P=0.08). IBTRs did not display features of higher aggressiveness than PTs. Neither clinical nor histological definition of a true recurrence could be established other than the conservation of the histological type.The characteristics of ipsilateral breast tumour recurrences (IBTRs) relative to those of their primary tumours (PTs) remain scarcely studied. Of 70 young (⩽40 years) premenopausal women with IBTRs, we studied a series of 63 with paired histological data. Median follow-up since IBTR was 10 years. Rates of histological types, grades or hormonal receptors were not significantly different in PTs and in IBTRs. The concordance between IBTRs and their PTs was good for histological types. IBTRs with conserved histological types tended to occur more locally, but not significantly sooner than others. These IBTRs had good concordance for hormone receptors. In discordant cases there were as many losses as appearances of the receptors. The concordance was weak for grades, with equivalent numbers of IBTRs graded lower as higher than their PTs. The 10-year overall survival rate was 70%. Neither the conservation of histological type, location, nor of the two combined were associated with deaths. Early (<2 years) IBTRs, tended to be associated with poorer survival (HR=2.24 (0.92–5.41);  The most important prognostic factor for local recurrence is the young age of the patient , as low when <11, moderate when (11–22) and high when >22 . Histological classification of the infiltrating carcinomas was done according to the World Health Organization criteria and histological grades were scored according to in situ in four (6%), involved with invasive carcinoma in five (8%). The reasons for the absence of re-excision were not always specified. When they were, it was because of the patient's choice not to undergo a new surgical procedure that could have been a mastectomy.Treatments of the primary tumour consisted, for all patients, in surgery with breast-conserving procedures as the first treatment. The quality of the surgical margin was stated as wide (>3 mm) in 40 patients (63%), close (⩽3 mm) in 14 (22%), involved with ductal carcinoma All patients received post-operative radiotherapy with a median dose of 54 Gy (45–62) to the breast. A boost to the tumorectomy bed was performed for 68% (36 patients) of the women with a median dose of 16 Gy (2–25). The median total dose to the tumorectomy bed was 62 Gy (52–76). There was no protocol to boost all young patients with negative surgical margins at that time and some of the patients reported in this series were accrued in the EORTC boost trial that randomised from 1989 to 1996 between boost and no boost (Systemic treatments were given in 30% (63 patients) of the women. At the time of treatment, the protocol for premenopausal women consisted of anthracycline-based polychemotherapy  without hormone therapy.2 paired analysis that tests the hypothesis that, among discordant cases, the evolution was more often of one kind than another. For instance, when applied to oestradiol receptors, there was a significant difference between the numbers of cases with a loss of ER and those with an appearance of ER (ER− in PT and ER+ in IBTR). When the analysed pathological feature had more than two classes, analysis was downsized to a 2 × 2 table.Comparisons of pathological characteristics between primary tumours and ipsilateral breast tumour recurrences were performed by McNemar χ2-test, while differences in quantitative measurements were tested by either the Student's t-test or Kruskal–Wallis test (depending on the distribution characteristics). Median follow-up was calculated using the reverse Kaplan–Meier estimator  or to the site of the local recurrence with respect to the primary tumour (same quadrant or different quadrant).Median time-lapse between the primary tumour and its ipsilateral breast tumour recurrence was 43 months (3–158). Median follow-up since ipsilateral breast tumour recurrence was 10 years (1–18).Histological details of both the primary tumour and its ipsilateral breast tumour recurrence are given in vs 53 months; P=0.8). The rate of ipsilateral breast tumour recurrences occurring within the first 2 years was not significantly higher for those that had recurred at the index quadrant than for the others .Ipsilateral breast tumour recurrences occurred in the index quadrant, the one where the primary tumour had arisen, in 70% (44 patients), in a different quadrant in 27% (17 patients) and in 3% of cases (two patients) the relative locations were unknown. The mean time-lapse before occurrence of the ipsilateral breast tumour recurrence was not significantly different in the case of a recurrence at the index quadrant or not  but not statistically sooner .As represented in vs 54 months; P=0.34).In 45 cases (71%) ipsilateral breast tumour recurrences shared both the same histological type and the same location (index quadrant) with their primary tumours. These ipsilateral breast tumour recurrences did not occur significantly sooner than the others  nor sooner  than others.In 33 cases out of 61 cases for which data were available (52%), the ipsilateral breast tumour recurrences had occurred not only with a conserved histological type, but also with features of equal or lesser differentiation  than their primary tumours. These cases did not occur significantly neither more locally .Of the 54 pairs for which both the primary tumours and the ipsilateral breast tumour recurrences were histologically graded, the grades remained the same in 21 patients (39%), weak concordance (κ 0.03). There was no statistically significant difference between patients for whom the IBTR was graded higher and those for whom it was graded lower than the primary tumour  and 70% (59–84).P=0.75), nor the conservation of location with ipsilateral breast tumour recurrence occurring at the index quadrant (hazard ratio=1.13 (0.43–2.95); P=0.8), nor the two combined, that is conservation of both histological type and location versus the others (hazard ratio=1.07 (0.43–2.7); P=0.88) nor those with conserved histological types and equal or lesser differentiation (hazard ratio=2.08 (0.85–5.2); P=0.11) were statistically associated with deaths. Early ipsilateral breast tumour recurrences, defined as occurring within the first 2 years, tended to be associated with a higher rate of death (hazard ratio=2.24 (0.92–5.41); P=0.08) than late recurrences. When comparing both the tumours and their ipsilateral breast tumour recurrences according to the free interval with a cutoff at 2 years, the only statistically significant differences were that primary tumours associated with early ipsilateral breast tumour recurrences (IBTRs) were more proliferating than the others and that early IBTRs (occurring within the first 2 years) had a higher degree of nuclear polymorphism than late IBTRs, as shown in Neither the conservation of the histological type in the ipsilateral breast tumour recurrence (hazard ratio=1.27 (0.29–5.56); The occurrence of an ipsilateral breast tumour recurrence is associated with poor prognosis because of the potential risk of lethality from its propensity to yield metastases  patients, we did not find that,  tumours . All feaIn some cases, the occurrence of an ipsilateral breast tumour recurrence can also reflect the evolution of the primary tumour and thus its resistance to the adjuvant therapies used in the first instance: that is radiotherapy with or without systemic treatments. These ipsilateral breast tumour recurrences are also called true recurrences, in opposition to new primaries. Their definition according to clinical and histological criteria remains a wild dream. All authors agree on a basic definition of a new primary that is, an ipsilateral breast tumour recurrence with a different histological type from its primary tumour  or distant recurrences.The arguments to oppose this theoretical assumption  are twofold. First, some adjuvant treatments such as chemotherapy could electively kill the most undifferentiated clones from the primary tumour and thus select those of higher differentiation, resistant to chemotherapy. The equivalent has already been shown in some studies where either the clonal heterogeneity found in the primary tumours (Contrary to others (The main drawback of this study is the relatively small size of our series under-powering the possibility of revealing real associations between the assessed factors. We conclude however that the definition of true recurrences according to clinical and histological criteria should rely solely on the conservation of the histological type with the added information brought by the location. There is no indication that true recurrences should have features of equal or lesser differentiation than their primary tumours.However, because of the overwhelmingly high rate of the ductal histological type among breast cancers, there is need to improve the definition of true recurrences by using new biological tools of clonal relation such as pan-genomic profiles (We hope that a better distinction of true recurrences will open new perspectives. So far, very little is known about the differential or similarity of the pan-genomic expression or nature of both new primaries and IBTRs. Ipsilateral breast tumour recurrences did not display features of higher aggressiveness than primary tumours. There was no indication that tumours more prone to be true recurrences because of conserved histological type, location or both, recur sooner or are deadlier than the others. No clinical nor histological definition of a true recurrence could be established other than the conservation of the histological type.


The formation of lung colonies by i.v. injected Lewis lung-tumour cells in syngeneic recipients was greatly enhanced by prior treatment of the mice with cyclophosphamide. The lung-cloning efficiency was linearly related to cyclophosphamide dose and the optimum time of treatment was 2-4 days before the injection of tumour cells. The resulting lung colonies had a similar size distribution to colonies in untreated recipients. Bleomycin, local thoraric irradiation and whole-body irradiation were much less effective in enhancing the lung-cloning efficiency. Cyclophosphamide also enhanced the take probability of i.m. implanted tumour cells.


Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2/neu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (t1/2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g(-1)) than in blood (6.7 %ID ml(-1)) or normal tissue ; lung, 7.1 %ID g(-1); kidney, 5.2 %ID g(-1)). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g(-1) or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g(-1). When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.


The serum of athymic nude mice bearing rat tumour xenografts has been examined for tumour-specific antigen. With a sarcoma and a hepatoma, tumour-specific antigen expression continued in xenograft growths, and sera of tumour-bearing mice contained free antigen, assayed by its ability to neutralise reactivity of tumour-immune rat sera against tumour target cells in an indirect membrane-immunofluorescence test. In contrast, no anti-rat antibody was detectable in sera of mice bearing the xenografts, or rejecting cells injected in admixture with BCG.


Drosophila.microRNA-263a/b confer robustness to sense organ development by controlling expression of the pro-apoptotic gene hid during apoptotic tissue pruning in  miR-263a/b are members of a conserved family of microRNAs that are expressed in peripheral sense organs across the animal kingdom. Here we present evidence that miR-263a and miR-263b play a role in protecting Drosophila mechanosensory bristles from apoptosis by down-regulating the pro-apoptotic gene head involution defective. Both microRNAs are expressed in the bristle progenitors, and despite a difference in their seed sequence, they share this key common target. In miR-263a and miR-263b deletion mutants, loss of bristles appears to be sporadic, suggesting that the role of the microRNAs may be to ensure robustness of the patterning process by promoting survival of these functionally specified cells. In the context of the retina, this mechanism ensures that the interommatidial bristles are protected during the developmentally programmed wave of cell death that prunes excess cells in order to refine the pattern of the pupal retina. hid, the expression of which triggers cell death. Specific mechanisms are needed to protect specialized cells which need to be maintained to ensure that only excess cells are eliminated. We report that a pair of related microRNAs, miR-263a/b, protect sense organs during this pruning process by directly acting upon and limiting the expression of the proapoptotic gene hid. This example, illustrates a novel function for miRNAs in ensuring developmental robustness during apoptotic tissue pruning.In spite of continuous challenges from the ever-changing environment, biological systems exhibit incredible stability in their developmental and physiological processes. In addition to extrinsic variability caused by environmental fluctuations, cells face intrinsic variability arising from the inherent noise of gene expression and of other molecular processes. microRNAs, which act as post-transcriptional regulators of gene expression, are beginning to be recognized for their ability to confer robustness to biological systems by buffering the effects of noisy gene expression. Although noise often is viewed as destabilizing, some biological processes make use of noise in order to make stochastic decisions. In this paper, we describe a role for microRNAs in preventing the stochastic elimination of excess cells in the developing fly retina. After the sense organs that make up the eye have been specified, pruning of excess cells occurs through the action of the gene  Organogenesis requires the organization of different cell types into precise spatial patterns. The Drosophila compound eye has proven to be a useful model system in which to investigate how such ordered patterns are established and maintained. The mature retina consists of ∼750 regular units, called ommatidia. Each ommatidium consists of eight photoreceptors, four cone cells, and two primary pigment cells. Individual ommatidia are separated by a layer of secondary and tertiary pigment cells. The “interommatidial” lattice also includes sense organs called interommatidial bristles (IOB). The IOB are mechanosensory hair cells, which may help the fly to avoid damage to the eye surface. IOB develop from a distinct set of sensory organ precursors (SOP), specified at discrete positions among the array of interommatidial cells In the developing eye imaginal disc, a field of naïve cells is produced by proliferation and the requisite number of ommatidial precursors is selected in a process of spatially patterned cell-type specification miR-7, which has been shown to contribute to the robustness of regulatory networks that ensure correct sense organ specification in Drosophila miR-7 is not required under normal conditions, SOP patterning was compromised when miR-7 mutant flies were subjected to environmentally challenging conditions. miRNAs act as post-transcriptional regulators that limit levels of target gene expression. This property makes them well suited to buffer fluctuating levels of gene activity. It may also make them well suited to serve a protective function during patterned tissue pruning.Mechanisms to ensure robustness are an important feature of developmental systems that can be subject to perturbation. MicroRNAs (miRNAs) have been proposed to play a role in conferring robustness during development miR-263a/b family of miRNAs contributes to the robustness of sense organ development. During apoptotic tissue pruning, functionally specified cells such as photoreceptors and mechanosensory organs are protected, while excess cells are eliminated. Mechanisms to ensure survival of specific cells are needed. Tissue pruning in the developing retina depends on activity of the pro-apoptotic gene hidmiR-263a/b sensory bristles are lost, like other cells, in a stochastic manner. Through a process of experimental validation we identify hid, among over 50 candidates examined in vivo, as a biologically important target of miR-263a/b in this context. While hid and other proapoptotic genes are targeted by other miRNAs, including bantam and the miR-2 family miR-263a/b may have a dedicated antiapoptotic role to ensure the robustness of sense organ development in a fluctuating developmental landscape.In this report we present evidence that the miR-263a is located near the bereft locus on chromosome 2L  by homologous recombination and confirmed that mature miR-263b was absent in the mutant X-21.2; miR-263a and miR-263b compared to miR-263a alone  on the head and thorax was reduced in miR-263a mutant flies compared to controls , its socket cell, a neuron, and its sheath cell bereft mutants, all four of these cells are present and properly specified upon completion of IOB cell fate determination miR-263a must act at a later stage, after the asymmetric division of the SOP.Because of the greater dependence of IOB development on miR-263a mutant retinas were indistinguishable from the controls and bristle shaft progenitor cells were present in normal numbers . These findings indicate that miR-263a acts to protect these sense organs from the wave of programmed cell death that sweeps over the retina during early pupal development.The loss of bristle shaft cells by 40 h APF raised the possibility that they might be eliminated during the normal wave of apoptotic pruning of interommatidial cells. To test this possibility, we made use of nt flies . Similar3a flies . We also3a flies . However arrows, . The tot arrows, , but themiR-263a has several hundred computationally predicted targets miR-263a expressing cells would be expected to result in IOB loss, phenocopying the miR-263a mutant phenotype. miR-263b-Gal4 was used to drive over-expression of target genes in this series of experiments because it has higher Gal4 activity than miR-263a-Gal4. We selected 56 predicted targets for analysis .analysis . Only twCyclin E has been shown to interfere with endoreplication Cyclin E over-expression is the cause of the bristle loss in miR-263a mutants, limiting their capacity to express Cyclin E should suppress this phenotype. Bristle loss occurs between 24 and 40 h APF in the mutants. RNA was prepared from pupae at 30 h APF, because the majority of the bristles are not yet lost at this stage. Cyclin E mRNA levels were elevated by ∼2.5-fold in RNA samples from miR-263a mutants . This was attributable to reduced luciferase mRNA levels , in the eye. Reducing miR-263a levels by removing one copy of the miR-263a gene led to fewer IOB, producing a more sparse appearance in the tuft of bristles (hid(Ala5) eyes is strongly perturbed, the effect of reducing the miRNA can be quantified most reliably by counting the number of empty sockets. Every socket should have an IOB hair cell, so the empty socket indicates a specified sense organ lacking the bristle shaft cell. Although the variance of this measure is high, there was a statistically significant increase of ∼2-fold in the proportion of empty sockets (p<0.005), indicating loss of IOB due to reduced miR-263a activity. Thus reduced activity of the miRNA enhanced the GMR:hid(Ala5) phenotype, suggesting that the miRNA has protective activity even in these extreme conditions of hid over-expression.It has been reported previously that excess bristles . BecausemiR-263a mutant flies was also a consequence of apoptosis due to elevated Hid activity. To address this we introduced the antimorphic allele of hid, 1W, into miR-263a miRNA mutant flies and found that mechanosensory bristle loss on head and thorax was also significantly suppressed (p<0.001). These observations suggest that miR-263a/b play a protective role, preventing the loss of mechanosensory cells due to hid-induced apoptosis.Finally, we asked whether the loss of the other mechanosensory bristles in ppressed ; p<0.001miR-263a/b in conferring robustness to sensory organ survival during a developmental pruning process.Robustness of a biological system can be thought of in terms of the mechanisms that ensure stability. In developmental terms, perturbation can come in the form of fluctuating levels of intercellular signaling and/or gene expression and can be intrinsic or of environmental origin. Gene regulatory networks have properties that can help to confer stability by buffering the effects of fluctuations in gene expression . We also did not find evidence that miR-263a/b act in the context of a gene regulatory network. Instead, miR-263a/b appears to function in a different context, acting as a buffer in a biological process that is inherently stochastic. In this way miRNA activity is used to ensure that apoptotic cell death is not allowed to compromise specific cells. It is noteworthy that as little as 20% of normal miR-263a levels are sufficient to support IOB development. This implies a considerable buffering capacity to ensure that the process of IOB formation is robust in a fluctuating developmental landscape.In the bantam miRNA functions during tissue growth and regulates hid, to prevent proliferation induced apoptosis bantam regulation of hid does not appear to impact upon apoptotic pruning or on survival of sense organs (our unpublished observation). miR-14 has also been reported to be anti-apoptotic miR-14 mutants do not impact on IOB apoptosis  being able to mimic the miRNA mutant phenotype when over-expressed in the miRNA expressing cells and (2) being able to suppress the miRNA mutant phenotype when its level was reduced in the miRNA expressing cells. Further investigation of the miR-263 family miRNA mutants may lead to identification of targets important for other aspects of the miRNA function, such as the reduced viability observed in the double mutants.The genetic evidence presented here identifies miR-263a and miR-263b are members of a conserved family of miRNAs, including mammalian miR-183, miR-96 and miR-182, and miR-228 in C. elegans. Interestingly, members of this family display conservation of expression in ciliated sensory organs in vertebrate and invertebrate organisms miR-183, miR-96, and miR-182 are expressed in sensory hair cells in mammalian auditory and vestibular organs, as well as in sensory cells of the eye and ear in zebrafish and chicken C. elegans miR-228 is expressed in chemosensory and mechanosensory sensilla miR-263a and miR-263b are expressed in sense organ precursors in embryos Drosophila miR-96 have been identified as the cause of auditory hair cell degeneration and non-syndromic progressive hearing loss in mice and humans dicer mutants in the mouse also leads to defects in inner ear hair cell development miR-263a/b in support of sensory hair development in Drosophila remains to be determined. Superficially the way in which these sense organs are lost appears to differ. In the fly, the mechanosensory cells are lost due to apoptosis in the miR-263a/b mutants. In the mammalian systems, mature differentiated sensory cells appear to be lost through degeneration. In the case of the miR-96 mutant this could be due to inappropriate regulation of genes that are not normally miR-96 targets due to the change in sequence of the mutant miRNA seed, but in the case of dicer conditional mutants it is more likely due to loss of normal miRNA mediated target regulation. Whether this involves apoptosis is not known. Intriguingly, there is evidence suggesting an anti-apoptotic role for miR-182 and related family members in human cancers Further support for the idea of conservation of function comes from the observation that mutations affecting hid UAS-RNAi strain was obtained from the VDRC. 24bft, 225bft, and 6Bbft were provided by Rolf Bodmer. 05014hid and GMR:hid(Ala5) were provided by Hermann Steller. 1W, Df(3L)X-21.2, UAS-p35, and UAS-Diap1 were obtained from the Bloomington stock centre. UAS-miR-263a and UAS-miR-263b lines were made by cloning a 300 base pair genomic fragment containing the miRNA hairpin into the 3′UTR of dsRed in pUAST, as described in hid 3′UTR reporters were made by cloning the 2.2 kb hid 3′UTR after GFP or luciferase, under control of the tubulin promoter hid UTR reporters with mutated miR-263a/b sites were generated by PCR using primers designed to change the seed region from TGCCA into TCCGA. PCR products were sequence verified.Canton-S flies were used as the wild-type control. EP lines were obtained from the Bloomington, Szeged, and Exelixis stock centres. The miR-263b is located in an intron of CG32150, we removed the intron-containing mini-white gene cassette and confirmed that splicing of CG32150 mRNA was not affected in the ΔmiR-263b mutants by comparing the level of spliced mRNA using qRT-PCR. miR-263a and miR-263b Gal4 knock-in alleles were made using a modified targeting vector Ends-out homologous recombination was performed as described Luciferase reporters and miRNA expression plasmids were expressed under the control of the tubulin promoter. S2 cells were transfected in 6-well plates with 1,000 ng of miRNA expression plasmid or empty vector, 500 ng of firefly luciferase reporter plasmid, and 500 ng of Renilla luciferase DNA as a transfection control. Transfections were performed with triplicate technical replicates in at least three independent experiments. At 60 h post-transfection, dual luciferase assays (Promega) were performed on a portion of the transfected cells. The other portion was pelleted and dissolved in Trizol reagent (Invitrogen) for total RNA extraction. For luciferase assays on pupal retinas, retinal tissue was dissected and immediately lysed in passive lysis buffer (Promega). Luciferase activity was normalized to total protein content, measured on the same sample using the Bradford method (Bio-Rad).AAAAAGATTGCGACCTCGAT and AAACCAACCTGGCTTACACC) or to the transfection control Renilla luciferase mRNA (CGGACCCAGGATTCTTTT and TTGCGAAAAATGAAGACCT). Primers for hid pre-mRNA: TGAAGGTGTTCTCCGATTCC and ATCTCACCCAGCGCTCTTTA. Primers for mature hid mRNA: GAGAACGACAAAAGGCGAAG and CAAAACGAAAACGGTCACAA. Firefly luciferase primers: CCGCCGTTGTTGTTTTG and CTCCGCGCAACTTTTTC. GFP primers: GCAGTGCTTCAGCCGCTA and AGCCTTCGGGCATGGC.Northern blots on small RNA were carried out according to Adult flies were fixed overnight in 2.5% glutaraldehyde at 4°C, washed 3×15 min with PBS, dehydrated in a series of ascending ethanol concentrations, critical point dried, mounted on stubs, and coated with gold. Eyes were imaged with a JSM-6360LV scanning microscope . The orientation, contrast, and brightness of the images were adjusted using ImageJ. For the quantification of bristle numbers, a high-resolution image of a whole eye was printed and the maximal visible surface delimited, usually 300–500 ommatidia. The number of visible IOBs was counted and divided by the total number of corners where IOBs would be expected or, for To stage pupae, white pre-pupae were collected and aged at 25°C until dissection. Pupal eye imaginal discs were dissected and fixed in 4% formaldehyde for 20 min on ice. The following primary antibodies were used: rat anti-Dcad2 1∶40 , rabbit anti-DPax2 1∶50 (a gift from Erich Frei and Markus Noll), and chicken anti-GFP 1∶1000 (Abcam). Fluorescent secondary antibodies were from Jackson Laboratories. Samples were mounted in Vectashield (Vector Laboratories). Detection of apoptotic cells in pupal eye discs was done using the Apoptag ISOL dual fluorescence kit (Chemicon). Immunofluorescence images were collected using a Leica SPE confocal microscope and processed using ImageJ. Quantification of apoptotic nuclei was done using z-projections of confocal sections. Total apoptotic nuclei as well as apoptotic shaft cell nuclei were counted and normalized to the total number of ommatidia analyzed.Figure S1miR-263a is absent in bft lines. Northern blot showing mature miR-263a in total RNA extracted from adult control flies (WT) and the three bft homozygous mutants described in Hardiman et al. 2002 (0.47 MB TIF)Click here for additional data file.Figure S2miR-263b contributes to IOB formation. (A) Aligned sequences of miR-263a and miR-263b. The three differing nucleotides are highlighted in red. The seed region (grey shading) comprises nucleotides 2 to 8 of the miRNA. (B) Normalized miR-263b levels in adult flies, measured by miRNA qPCR. Δ263b: miR-263b knockout allele, Δ263b-G4/Def: miR-263b-Gal4 knock-in allele in trans with the genomic deficiency Df(3L)X-21.2. (C) SEM of adult eyes from miR-263b single mutant (two representative examples) and miR-263a miR-263b double mutant flies. Scale bars = 50 µm.(0.45 MB TIF)Click here for additional data file.Figure S3Absence of miR-263 causes loss of bristles on head and thorax. Quantification of macrochaetae on head and thorax of adult flies: wild-type (WT), miR-263a mutant (Δ263a/bft), miR-263a mutant expressing an UAS-miR-263a transgene , miR-263a miR-263b double mutant X-21.2), miR-263a mutant with one copy of the antimorphic hid allele 1W . Error bars represent mean ± SD for N = 50 flies per genotype. [*] = p<0.001, using two-tailed unpaired Student's t test comparing to Δ263a/bft flies. Macrochaetae numbers in the miR-263a miR-263b double mutant differed slightly, but not statistically significantly, from those in miR-263a mutants. Single mutant: 95.3%, double mutant 94.4%, p = 0.12 using two-tailed unpaired Student's t test comparing the single and double mutants.(0.11 MB TIF)Click here for additional data file.Figure S4Viability of miR-263a and miR-263b mutants. Viability of different miR-263a and miR-263b mutant lines. Numbers indicate the percentage of flies observed relative to what is expected if fully viable. Hatched: percentage of embryos that hatched (n = 500 embryos counted); pupated: percentage of the resulting first instar larvae that pupated; eclosed: percentage of adult flies that emerged from these pupae. 1-d-old is the percentage of adult flies surviving 1 d after eclosion. For ease of comparison, the numbers in each category for 1118w were set to 100%. 1118w flies were used as a control. Δ263a/bft: miR-263a mutant, Δ263b/Def: miR-263b mutant, where Def represents the genomic deficiency Df(3L)X-21.2, Δ263a/bft; Δ263b/Def: miR-263a miR-263b double mutant.(0.38 MB TIF)Click here for additional data file.Figure S5CycE over-expression does not cause the IOB phenotype. (A) Normalized Cyclin E mRNA levels in flies with the indicated genotype. RNA was extracted from whole 30 h APF pupae. WT: wild-type; Δ263a/bft: trans-heterozygous miR-263a mutant; Δ263a,CycEAR95/bft: miR-263a mutant carrying one copy of AR95CycE, a null allele of CycE. Bars represent mean ± SD of three independent batches of pupae. (B) SEM of adult eyes from flies with the indicated genotype. Scale bars = 50 µm. CycE is elevated in miR-263a mutants. Reducing the dosage of CycE to wild-type levels does not rescue bristle loss, which indicates that over-expression of CycE is not the cause of the miR-263a phenotype.(0.44 MB TIF)Click here for additional data file.Figure S6miR-263b target sites in the hid 3′UTR. Predicted miR-263b target sites in the hid 3′UTR. Pairing to the miRNA seed sequence is shaded in grey. Nucleotides changed to generate the target site mutant UTR are shown in red.(0.59 MB TIF)Click here for additional data file.Figure S7miR-263a regulates a GFP transgene carrying the hid 3′UTR. Normalized GFP mRNA levels measured by qRT-PCR. RNA was extracted from 30 h pupal eye imaginal discs from flies expressing a GFP reporter carrying the hid 3′UTR or a mutated version of it, in a miR-263a mutant or wild-type control background. Bars represent mean ± SD of three independent experiments. [*] p<0.001 using two-tailed unpaired Student's t test comparing to the control levels.(0.11 MB TIF)Click here for additional data file.Figure S8A predicted feed-forward regulatory network involving mR-263a, hid, and the MAPK pathway. (A) Topology of the predicted feed-forward network: Negative regulation of RAS by the miRNA would repress MAPK activity and alleviate repression of hid transcription and of HID protein activity. In other words the effect of the miRNA on the MAPK branch would be to increase hid transcription and Hid protein activity. In the miR-263a mutant (illustrated at right), the predicted elevation of MAPK activity should lower hid activity, acting in opposition to the increase in hid mRNA levels caused by the miR-263a mutant. (B) Ras85D mRNA levels in pupal eye discs of control and miR-263a mutants. Ras85D is on the list of predicted miR-263a targets (but not on the miR-263b list due to differences in the seed sequence). Ras mRNA levels were measured by Q-RT-PCR on RNA from pupal eye discs dissected from control animals and miR-263a mutants, as well as rescued mutants. There was no significant difference.(0.10 MB TIF)Click here for additional data file.Table S1List of tested candidate genes, with the corresponding EP lines and results (IOB loss: yes or no) when expression is driven with miR-263b-Gal4.(0.09 MB DOC)Click here for additional data file.


Unilamellar liposomes of dipalmitoyl-phosphatidylcholine can incorporate various porphyrins in either the phospholipid bilayer or the internal aqueous compartment depending on the water-/lipo-solubility of the drug. Intraperitoneal injection of the liposome-bound porphyrins to mice bearing a MS-2 fibrosarcoma results in remarkably more efficient tumour targeting than that obtained by administration of the same porphyrins dissolved in homogeneous aqueous solution. Moreover, also water-insoluble porphyrins can be transported to the tumour via liposomes. Fractionation of liver and neoplastic cells indicates that the subcellular distribution of liposome-delivered porphyrins is also dependent on their solubility properties: thus, relatively polar porphyrins, such as tetra(4-sulfonatophenyl)porphine and uroporphyrin, are mainly recovered from the soluble fraction, whereas hydrophobic porphyrins, such as haematoporphyrin or porphyrin esters, preferentially partition in the cytoplasmic membrane. As a consequence, different subcellular sites can be targeted by porphyrins and possibly photodamaged through a suitable choice of the drug-carrier system.


Two transplantable, highly immunogenic syngeneic C57BL fibrosarcomas, FS1 and FS6, were shown to have tumour-specific rejection antigens, as shown by excision of the primary tumours and i.p. or i.m. injection of graded doses of the specific and unrelated tumour cells. I.p. challenge with tumour cells induced a large and relatively long-lasting increase in numbers of peritoneal leucocytes. Macrophage monolayers prepared from such exudates were, in general, non-specifically cytotoxic, though occasional specific cytotoxicity was detected. T lymphocytes isolated from exudates were shown to kill in a specific manner. When immunized mice were challenged with the specific tumour cells to elicit large numbers of peritoneal cytotoxic cells, and with graded doses of the non-cross-reacting tumour cells at the same time or at various times thereafter, growth of the non-related tumours occurred in all cases and only the specific tumour was rejected. Moreover, Winn tests, in which the inflammatory cells were mixed with unrelated tumour cells and implanted i.m., did not delay tumour growth. The relevance of these findings to the role of macrophages and lymphocytes in syngeneic tumour rejection is discussed.


Experiments are described in which dose-response data have been obtained for EMT6 mouse tumour cells growing in vivo and exposed to various cytotoxic agents. A comparison has been made of the response of solid tumours in the flank and of rapidly growing lung nodules. The results are discussed with regard to their cell kinetic implications and compared with our results for the chemosensitivity of EMT6 cells in exponential and plateau phase growth in vitro.


Tumour angiogenesis is a complex multistep process regulated by a number of angiogenic factors. One such factor, platelet-derived endothelial cell growth factor has recently been shown to be thymidine phosphorylase (TP). TP catalyses the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine. Although known to be generally elevated in tumours, the expression of this enzyme in breast carcinomas is unknown. Therefore, we used ribonuclease protection assays and immunohistochemistry to examine the expression of TP in 240 primary breast carcinomas. Nuclear and/or cytoplasmic TP expression was observed in the neoplastic tumour epithelium in 53% of tumours. Immunoreactivity was also often present in the stromal, inflammatory and endothelial cell elements. Although endothelial cell staining was usually focal, immunoreactivity was observed in 61% of tumours and was prominent at the tumour periphery, an area where tumour angiogenesis is most active. Tumour cell TP expression was significantly inversely correlated with grade (P = 0.05) and size (P = 0.003) but no association was observed with other tumour variables. These findings suggest that TP is important for remodelling the existing vasculature early in tumour development, consistent with its chemotactic non-mitogenic properties, and that additional angiogenic factors are more important for other angiogenic processes like endothelial cell proliferation. Relapse-free survival was higher in node-positive patients with elevated TP (P = 0.05) but not in other patient groups. This might be due to the potentiation of chemotherapeutic agents like methotrexate by TP. Therefore, this enzyme might be a prediction marker for response to chemotherapy.


The grant number from the funder University of South Bohemia Ceske Budejovice was listed incorrectly. The correct grant number is: MSM6007665801.


A second group was acutely exposed to inspiratory hypoxia for only 20 min prior to the measurements (acute hypoxia), whereas animals housed under normal atmospheric conditions served as controls. Acute hypoxia reduced the median oxygen partial pressure (pO2) dramatically (1 vs 10 mmHg in controls), whereas in chronically hypoxic tumours the pO2 was significantly improved (median pO2=4 mmHg), however not reaching the control level. These findings reflect the changes in tumour perfusion where acutely hypoxic tumours show a dramatic reduction of perfused tumour vessels . In animals under chronic inspiratory hypoxia, the number of perfused vessels increased (compared to acute hypoxia), although the perfusion pattern found in control tumours was not reached. In the chronically hypoxic animals, tumour cell proliferation and tumour growth were significantly reduced, whereas no differences in VEGF expression and vascular density between these groups were observed. These results suggest that long-term adaptation of tumours to chronic hypoxia in vivo, while not affecting vascularity, does influence the functional status of the microvessels in favour of a more homogeneous perfusion.This study investigated long-term microenvironmental responses  expression and vascularisation) to chronic hypoxia in experimental tumours. Experiments were performed using s.c.-implanted DS-sarcomas in rats. In order to induce more pronounced tumour hypoxia, one group of animals was housed in a hypoxic atmosphere (8% O Howeverc agents . Many ofc agents , 2002. Mc agents . In partc agents , 1995 poc agents . Thus, tc agents .2 supply. In addition, tumour blood flow in these newly formed vessels has been seen to undergo temporal fluctuations with intermittent flow stops in individual vessels . Subsequently, oxygenation, metabolic and bioenergetic parameters of the tumours were determined. In order to study the underlying mechanisms by which the oxygen and nutrient supply of the tissue was modified, vascularisation and perfusion were evaluated.The aim of the present study was to assess changes in oxygenation and metabolic and bioenergetic parameters as a consequence of an increase in tumour hypoxia. The Oad libitum. Experimental tumours grew following subcutaneous injection of DS-ascites tumour cells  into the dorsum of the hind foot, as flat, spherical segments and replaced the subcutis and corium completely. The volume was determined by measurement of the three orthogonal diameters of the tumour and using an ellipsoid approximation with the formula V=d1d2d3π/6. Tumour volume doubling time was calculated from growth curves during the exponential growth phase (days 4–12 after inoculation). Microenvironmental measurements were performed when tumours reached a target volume of 0.5–3.0 ml approximately 6–14 days after inoculation. Animals were housed either under normoxic ambient conditions  or in a hypoxic atmosphere containing 8% O2 (chronic hypoxia) continuously for the whole period of tumour growth and during measurements (e.g. oxygenation measurements). For experiments analysing the impact of acute hypoxia, animals were housed under normoxic conditions during tumour growth but breathed the hypoxic gas mixture (8% O2) for 20 min prior to and during the measurements. Studies had previously been approved by the regional ethics committee and were conducted according to UKCCCR guidelines  housed in our animal care facility were used in this study. They received a standard diet and acidified water pO2) was measured polarographically using steel-shafted microelectrodes (outer diameter: 300 μm) and the pO2 histography system  to ensure that values for the arterial blood gases were within the physiological range during the measurement period. Mean arterial blood pressure (MABP) was continuously monitored through connection of an arterial catheter placed in the left carotid artery to a Statham pressure transducer .The distribution of tumour oxygen tensions , and ATP, ADP and AMP were measured by HPLC , sonicated five times on ice and centrifuged. The protein content of the supernatant was determined using Bradford reagent . For each measurement, approximately 50 μg of protein was analysed. For this, 96-well microplates were coated with a mouse anti-rat VEGF antibody. The supernatant of the tissue preparation was diluted in PBS+1% BSA (1 : 10 v v−1) and incubated in the wells for 2 h at room temperature. After washing, the detection antibody (goat anti-rat VEGF) was added and incubated for a further 2 h at room temperature. The optical density was determined on a microplate reader at a wavelength of 450 nm. The VEGF concentration determined for each well was normalised to the protein content in the sample. In contrast to all other experiments, in the VEGF study, the animals in the group ‘acute hypoxia’ were exposed to the hypoxic atmosphere (8% O2) for 18 h (not just for 20 min) prior to tumour excision in order to leave sufficient time for the in vivo upregulation of VEGF expression to take place.Vascular endothelial growth factor was determined by an ELISA kit detecting rat VEGF  according to the manufacturer's instructions. In brief, tumours were excised and cut into small pieces. Approximately 0.5 g of tissue was suspended in 1.5 ml RIPA buffer . Sections were rinsed in PBS (pH 7.4) and thereafter with 3% H2O2. Sections were washed in PBS between all the following steps. The specimens were incubated in normal horse serum  for 20 min and incubated overnight in CD31 antibody  at room temperature in humidified chambers. Biotinylated anti-mouse IgG  was applied as a secondary antibody for 30 min, followed by ABC complex  for 30 min. Immune complexes were made visible by incubation with AEC chromogene  for 15 min. The specimens were then counterstained with haematoxylin (1 : 2) for 2 min and rinsed in tap water. Finally, using aquatex mounting medium , specimens were mounted with a coverslip.Endothelial cells were stained with a CD31 antibody. Cryosections with a thickness of 7 All morphometric analyses were performed single-blinded by the same observer using a computerised digital image analysis system  connected with a Zeiss Axiophot microscope . In each tumour, the areas with the highest microvessel density (hot spots) were identified. Quantification of the microvessels was performed in five hot spots in the tumour periphery and five hot spots in the tumour centre. The vessel counts were averaged separately for the tumour centre and periphery. Analysis of the fraction of viable tumour tissue was performed in the haematoxylin and eosin-stained slices by marking necrotic areas and calculating the fraction of necrosis in the image.−1 body weight. For this, the dye was dissolved at a concentration of 10 mg ml−1 in isotonic saline and approximately 0.5 ml of this stock solution was rapidly injected into a tail vein. At 60 s after injection, animals were killed by an overdose of anaesthetic and the tumours were rapidly frozen in liquid nitrogen. Following subsequent tumour removal, cryosections were prepared. Using fluorescence microscopy  at low resolution (range of vision 3 × 2.5 mm), images of perfused vessels were taken from two to three different regions of each cryosection. Approximately three to four sections from each tumour were analysed resulting in 9–10 images per tumour. In these images, each vessel was marked and the mean density of perfused vessels, the mean distance between neighbouring vessels and the area of vascular domains were calculated using image analysis software .In order to assess parameters related to the perfusion distribution within a tumour, the fluorescent dye Hoechst 33342  was injected i.v. at a dose of 25 mg kg−1 and injected i.p. (150 mg kg−1 body weight). After 120 min, tumours were excised and dissipated into a single cell suspension. Incorporated BrdU was detected using monoclonal antibodies and DNA content was determined by propidium iodide staining . For this, BrdU  was dissolved in PBS at a concentration of 10 mg mlμl) taken from a tail vein.Blood cell parameters were assessed using a multiparameter, automated haematology analyser , whereby erythrocyte, white blood cell and platelet counts together with the mean red blood cell (RBC) volume (MCV) were measured by impedance and the haemoglobin concentration was measured by a photometric method. In addition, the analyser uses the measured values to calculate several other parameters ). All measurements were performed using a sample of venous blood . Hypoxia led to a significant reduction in arterial pO2 followed by a stimulation of ventilation as indicated by a lower arterial pCO2 and a higher pH , possibly indicating an adaptation of ventilation to a chronically reduced inspiratory O2 fraction.Exposing rats to an atmosphere containing a reduced oxygen fraction (8%) over a prolonged period of time (6–14 days) affects several systemic parameters. As might be expected, inspiratory hypoxia markedly stimulated erythropoiesis, as indicated by a significantly higher haemoglobin concentration and haematocrit as well as an increased number of RBCs , the DS-sarcoma used in the study had a volume doubling time of 2.4 days , whereas tumours growing under inspiratory hypoxia had a significantly longer volume doubling time  to 1 mmHg within 20 min , the worsening of the O2 status was less pronounced: the median pO2 was 4 mmHg and the fraction of hypoxic pO2 values ⩽2.5 mmHg was 44% , the lactate level in the group acutely exposed to inspiratory hypoxia was significantly higher compared to control animals and to chronically hypoxic tumours .Although the results of tumour oxygenation and metabolic parameters indicate that the O2 deficiency in the tumour) on the induction of vessel formation, the vascular densities in areas of high vascular density (hot spots) in the tumour centre and periphery were determined. However, neither the size of the hot spots nor the mean or maximum vascular density within the hot spots (in vitro (data not shown), no marked differences in the VEGF level in tumours of the different groups were seen in the in vivo situation  to values far greater than the oxygen diffusion distance. In tumours under chronic inspiratory hypoxia conditions, the mean number of perfused vessels was also reduced to approximately the same extent as during acute hypoxia . These data indicate a trend to a more uniform perfusion compared to acute hypoxia, although values of control tumours were not reached (134±3 μm). This trend also becomes obvious when the fraction of vessels that are more than 350 μm away from the next perfused vessel is calculated (P=0.0052 vs acute hypoxia).Even though the number of vessels (as indicated by CD31 staining) was not different in chronically hypoxic tumours as compared to controls , the pat7A hypoxia . However hypoxia . In chrolculated . One pos2 deficiency even under control conditions , were artificially made even more hypoxic by exposing the animals to a hypoxic atmosphere containing 8% oxygen for the period of tumour growth. However, this procedure induces severe hypoxia systemically in the whole animal rather than just in the tumour. Therefore, several other processes that are not directly related to tumour hypoxia and that in turn may affect the tumour microenvironment might be induced. For instance, the measured blood gas parameters (2 to haemoglobin (as indicated by a left shift of oxyhaemoglobin dissociation curve). This mechanism may contribute to the worsening of the O2 supply to the tumour and by this increase tumour hypoxia. On the other hand, hypoxaemia induces erythropoietin (EPO) formation in the kidney resulting in forced erythropoiesis , the status of this checkpoint is not known. For this reason, it might be possible that the lack of hypoxia-induced cell cycle arrest results from a deficient G1/S checkpoint. Since the oxygenation status of a tissue results from a dynamic steady state between O2 delivery and consumption, the reduced number of proliferating cells may also contribute to the better oxygenation under these conditions . For this reason, other adaptive mechanisms have to take place in addition to the reduction of the O2 diffusion distance.2 to haemoglobin and by this improved the oxygen supply to the tumour cells. Even though 2,3-DPG levels were not determined in the present study, this mechanism may also contribute to the long-term improvement of the O2 status to chronic hypoxia. Finally, the arterial pO2 in chronically hypoxic animals was slightly higher than during acute hypoxia  contribute to the higher tumour pO2 observed.In the experimental setting used, improvement of tumour hypoxia during chronic inspiratory hypoxia may also be the result of the increased haemoglobin concentration seen , which w hypoxia , indicatα or EF5) and the production of VEGF was found (in vitro experiment that tumour cells – and even more so tumour stroma-derived fibroblasts – increase their VEGF secretion rate when they are kept under hypoxia (pO2 <1.5 mmHg for 24 h). Under these conditions, tumour cells were able to increase the VEGF production by up to 10-fold compared to the basal secretion found under normoxia (pO2=150 mmHg). It should be noted, however, that these results varied between the different cell lines used. Fibroblasts were able to increase VEGF secretion by a factor of four. DS-sarcoma cells used in the present study produce small quantities of VEGF even under normoxic conditions (approximately 24 pg h−1 10−6 cells). When these cells were kept under hypoxia (pO2 <1.5 mmHg for 24 h), VEGF secretion increased by a factor of six indicating that, in the tumour cell line used in the present study, hypoxia is able to pronouncedly induce VEGF formation in vitro. However, in vivo, the VEGF concentration in the tumour was not markedly different in animals kept under chronic hypoxia compared to normoxic conditions  to hypoxic conditions has to be ruled out in the present study. It has been postulated that severe hypoxia may induce the formation of new tumour blood vessels necessary for further tumour growth . In manynditions . Since tin vivo as compared to cell culture experiments have to be taken into account. Since the DS-sarcomas show pronounced hypoxic regions with almost 50% of pO2 values below 2.5 mmHg  for several hours  but from a functional adaptation of the perfusion pattern. Acute hypoxia results in pronounced heterogeneity of the tumour blood flow distribution, whereas chronic hypoxia leads to a more homogeneous pattern with significantly smaller distances between perfused vessels resulting in a better diffusive O2 supply to the cells. In the tumour model used, functional changes in physiological parameters appear to play a more important role in the adaptation to chronic hypoxia than hypoxia-induced alterations in VEGF expression and morphological changes of vasculature.In conclusion, pronounced long-term hypoxia in an experimental tumour induces adaptive processes resulting in an improvement of the oxygenation status (as compared to acute hypoxia). However, the increase in median


Tumour-inhibitory effects of a new antagonist of growth hormone-releasing hormone (GH-RH), MZ-4-71, were evaluated in nude mice bearing androgen-independent human prostate cancer cell lines DU-145 and PC-3 and in Copenhagen rats implanted with Dunning R-3327 AT-1 prostatic adenocarcinoma. After 6 weeks of therapy, the tumour volume in nude mice with DU-145 prostate cancers treated with 40 microg day(-1) MZ-4-71 was significantly decreased to 37 +/- 13 mm3 (P < 0.01) compared with controls that measured 194 +/- 35 mm3. A similar inhibition of tumour growth was obtained in nude mice bearing PC-3 cancers, in which the treatment with MZ-4-71 for 4 weeks diminished the tumour volume to 119 +/- 35 mm3 compared with 397 +/- 115 mm3 for control animals. Therapy with MZ-4-71 also significantly decreased weights of PC-3 and DU-145 tumours and increased tumour doubling time. Serum levels of GH and IGF-I were significantly decreased in animals treated with GH-RH antagonist. In PC-3 tumour tissue, the levels of IGF-I and IGF-II were reduced to non-detectable values after therapy with MZ-4-71. The growth of Dunning R-3327 AT-1 tumours in rats was also significantly inhibited after 3 weeks of treatment with 100 microg of MZ-4-71 day(-1) i.p. as shown by a reduction in tumour volume and weight . Specific high-affinity binding sites for IGF-I were found on the membranes of DU-145, PC-3 and Dunning R-3327 AT-1 tumours. Our results indicate that GH-RH antagonist MZ-4-71 suppresses growth of PC-3, DU-145 and Dunning AT-1 androgen-independent prostate cancers, through diminution of GH release and the resulting decrease in the secretion of hepatic IGF-I, or through mechanisms involving a lowering of tumour IGF-I levels and possibly an inhibition of tumour IGF-I and IGF-II production. GH-RH antagonists could be considered for further development for the therapy of prostate cancer, especially after the relapse.


Sir,R=0.951) when compared with the intact liver, and in other series of experiments when liver growth was induced by partial hepatectomy, metabolic changes of the same protein fractions in regenerating liver also correlate strongly with those in ascites hepatoma (R=0.954)  expression proceeds not only in the tumour, but also in adjacent non-tumour tissues in gastro-oesophageal malignancy (R=0.954) . One canR=0.954) . In the R=0.954) . Thus, t


The study included 99 stage IB-IV cervical cancer patients: immunostaining of tumour tissue sections was performed with rabbit antiserum against cyclooxygenase-2. CD3, CD4, CD8, CD25, Mast Cell Tryptase monoclonal antibodies were used to characterise stroma inflammatory cells in nine cervical tumours. An inverse relation was found between cyclooxygenase-2 levels (cyclooxygenase-2 IDV) of tumour vs stroma compartment . The percentage of cases showing high tumour/stromal cyclooxygenase-2 IDV ratio was significantly higher in patients who did not respond to treatment (93.3%) with respect to patients with partial (60.5%), and complete (43.7%) response (P= 0.009). Cases with a high tumour/stroma cyclooxygenase-2 IDV ratio had a shorter overall survival rate than cases with a low tumour/stroma cyclooxygenase-2 IDV (P<0.0001). In the multivariate analysis advanced stage and the status of tumour/stroma cyclooxygenase-2 IDV ratio retained an independent negative prognostic role. The proportion of CD3+, CD4+, and CD25+ cells was significantly lower in tumours with high tumour/stroma cyclooxygenase-2 IDV ratio, while a higher percentage of mast cells was detected in tumours showing high tumour/stroma cyclooxygenase-2 IDV ratio. Our study showed the usefulness of assessing cyclooxygenase-2 status both in tumour and stroma compartment in order to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant therapy and unfavourable prognosis.This study aims at investigating the relationship between cyclooxygenase-2 expression in tumour British Journal of Cancer (2002) 87, 1145–1152. doi:10.1038/sj.bjc.6600578www.bjcancer.comCancer Research UK© 2002  Cyclooxygenase (COX), the key enzyme in the conversion of arachidonic acid to prostaglandins, exists in two isoforms namely COX-1, constitutively expressed in most tissues, and COX-2, which is inducible by growth factors, prostaglandins, and tumour promoters and associated with the inflammatory response . It has There is some evidence that, besides the expression in tumour cells, COX-2 is also expressed in stromal cellular elements such as endothelial cells, macrophages, and fibroblasts  the possible clinical role of COX-2 expression according to the cellular compartment of staining.Based on our preliminary observations about the presence of COX-2 staining in the stroma compartment of cervical tumours we were then prompted at providing a more in depth analysis of (i) COX-2 content in the stroma inflammatory cellular elements of this neoplasia, (ii) the relationship between COX-2 expression in tumour cells In addition, an immunophenotypic characterization of stroma inflammatory cells was carried out in a series of cervical tumours.n=24), whereas locally advanced cancer cases (n=75) were first administered neoadjuvant cisplatin-based treatment (cisplatin dose 100 mg m2 every 3 weeks for 2–3 courses). In case of clinical response, assessed by the above described procedures and recorded according to WHO response evaluation criteria (n=61): operative technique consisted of type II–IV radical hysterectomy, and systematic pelvic lymphadenectomy. An anterior exenteration was performed in two cases with persisting involvement of the bladder after neoadjuvant treatment. Para-aortic lymphadenectomy up to the level of inferior mesenteric artery was performed in high risk patients as previously reported . The clinico-pathological characteristics are summarised in Table 1criteria , LACC paThe following monoclonal and policlonal antibodies were used: anti-CD3 , anti-CD4 , anti-CD8 , anti-CD25 , all from Ylem ; anti-Mast Cell Tryptase ; anti-COX 2 rabbit policlonal antibody .Tumour tissues biopsies were performed under colposcopic examination. Immunohistochemistry of COX-2 was performed as previously described , and washed with PBS. The slide was immersed for 10 s in 99.8% (vol/vol) methyl alcohol and then exhaustively washed with PBS. The section was treated in microwave owen using the Dako ChemMate detection kit (DAKO) and then incubated with anti-mast cell tryptase antibody. The immunoreaction was developed by fast blue and the slide was remounted. The exact area on the same slide was acquired by the digital camera and compared with the first image. As a control, after the first immunostaining with anti-COX-2 antibody, the slide was processed, as reported above, and then treated with the streptavidin-biotin-alkaline phosphatase complex kit, omitting the anti-mast cell tryptase antibody.Double-labelling subtraction immunostaining was carried out as previously reported . The number of immunostained cells, relative to each phenotype, was calculated by averaging cell counts from two grid areas from five ×20 fields (n=10). The results were reported as mean±s.e.The intensity of immunohistochemical staining was evaluated as previously reported  and Statview survival tools .Figure 1Apartment .In the whole series, COX-2 integrated density values in the tumour component ranged from 1.2 to 82.3 with mean±s.e. values 25.5±2.2. COX-2 integrated density values in the stromal component range from 0.9 to 96.0 with mean+s.e. values of 20.0±1.9.vs COX-2 IDV in the stroma compartment  (data not shown).A statistically significant inverse relation was found between COX-2 IDV of tumour vs COX-2 IDV in the stroma component was used in order to normalise the COX-2 expression in each case, and to categorise tumours according to low vs high COX-2 content. The tumour/stroma COX-2 IDV ratio range from 0.03 to 48.2 (mean±s.e.=5.1±0.9). The ratio of ⩽1 was used to indicate cervical tumours with COX-2 expression in the tumour component lower or equivalent to COX-2 expression in the stroma.For this reason, the ratio between COX-2 IDV in the tumour According to the chosen cut off value, 56 out of 99 (56.6%) were scored as having a high (>1) tumour/stroma COX-2 IDV ratio.High COX-2 IDV in the tumour compartment were shown to be significantly associated with larger volume of the tumour and more aggressive histotype while COX-2 IDV in the tumour stroma showed the opposite pattern (data not shown).P value=0.029). Moreover, cases with high tumour/stroma COX-2 IDV ratio were more frequently observed in cases with tumour volume ⩾4 cm than in smaller tumours (66.1% vs 40.5%) . No association with age, and grade of differentiation was found , in adenocarcinoma and adenosquamous carcinoma versus squamous cell , in tumours ⩾4 cm vs smaller tumours .Similarly, higher tumour/stroma COX-2 IDV ratio was found in stage III-IV with respect to stage I–II cases (Metastatic lymph node involvement was found in 14 out of 69 (20.3%) cases: the percentage of COX-2 tumour positivity was 28.6% in lymph node positive with respect to 35.7% in lymph node negative cases (difference not significant).P=0.003). Similar results were found considering COX-2 IDV ratio as covariate, according to clinical response (P=0.007) (data not shown). On the other hand, the percentage of cases showing COX-2 IDV positivity in the stroma was not correlated per se with response to treatment (data not shown).The percentage of cases showing tumour COX-2 positivity was significantly higher in patients who did not respond to treatment (87.5%) with respect to patients with partial (46.5%), and complete (31.2%) response (P=0.009). Similar results were found considering mean COX-2 IDV ratio according to clinical response  with respect to patients with partial (60.5%), and complete (43.7%) response (response .vs no response). When logistic regression was applied, FIGO stage  and tumour/stroma COX-2 IDV ratio  retained an independent role in predicting a poor chance of response to treatment.In the univariate analysis advanced FIGO stage, and high tumour/stroma COX-2 IDV ratio proved to associated with poor chance of response to neoadjuvant therapy . During the follow up period, 21 out of 99 (21.2%) patients died of disease. For analysis of survival COX-2 status in tumour cells was defined according to the cut-off used in our previous study (P=0.0001). COX-2 IDV in tumour cells were directly associated with risk of death, using COX-2 values as a continuous covariate  (data not shown).Cases showing COX-2 positive reaction in tumour cells   (data not shown).On the other side, cases showing COX-2 positive reaction in stroma inflammatory cells showed a longer OS than COX-2 negative cases: in particular, the 3-year OS was 91% (CI 95%=79–101) in COX-2 positive with respect to COX-2 negative cases who had a 3-year OS of 59% (CI 95%=46–72) (P=0.010) . COX-2 IP<0.0001)  using the ratio between tumour/stromal COX-2 IDV as continuous covariate  . SimilarP=0.0004), while the positivity of stromal COX-2 showed a trend toward a favourable role (P=0.056). Similar results were obtained when using tumour COX-2 and stromal COX-2 as continuous variables (data not shown).In multivariate analysis the positivity of tumour COX-2 retained an independent negative prognostic role for OS As shown in Table 2vs tumour/stroma COX-2 IDV positivity was also analysed in order to test whether a statistically significant difference exists beween the two coefficients. Indeed, in the Cox's proportional hazard model we found that the relative risk of death in patients having a tumour/stroma COX-2 IDV ratio >1 positivity showed a trend to be higher  than that of patients with tumour COX-2  .Moreover, the comparison between the coefficients of relative risk of death for tumour COX-2 positivity vs low (⩽1) tumour/stroma COX-2 IDV ratio. The total amount of cells observed in the stromal inflammatory compartment per unit area was significantly lower in cervical cancer showing high vs low tumour/stroma COX-2 IDV ratio . Moreover, analysis of the data of CD3, CD4, CD25, and tryptase immunostaining, represented in Figure 4+, CD4+, and CD25+ cells is significantly lower in tumours showing COX-2 positive vs COX-2 negative tumour cells . On the contrary, a higher percentage of tryptase positive mast cells was detected in stromal inflammatory cells from tumours with COX-2 positive vs COX-2 negative tumour cells (P=0.013). Interestingly, mast cells endowed with positive tryptase immunostaining were more frequently observed in tumours showing high versus low tumour/stroma COX-2 IDV ratio (P=0.014).Quantification of the total amount of cells as well as the expression of CD3, CD4, CD8, CD25 and tryptase in the stromal inflammatory compartment were analysed in nine squamous cervical cancers randomly chosen among cases with high (>1) Double labelling subtraction consecutive immunostaining of the same COX-2 positive tumour section by anti-COX-2 and anti-tryptase antibodies showed that the vast majority (>70%) of tryptase positive mast cells also express COX-2 (Figure 5In this study, we first reported that in cervical tumours the expression of COX-2 in the stroma inflammatory cells and its relationship with COX-2 expression in tumour cells can be clinically relevant. An inverse relationship between COX-2 expression in tumour cells and the amount of the stroma inflammatory infiltrate in the tumour has been detected.+, CD4+ and CD25+ T cells in tumours with COX-2 positive immunostaining in tumour cells suggesting that the expression of high levels of COX-2 in tumour cells can play a major role in inhibiting host immune functions. In this context, it is noteworthy that COX-2 can up-regulate the immunosuppressive cytokine IL10, while reducing the production of IL12, which is critical for cell mediated anti-tumour immunity (Moreover, COX-2 positive tumours showed a lower percentage of COX-2 positive stromal cells/unit area. This finding seems to be a distinctive morphologic feature of cervical tumours since we could not find such histopathological pattern in ovarian  and assovs low COX-2 staining in tumour cells. Moreover, a large proportion of stromal mast cells was found to express COX-2, suggesting that they are in an activated state, as supported by the observations that induction of COX-2 at mRNA and protein level occurs in antigen-activated mast cells (We also found a higher number of tryptase positive mast cells in tumours showing intense Tryptase positive mast cells have been reported to increase from normal cervical tissue through cervical dysplasias to invasive carcinoma (Therefore, the presence of high COX-2 expression in cervical tumour cells seems to be associated with a scarce cellular infiltrate in the stroma and, notably, with a lower proportion of immunoregulatory cells and a high proportion of tryptase positive mast cells. At present, our data do not allow to indicate the mechanisms and direction of tumour/host interaction relative to COX-2 expression. The possible active role of COX-2 expression in the stromal compartment of the tumour in terms of tumour cell/ stromal cell cross-talk has been recently highlighted . In partFrom a clinical point of view, it is noteworthy that, while COX-2 in tumour cells identifies cervical cancer patients with unfavourable prognosis, COX-2 expression in stroma inflammatory compartment is associated with better clinical outcome, suggesting that COX-2 positive stromal cells can play a role in reducing tumour cell aggressiveness. Therefore, the balance between COX-2 expression in the tumour and in the stroma area, rather than the independent evaluation of each compartment, could offer additional prognostic information. In this context, the use of the ratio between COX-2 in the tumour cells and COX-2 in the stroma cells, seems to be a more effective estimate of the cross-talk occurring between tumour tissue and its stromal compartment (vs high risk of death of disease both in univariate and multivariate analysis. Finally, a very strong correlation between both tumour COX-2 expression and tumour/stroma COX-2 IDV ratio were shown to be highly correlated with response to chemotherapy while, although high COX-2 expression in the stroma was significantly associated with better survival, it failed to directly correlate with response to treatment.Indeed, we showed that the ratio between COX-2 in the tumour cells and COX-2 in the stroma cells was very effective in distinguishing patients with low In conclusion, our study showed the usefulness of assessing COX-2 status both in tumour and stroma compartment in order to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant therapy and unfavourable prognosis. This group of patients could possibly benefit of more individualised treatments, such as selective COX-2 inhibitors, which are already approved for treatment of familial colorectal adenomatous polyposis, and started to be explored in Phase I–II studies .


A new semisynthetic anti-tumour bis-indol compound, KAR-2  with lower toxicity than vinca alkaloids used in chemotherapy binds to calmodulin but, in contrast to vinblastine, does not exhibit anti-calmodulin activity. To investigate whether the modest chemical modification of bis-indol structure is responsible for the lack of anti-calmodulin potency and for the different pharmacological effects, new derivatives have been synthesized for comparative studies. The synthesis of the KAR derivatives are presented. The comparative studies showed that the spiro-oxazolidino ring and the substitution of a formyl group to a methyl one were responsible for the lack of anti-calmodulin activities. The new derivatives, similar to the mother compounds, inhibited the tubulin assembly in polymerization tests in vitro, however their inhibitory effect was highly dependent on the organization state of microtubules; bundled microtubules appeared to be resistant against the drugs. The maximal cytotoxic activities of KAR derivatives in in vivo mice hosting leukaemia P388 or Ehrlich ascites tumour cells appeared similar to that of vinblastine or vincristine, however significant prolongation of life span could be reached with KAR derivatives only after the administration of a single dose. These studies plus data obtained using a cultured human neuroblastoma cell line showed that KAR compounds displayed their cytotoxic activities at significantly higher concentrations than the mother compounds, although their antimicrotubular activities were similar in vitro. These data suggest that vinblastine/vincristine damage additional crucial cell functions, one of which could be related to calmodulin-mediated processes. © 1999 Cancer Research Campaign


The responses of four murine lymphomata, transplanted in their syngeneic hosts and differing widely in their biological properties such as tendency to metastasize and “strength” of tumour specific antigen, to immunotherapy, were investigated. Following the injection of a known number of tumour cells, the mice were treated either by administration of irradiated tumour cells, living BCG or both. In general, the response to BCG alone was small even in the most responsive tumours, irradiated cells were more effective and the best results were obtained by a combination of the two procedures. “Cures” were only obtained with the most antigenic of the tumours tested and then only when the live tumour cells were inoculated intraperitoneally. The effect of these treatments on the rate of growth of tumour when the cells were given subcutaneously was small but the rate at which metastatic spread occurred from the s.c. site was slowed and for one of these tumours this was quite marked.


MUC1 is a membrane bound, polymorphic epithelial mucin expressed at the luminal surface of glandular epithelium. It is highly expressed in an underglycosylated form on carcinomas and metastatic lesions and is, therefore, a potential target for immunotherapy of cancer. The monoclonal antibody HMFG1 binds the linear core protein sequence, PDTR, contained within the immunodominant domain of the tandem repeat of MUC1. The efficacy of murine and humanized HMFG1 (Ab1) used as an anti-idiotypic vaccine was examined in mice transgenic for human MUC1 (MUC1.Tg) challenged with murine epithelial tumour cells transfected with human MUC1. Humoral idiotypic cascade through Ab2 and Ab3 antibodies was observed in MUC1.Tg mice following multiple antibody inoculations in the presence of adjuvant. Impaired tumour growth at day 35 and highest Ab3 levels were found in mice that had received mHMFG1 with RAS adjuvant. However, comparison of Ab3 levels in individual mice with tumour size in all treatment groups did not show a correlation between smaller tumours and increased levels of anti-idiotype antibody. This suggests that the anti-tumour effects of anti-idiotype vaccination are not solely related to the induction of idiotypic antibody cascades and probably involve other mechanisms. © 2000 Cancer Research Campaign


In the present study the tube LAI assay was used to monitor the isolation of the TSA of 4 different types of human cancers. Each tumour antigen was found to be specific for tumours arising in the organ from which the TSA was initially derived and which were histopathologically similar. Immunochemical studies revealed that these molecules co-isolate with normal human HLA antigens and are associated with beta2m. On Sephadex G-150, the majority of the papain-solubilized tumour antigen eluted in the mol. wt range 70,000-150,000. Analysis of this material by SDS-PAGE and 6M guanidine-HC1 column chromatography indicated that the material is composed of smaller subunits with prominent peaks at approximately 40,000, 25,000 and 12,000 mol. wt. Immunoadsorbent affinity chromatography of the solubilized tumour-membrane constituents on AH-Sepharose-linked horse anti-human-beta2m indicated that the tumour antigens, like HLA molecules, contain a beta2m subunit. The specificity of binding of TSA to the immunoadsorbent columns and the immunologically specific abrogation of LAI reactivity were clearly shown. The present study, therefore, indicates that by the isolation of beta2m, human tumour antigens can also be isolated, since human tumour antigens are associated with beta2m. Whether human TSAs may perhaps be modified histocompatibility antigens remains to be answered. Although the change upon malignant transformation in the pattern of the cell-surface proteins expressing the TSA determinant remains obscure, it would appear that for tumours arising within a given organ, a consistent alteration of cell-surface proteins occurs.


Although breast cancer is perceived to be relatively chemosensitive, cytotoxic drug therapy only leads to cure in the adjuvant setting. In advanced disease, primary resistance and inadequate cell kill may be important in determining the lack of a durable response to cytotoxics, but for an individual patient's tumour there is no consistent way of determining the importance of these two factors. An adaptation of Skipper's log cell kill model of tumour response to chemotherapy was applied to serial tumour measurements of 46 locally advanced primary breast carcinomas undergoing neoadjuvant chemotherapy. Assuming a log-normal distribution of errors in the clinically measured volumes, the model produced, for each tumour separately, in vivo estimates of proportional cell kill, initial resistance and tumour doubling times during therapy. After 4 weeks' treatment, these data could then be used to predict subsequent tumour volumes with good accuracy. In addition, for the 13 tumours that became operable after the neoadjuvant chemotherapy, there was a significant association between the final volume as predicted by the model and the final pathological volume (P < 0.05). This approach could be usefully employed to determine those tumours that are primarily resistant to the treatment regimen, permitting changes of therapy to more effective drugs at a time when the tumour is clinically responding but destined to progress.


Chromosome studies have been made of 2 human colonic tumour lines maintained as xenografts in immune-deprived mice. In both tumours human karyotypes were retained, although progressive changes occurred during serial passage. In one tumour, independent gain of a chromosome 19 was found in the stemline and 2 sidelines. In the other tumour there was selection for a sideline containing a particular deleted marker chromosome. The advantages of chromosome analysis in a xenograft system, both for the study of human solid tumour karyotypes and for monitoring the continued presence of the human genome, are discussed.


An accurate forecast of global demand is essential to stabilize the market for artemisinin-based combination therapy (ACT) and to ensure access to high-quality, life-saving medications at the lowest sustainable prices by avoiding underproduction and excessive overproduction, each of which can have negative consequences for the availability of affordable drugs. A robust forecast requires an understanding of the resources available to support procurement of these relatively expensive antimalarials, in particular from the Global Fund, at present the single largest source of ACT funding.Predictive regression models estimating the timing and rate of disbursements from the Global Fund to recipient countries for each malaria grant were derived using a repeated split-sample procedure intended to avoid over-fitting. Predictions were compared against actual disbursements in a group of validation grants, and forecasts of ACT procurement extrapolated from disbursement predictions were evaluated against actual procurement in two sub-Saharan countries.Quarterly forecasts were correlated highly with actual smoothed disbursement rates . Additionally, predicted ACT procurement, extrapolated from forecasted disbursements, was correlated strongly with actual ACT procurement supported by two grants from the Global Fund's first  and fourth  funding rounds.This analysis derived predictive regression models that successfully forecasted disbursement patterning for individual Global Fund malaria grants. These results indicate the utility of this approach for demand forecasting of ACT and, potentially, for other commodities procured using funding from the Global Fund. Further validation using data from other countries in different regions and environments will be necessary to confirm its generalizability. Plasmodium falciparum, the parasite species responsible for a majority of malaria-related mortality worldwide. Modern artemisinin drugs were first developed by the Chinese for the Viet Cong during the Vietnam-American War, on the basis of ancient Chinese fever remedies involving Artemisia annua, the sweet wormwood plant. Artemisinin-based combination therapy (ACT), which utilizes artemisinin along with an additional partner drug, such as amodiaquine, lumefantrine or mefloquine, is now widely considered the most effective treatment for uncomplicated malaria [Since the 1970s, the rise of global resistance to the cheap, ubiquitous antimalarial chloroquine (CQ) has made it imperative to find new, effective drugs to fight There is widespread agreement that accurate forecasts of demand are essential to the success of procurement operations. Agreement is equally widespread that accurately forecasting the demand for antimalarials is nearly impossible"[Following a 2003 critique in the Lancet , and thepossible".The environment surrounding forecasting today remains dynamic, with evolution of national treatment guidelines  and contArtemisia annua to ACT production [Accurate forecasting of demand is critical for ACT, because the key ingredient, artemisinin, is a natural product derived from a plant, creating supply chain challenges . Productoduction . This tioduction  by avoidoduction , and losACT is significantly more expensive than conventional antimalarial medicines , necessiDuring each round, countries apply to the Global Fund for funding by submitting a proposal. Funding for approved proposals is transmitted to recipient countries through a series of disbursements. The magnitude and timing of each disbursement of funding is contingent upon the country's performance in meeting key milestones  as stated in the approved grant agreement, as well as the country's compliance with the Global Fund's policies. Disbursement of awarded funding may slow or be halted if a country's progress is deemed insufficient. Thus, it is hypothesized that the rate at which a grant is disbursed can be considered an indicator of grant progress. For example, if a substantial portion of a particular grant is intended for ACT procurement, a large disbursement rate may be considered indicative of successful procurement , and the magnitude and timing of that procurement can be estimated as a function of the supporting grant's disbursement rate.et al [Past research has investigated factors associated with grant performance. Lu et al  used ranet al  identifiThe analysis presented here describes the derivation of predictive regression models to forecast the rate at which malaria grant funding is disbursed from the Global Fund to recipient countries. The utility of these predictions for estimation of future ACT procurement then is demonstrated using importation records from two sub-Saharan African countries.Data on Global Fund grant awards  and disb(1) The rate at which funding was disbursed from the Global Fund to the recipient country (the disbursement slope)(2) The date on which disbursement began (the disbursement line intercept)Predictive regression models may include variables that are not statistically significant and exclude others that are, since statistical association does not necessarily indicate useful predictive value . To prevTwo models were constructed, one to predict the disbursement slope, and the other to predict the intercept of the disbursement line. Variable selection for each model occurred through a series of 99 model fitting and testing repetitions Figure . In eachAfter variable selection, final models predicting slope and intercept were fit using all 97 grants in the derivation group. These multivariate regression models then were used to predict the disbursement slopes and intercepts for the unused 33 grants in the validation dataset. Each grant's predicted slope and intercept were used to generate a line representing estimated funding disbursements, extending until either reaching the grant's maximum potential or expiration date, whichever came first. The expiration date was calculated by adding the planned length of the grant to the grant start date. Predicted quarterly disbursements for 2003–2007 were summed across all grants, and these predictions were compared against summations of the actual disbursement lines fit to grant disbursement history  as well To examine how actual grant performance differed from planned disbursement, the amount of funding requested by recipient countries from the Global Fund for each year of the grant was extracted from the 146 approved malaria grant proposals listed on the Global Fund website . Since tEach approved malaria Global Fund proposal was read to determine the proportion of each year's request intended for procurement of drugs for uncomplicated malaria. The percentage of the grant intended for procurement of drugs for uncomplicated malaria was then calculated separately for Phase 1 (years 1–2) and Phase 2 (years 3–5) of the grant, if applicable. If an itemized drug budget was not available in a particular proposal , average values were substituted from either the applicant country's other grant proposals, when possible, or otherwise from all grants in that funding round.Multiplying predicted disbursements by the percentage intended for procurement of drugs for uncomplicated malaria resulted in a prediction of the amount of funding available for ACT procurement for each grant. Because ACT orders are customarily delivered to countries in staggered shipments over the course of a year, forecasted demand for ACT was lagged one year behind predicted disbursements from the Global Fund to recipient countries. For example, hypothetical country × might receive a disbursement of $1 M on 1 Jan 2005; of this $1 M, the Global Fund grant calls for 80% of 2005 funds to be used for purchase of drugs, and therefore, the country immediately places an $800,000 ACT order to cover yearly drug requirements. ACT arrives in staggered shipments throughout the year, until all $800,000 worth have been delivered by the end of 2005. Once the amount of funding for drugs had been predicted for each grant, amounts were summed across all grants for a particular country to determine predicted funding available for ACT procurement in that country, and globally, to examine the amount of ACT procurement the Global Fund may fund each year.To validate predictions of country-specific ACT demand generated using this approach, data were collected on ACT importation supported by specific Global Fund grants. Validation requires a complete set of importation records for all the ACT procured using Global Fund grants, including the date of all deliveries, total volumes, and their prices. Although partial records were obtained from several countries, comprehensive importation records were compiled for only two grants: one awarded during the first funding round to a Southern African country and one awarded in Round 4 to a country in East Africa. Data on all ACT orders placed and shipments received were obtained from sources including the National Medical Stores, the National Drug Authorities, and the Ministries of Health or Malaria Control Programmes. Cumulative dollars spent on ACT over time were summed from these records and compared to forecasted procurement.Through Round 7, 143 malaria grants had been awarded to 158 principal recipients (15 grants had two recipient organizations) in 75 countries or multi-country cooperatives. Fitting regression lines to each grant's cumulative disbursement resulted in smoothed quarterly estimates of total disbursements from the Global Fund to recipient countries . Disbursement began later for grants in the Southeast Asian WHO region, grants requested for longer periods of time , and for countries with greater numbers of malaria grants.Using these models, disbursement slopes and intercepts were predicted for the 33 grants in the validation set. These predictions were compared against the smoothed actual disbursement lines and the disbursements planned in the grant application Figure . PredictThe antimalarial procurement plans outlined in grant proposals were used to estimate the fraction of disbursements funding ACT procurement for each grant. Multiplying these fractions by the predicted yearly disbursement amounts for each funding round produced the round-specific global funding estimates depicted in Table To examine whether the model could successfully predict actual procurement supported by individual Global Fund grants, ACT procurement predictions were validated against procurement records from two sub-Saharan countries, one supported by a Round 4 grant and the other by a Round 1 grant. Actual procurement records indicated that the Round 4 grant Figure  supporteAn accurate forecast of global demand is dependent upon a thorough understanding of the availability of resources to support antimalarial procurement, in particular from the Global Fund, at present the single largest funding source. This analysis derived predictive regression models that successfully forecasted the rate and timing of malaria grant disbursement from the Global Fund to recipient countries. Additionally, it was demonstrated that smoothed disbursement rates provided accurate prediction of ACT procurement funded by specific malaria Global Fund grants in two countries. More data from other countries in different regions and epidemiological and political contexts will be necessary to further validate this approach.This investigation constructed models to predict the rate at which grants were disbursed from the Global Fund to recipient countries, rather than the exact days and magnitudes of discrete disbursements. By predicting disbursement rate, rather than discrete disbursements, it is acknowledged that precise prediction of actual quarterly disbursements – with their stochastic temporal patterning Figure  – is impOne potential problem with using the disbursement rate as a proxy for the ACT procurement rate is that the Global Fund considers the entirety of a grant when determining performance, so an underperforming component  may delay disbursement of a grant even if ACT procurement is in fact on schedule. However, the subsequent funding delays also will affect all activities funded by the grant, including ACT procurement, so a slower rate of disbursement will remain associated with the procurement rate even though the causality of the association is reversed. Additionally, modeling each grant individually and aggregating for a global total allows a "portfolio" approach to forecasting, in which random error in estimation of funds in one country is balanced by the random error in another.Some, although not all, of the factors identified here, like measures of government effectiveness and grant size, were similar to those identified in prior investigations ,13. CertThis methodology provides a means of forecasting ACT procurement driven by Global Fund grants over their lifespan which appears to be more accurate than reliance upon planned funding, although the magnitude of the increase in predictive accuracy varies by year Figure . Past exUltimately, a global forecast useful for planning drug production and artemisinin cultivation must predict demand for ACT on a product- and dosage-specific basis. The Global Fund-driven ACT funding predicted by this model provides a foundation for such a robust, accurate demand forecast. Conversion of ACT funding into product- and dosage-specific treatments, and subsequently into active pharmaceutical ingredients and artemisinin requirements, may be performed using information contained in Global Fund proposals specifying exact products to be procured, country malaria guidelines dictating public sector product choice, and epidemiological and demographic characteristics which, in the absence of other information on planned procurement, can be used to estimate the relative age-specific breakdown of dosages required. These calculations must include consideration for how the price of ACT may have decreased since the country planned its ACT procurement in its Global Fund proposal, thus altering the volume of drugs that can be purchased with the predicted disbursements.An accurate forecast of future Global Fund supported ACT procurement is an important component of robust global demand estimation. However, estimating total global demand for antimalarials must also consider the role played by other important public sector funding organizations, including the World Bank, the United States President's Malaria Initiative, and UNITAID, as well as the growing importance of private sector procurement. Additionally, the methods described here cannot predict procurement funded by grants not yet awarded by the Global Fund. These components of the global demand require separate forecasts.This report describes an approach to predicting the size and timing of the Global Fund monetary disbursements that will continue to be used to drive the bulk of ACT purchases, at least until implementation of a global subsidy for ACT, currently being discussed by the international malaria community, becomes a reality. The evidence suggests that this approach is more accurate than forecasting based on planned funding from the grant proposals. Approaches like this one are useful tools for building comprehensive, robust models of global ACT demand that can help ensure affordable, universal access to these life-saving drugs.JMC participated in study conception, study design, literature review, model development, statistical analysis, analysis of results, and preparation and editing of the final report. IS participated in study conception, study design, analysis of results, and editing of the final report. MEO participated in study conception, study design, model development, analysis of results, and editing of the final report.Statistical models predicting the rate of grant disbursement and data of initial disbursement. The data provided represent the final predictive regression models selected through the repeated split-sample model fitting procedure.Click here for file