMG participated in writing the manuscript and all authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Some drugs have been evaluated in this system. When drug treatment is combined with antibody treatment much greater protection can be obtained than when the same amounts of drug or antibody are used alone. It is preferable to administer drug before antibody and with the combined schedule it is possible in the test model to protect all mice from tumour growth, even allowing the tumour up to 48 h “get-away” time before starting treatment.Rabbit anti-mouse tumour cell serum can be made tumour specific by absorption with normal mouse cells and in an


To study the impact of these factors, we measured the uptake of fluorochrome-labelled IgG using confocal laser scanning microscopy, interstitial fluid pressure by the ‘wick-in-needle’ technique, vascular structure by stereological analysis, and the content of the extracellular matrix constituents collagen, sulfated glycosaminoglycans and hyaluronan by colourimetric assays. The impact of the microenvironment on these factors was studied using osteosarcomas implanted either subcutaneously or orthotopically around the femur in athymic mice. The uptake of IgG was found to correlate inversely with the interstitial fluid pressure and the tumour volume in orthotopic, but not subcutaneous tumours. No correlation was found between IgG uptake and the level of any of the extracellular matrix constituents. The content of both collagen and glycosaminoglycans depended on the site of tumour growth. The orthotopic tumours had a higher vascular density than the subcutaneous tumours, as the vascular surface and length were 2–3-fold higher. The data indicate that the interstitial fluid pressure is a dominant factor in controlling the uptake of macromolecules in solid tumours; and the site of tumour growth is important for the uptake of macromolecules in small tumours, extracellular matrix content and vascularization.© 2001 Cancer Research Campaign


It has been proposed that the generation of O2 during photodynamic therapy (PDT) may lead to photochemical depletion of ambient tumour oxygen, thus causing acute hypoxia and limiting treatment effectiveness. We have studied the effects of fluence rate on pO2, in the murine RIF tumour during and after PDT using 5 mg kg(-1) Photofrin and fluence rates of 30, 75 or 150 mW cm(-2). Median pO2 before PDT ranged from 2.9 to 5.2 mmHg in three treatment groups. Within the first minute of illumination, median tumour pO2 decreased with all fluence rates to values between 0.7 and 1.1 mmHg. These effects were rapidly and completely reversible if illumination was interrupted. During prolonged illumination (20-50 J cm(-2)) pO2 recovered at the 30 mW cm(-2) fluence rate to a median value of 7.4 mmHg, but remained low at the 150 mW cm(-2) fluence rate (median pO2 1.7 mmHg). Fluence rate effects were not found after PDT, and at both 30 and 150 mW cm(-2) median tumour pO2 fell from control levels to 1.0-1.8 mmHg within 1-3 h after treatment conclusion. PDT with 100 J cm(-2) at 30 mW cm(-2) caused significantly (P = 0.0004) longer median tumour regrowth times than PDT at 150 mW cm(-2), indicating that lower fluence rate can improve PDT response. Vascular perfusion studies uncovered significant fluence rate-dependent differences in the responses of the normal and tumour vasculature. These data establish a direct relationship between tumour pO2, the fluence rate applied during PDT and treatment outcome. The findings are of immediate clinical relevance.


T2*, which is sensitive to tissue (deoxyhaemoglobin). Tumour IAUC and R2* (=1/T2*) decreased post-treatment with ZD6126 in a dose-dependent manner. In the rat model, lower doses of ZD6126 reduced the IAUC close to zero within restricted areas of the tumour, typically in the centre, while the highest dose reduced the IAUC to zero over the majority of the tumour. A decrease in both MRI end points was associated with the induction of massive central tumour necrosis measured histologically, which increased in a dose-dependent manner. Magnetic resonance imaging may be of value in evaluation of the acute clinical effects of ZD6126 in solid tumours. In particular, measurement of IAUC by DCE MRI should provide an unambiguous measure of biological activity of antivascular therapies for clinical trial.ZD6126 is a vascular targeting agent that disrupts the tubulin cytoskeleton of proliferating neo-endothelial cells. This leads to the selective destruction and congestion of tumour blood vessels in experimental tumours, resulting in extensive haemorrhagic necrosis. In this study, the dose-dependent activity of ZD6126 in rat GH3 prolactinomas and murine RIF-1 fibrosarcomas was assessed using two magnetic resonance imaging (MRI) methods. Dynamic contrast-enhanced (DCE) MRI, quantified by an initial area under the time–concentration product curve (IAUC) method, gives values related to tumour perfusion and vascular permeability. Multigradient recalled echo MRI measures the transverse relaxation rate  A functioning vascular network is a prerequisite for tumour growth, providing tumour cells with a nutritive blood supply and facilitating the removal of toxic waste products of metabolism. It is well known that the architecture of tumour blood vessels differs considerably from that of normal tissues . For exaN-acetylcolchinol (NAC), a tubulin binding agent that inhibits tubulin polymerisation and causes microtubule destabilisation.ZD6126 is a novel vascular targeting agent that has been shown to have significant antitumour activity against a broad range of human xenografts in rodent model systems, including early-phase lung cancer metastases  MRI, in which the tumour uptake of exogenously administered Gd-DTPA-BMA (gadolinium diethylenetriamine penta-acetic acid bismethylamide or gadodiamide) is monitored (R2* (=1/T2*), which is dependent on the tissue deoxyhaemoglobin concentration  is one approach that affords such biomarkers of response to antivascular therapies, and drug efficacy has been demonstrated in both preclinical (onitored . In a se−1(rat) or 10 ml kg−1 (mouse) intraperitoneal injection of fentanyl citrate (0.315 mg ml−1) plus fluanisone (10 mg ml−1) , midazolam (5 mg ml−1)  and water (1 : 1 : 2). This anaesthetic mixture has been shown to have a minimal effect on tumour blood flow  was formulated in 20% of 5% sodium carbonate and 80% phosphate-buffered saline yielding a clear solution at ∼pH 7, and was rapidly administered intravenously as a bolus injection via a tail vein. Control animals were treated with vehicle alone. Doses of 12.5, 25 or 50 mg kg−1 ZD6126 for rats and 100 or 200 mg kg−1 ZD6126 for mice were used which, when taking into account the surface law formula, are similar and well-tolerated host dose regimes (ZD6126 (n=6), 12.5 mg kg−1 (n=5), 25 mg kg−1 (n=5) or 50 mg kg−1 (n=5) ZD6126 administered intravenously. The mean tumour volume for this study was 3.49±0.3 cm3 (range 2.54–5.38 cm3). Rats were anaesthetised, a butterfly catheter inserted into a tail vein for contrast agent administration, and placed supine into a 63 mm quadrature birdcage coil. Image sets were acquired on a Varian Unity Inova system interfaced to a 4.7 T horizontal magnet from four contiguous 2 mm slices through the tumour and one through the abdomen to provide a muscle tissue reference, using a spin-echo sequence with repetition time TR=120 ms, echo time TE=10 ms and a 128 × 128 matrix, giving an acquisition time of 15.4 s per image set. Prior to DCE MRI, spin-echo images were acquired from the same slices and with the same field of view and spatial resolution as the time course data to provide baseline T1 data. The images were acquired with TE=120 ms and TR=0.12, 0.5, 2 and 10 s. In some cases, these data were either missing or were too noisy, so mean data from the remainder of the cohort were used. The mean baseline R1 (=1/T1) obtained was 0.53±0.07 s−1 (mean±s.d.). Since the change in R1 in well-vascularised pixels was typically three times this value, it is unlikely that serious errors were introduced by the use of this mean value. Five image sets were acquired prior to and 40 sets after bolus injection of 0.1 mmol kg−1 gadodiamide (Gd-DTPA-BMA) . The total DCE MR imaging time postinjection was 10 min 15 s.The dose–response of rat GH3 prolactinomas to ZD6126 was determined using DCE MRI, performed 24 h prior to and 24 h post-treatment with phosphate-buffered saline  for the first 150 s after administration of gadodiamide was then integrated, and the tumour data normalised to the median muscle IAUC to account for differences in the arterial input function between animals , and running under IDL 5.3/5.4 . For each tumour slice, a region-of-interest (ROI) was drawn over the whole tumour, but excluding the surrounding skin/muscle. A similar ROI was drawn over back muscle in the abdominal slice. The five preinjection images were averaged to increase signal-to-noise ratio and subtracted from the postinjection images to give enhancement images. Tumour and muscle  animals . This meAfter DCE MRI, the tumours were excised, fixed in formal saline, cut in the same plane/orientation as for the MRI, and stained with haematoxylin and eosin. Tissue sections were assessed for necrosis, using a scale from grade 1 (0–10% necrosis) to grade 10 (>90–100% necrosis) (−1) and mice bearing RIF-1 fibrosarcomas  (n=3 per treatment group). The mean tumour volume for the rat GH3 prolactinomas was 3.61±0.41 cm3 (range 2.92–4.3 cm3), and for the murine RIF-1 fibrosarcomas was 0.7±0.13 cm3 (range 0.53–0.91 cm3). 1H MRI was performed 24 h later at 4.7 T using a 3-turn, 25 mm coil for rats or a 2-turn 12 mm coil for mice. The anaesthetised animals were positioned so that the tumour hung vertically into the radiofrequency coil and covered with a warm water blanket to maintain the core temperature at 37°C. Field homogeneity was optimised by shimming on the water signal for each tumour to a linewidth of 30–50 Hz.ZD6126 was administered intravenously to rats bearing GH3 prolactinomas  maps for each slice were generated using all eight gradient-echo image sets by fitting an exponential model on a pixel-by-pixel basis. For each slice, R2* was determined from an ROI encompassing the whole tumour but excluding the surrounding skin/muscle, and the mean R2* for each tumour determined from all five slices  decreased with treatment. R2* was associated with the induction of extensive central necrosis induced by ZD6126, as measured by histology. The results of this study are summarised in Calculated tumour The development and validation of quantitative, clinically applicable MRI end points to assess parameters associated with tumour vasculature is critical for determining the activity of vascular-targeting agents, where access to tumour tissue following therapy is not usually possible. Preclinical models have shown that a single dose of ZD6126 demonstrates significant antitumour activity, causing extensive tumour necrosis, but that this does not necessarily translate into a significant growth delay in human tumour xenografts (−1) reduced the IAUC close to zero within restricted areas of the tumour, typically in the centre of the tumour, while the IAUC remained at pretreatment levels elsewhere. The highest dose (50 mg kg−1) reduced the IAUC to zero over the majority of the tumour. This heterogeneous response contrasts with the response seen with other therapeutic approaches to the tumour vasculature, for example, inhibition of VEGF signalling, where the histogram shifts to the left but the shape remains unchanged (The dose–response of rat GH3 prolactinomas to ZD6126 was measured using DCE MRI and histological assessment of necrosis. Incorporation of a reference muscle tissue permitted an accurate assessment of the tumour IAUC, without direct measurement of the arterial input function. This approach was used to monitor the effects of ZD6126 on the perfusion of GH3 prolactinomas. The proportion of highly enhancing pixels within the tumour was significantly reduced in a dose-dependent manner by ZD6126. These data are consistent with the tumour vasculature being compromised by ZD6126 and subsequent induction of necrosis, which, according to histological analysis, increased in a dose-dependent manner. Lower doses of ZD6126 (12.5–25 mg kgnchanged .R2* in an efficient and accurate manner. Furthermore, it is completely noninvasive, relying on intrinsic susceptibility contrast mechanisms rather than injection of a contrast agent. An increase in R2* may reflect an increase in the tissue content of paramagnetic Fe species, primarily deoxyhaemoglobin within erythrocytes (R2* is consistent with increased tissue water associated with oedema or necrosis. We hypothesised that following treatment with ZD6126, haemoglobin within erythrocytes would deoxygenate, resulting in an increase in R2*. This hypothesis was not supported by the data, which showed a decrease in R2* 24 h after treatment.Activity of ZD6126 against rat GH3 prolactinomas and murine RIF-1 fibrosarcomas was also evaluated by MGRE MRI. Multi-gradient recalled echo MRI allows the quantification of the transverse relaxation rate R2* can be related to tissue vascularity and blood oxygenation by (Tumour A is a constant dependent on blood vessel size and distribution, BV is the fractional blood volume, Hct the haematocrit, [dHb] the blood deoxyhæmoglobin concentration and R2, tissue* the intrinsic contribution of the tissue to the tumour R2*. The decrease in tumour R2* that we measured could be because of one of several factors. ZD6126 induces thrombosis and congestion of erythrocytes in tumour blood vessels 24 h after treatment resulting in extensive central tumour necrosis (dHb], and hence an increase in R2*. However, if there is an agglomeration of erythrocytes into small focal areas (A that more than compensates for the [dHb] increase, decreasing R2*. There could also be vessel collapse prior to necrosis, which would decrease R2* via the term BV. Lastly, any oedema formed, which may be more fluid than viable tumour tissue, would cause a decrease in R2, tissue*. Although further work is needed to elucidate the exact mechanisms responsible, a change in tumour R2* measured by MGRE MRI may be a simple and convenient alternative end point for detecting acute changes induced by antivascular therapies.where Histological assessment of ZD6126-treated tumours showed massive necrosis in the centre of the tumour while the peripheral tissue remained viable. This response pattern has been commonly observed in a wide range of different rodent tumour models with both ZD6126 .R2*, measured by MGRE MRI, are complex and require further validation, but may also provide a useful biomarker of response to antivascular therapies. ZD6126 is effective in attacking the tumour vasculature and is a promising agent for the treatment of cancer.In summary, we have shown a strong inverse correlation of decreased IAUC with increased tumour necrosis following treatment with ZD6126. Measurement of IAUC by DCE MRI should provide an unambiguous primary end point of biological activity of antivascular therapies for clinical trial, and also a marker for dose scheduling and timing for adjuvant therapy. As part of the ongoing phase I study of ZD6126, the same DCE MRI approach is being used to measure the antivascular effects in human tumours, and in which an apparent IAUC dose–response effect has been observed (


Nevertheless, infected NB41A3 cells were lysed efficiently by influenza-specific CTLs. These results demonstrate that all steps of the antigen-processing pathway function properly in the NB tumour cells, and that the limited MHC class I expression suffices for efficient recognition by CTLs. In addition, lysis of the NB tumour cells shows that the cells are susceptible to CTL-induced apoptosis, a pathway that is often impaired in tumour cells. These characteristics make neuroblastoma a suitable target for immunotherapy. The presented assay allows evaluation of various immunological properties of tumour cells and, thus, represents a valuable tool to assess whether a given tumour will be susceptible to immunotherapy or not. Copyright 2000 Cancer Research Campaign. © 2000 Cancer Research CampaignImmunotherapy of tumours by induction of tumour-specific cytotoxic T-lymphocytes (CTLs) will only be effective for tumours with a functional antigen processing and presentation machinery. However, many tumours are known to down-regulate expression of major histocompatibility complex (MHC) class I molecules and/or to impair antigen processing. It is therefore desirable to evaluate the ability of a given tumour to present antigenic epitopes before developing an immunotherapy protocol. In this study we have used influenza virus as a tool to determine the antigen-presenting capacities of the murine neuroblastoma C1300 cell line NB41A3, a frequently used model for human neuroblastoma. Immunofluorescence analyses revealed low and moderate expression of MHC class I molecules D


The haematopoietic system is sensitive to cytotoxic damage and is often the site of dose-limiting toxicity. We previously reported that swainsonine, an inhibitor of protein glycosylation, reduced the bone marrow toxicity resulting from a single dose of anticancer drugs in otherwise healthy mice. However, more important questions are (1) can swainsonine protect tumour-bearing mice without interfering with the anti-tumour effects of the drugs, and (2) can swainsonine stimulate haematopoietic activity of human, as well as murine, bone marrow. We demonstrate here that swainsonine protects C57BL/6 mice bearing melanoma-derived tumours from cyclophosphamide-induced toxicity without interfering with the drug's ability to inhibit tumour growth. Similar results were obtained in vivo with 3́-azido-3́-deoxythymidine (AZT), a myelosuppressive agent often used in therapy for acquired immune deficiency syndrome. Swainsonine increased both total bone marrow cellularity and the number of circulating white blood cells in mice treated with doses of AZT that typically lead to severe myelosuppression. Swainsonine also increased the number of erythroid and myeloid colony forming cells (CFCs) in short-term cultures of murine bone marrow, restoring the number of progenitor cells to the control level in the presence of AZT doses that reduced CFCs by 80%. With respect to the sensitivity of human haematopoietic cells to swainsonine, we show that swainsonine protected human myeloid progenitor cells from AZT toxicity in vitro. These results suggest that swainsonine may be useful as an adjuvant in several types of human chemotherapy. © 1999 Cancer Research Campaign


Zinc phthalocyanine (ZnPc) is a second-generation photosensitiser for the photodynamic therapy (PDT) of cancer. Unsubstituted ZnPc is, however, highly insoluble in most common solvents, and for clinical applications the material needs to be incorporated in liposomes. We report a simple, alternative procedure to formulate ZnPc through non-covalent binding to bovine serum albumin (BSA). Intravenous administration of ZnPc-BSA preparations, at a molar ratio of 11:1 and at a ZnPc dose equivalent to 0.5 mol kg-1, to tumour-bearing mice followed 24 h later by PDT was shown to provide tumour control in two different models, the EMT-6 tumour in Balb/c mice and the human colon T380 carcinoma in nude mice. Analysis of serum fractions from treated animals showed that ZnPc readily redistributes over the serum high-density lipoprotein (HDL) fraction. We also demonstrated the absence of hepatic toxicity of the ZnPc-BSA preparation by monitoring the hepatic cytochrome P450 activity in treated animals and the viability of human cultured hepatocytes.


This case report describes outpatient psychological treatment targeting adherence to fluid restrictions in a hemodialysis patient. The consequences of nonadherence to fluid restrictions in hemodialysis patients range from minor discomfort to increased hospitalizations and mortality rates. In addition, when patients chronically fail to adhere, they may no longer be candidates for kidney transplant. The interventions focused on polydipsia, characterized by excessive fluid intake. The methods involved 11-sessions of individual psychotherapy incorporating strategies including increasing awareness, decreasing motivation, increasing effort, engaging in competing events, conducting thought stopping, breaking repetitive routines, eliciting social support, and receiving reinforcement. Results demonstrated that the patient successfully restricted his fluid intake at or below recommended levels 83% of days after fading of treatment began. This case report demonstrates the success of cognitive behavioral treatment strategies with a nonpsychiatric hemodialysis patient. In the recent years, patients with end-stage renal disease (ESRD) have increased life expectancies due to advances in hemodialysis.Hemodialysis as used in the case of kidney failure involves dialysis of the blood to remove toxic substances or metabolic wastes from the bloodstream. Such procedures serve as a bridge to kidney transplant or a means to manage ESRD in a way that increases both the length and quality of life for patients who are not candidates for transplant.Hemodialysis involves a complex treatment regimen, and to be successful requires a number of self-management tasks, including maintenance of a polypharmacological treatment regimen, careful monitoring and control over dietary and fluid intake, and scheduling, attending, and completing the dialysis sessions themselves . It has been estimated that more than 50% of hemodialysis patients are nonadherent in some manner within a one-month time period . These rP < .001) at 10-week followup, suggesting that treatment improved adherence over time. Similarly, Hegel et al. [n = 4) of male hemodialysis patients. Both interventions were equally successful during the treatment phase, but the effects of behavioral strategies resulted in maintenance of adherent behavior at a two-month followup. Furthermore, rates of adherence did not improve with a combination of cognitive and behavioral interventions. Unfortunately, the data from these studies are not yet sufficient to suggest a standard of practice in improving adherence to fluid restrictions in hemodialysis patients.Several attempts have been made to improve patient adherence to hemodialysis treatment recommendations regarding fluid restrictions. The most rigorous study to date conducted a randomized controlled trial of group cognitive behavioral therapy to improve adherence to fluid restrictions . Althougl et al.  comparedIn the current case report, cognitive behavioral strategies were used to improve fluid adherence in a hemodialysis patient. The main treatment goal for the patient involved decreasing a behavior, namely, fluid intake. The current patient was unwilling to engage in emotionally focused psychotherapy, and he specifically requested behavioral treatment. Therefore, the researchers chose to use interventions consistent with habit reversal strategies. There are many similarities between habit reversal strategies  and typiThe patient was a 60-year-old, married, Caucasian male. He was a highly educated college professor in a health related field. The patient had been diagnosed with type II diabetes, ESRD, and congestive heart failure (CHF). The patient had been dependent on three weekly hemodialysis sessions for four years. He was a candidate for kidney transplant, but his status was deactivated due to chronic nonadherence to fluid restrictions resulting in fluid overload. He presented for cognitive behavioral treatment due to difficulties tolerating the fluid restrictions set forth by his renal specialist.The patient's doctors recommended that he consume less than 48 to 60 ounces of fluid per day. Upon entering cognitive behavioral treatment, the patient reported fluid consumption of approximately 90 to 150 ounces per day, which was 2 to 4 times greater than the recommended amount. Ninety percent of these liquids included caffeinated sodas and water. The other ten percent included foods such as soup and gelatin.The patient reported that he typically consumed fluid excessively during repetitive tasks such as grading papers, lecturing, driving, or watching television. Prior to fluid consumption, he stated that he generally felt an urge to “quench his thirst” due to sensations of a dry throat or mouth. In addition, he also engaged in fluid consumption “out of habit,” as he had arranged his environment in such a way that fluid intake required little response effort .As previously noted, treatment consisted of eight main components to focus on decreasing the patient's excessive consumption of liquid. A detailed description of treatment sessions is available from the first author.Awareness training was introduced during session 1 and adjusted as necessary throughout subsequent sessions. The purpose of awareness training was to assist the patient in becoming attentive to his fluid intake behavior including associated stimuli, environments, and thoughts . InitialIn addition to daily fluid monitoring, the patient completed thought records associated with urges to consume fluid. These thought records emphasized rational emotive behavior therapy approaches , 9. The Finally, a fluid intake schedule was created in an effort to weaken the association between thirst and fluid consumption. The patient and therapist created a daily fluid intake schedule based on the patient's monitoring of fluid consumption throughout his day. This schedule involved having the patient consume predetermined amounts of fluid at scheduled intervals throughout the day, rather than in response to internal  or external  antecedents. The patient reported the schedule to be successful in keeping him alert to his fluid consumption and helped him “stay on track” throughout the day.Decreasing motivation was introduced during session 2. Caffeinated soda composed a large volume of the patient's consumed liquids. The patient was asked to eliminate caffeine from his diet due to the dehydrating effects of caffeine . Once thIncreasing effort  was intrEven with drinking containers removed from leisure areas, the patient continued to go to the refrigerator in his home for drinks often. To assist in decreasing trips to the refrigerator, the patient self initiated “barricading” the refrigerator with chairs and used visualizations such as warning/danger signs.Competing events were introduced during session 2 and adjusted as necessary throughout subsequent sessions. Competing events involve patients engaging in responses that are mutually exclusive from the behavior excess . The patThought stopping was introduced during session 7. Thought stopping is a method used to interrupt undesirable or unproductive thoughts . The theBreaking repetitive routines, or competing response training  and precEliciting social support  was intrReinforcement was introduced during session 2. The patient was asked to reward himself with pleasurable activities at the end of days he was able to successfully keep his fluid intake under the recommended amount . The mosThe patient appeared to be motivated and committed to the cognitive behavioral intervention. He readily engaged in assessment, intervention, and evaluation with the therapist during each session and expressed pride in his improvement over the course of the intervention. Initial self-report indicated that the patient was consuming 90 to 150 ounces of fluid each day. According to his subsequent self-monitoring reports and as illustrated in The current case report illustrates the successful use of cognitive behavioral therapy intervention strategies with a patient diagnosed with end-stage renal disease who presented for treatment due to difficulties tolerating fluid restrictions set forth by his renal specialist. The treatment was implemented successfully in an outpatient setting with 1150-minute sessions. This case study clearly demonstrates the successful application of a combination of well researched interventions , 13, 18 In the previous studies researchers have attempted to identify underlying cognitive and emotional processes to investigate factors associated with nonadherence to medical regimens such as perceived locus of control  and waysThe patient in this case study demonstrated reduction in fluid intake early in treatment. This is in contrast to Sharp et al.  where noThe patient in this case report also responded positively to the incorporation of cognitive and behavioral strategies, which is similar to Hegel et al.'s  finding Another weakness of the current case report is the absence of followup data following termination of treatment. Additional studies are required to determine the most effective treatment strategies in assisting dialysis patients in adherence to fluid restrictions as well as the best strategies to maintain their gains. A final weakness of the current report is the lack of interdialytic weight gain (IWG) measures, which is the accepted manner of evaluating adherence to fluid restrictions. There is an inherent danger in relying solely on patient report of consumed liquid as we did in this case due to the possibility of biased or otherwise erroneous reporting. However daily self-report, as opposed to typical weekly self-report, increases reporting accuracy –26. It wThe current case report highlights the efficacy of combining cognitive and behavioral interventions. After cognitive and behavioral treatment, the patient's eligibility to be considered for a kidney transplant was reactivated, as he successfully demonstrated control over his fluid intake. This case demonstrates the utility and efficacy of applying cognitive behavioral techniques to improve response to medical interventions.


We examined the microvascular effects of competitive nitric oxide synthase (NOS) inhibition with NG-monomethyl-L-arginine (MeArg), followed by L-arginine, on R3230Ac mammary adenocarcinoma perfusion. In window preparations containing tumours, superfusion of 50 microM MeArg reduced diameters of central tumour venules by 13%, of peripheral tumour venules by 17% and of normal venules near tumours by 16% from baseline. MeArg reduced red blood cell (RBC) velocity in central tumour venules by 25%, and increased intermittent flow and stasis frequency by 20% in central tumour venules. Subsequent superfusion of 200 microM L-arginine did not restore diameters or RBC velocity of any tumour preparation venules, and decreased length density in both central tumour venules and peripheral tumour venules. In contrast, MeArg reduced control preparation venule diameter by 30% and RBC velocity by 66%, but did not decrease length density or increase intermittent flow or stasis frequency. Unlike tumour preparation venules, L-arginine restored control venule diameters and velocities. NOS inhibition reduces both tumour and control venule perfusion, but the effect is blunted in the vicinity of tumours, possibly because of increased NOS levels. Perfusion can be subsequently restored in control, but not tumour, venules with L-arginine. Tumour NOS inhibition, followed by normal tissue rescue with L-arginine, may provide a novel means to achieve the goal of selective tumour hypoxia.


A method is presented in this report for concurrent analysis of vascular architecture, blood perfusion and proliferation characteristics in whole-tumour cross-sections of human larynx carcinoma and glioblastoma xenografts. Tumours were implanted subcutaneously in nude mice. After i.v. injection with Hoechst 33342 and bromodeoxyuridine (BrdUrd) as perfusion and proliferation markers, animals were killed. An antiendothelial antibody (9F1) was used to delineate vascular structures. Cross-sections were analysed by a multistep immune staining and a computer-controlled microscope scanning method. Each tumour section was stained and scanned four times . When these images were combined, vasculature, perfusion and proliferation parameters were analysed. The labelling index (LI) was defined as the ratio of the BrdUrd-labelled area to the total nuclear area. The LI based on manual counting and the LI calculated by flow cytometry (FCM) were in good agreement with the LI based on surface analysis. LI decreased at increasing distance from its nearest vessel. In the vicinity of perfused vessels, the LI was 30-70% higher than near non-perfused vessels. This method shows that both vasculature/perfusion and proliferation characteristics can be measured in the same whole-tumour section in a semiautomatic way. This could be applied in clinical practice to identify combined human tumour characteristics that predict for a favourable response to treatment modifications.


The mechanisms of non-specific resistance to syngeneic methylcholanthrene-induced fibrosarcomas of mice were investigated. Results showed that a small tumour graft of 0.05 X 10(5) cells is greatly enhanced in growth when admixed with large numbers of cell fragments, killed cells or viable non-replicating cells. The enhancement of small tumour grafts in cell mixtures was found to be non-specific. Carrageenan, a known anti-macrophage agent, significantly increased tumour growth in normal mice. However, it did not enhance the increased tumour growth of 0.05 X 10(5) cells mixed with 10(6) viable, non-replicating mitomycin C-treated tumour cells. The latter observation indicates that carrageenan and admixed cells interfere with the same tumour-inhibitory mechanism and therefore cannot produce additive effects. The results give support to the concept of a non-specific macrophage "surveillance" system which appears crucial in controlling tumour growth, since it determines the establishment of small numbers of tumour cells while they can still be easily destroyed.


P < 0.05). However, the tumour weight (0.156 ± 0.067 g) in the remaining liver following 60% hepatectomy was significantly lower than that in sham animals (P < 0.005). The number of TdT-mediated dUTP nick-end labelling (TUNEL) positive tumour cells was significantly increased in 60% hepatectomy as compared with the sham and 30% hepatectomy group. The mRNA expression of TGF-β1, TNF-α and Fas in the tumour portion of 60% hepatectomy, was higher than that in 30% hepatectomy group. Plasma levels of TGF-β1 were inversely correlated with intra-hepatic tumour weights. These results suggest that major hepatic resection may lead to an increased induction of apoptosis for the remaining hepatic tumour. © 2000 Cancer Research CampaignLittle is known as to how hepatectomy is associated with the growth of hepatic tumours, which may reside in the remaining liver after curative resection for hepatocellular carcinoma. Using an intra-hepatic tumour implantation model in rats, the effects of hepatectomy on tumour growth in the remaining liver were investigated. On post-operative day 7, the tumour weight in the remaining liver following 30% hepatectomy was 0.321 ± 0.058 g (mean ± SD) which was significantly greater than that (0.245 ± 0.040 g) in sham operations (


Extracts of ovine squamous-cell carcinoma (OSCC) prepared by different procedures, were injected at varying concentrations into 184 tumour-bearing sheep. At a total protein of 0.5 mg and greater, there was significant enhancement and metastasis in all 120 sheep examined. Extracts of OSCC containing less than 0.5 mg protein, or of human squamous-cell carcinoma and normal sheep skin containing high levels of protein, had no effect on subsequent tumour development. Extracts of foetal sheep skin at the 3mg-protein level produced significant enhancement and metastasis. The degree of enhancement was inversely proportional to the developmental stage of the tumour at the time of treatment.


LM985 is one of a series of compounds based on the flavone ring structure and selected for clinical trial primarily for its activity in colon 38 as part of the NCI screen. We have investigated the anti-tumour activity against three differing transplantable adenocarcinomas of the mouse colon (MAC). Single i.p. injection at maximum tolerated dose showed no activity against the ascitic tumour MAC 15A, moderate activity against subcutaneous tumours MAC 13 and MAC 15A and produced a significant growth delay against MAC 26. These responses against s.c. tumours were considerably enhanced by repeated injection 7 days later when greater than 90% tumour inhibition was seen in MAC 13 and cures were achieved in MAC 26. Pharmacokinetic studies confirm the rapid degradation of LM985 to LM975, the possible active principle. Analysis of area under the curve for LM975 indicated a good relationship with administered doses of LM985 and tumour responses. The MAC system shows a good correlation with human large bowel cancer and these preliminary observations with LM985 would suggest that it or its metabolite LM975 may have a value in the management of large bowel cancer.


Lonidamine or 1-[ methyl]-1H-indazole-3-carboxylic acid, studied in a battery of in vitro and in vivo tests currently used for the screening of anti-tumour agents affecting cell division, has been shown to have a narrow spectrum of anti-tumour activity. The significance of this finding is discussed in the light of previous investigations suggesting that lonidamine affects mitochondrial function and not cell replication. Hyperthermia has been shown to sensitize tumour cells to lonidamine. This observation indicates that in combination with hyperthermia lonidamine has some potential for the treatment of cancer; moreover, it suggests that hyperthermia might reproduce a metabolic condition occurring in some stages of the disease. The blood levels corresponding to the anti-tumour action of lonidamine in animals are in the range of those detected in patients treated with the drug.


Several studies have shown that dietary lipid exerts an effect on carcinogenesis. We report here that progression to malignancy in vitro is associated with changes in the response to fatty acids (FAs). Tumorigenic (THKE) cells were more sensitive to the n-3 FAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) than immortalised (IHKE) cells. The growth of THKE cells was inhibited 25% more than the growth of IHKE cells at 80 microM EPA (P < 0.01) and 35% more at 40 microM DHA (P < 0.001). Furthermore, the results indicate that there is a wide cell type variation in the response to FAs. We found that the in vitro inhibition by FAs correlated with the reduction in the growth rate of the tumour in nude mice fed K85 (55% EPA and 30% DHA). A significant difference in tumour latency was observed for the A427 cell tumour groups . Tumours in the animals fed n-3 FA exhibited significantly higher levels of EPA and DHA; the level of arachidonic acid (ARA) was significantly lower in THKE tumours and the level of linoleic acid (LA) was significantly lower in A427 tumours than in controls fed corn oil. The higher sensitivity of the A427 cell line was not explained by higher uptake of EPA/DHA.


Drug resistance is one of the problems severely limiting chemotherapy in cancer patients. Thus, it is very important to develop new drugs that are effective against drug-resistant tumour cells. The novel anti-tumour agent NK109 has been developed from benzo[c]phenanthridine derivatives by Nippon Kayaku . We have confirmed that NK109 shows anti-tumour effects against a number of human tumour cell lines by inhibiting DNA topoisomerase II activity through the stabilization of the cleavable complex. Further, its efficacy against several drug-resistant tumour cell lines was also shown. NK109 showed potent anti-tumour activity against doxorubicin-resistant human tumour cell lines that have a typical multidrug resistance phenotype caused by P-glycoprotein. NK109 was not pumped extracellularly by P-glycoprotein and, consequently, NK109 accumulated in resistant cells. Cisplatin-resistant human tumour cell lines, which demonstrated decreased cisplatin accumulation, were sensitive to NK109. NK109 non-cross-resistance was confirmed using xenografts of tumour cells that were resistant to cisplatin in SCID mice. Furthermore, etoposide-resistant cells, with decreased topoisomerase II activity, were markedly sensitive to NK109 when compared with their parent cells, suggesting the possibility that the cytotoxic mechanism of NK109 differs from that of etoposide. In conclusion, NK109 is a very promising new anti-tumour drug for clinical use, because the efficacy of NK109 is not susceptible to several known molecular alterations that are associated with drug resistance. A clinical study of this compound is now in progress in Japan.


Of the four identified PHD enzymes, PHD2 is considered to be the key oxygen sensor, as knockdown of PHD2 results in elevated HIF protein. Several recent studies have highlighted the importance of PHD2 in tumourigenesis. However, there is conflicting evidence as to the exact role of PHD2 in tumour angiogenesis. The divergence seems to be because of the contribution of stromal-derived PHD2, and in particular the involvement of endothelial cells, vs tumour-derived PHD2. This review summarises our current understanding of PHD2 and tumour angiogenesis, focusing on the influences of PHD2 on vascular normalisation and neovascularisation.Originally identified as the enzymes responsible for catalysing the oxidation of specific, conserved proline residues within hypoxia-inducible factor-1 The rapid growth of tumours results in their surpassing the capacity of the established circulatory system to supply the developing tumour with oxygen and nutrients as well as remove waste products. For a tumour to expand, it must respond to these assaults. Thus, a necessary step in the progression of any solid tumour is adaptation to low oxygen or hypoxic conditions. Tumour cells can respond to hypoxia by increasing oxygen delivery or acclimating to decreased oxygen availability. The hypoxia-inducible factor (HIF) family of transcription factors mediates both the systemic and cellular response to hypoxia.β-subunit  and an oxygen-sensitive α-subunit. The α-subunit is hydroxylated on two conserved proline residues by a family of prolyl hydroxylases  E3 ubiquitin ligase to interact, adding ubiquitin ladders and targeting the α-subunit to the proteasome for degradation (α and Asp 831 of HIF-2α) prevents the interaction of HIF with p300, a transcriptional coactivator  that modify HIF to be recognised by VHL. The prolyl hydroxylases require oxygen and 2-oxyglutarate as substrates and Fe(II) and ascorbate as cofactors, resulting in oxidation of two highly conserved proline residues . RecentlPHD2 through RNA interference increased HIF-1α levels under normoxic conditions. This effect was not observed with either PHD1 or PHD3. Furthermore, several groups independently generated genetic knockout mice of the different Phds  and IL-8, two known soluble pro-angiogenic factors. Conditioned media from HCT116 cells that had PHD2 knocked down had elevated protein levels of ANG and IL-8. Notably, the levels of VEGF were unchanged by PHD2 levels. Silencing either ANG or IL-8 impaired both angiogenesis and tumourigenesis. These results suggested that PHD2 normally functions to inhibit angiogenesis and that silencing PHD2 promotes angiogenesis.We then investigated whether the PHD2 knockdown cells were secreting factors capable of influencing angiogenesis. Using a standard de novo production of new blood vessels. Angiogenesis is the formation of new blood vessels from pre-existing vessels. This local angiogenesis can also send signals to the bone marrow, which can in turn release precursor or progenitor cells to induce neovascularisation. This process of blood vessel formation by de novo production of endothelial cells, or vasculogenesis, requires tumour cells to interact with stromal cells and circulating bone marrow-derived cells (BMDCs). We then investigated whether PHD2 knockdown affected recruitment of BMDCs to the growing tumour vasculature. To determine this we stained for two BMDC markers, CD11b and CD45. CD11b is a marker of myeloid–monocytic precursors, whereas CD45 is a myeloid marker. PHD2 silencing increased mobilisation of BMDCs to the growing tumour, whereas silencing of ANG and IL8 impaired this mobilisation. Thus, PHD2 functions to regulate both angiogenesis and vasculogenesis through ANG and IL-8.Tumour vasculature is actually regulated by two complementary processes: angiogenesis, which is local sprouting, and vasculogenesis, which is the β, an inhibitor of NF-κB, which suggested that PHD2 might also regulate NF-κB. In PHD2-silenced cells, NF-κB activity was elevated and mutation of the NF-κB sites in the promoters of ANG and IL-8 impaired NF-κB activation. Furthermore, IL-8 is a well-characterised NF-κB target and NF-κB bound to the promoter of ANG, as determined by chromatin immunoprecipitation, showing that the influence of PHD2 on the tumour vasculature is mediated through NF-κB activity on ANG and IL-8, two known pro-angiogenic factors. Transient silencing of p65, an essential subunit of the NF-κB complex, impaired in vitro angiogenesis. Using breast cancer data sets, there was a strong, inverse correlation between PHD2 mRNA levels and NF-κB activity profiles as well as negative relationship between PHD2 and CD31 mRNA levels, as determined by microarray analysis. Furthermore, NF-κB activity profiles positively correlated with an increase in CD31 mRNA. Taken together, these data illustrate that PHD2 can regulate the tumour vasculature through an HIF-independent mechanism but relies in part on inhibition of NF-κB and the downstream targets of ANG and IL-8.The PHD2 regulation of tumour vasculature through ANG and IL-8 is independent of HIF. Phd2, they saw no difference in ectopic, primary tumour growth. In an orthotopic, pancreatic model, tumours grown in wild-type mice were more invasive and had more metastatic disease compared with Phd2+/− mice. This phenotype was attributed to higher intravasation of tumour cells into blood vessels of wild-type vs Phd2 heterozygous animals. The Phd2 heterozygous animals had higher expression of VE-cadherin and less hypoxia based on pimonodozole staining, oxymetry, and HIF protein levels. Tumours implanted into Phd2+/− mice also had less necrosis and lower metabolic rates. The same researchers showed that vessels of tumours implanted in heterozygous mice were normalised compared with those of wild-type mice. The vessel normalisation of tumours in Phd2 heterozygous mice resulted in less intravasation of tumour cells and consequently less metastasis.In a parallel study, Phd2 and wild-type mice. However, despite equal numbers of tumour vessels, the tumours of the Phd2+/− mice had smoother, more regular, and more mature blood vessels that were not as malformed or as leaky as tumours implanted in wild-type mice. These researchers suggested that the tumour vessels formed in the Phd2 heterozygous mice were ‘normalised,’ comparable to normal blood vessels. Examining the angiogenic profile of Phd2 heterozygous endothelial cells as well as tumour endothelial cells from tumours implanted into Phd2+/− mice, they found higher soluble Flt1 and VE-cadherin expression at the mRNA and protein levels, respectively. Soluble Flt1 and VE-cadherin are two angiogenic factors implicated in vessel normalisation and more specifically, cell motility and cytoskeletal reorganisation. Interestingly, in response to VEGF, endothelial cells heterozygous for Phd2 proliferated slower, had decreased motility, less lamellipodia formation, and less apoptosis. The effects of sFlt1 and VE-cadherin were dependent on HIF2 and not HIF1, as transient silencing of only HIF2 decreased levels of both angiogenic factors. Taken together, In contrast to our findings, this group (Phd2 mice. Our study examined the role of PHD2 in a tumour to contribute to its growth through angiogenesis and recruitment of bone marrow-derived cells (The simplest explanation for these apparent discrepancies between our study and that of ed cells . The comed cells . Simulta


TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.In advanced breast cancers,  Breast cancers are a heterogeneous group of tumours. Whereas most advanced breast cancer patients receive neoadjuvant chemotherapy, less than 20% of them will fully benefit from this type of treatment and reach complete pathological response, which is closely linked with longer survival  more than 15% of the yeast colonies were red and (ii) analysis using the split versions of the test could identify the defect in the 5′ or 3′ part of the gene, confirming the initial determination and (iii) sequence analysis from mutant yeast colonies could identify an unambiguous genetic defect .After tumour RNA extraction , TP53 stTP53 status with the yeast functional assay in all 29 tumours before and after chemotherapy, whenever sufficient frozen material was available for RNA extraction (18 tumours).We determined the TP53 mutation before treatment, frozen tumour tissue was available after treatment in six cases and used to perform the yeast functional test as well as TP53 gene sequencing. For the other three mutated cases, we tried to use formalin-fixed paraffin-embedded tissue, but failed to obtain good enough RNA to perform the yeast functional assay or long enough DNA fragments (>300 bp) to perform gene sequencing.For the nine cases with For all nine mutated cases, TP53 immunostainings on paraffin-embedded samples were obtained before and after treatment using DO-7 monoclonal mouse anti-human antibody , diluted 1/50 in an automated immunostainer . The percentage of stained cells (nuclear staining) was noted.μm thick paraffin sections were spread on membrane-coated slides and stained with H&E (μl of buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5% Tween 20, 0.2 mg ml−1 proteinase K). After an incubation of 24–48 h at 56°C, proteinase K was inactivated at 95°C for 10 min. No further DNA extraction was performed before PCR analysis for these microdissected samples , EDTA 1 mM)  were also analysed with DNA extracted from frozen whole tumour tissue. Briefly, frozen tumour sections were immersed in a buffer containing 8 M MgCl2, 0.2 mM dNTP, 0.2 μM labelled forward primers  or VIC™ for tumour) and 0.2 μM non-labelled reverse primers. The PCR final volume was 20 μl. Thirty-five cycles of PCR were performed.For microdissected cells, a volume of lysis buffer accounting for 500 cells was added in each PCR vial. For DNA extracted from blood cells, 10 ng DNA was used for each PCR. The following 10 microsatellite dinucleotide repeats were used : D3S1573After denaturation, the PCR products were run on ABI PRISM 310 Genetic Analyzer. The analysis of the migration data were performed with Genescan 3.1 software (Applied Biosystems).Fluorescent allelic ratios obtained from microdissected tumour tissue were compared with fluorescent allelic ratios obtained from control DNA, allowing the measurement of allelic imbalances (AI). Loss of heterozygosity (LOH) was defined as AI greater than 50% for one tumour allele.AI observed after chemotherapy were then compared to AI observed before chemotherapy, showing locus with unchanged AI, decreased AI or increased AI. Decreased or increased AI were considered significant only when allelic peak heights differed by at least 50%. This threshold was chosen to minimize the risk of false-positive results induced by PCR variations. All these significant pre-post-treatment differences were verified with a second round of PCR performed with microdissected tissue and a third round of PCR performed with frozen whole tumour DNA.Among a total of 290 ‘before chemotherapy’ PCR (10 markers performed in 29 tumours), 63 allelic profiles were homozygote (non-informative) and 15 PCR could not be analysed due to technical issues. The rates of informativity for these markers were similar to those expected from databases  and 3. TAmong a total of 290 ‘after chemotherapy’ PCR , 63 allelic profiles were homozygote (non-informative) and 24 PCR could not be analysed due to technical issues. The 203 informative profiles revealed 78 (38%) LOH and 125 (62%) retentions of heterozygosity.  and 3* cells).Altogether, 191 pairs of PCR were informative and analysable before and after chemotherapy. In 180 pairs (94%), allelic profiles before and after chemotherapy were not different  or after treatment .The overall rates of LOH at all loci were similar in tumours with or without TP53 intragenic IGP53 locus before treatment was observed in 4 out of these 9 tumours with TP53 mutation (44%) and in 5 out of 20 tumours without TP53 mutation (20%). Eighteen tumours had sufficient post-treatment frozen material to allow analysis with the yeast functional assay after chemotherapy. Among them, all 12 tumours that did not bear TP53 mutation before treatment did not show post-treatment TP53 mutation.LOH at the TP53 mutation before treatment had good quality post-treatment tissue available and have been further analysed for post-treatment TP53 status  or a single small (2 mm) residual focus of tumour cells (patient 8).Six out of nine tumours with 3 status . Among tTP53 status of the tumours.The distribution of the 11 post-chemotherapy profile changes was not linked to any other clinicopathological parameter  and was not linked to the pre-treatment We found that genetic changes are detectable early after chemotherapy in breast cancers.All 29 patients had poor prognosis breast tumours treated with the same dose–dense epirubicin/cyclophosphamide regimen. The large amount of tumour tissue obtained before  and after (mastectomy) treatment allowed reliable assessment of response to treatment. Obtaining such homogeneous pre- and post-chemotherapy tissue samples is difficult and explains why we could study only 29 patients.Advanced breast cancers are often high grade with necrotic areas, and become more heterogeneous after treatment, with inflammatory infiltrate, oedema, fibrosis and necrosis. To overcome tissue heterogeneity, we used laser tissue microdissection, which allows a targeted sampling of tumour cells with very little contamination. In this regard, 20/29 tumours in our study had at least one allelic loss greater than 80%, indicating that tumour tissue samples were made of almost pure tumour cells. Although laser-microdissection is a highly efficient sampling method, it yields limited amounts of tissue material, and we took special care of sampling at least 5000 cells for each tumour. Indeed, allelic profiling must take into account allelic dropout artefacts that are artificial allelic imbalances occurring when DNA concentration is too low , the TP53 mutation found before chemotherapy was not found after  that showed a low post-treatment stromal cell density as well as an important decrease of the proportion of cells with TP53 LOH  is clearly in favour of clonal selection. Yet, whatever the mechanism, these treatment-induced events should favour the emergence of therapy-resistant tumour subclones and subsequent tumour recurrences.In this study, we demonstrate that genetic changes do occur early after chemotherapy in breast carcinomas. The most likely explanation for our observations is chemotherapy-driven selection of tumour subclones: breast cancer tumour cells are well known to be highly heterogeneous (


The purpose of this study was to test whether pretreatment with imatinib increases the efficacy of traditional chemotherapy in mice bearing non-small cell lung carcinoma xenografts, and to investigate the effects of imatinib on liposomal drug delivery. Efficacy treatment groups included (n=9–10): saline control, imatinib alone , docetaxel alone (10 mg kg−1 i.p. 2 × /week until killing), and imatinib plus docetaxel (started on day 7 of imatinib). Tumours were monitored until they reached four times the initial treatment volume (4 × V) or 28 days. A separate experiment compared tumour doxorubicin concentrations (using high performance liquid chromatography) 24 h after treatment with liposomal doxorubicin alone  or imatinib plus liposomal doxorubicin (n=16). Imatinib plus docetaxel resulted in significantly improved antitumour efficacy  when compared to docetaxel alone  or imatinib alone . Pretreatment with imatinib also significantly increased tumour concentrations of liposomal doxorubicin. Overall, these preclinical studies emphasise the potential of imatinib as an adjunct to small molecule or liposomal chemotherapy.Imatinib, an inhibitor of PDGF-R Non-small cell lung carcinoma (NSCLC) has a limited response rate to current chemotherapeutics, with tumour shrinkage in only 20% of patients (by the RECIST criteria) and 2-year survival rates of 10–16% . This chSystemic chemotherapeutics encounter a number of obstacles that limit transport to solid tumours . Vesselsβ) and decompression of vessels by induction of tumour cell death  significantly decreases IFP and microvascular density, lowers VEGF levels, and improves tumour oxygen delivery  is a synthetic small molecule that inhibits phosphorylation of a number of tyrosine kinase receptors including PDGFR, c-kit, and bcr-abl . In coloImatinib  was obtained from the hospital pharmacy in a 100-mg tablet form. Tablets were crushed and re-suspended in 0.9% NaCl. Liposomal doxorubicin (Doxil) was obtained from Ortho Biotech Products , and docetaxel (Taxotere) was obtained from Sanofi Aventis (Bridgewater). A human NSCLC line, A549, was purchased from American Type Culture Collection .ad libitum. Human NSCLC cells (A549) were grown in RPMI 1640 media supplemented with 5% bovine calf serum. The right hind limb of each mouse was inoculated subcutaneously with ∼5.0 × 105 A549 cells, obtained from exponentially growing cell cultures, suspended in 200 μl of HEPES-buffered saline. Xenograft tumour growth was monitored twice per week until tumours reached an average volume of 100 mm3. Xenograft volume (V) was determined by the following equation: V=(L × W2) × 0.5, where L is the length and W is the width of the flank tumour.Homozygous adult female athymic nude mice were housed in isolated caging with sterile rodent food and water 3), tumour-bearing animals were used in three separate studies. The first study compared vascular maturity (by immunohistochemistry) after treatment with imatinib (n=4) vs control . Imatinib was administered via gavage once a day at a dose of 100 mg kg−1 for 7 days, and saline was administered following the same schedule. Animals were killed 24 h after the last imatinib injection, and tumours were harvested and snap frozen at −80°C.After xenografts reached treatment volume (100 mmn=9–10 mice per group). Docetaxel was administered intraperitoneally at a dose of 10 mg kg−1 twice per week until killing, and saline was administered following the same schedule. Injections of docetaxel (docetaxel±imatinib groups) or intraperitoneal saline  were initiated on day 7 of imatinib or saline pretreatment. After therapy, animals were monitored as they recovered from anaesthesia. Tumour volumes were measured three times per week for 28 days or until the tumour volume reached four times the volume on the first day of treatment.The second study compared tumour growth times after treatment with control , imatinib alone , docetaxel alone , or imatinib plus docetaxel (n=9) vs imatinib plus liposomal doxorubicin . Liposomal doxorubicin was delivered intravenously as a single dose (6 mg kg−1). When combined with imatinib, liposomal doxorubicin was delivered on day 7 of imatinib treatment. Animals were killed 1 day (24 h) after liposomal doxorubicin injection, and tumours were harvested and snap frozen at −80°C. All animal protocols were approved by the Duke University Animal Care and Use Committee. Experiments were performed in accordance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education , as well as the UKCCCR guidelines . CD31 was used to stain all endothelial cells, whereas CD105 was used to indicate immature endothelial cells. Smooth muscle α-actin was used to stain pericytes covering microvessels. Frozen tumours harvested from control and imatinib alone groups were cut into 10–12 μm sections using a LEICA CM 1850 cryotome . Consecutive tissue sections were cut at the largest circumference of the tumour and placed onto poly-L-lysine-coated slides . Cryosections were then split into two sets for staining. All antibodies were diluted in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin and 0.1% Tween-20 (PBS-BT).Vascular markers for this study included the endothelial cell markers CD31 and CD105, as well as smooth muscle alpha-actin  and α-SMA . After rinsing again with PBS, primary antibodies were respectively revealed with secondary antibodies coupled to TRITC  or FITC  .In the first set, sections were stained for CD31 and In the second set, sections were stained for CD31 and CD105. Sections were fixed for 1 h in 4% paraformaldehyde in PBS (pH 7.2). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 min, and tumour sections were then blocked with 10% normal donkey serum for 15 min and rinsed three times for 2 min in PBS. Consecutive sections were incubated overnight at 4°C with rat monoclonal antibody to CD31 diluted 1 : 200 or CD105 diluted 1 : 400 . After rinsing again with PBS, primary antibodies were enhanced with biotinylated donkey anti-rat secondary antibody (diluted 1 : 100) for 30 min at room temperature (Jackson Immunoresearch). Finally, an avidin-biotin complex was applied . Markers were visualised with 3,3′-diaminobenzidine tetrahydrochloride .vs α-SMA), digitised images were acquired with both TRITC and FITC filters, and images were overlaid using the Metamorph software. Microvessel density was determined by counting CD31-positive structures in five random fields per tumour, and α-SMA-positive vessels were counted from among these structures.Tumour sections were quantitatively analysed using a semiautomatic computerised digital image analysis system . A high-resolution intensified solid-state camera was mounted on a fluorescence microscope  with a computer-controlled motorised stepping stage. For the first set (CD31 vs CD105), quantification of vessels was performed using the Image J ‘Analyze Particles’ function by a blind observer without any manipulation in brightness or contrast. The threshold intensity was manually set above the background staining intensity, and the Image J function selected for stained vessels with a minimum size of 10 pixels per vessel. The average number of CD31-stained vessels per region of interest  was calculated as the microvessel density. The average number of CD105-stained vessels in the same regions was calculated from the consecutive section.For the second set  with fluorimetry  was analysed using the Cox proportional hazards model to compare groups. Censoring was taken into account for animals that did not reach four times the original treatment volume by the time they were killed (at 28 days). P-values less than 0.05 were considered to be statistically significant.Descriptive summary statistics are presented as mean±s.e. of the mean, except for the time-to-four-times-tumour-volume data, which are expressed as median values with 95% confidence intervals. Statistical analyses of doxorubicin concentration and histologic data were performed using the two-sample Student's β and decrease IFP in solid tumours, it has the potential to ‘normalise’ the tumour microenvironment and thereby improve delivery and efficacy of chemotherapeutic agents. With this rationale, we performed three in vivo translational studies on the effects of imatinib in NSCLC xenografts. We first used histology to test the vascular effects of imatinib, specifically assessing microvessel density (CD31 positivity), pericyte coverage (α-SMA positivity), and endothelial cell immaturity (CD105 positivity). Then, we followed up on recently published data that demonstrate that pretreatment with imatinib increases delivery of small molecules to tumours (β-mediated effects of imatinib in NSCLC.Since imatinib is known to inhibit PDGF-Rα-SMA (green) are shown in P=0.01, α-SMA-stained microvessels, representing mature pericyte-covered vessels, also decreased significantly, from 14.1±1.2 to 7.1±1.3 (P=0.02). The percentage of total vessels that were positive for α-SMA lowered from 28 to 23% (Representative images of NSCLC microvessels stained for CD31 (red) and 8 to 23% , indicatP=0.05). The density of immature CD105-stained endothelial cells in the tumour decreased from 19.5±2.5 to 8.7±2.0 after treatment with imatinib (P=0.01), and the ratio of CD105- to CD31-stained  vessels was used as an indicator of vascular immaturity. This ratio was 0.95±0.09 in the control tumour and 0.85±0.21 after imatinib therapy  by 28 days after therapy. In comparison, three out of nine animals receiving docetaxel alone reached 4 × V by 28 days, showing a significantly higher likelihood ratio for reaching 4 × V (P=0.014 vs combination). Almost all (9 out of 10) animals receiving imatinib alone reached this end point, with a median tumour growth time of 22.5±3.0 days (95% CI). Again, imatinib alone showed a significantly higher likelihood ratio for reaching 4 × V (P=0.025 vs combination).−1 liposomal doxorubicin (without imatinib) resulted in a mean tumour doxorubicin concentration of 530.4±53.5 ng doxorubicin per mg protein at 24 h after injection (n=9). Pretreatment with 7 days of 100 mg kg−1 imatinib gavage significantly increased the tumour doxorubicin concentration to 779.2±68.8 ng doxorubicin per mg protein .The assessment of imatinib plus liposomal doxorubicin showed that prior treatment with imatinib improves delivery of liposomal doxorubicin to NSCLC tumours . A singl51Cr-EDTA, and lowering VEGF and microvessel density  drug delivery by reduction of IFP . Macromoβ; these changes in the microenvironment, including decreased IFP and modified vasculature, result in improved drug delivery and efficacy in the preclinical setting. Currently, this hypothesis is being further tested in clinical trials in which patients with metastatic NSCLC are treated with standard cisplatin and docetaxel plus concurrent imatinib. All enrolled patients must be PDGFR-β positive by immunohistochemistry, and treatment response is monitored by the RECIST criteria. Patients also participate in a lead-in portion of the study in which dynamic contrast-enhanced (DCE) MRI is performed before and after a 7-day course of imatinib alone, to explore DCE-MRI as a biomarker for changes in the tumour microenvironment. Preliminary data have revealed that DCE-MRI is feasible and demonstrates a decrease in tumour leakage space  after treatment with imatinib. This suggests a decrease in IFP that may imply improvement in tumour drug delivery. However, a major challenge to clinical research in drug delivery is the lack of non-invasive drug imaging methods (Overall, the observed effects of imatinib in NSCLC are consistent with the inhibition of PDGFR-


It is clear therefore that hsps are overexpressed in patients with malignant tumours compared with healthy controls and this overexpression does show some correlation with disease features. Furthermore, expression of hsps has been reported on the cell surface of tumour cell lines. This could be associated with the immune response which has been reported with hsp90 and which also correlates with some disease features. It now appears that hsps may be involved in the presentation of tumour antigens leading to the possibility of hsps being used as a means of therapy. Hsp65 expression has not been investigated in patients with breast cancer. However, transfection of bacterial hsp65 into a tumour cell line resulted in the hsp65-expressing tumour cells losing their tumorigenicity in mice . Thus, hsps and the immune response to them are of interest as diagnostic and prognostic tools as well as a novel form of immunotherapy.


Several radiotherapeutic schedules compatible with continued structural-functional integrity of the gastrointestinal (GI) mucosa were compared utilizing the P815X2 murine mastocytoma grown as a solid subcutaneous tumour. Both the tumour and underlying normal tissues were irradiated during the treatments. The tumour exhibited a Do that increased from 210 rad to 397 rad as the tumour aged and in all instances demonstrated minimal shoulders in survival curves. In spite of a relative radioresistance of cells within the solid tumour, quite effective control of localized disease could be accomplished with radiotherapy schemes compatible with GI tolerance limits. Schedules evaluated utilizing this model included acute exposures to 1122 rad, daily exposure to 187 rad, 5 days/week exposures to 281 rad, twice weekly exposures (561 rad on Mondays and 374 rad on Thursdays) and a high dose, two fractions per day, schedule. Tumours were followed for changes in growth patterns during these schedules. Efficacy of tumour control was determined and schedules were compared on this basis. Aggressive radiotherapy approaching the tolerance limits of any of the fractionation schemes proved most effective.


Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.