There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated – NCAM1 expressing – “blastema” phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets. This has allowed, for the first time, a robust definition of the CIC/CSC phenotype leading to discovery of therapeutic targets for WT eradication. Molecular analysis of CIC/CSCs in WT Xn revealed distinct genes, signaling elements and miRNAs likely relevant to their function as undifferentiated “blastema” cells. The generation of xenografts carrying cell-repopulating human developmental malignancies with aggressive phenotypes may serve as a more reliable platform compared to cell-line-based xenografts or in vitro propagation for the discovery of CICs and future targeted therapies.Herein, we have propagated human WT 7 cells) we found an extremely low frequency of xenograft formation: 2/20 mice injected with tumour cells from four different WT sources developed tumours 6 months after transplantation. These numbers are in line with previous reports  to initiate tumour Xns in mice . In contrast, primary Wilms' tumour fragments (2 × 2-mm-minced pieces) derived from 10 different WT patients resulted in robust Xn formation upon grafting; graft take was observed for 8 out of the 10 WT sources within 2–6 months . We therefore used these xenograft models containing primary human WT to determine the existence of CIC/CSCs. Xenografts are critical for this study because of the difficulty to routinely obtain paediatric primary solid tumours, which are less frequent than adult cancers.To study human WT initiating activity, we injected primary WT cells into NOD/SCID immunodeficient recipient mice. Primary WT cells were obtained from a cohort of tri-phasic favourable histology WT lacking mutations in β-catenin, WT1 or WTX, which represent genetic alternations in a subset of WT  were mostly sufficient to initiate later generation xenografts (>4th generation). Overall, these Xns, termed propagatable WT Xns (p-WT Xn) showed increased WT-initiating activity, serving as a WT reservoir for the study of tumour-initiating cells. The experimental outline for Xn generation is depicted in Supporting Information Having established 1st generation Xns from tissue samples obtained from eight different WT sources, we then analysed whether these Xns could be initiated and propagated following dissociation of Xn tissue and injection of the derived single cell suspension. Interestingly, we found that 1st generation Xns established from four WT sources  could be readily initiated and propagated in mice through the injection of 0.5–1 × 10in vitro assays of primary cell cultures of WT  in WT blastema and non-blastema components revealed significantly higher numbers of Ki-67+ cells in the blastema. This observation was repeated in both primary WTs and their derived Xn  revealed all WT tri-phasic components  but with enriched blastema compared to parental tumour, indicating the generation of a blastema-predominant WT. Interestingly, patients with recurrent blastema-predominant WT have been classified at very high risk with low survival rates  were continually used. In contrast, 100-fold fewer CD-133 negative cells were required to propagate and sustain late generation Xns of p-WT. Finally, proteasome and proliferating cell nuclear antigen (PCNA) protein levels increased in p-WT Xn compared to parental tumour tissue , showed an opposite phenotype: np-WT Xn did not expand the blastema and featured decreased NCAM, WT1 and Ki67 immunostaining  and loweAll subsequent experiments were performed on p-WT Xn.in vivo WT formation  and left (NCAM−) flanks of NOD/SCID mice. Strikingly, only the NCAM+ fraction was able to form tumours in NOD/SCID mice , while histology and immunostaining of these tumours disclosed a blastema compartment that expressed NCAM, but also NCAM− differentiated epithelial-like structures and glumeruloid bodies, recapitulating the histology of both the parental WT and the p-WT Xn from which they were derived , stemness factors  and WT poor prognostic factors  compared to NCAM− cells  on extracts of p-WT Xns early after initiation by sorted NCAM+ cells, un-sorted p-WT Xn and normal kidneys , the embryonic form of NCAM . We also observed NCAM+ALDH1+ cells to overexpress PBX1, a renal stromal lineage marker  and a large fraction of ALDH1− cells supporting the differentiation capacity of injected ALDH1+ cells. (+ALDH1+ compared to NCAM+ALDH1− cells in accordance with the CSC phenotype (+ALDH1+ cells were further sorted into NCAM+ALDH1+ and NCAM+ALDH1− WT cells and injected to secondary recipients (s.c. in NOD-SCID or NOG mice), in serial dilutions. Consequently, only the NCAM+ALDH1+ were capable of tumour initiation suggestive of their in vivo self-renewal capacity  with c-MYC elevated in NCAM+ALDH1+ tumour. Other proteins, such as NQO1, and to a lesser extent P53, were increased in control paediatric rhabdoid tumour, further indicating the specificity of the pathways active in WT and importantly in the NCAM+ALDH1+ CIC derived tumour xenografts. Thus, highly enriched WT CIC/CSCs are likely to feature signaling molecules and oncogenes distinct from those generally observed in WT and other closely related paediatric tumours.We next determined that biological properties of the NCAMTpSer473 , the proental WT . Moreoveental WT . c-Jun s+ALDH1+ derived p-WT Xn and human foetal kidney, resulting in the identification of 71 statistically significant, differentially expressed microRNAs  populations in which approximately 80% of cells express NCAM. As an antigen-negative control, we used the DLD cell line, which does not express NCAM  or with saline only  with high NCAM expression levels (HNWT), with an average of 90, 72 and 84% positive cells, respectively, and one (W016 Xn) with a relatively low NCAM expression level (LNWT) with an average of 50% NCAM-positive cells. For the initial experiments mice bearing tumours of each tumour type were divided into three treatment groups: (1) high doses of lorvotuzumab-mertansine (360 µg DM1/kg or 18 mg conjugate/kg); (2) low doses of lorvotuzumab-mertansine (180 µg DM1/kg or 9 mg conjugate/kg); (3) saline control. All groups were treated intravenously once a week for three consecutive weeks and monitored for tumour growth once-twice a week. Preliminary results determined the optimal dose (360 µg DM1/kg or 18 mg conjugate/kg), which was used in all subsequent larger scale experiments. To determine the effect of lorvotuzumab-mertansine on both HNWT and LNWT, mice were randomized into saline treated and lorvotuzumab-mertansine treated groups (n = 9) injected with saline, both HNWT and LNWT tumours grew in an exponential pattern. A complete to nearly complete regression of tumours was observed in mice bearing HNWT (n = 10) after 3 weeks of lorvotuzumab-mertansine injections (n = 4), we detected an initial significant reduction of tumour size (>50%) followed by a plateau, possibly due to residual NCAM− cells that remained post treatment but lacked WT-initiating activity and hence failed to resume tumour growth (n = 15); (2) unconjugated antibody ; (3) saline as a control (n = 11). Tumour treated with lorvotuzumab-mertansine were completely eradicated following 3 weeks of treatment while tumours treated with HuN901 or saline showed rapid tumour growth , close examination of the propagatable xenografts revealed a shift towards highly proliferating blastemal predominant tumours that overexpressed renal stemness genes and those known to predict poor outcome in WT patients. The validity of using these xenografts is supported by clinical observations that tumours with a predominant blastemal component are aggressive, while tumours with blastemal predominance after chemotherapy are classified as “high risk” and patients with blastemal predominant tumours after chemotherapy have a high relapse and low survival rates . These microRNAs have been closely linked with epithelial differentiation or maintenance and regulation of EMT in several malignancies  alongside the acquisition of stemness through expression of pluripotent (OCT4) and Polycomb  genes /Akt signaling signified in all of our analyses. Of note, it is now apparent that the Akt/mTOR signaling network plays a key role in cancer stem cell biology and that CIC/CSCs display preferential sensitivity to the inhibition of this pathway  and that there was an intimate link between WT-initiating activity and enrichment level for these genes. In addition, a number of the genes found to be elevated in the WT CIC population  are expressed in the Osr1+ metanephric mesenchyme and are not restricted to the nephron progenitor population , one can assume a common undifferentiated renal mesenchymal cell of origin for all three cell types. Indeed, it is generally accepted that both the stroma and the nephron progenitors arise from the metanephric mesenchyme  CSCs, isolated more than a decade ago  of WT cell cultures that varied in the extent of NCAM expression and the in vivo targeting of multiple WT xenograft models lead to dramatic results: WT bearing high NCAM expression were completely or significantly eradicated (n = 10). Two tumours that relapsed were associated with elevated NCAM levels and a residual NCAM+ALDH+ fraction and were completely eradicated by an additional course of lorvotuzumab-mertansine suggesting the NCAM+ cells were indeed responsible for tumour recurrence. Treatment of low NCAM expressing WT xenografts (LNWT) with low dose lorvotuzumab-mertansine resulted in attenuation of tumour growth, however without reduction in tumour size, possibly due to incomplete abolition of NCAM+ cells harbouring WT-initiating capacity. However, treatment of LNWT with high dose lorvotuzumab-mertansine resulted in reduction of tumour size followed by a plateau suggesting that once all NCAM+ cells were eliminated, the remaining NCAM− cells comprising most of these xenografts lacked CIC capacity. Since LNWT xenografts and primary WT mostly harbour similar NCAM levels, utilization of the immunoconjugate to eliminate WT-initiating activity and conventional chemotherapy for non-CIC cancer cells is likely to be more clinically relevant.Perhaps the most important translational contribution of human CSC/CIC research is that it unveils specific biomarkers that can be targeted e.g. hepatoblastoma, neuroblastoma and mesodermal tumours as rhabdomyosarcoma) recapitulate development, NCAM targeting might prove useful in a wide spectrum of developmental malignancies.We propose that our findings may be relevant to other paediatric solid tumours representing a distinct biological entity, with great similarities among themselves and marked differences from adult counterparts. Accordingly, taking into account that NCAM marks stem/progenitor cell compartments in various developing human organs  subcutaneously into the flanks of 5–8-week old NOD/SCID mice. This procedure was attempted for tumour cells dissociated from the primary tumour as well; however, engraftment rates were less than 10%, in contrast to the 100% engraftment obtained with dissociated cells from WT propagated in Xn. In an attempt to optimize conditions for WT, Xn formation in immunodeficient mice from dissociated single cells, in addition to transplanting freshly dissociated cells, Xn-derived cells were cultured as adherent cultures in Iscove's Modified Dulbecco's Media (IMDM) supplemented with 10% foetal bovine serum (FBS) or as in suspension as “spheres” in serum-free medium . Cells that had been grown as adherent cultures and those grown as spheres were resuspended in 100 µl SFM/Matrigel and injected into either NOD/SCID or SCID/BEIGE mice in triplicate, as described above. To assess tumourigenicity of WT Xn cell subpopulations dissociated tumour cells were counted, sorted according to the chosen markers, then re-suspended in 100 µl 1:1 SFM/Matrigel, and injected s.c. in serial dilutions into the right (+ cells) and left (− cells) flanks of 5–8 week old female NOD/SCID mice. Engrafted mice were inspected bi-weekly for tumour appearance by visual observation and palpation. Mice were sacrificed by CO2 inhalation at a tumour diameter of 1.5 cm or at 6-month post transplantation. Following resection, WT xenograft tumours were divided into five pieces, one was fixed and paraffin embedded for histologic staining of NCAM, WT1 and Ki67 to determine recapitulation of the parental tumour phenotype, three small pieces were kept in liquid nitrogen for RNA, DNA and protein extraction and the fifth piece was taken for formation of single cell suspension, followed by serial injections into the flanks of secondary mice (n = 3 in each passage). This was repeated at least three times. All animal experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of animals and with an approved animal protocol from the Sheba Medical Center Animal Care and Use Committee.Initial WT xenografting to 5–8 weeks old, female, nonobese nondiabetic immunodeficient mice (NOD/SCID) was performed as previously described  at 20,000 cells/well in serum-free DMEM-F12 (Invitrogen), supplemented with 10 ng/ml basic fibroblast growth factor (bFGF) and 20 ng/ml epidermal growth factor . For adherent growth, cells were cultured on conventional tissue culture plastic in IMDM medium supplemented with 10% FBS and the following growth factors: 50 ng/ml bFGF, 50 ng/ml EGF and 5 ng/ml.Single cell suspensions from primary WT or WT Xn tissues were grown in two culture conditions as mentioned above: in SFM under low attachment conditions where they formed free-floating spheres, or in SCM as adherent cultures for PROBLEM:in vivo tumourigenicity of primary WT cells and lack of cell lines, hampering the ability to functionally define CICs via their enriched capacity to regenerate cancers in xenograft mouse models.Cancer initiating cells/cancer stem cells (CICs/CSCs) are thought to be primarily responsible for tumour initiation, their persistence after conventional chemo/radiotherapy may lead to disease recurrence and hence their therapeutic targeting may be essential to ensure complete tumour eradication. WT, the most frequent tumour of the genitourinary tract in children, has served for decades as a general biologic model to study the intersection of cancer and development. Given the considerable differences in tumour biology between adult and paediatric cancers, the existence of cancer initiating and propagating cells in paediatric solid tumours including in WT is questionable. Identification of CICs in human WT is especially challenging due to minimal RESULTS:Our article reports the establishment of a human WT xenograft model system mimicking aggressive malignancy in patients and affording the opportunity to isolate undifferentiated “blastemal” CICs in WT, their prospective isolation in line with the functional criteria of CIC/CSCs and in depth molecular characterization showing elevated transcript levels of renal “stemness” and poor WT prognosis genes, preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family a known regulator of EMTs. Finally, targeting of a cancer cell population enriched for CIC activity leads to dramatic tumour eradication or attenuation in human WT xenograft models.IMPACT:In this paper, we report for the first time the discovery of CIC/CSCs in human WT xenografts, their characterization and therapeutic targeting. This both provides molecular insights into tumour biology and opens practical new venues to explore in the treatment of WT patients; Even though there have been successes in good prognosis WT treatment, our ultimate goal is to reduce the burden of conventional chemotherapy so as to decrease its long-term complications in this patient group. For the group of patients with relapsing bad prognosis disease treatments developed to eradicate CICs may have direct impact on morbidity and mortality.Prkdcscid/NcrCrl) by s.c. inoculation of Xn-derived freshly dissociated cells with either high  or low (LNWT – W016 WT Xn) NCAM expression. Four weeks later, mice bearing palpable tumours of each tumour type were randomized in the first series of experiments into three groups: saline treated, high dose lorvotuzumab-mertansine treated (360 µg DM1/kg or 18 mg conjugate/kg) and low dose lorvotuzumab-mertansine treated (180 µg DM1/kg or 9 mg conjugate/kg). Each mouse received bolus injections into the tail vein once a week for three consecutive weeks. Tumour size was calculated in three dimensions: L (length) × W (width) × H (height), using a caliper. Tumour dimensions were measured twice a week from the first conjugate injection until either tumours size reached 7000 mm3 or mouse death. Following determination of the preferred dose of lorvotuzumab-mertansine (360 µg DM1/kg or 18 mg conjugate/kg), in all the following larger scale experiments, mice were treated and followed as described above. To determine the effect of lorvotuzumab-mertansine on HNWT and LNWT, mice bearing tumours were randomized into two groups, either treated with lorvotuzumab-mertansine or with saline as control. To compare the effect of lorvotuzumab-mertansine with that of the unconjugated form of this Ab (HuN901) as well as to determine possible toxic effects, mice bearing palpable tumours were randomized into three treatment groups: saline, lorvotuzumab-mertansine (360 µg DM1/kg), and unconjugated Ab (HuN901-13.3 µg/kg). During the experiment, mice weights were measured and documented.WT xenografts were formed in 5–8 week old, female NOD-SCID mice  between WT cell populations were evaluated using the non-parametric, one-sided Sign test. Statistical differences of all the “


Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO2] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods , preparations of CoxD revealed a mean KM value of 0.69±0.14 mM ATP and an apparent Vmax value of 19.3±2.3 nmol ATP hydrolyzed min−1 mg−1. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states , preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10–16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO2] cluster assembly are discussed.CoxD of the α-proteobacterium  After the cell-harvest in the late exponential phase, the bacteria were stored at −80°C until use.Wild-type and mutant strains were grown in 70-liter fermentors  in a mineral medium E. coli K38 pGP1-2/pETMW2, which carries the coxD gene on the plasmid pETMW2 −1) and kanamycin (100 mg l−1). The expression of CoxD was induced at an optical density (OD600) of 0.7 with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and a subsequent heat shock (1.5 h at 42°C). Due to the high-level expression, CoxD appeared entirely in inclusion bodies. Crude extracts were centrifuged to sediment the inclusion bodies . Contaminating material was removed from the inclusion bodies employing two washing steps. For that purpose they were homogenized in 20 mM Tris/HCl (pH 8.0) supplemented with 1% Triton X-100 and 150 mM NaCl, and centrifuged as above. Finally, the detergent was removed by two washes in Tris/HCl.The construct 2EDTA, 1 mM glycine, and 100 mM Tris and stirred magnetically at 4°C for 1 h. None solubilized material was removed by centrifugation  followed by ultra-filtration employing polyvinylidene fluoride membranes (0.22 µm). Solubilized CoxD was refolded by ∼50-fold dilution employing pulsed renaturation. For that purpose, the following standard protocol or variations thereof was employed. CoxD solution (60 ml) was added drop wise (∼ 50 µl) to 3,000 ml of ice cold, filtered, magnetically stirred 20 mM Tris. Immediately after the dilution step the pH was adjusted to 9.0 with 1 N HCl and kept for 24 h at 4°C. Then, the pH was gradually decreased to 8.0 in increments of 0.2 per 24 h. The resulting solution composed of 2 mM Tris, 160 mM urea, 200 µM β-mercaptoethanol, 200 µM DTT, 20 µM reduced glutathione, 20 µM EDTA, and 20 µM glycine is being referred to as refolding buffer. Aggregation of CoxD in the course of refolding was checked regularly through the inspection of its UV/VIS-absorption spectrum for the appearance of turbidity.In order to solubilize CoxD, washed inclusion bodies were suspended in a mixture composed of 8 M urea, 10 mM mercaptoethanol, 10 mM dithiothreitol, 1 mM reduced glutathione, 1 mM Na2PO4/KOH (pH 8.0) contained in a quartz cell  was positioned under N2 in a circular-dichroism (CD) spectrometer  equipped with an Jasco CDF-426S Peltier thermostat, set to 20°C. CD spectra were acquired from 176 nm to 260 nm using the step mode with a step size of 0.2 nm, a band width of 1 nm, and an integration time of 2 s. Solvent spectra, obtained under same conditions, were subtracted and the data were smoothed using a Savitzky Golay filter CoxD in 10 mM KH2ATP) or 1 mM disodium guanosine-5′-triphosphate (Na2GTP). The hydrolysis of MgATP or MgGTP with time was assayed in two ways. The inorganic phosphate released was determined according to published procedures 340, NADH = 6.22 mM−1 cm−1) employing a modified MgATP-recycling enzyme linked assay 2ATP, 1 mM Mg-acetate, 2 mM phosphoenolpyruvate, 0.2 mM nicotinamide adenine dinucleotide (NADH), pyruvate kinase (1.75 U ml−1), and lactate dehydrogenase (2.5 U ml−1). Reactions were started through the addition of refolded CoxD.NTPase activity was assayed at 25°C in 100 mM Tris/acetate (pH 8.0) supplemented with 1 mM magnesium acetate and 1 mM disodium adenosine-5′-triphosphate . As a reference for molecular mass, a sucrose gradient in a separate tube received 2.5mg of CO dehydrogenase of O. carboxidovorans  and was included in the same run. After fractionation the contents of protein O. carboxidovorans CO dehydrogenase , yeast alcohol dehydrogenase , bovine liver catalase , and bovine serum albumin  were used as molecular mass standards.Linear sucrose gradients  were formed in polycarbonate tubes. Refolded protein solutions were concentrated by ultra-filtration at 4°C employing membranes with a nominal molecular weight limit of 5,000 , layered on top of gradients and centrifuged for 20 h at 130,000× −1 in 100 mM Tris/acetate (pH 8.0) were applied to freshly glow-discharged holey carbon grids  covered with an additional thin layer of continuous carbon. After 45 s at room temperature excess of liquid was blotted, and the sample was negatively stained using 2% (w/v) uranyl acetate for 15 s. Samples were evaluated at 28,500-fold magnification using a Philips CM100 electron microscope operated at 100 kV. For 2D analysis, data was collected using a Tecnai Spirit electron microscope equipped with a 2 k Eagle CCD™ camera  operated at 120 kV. Images were recorded automatically using the Leginon system For TEM imaging, 3.5 µl of refolded CoxD  and the program package MODELLER Rhodobacter capsulatus61 of CoxD; deletion of 25 aa (which are absent from BchI) in the H2-insert of CoxD  yielded the cell free crude extract.Bacteria were disintegrated by 5–15 passages through a French pressure cell  at maximum pressure. Subsequent centrifugation  = 30.035 mM−1 cm−1]. All chemicals employed were of analytical grade and purchased from usual commercial sources.Samples for analysis on SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis) were prepared by boiling for 4 min in 160 mM Tris/HCl (pH 6.8) containing 1% (w/v) SDS and 2.5% (w/v) 2-mercaptoethanol. Protein samples separated by SDS-PAGE For sequence alignments the UVa FASTA Server (fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml) was employed.coxD, coxE and coxF, which are essential for the posttranslational maturation of the [CuSMoO2] active site cluster of CO dehydrogenase 2 plus CO2 or with organic substrates. CoxD was synthesized under chemolithoautotrophic conditions with CO as a sole source of energy and CO or CO2 as carbon source  for solubilization (data not shown). We have not been able to identify suitable detergents and conditions to solubilize CoxD in a folded state from membranes of its native host O. carboxidovorans. Tween 20, Triton X-100, n-dodecyl-β-D-maltoside, and n-octyl-β-D-glucopyranoside were not effective in the removal of the protein from cytoplasmic membranes. CoxD is only a minor component in the cytoplasmic membrane of O. carboxidovorans . Facing this situation, attempts were made to overexpress CoxD in E. coli, which was anticipated to be more convenient and to give better yields of functional and stable protein than detergent solubilization of native CoxD from cytoplasmic membranes of O. carboxidovorans.The clustered genes n source , lane A ynthesis , lane B.D mutant , lane D.D mutant , it appeD mutant , lanes PD mutant , lanes SE. coli K38 pGP1-2/pETMW2. The protein entirely appeared in inclusion bodies, which might be ascribed to its high expression level. Adjustments of the bacterial cultivation to turn down the expression  did not lead to a soluble product. We thus decided to take advantage of the high productivity of E. coli under the established conditions and of the high degree of purification of the recombinant CoxD in inclusion bodies. The molecular mass of the recombinant CoxD polypeptide of 33 kDa apparent from SDS-PAGE  at 4°C was perfectly stable for at least one month. Refolded CoxD kept in 20 mM Tris/HCl (pH 8.0) or in 100 mM Tris/acetate (pH 8.0) remained soluble and did not develop aggregates in the tested temperature range of 4–30°C. On the other hand, our research was hampered to a certain extent by a pronounced sensitivity of refolded CoxD to different salts and chemical compounds which led to precipitation of CoxD within minutes to hours . CoxD kept at pH 8–10 in 20 mM Tris/HCl containing 10 mM MgCl2 at 25°C was completely precipitated after 20 min.; at 4°C the stability of CoxD could be significantly improved, and complete precipitation took about 10 hours. To maintain CoxD in solution it was also essential to add the protein to the salt solution and not vice versa.Refolding of CoxD from inclusion bodies could be achieved by solubilization with urea followed by stepwise dilution using the protocol detailed in the methods section. The absence of any turbidity in the absorption spectra of CoxD recorded at the beginning and at the end of the refolding process indicated that the protein did not precipitate under these conditions . CoxD kei.e. less α-helix and more β-sheet), present a molten globule state or an intrinsically disordered state in coexistence with CoxD refolded into the ordered structure. The inspiring review article by Dunker et al. i.e. ATPase activity, oligomerization) can arise from any of these states remains to be elucidated.The presence of secondary structural elements in CoxD solubilized from inclusion bodies and refolded was apparent from circular dichroism (CD) spectroscopy . The CD 2+-ions  and Vmax values (nmol ADP min−1 mg−1) were 0.58 and 17.72, 0.63 and 18.18, and 0.85 and 21.9, respectively. From this data a mean KM of 0.69±0.14 mM ATP and a mean Vmax of 19.3±2.3 nmol ATP hydrolyzed min−1 mg−1 can be calculated. ATP-γ-S is a slowly hydrolysable analogue of ATP. It very effectively competed with ATP for CoxD  favorably compare to the activities of recombinant ChlI from Arabidopsis thaliana  from [Cu(I)(thiourea)3]Cl, under reducing conditions This report has established CoxD as a AAA+ ATPase  and 6A w


Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup.The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys.Both species-specific and antigen-specific binding of the conjugates were demonstrated 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies.We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the  Squamous cell carcinoma of the head and neck region (HNSCC) makes up approximately 5% of all solid cancers in Europe and North America. In 2008, the worldwide incidence of oral cavity cancer was estimated to 263,900 cases . SquamouAlready at diagnosis, 60% of HNSCC patients have metastatic disease .A cornerstone in the work-up of patients with HNSCC is accurate imaging of the primary tumour and metastases, since tumour stage strongly correlates to prognosis and hence the chosen treatment. The imaging methods available in clinical use today are computed tomography (CT), magnetic resonance imaging (MRI), ultrasound and fludeoxyglucose-positron emission tomography/CT (FDG-PET/CT). FDG-PET/CT is a valuable method when searching for metastases and primary tumours. However, its specificity is limited due to increased FDG uptake in benign lymphadenitis, other inflammatory processes or after treatment. Also, small clusters of cancer cells can escape diagnosis due to the limiting size factor of the PET/CT technique . More sp2 fragment antibodies have two antigen-binding Fab portions linked together by disulphide bonds and therefore are divalent with a molecular weight of about 110 kDa. Fragments of the intact antibody may exhibit shorter half-life, faster blood clearance and better tumour penetration, resulting in faster and higher signal-to-noise ratio. Furthermore, the removal of the Fc segment could reduce the non-specific distribution in vivo of the mAb via Fc receptors found on normal cells [One promising method that may improve the management of patients with head and neck malignancies is radio-immunodiagnostics, which combines the high sensitivity and resolution of a PET or single-photon emission tomography (SPECT) camera with the tumour specificity of an antibody or antibody fragment. Such radiolabelled monoclonal antibodies (mAbs) can be utilized not only in the detection of metastatic lesions and primary tumours but also in applications that include monitoring of tumour response to therapy, dosimetric calculations, and therapy . Althougal cells . Howeveral cells , and theal cells .2 fragments of U36 radiolabelled with 125I were characterized in vitro and in vivo and compared to the intact antibody. Tumour-to-blood ratios and tumour penetration were increased for Fab and Fab2 compared with the intact antibody [Receptors on the surface of cells can serve as targets for antibodies and antibody fragments, and if they are expressed specifically by tumour cells, they are excellent targets for radio-immunodiagnostics. There are several promising receptors for radio-immunodiagnostics such as EGFR and isoforms of CD44. CD44 belongs to a family of glycoproteins serving as surface receptors for extracellular matrix components, mainly hyaluronic acid. The receptors are involved in migration and adhesion of cells. Twenty exons encode CD44, and exons 6 to 15, namely variable exons 1 to 10 (v1 to v10), can be alternatively spliced with diverse end products . Most tiin vitro characterized fully human Fab fragments, derived from the HuCAL PLATINUM library, which specifically recognize v6-containing isoforms of CD44 [Escherichia coli. Furthermore, the recombinant origin of the antibody fragments enables future reformatting to provide molecules that are optimized for either diagnostic or therapeutic applications [To date, few antibody fragments toward CD44v6 have been reported, and none of them are fully human with a thoroughly characterized binding site. Thus, to facilitate improved targeting of CD44v6, we have selected  of CD44 . Clones ications . One maiications .in vivo tumour targeting capabilities of the novel, fully human, CD44v6-targeting antibody fragment AbD15179. The Fab fragment was first evaluated for species specificity using surface plasmon resonance (SPR) and was then labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Specific binding and internalization of labelled conjugates was evaluated in CD44v6-expressing SCC cells in vitro, and in vivo binding specificity and biodistribution studies were then performed using 111In- or 125I-labelled Fab fragments in a dual-isotope study in tumour-bearing mice with xenografts of varying CD44v6 expression.The aim of this study was primarily to assess the 111In-labelling was separated by size-exclusion chromatography on a NAP-5 column  pre-equilibrated with purified  water. It was then freeze-dried and stored at −20°C before use in order to facilitate buffer exchange and concentration adjustments.The CD44v6-binding Fab fragment AbD15179 was supplied from AbD Serotec . It was selected from an array of 13 different human antibody fragments, all recognizing CD44v6. The selection and production of this antibody fragment have been described previously . The natTwo 16-mer peptides corresponding to the human (CGNRWHEGYRQTPREDS) and mouse (CQNGWQGKNPPTPSEDS) forms of the CD44v6 antigen used to generate AbD15179 were produced by peptide synthesis . The peptide antigens were individually conjugated to bovine serum albumin (BSA) through N-terminal cysteine residues. Purified recombinant CD44v3-10 . H314 has demonstrated an average antigen density of approximately 650,000 CD44v6 receptors [2. The cells used in the monolayer culture experiments were trypsinized and seeded in separate dishes used for experiments 2 to 3 days prior to the studies.The human HNSCC cell line H314 , derived from floor of mouth, was cultured in a one-to-one mixture of Ham's F12 and Dulbecco's modified eagle medium (DMEM), supplemented with 10% foetal calf serum, 2 mM eceptors . As a ne125I- and 111In-labelled AbD15179 have previously been extensively evaluated in vitro[125I  using direct chloramine T labelling [125I was mixed with 100 μl PBS, and 100 μg of the Fab fragment  was added. The reaction was initiated by the addition of chloramine T (Sigma-Aldrich) in water  and was quenched after a 5-min incubation on ice by the addition of sodium metabisulphite  in water . Each labelled protein was separated from non-reacted 125I and low-molecular-weight reaction components on a NAP-5 column pre-equilibrated with PBS. 125I-labelled AbD15179 is referred to as 125I-Fab in this paper. in vitro. In the abelling   was done using CHX-A″-DTPA labelling as described previously [111In labelling exhibits high affinity for several isotopes that are suitable for either diagnostic or therapeutic purposes, including 111In, 86Y, 90Y and 177Lu. In short, to 75 μg AbD15179 solution , 4.1 μl of CHX-A″-DTPA solution  was added, corresponding to a chelator-to-AbD15179 molar ratio of 5:1. The reaction mixture was incubated overnight at room temperature, and the conjugated antibody was then separated from free CHX-A″-DTPA using a NAP-5 column equilibrated with acetate buffer . Approximately 10 MBq of 111In in acetate buffer was then added to the conjugated AbD15179, and the reaction mixture was allowed to incubate for 30 min at room temperature. Labelled Fab fragment was then separated from the non-reacted radionuclide and low-molecular-weight reaction components by using a NAP-5 column pre-equilibrated with PBS. 111In-labelled AbD15179 is referred to as 111In-Fab in this paper.Labelling of AbD15179 with eviously ,34. The 125I- and 111In-labelled conjugates. The purity of the samples taken immediately, as well as stored at 4°C for 48 h, was analysed. Approximately 1 μl of the conjugate was placed on a chromatography strip  and placed into a ‘running buffer’ , followed by measurements on a Cyclone Storage Phosphor System (PerkinElmer). The data were analysed using the OptiQuant image analysis software (PerkinElmer). Furthermore, stability in murine serum was evaluated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (200 V constant) on NuPAGE® 4-12% Bis-Tris gel . Briefly, 1.8 μg of 125I-Fab or 111In-Fab was incubated in 120 μl of murine serum  for 60 min at 37°C. After incubation, the samples were treated with NuPAGE® LDS Sample Buffer according to the manufacturer's instructions and loaded on the gel. One well was loaded with 4 μl of HiMark™ Pre-Stained Protein Standard (Novex®). Measurements were performed on a Cyclone Storage Phosphor System, and the data were analysed using the OptiQuant image analysis software.To determine the purity and stability of the labelled antibody fragments, instant thin-layer chromatography (ITLC) was performed on In vitro binding and specificity were assessed for 125I-Fab, and binding, specificity and internalization were assessed for 111In-Fab using both H314 and A431 cells. For binding and specificity measurements, 10 nM 125I-Fab or 111In-Fab was added to each dish and incubated for 2 to 24 h. To some of the dishes, a 100-time molar excess of unlabelled antibody fragment was simultaneously added for assessment of binding specificity. After incubation, the cells were washed with serum-free medium to remove unbound antibody fragment, detached with 0.5 ml trypsin at 37°C and resuspended in 1 ml complete medium. A sample of 0.5 ml of the cell solution was taken for cell counting, and the remaining 1 ml was collected for radioactivity measurements.111In-Fab was added to each dish and incubated for 2 to 24 h using the following conditions: (i) incubation at 37°C to allow internalization of the conjugate, (ii) incubation at 37°C in the presence of a 100-time molar excess of unlabelled antibody for assessment of binding specificity and (iii) incubation at 0°C for correction of acid-resistant surface binding at non-internalizing conditions. After incubation, the cells were washed with serum-free medium to remove unbound antibody and incubated at 0°C with 20 mM HCl/150 mM NaCl (pH 1.7) for 15 min to release antibodies bound to the cell surface. The supernatant was collected, and the radioactivity was counted in a gamma counter to assess the amount of membrane-bound conjugates. Finally, the cells were lysed with 0.1 M NaOH/1% Triton X-100, and the radioactivity was measured in a gamma counter to assess the amount of internalized conjugates. After correction for acid-resistant surface binding, the internalization at 37°C was calculated as the percentage of the total amount of radioactivity specifically bound  to the cells.For internalization measurements, 10 nM 6 A431 cells (high CD44v6 expression) suspended in 300 μl injected medium in the right posterior leg, and approximately 8 × 106 MDA-MB-231 cells (no CD44v6 expression) in the left posterior leg, in five nude balb/c (nu/nu) female mice. The mice were housed in a controlled environment and fed ad libitum. All experiments complied with the current Swedish law and were performed with permission granted by the Uppsala Committee of Animal Research Ethics.Tumour xenografts were formed by subcutaneous inoculation of approximately 8 × 10125I-Fab  in the tail vein. After 24 h, the animals were euthanized with a mixture of ketamine and xylazine followed by heart puncture, and blood was aspirated and weighed. Tumours were excised and weighed. Radioactivity content of the blood and tumours was measured in a gamma counter, together with three injection standards of 200 μl 125I-Fab (volume of injection). A mean injected dose was calculated from the injection standards, and the injected activity of 125I-Fab for each mouse was calculated by subtracting the residual activity in the syringe from the mean injected dose. Radioactivity uptake in the organ was calculated as percent of injected activity per gram of tissue (%ID/g). Tumour-to-organ ratio was calculated as activity/gtumour divided by activity/gorgan.After allowing the tumours to grow for 2 weeks, the animals were injected withad libitum. The experiments complied with the current Swedish law and were performed with permission granted by the Uppsala Committee of Animal Research Ethics.Tumour xenografts of A431 and H314 cells  were formed in 20 nude balb/c (nu/nu) female mice as described above. The mice were housed in a controlled environment and fed 125I-Fab  and 111In-Fab , totally 5 μg Fab fragments in 200 μl PBS. At 6 h (N = 5), 24 h (N = 5), 48 h (N = 5) and 72 h (N = 5) post injection, the animals were euthanized with a mixture of ketamine and xylazine followed by heart puncture, and blood was aspirated and weighed. The tumours, salivary glands, thyroid (en bloc with larynx), tongue, liver, kidneys, spleen, urinary bladder, colon, upper gastrointestinal tract, skin, bone and muscle were excised and weighed, and radioactivity was measured in a gamma counter. The tail and the rest of the body were also separated, weighed and measured in a gamma counter, together with three injection standards for each group. The injected dose was calculated as described above. Radioactivity uptake in the organ was calculated as %ID/g. Uptake was calculated as percent of injected activity per organ. Tumour-to-organ ratio was calculated as activity/gtumour divided by activity/gorgan.Three weeks after tumour cell injection, the animals received an intravenous injection via the tail vein withhttp://www.graphpad.com). For in vivo specificity studies, the differences in 125I-Fab uptake between MDA-MB-231 tumours and A431 tumours were assessed using a two-tailed paired t test and were considered statistically significant if P < 0.05. In the biodistribution studies, the data are presented as the mean ± standard deviation (SD). The significance of differences between the groups over time was tested with one-way analysis of variance (ANOVA) with Newman-Keuls multiple comparison test. The differences were considered statistically significant if P < 0.05. The differences in 125I-Fab or 111In-Fab uptake between H314 tumours and A431 tumours were assessed using repeated measures ANOVA with Newman-Keuls multiple comparison test and were considered statistically significant if P < 0.05.Statistical analyses were performed using GraphPad Prism Version 5.02 for Windows  of AbD15179 reported previously [The Fab fragment AbD15179 bound strongly to the human v6 peptide, whereas no binding was detected for the murine form of the target  and was unchanged according to the ITLC analysis. SDS-PAGE analysis of the sample incubated in serum for 1 h at 37°C showed a single radioactivity peak corresponding to the size of the intact Fab fragment for both 125I-Fab and 111In-Fab, indicating high stability of the fragments.Labelling yields for 111In-Fab and 125In-Fab increased with time, levelling out at approximately 6 h in both cell lines. Furthermore, binding of 111In-Fab and 125In-Fab was blocked by an excess of unlabelled AbD15179 in both cell lines, demonstrating specific binding of the conjugates  for the high-affinity population  was approximately 8 ± 2 nM and did not significantly differ between 125I- or 111In-labelled AbD15179 on the H314 or A431 cells [Cellular binding of in vivo by comparing the uptake of 125I-Fab in CD44v6-negative (MDA-MB-231) and CD44v6-positive (A431) tumours 24 h p.i. in tumour-bearing mice. At the time of the experiments, median tumour weight for MDA-MB-231 tumours was 131 mg, generally ranging between 90 mg (lower quartile) and 338 mg (upper quartile). For the A431 tumours, the median weight was 62 mg, generally ranging between 43 and 95 mg. There was no correlation between the tumour weight and %ID/g of 125I-Fab. Tumour uptake differed significantly between the CD44v6-negative and CD44v6-positive tumours, with the CD44v6-positive A431 tumours displaying more than two times higher uptake than the CD44v6-negative MDA-MB-231 tumours (0.66 ± 0.13 (%ID/g ± SD) and 1.34 ± 0.26 %ID/g for MDA-MB-231 and A431 tumours, respectively).The specificity of AbD15179 was assessed in vivo by comparing the uptake of 125I-Fab and 111In-Fab in mice bearing both CD44v6-moderate (H314) and CD44v6-positive (A431) tumours. At the time of the experiments, the median tumour weight for the H314 tumours was 23 mg, generally ranging between 17 mg (lower quartile) and 33 mg (upper quartile), and the median tumour weight for the A431 tumours was 105 mg, generally ranging between 86 and 294 mg. There was no correlation between tumour weight and %ID/g of 125I-Fab or 111In-Fab. The biodistribution data of the selected organs of the 125I-Fab and 111In-Fab fragments in tumour-bearing mice are shown in Figure 111In-Fab displayed not only higher uptake of radioactivity and slower clearance in most organs compared with 125I-Fab but also a higher tumour-to-blood ratio and a clearer discrimination between high- and moderate-CD44v6-expressing tumours. The biodistribution data are described in more detail below.The biodistribution of AbD15179 was assessed oderate H14 and CD111In-Fab, the activity in tumours remained relatively stable over time in both the moderate (H314) and high (A431) CD44v6-expressing tumour models, with no significant changes in tumour uptake over time  throughout the study. At 6 h p.i., the percentage of the injected dose per gram of 111In-Fab in the moderate-CD44v6-expressing H314 tumours was 2.4 ± 1.1 (SD) and remained at this level. The uptake in the high-CD44v6-expressing A431 tumours was significantly higher, being 4.6 ± 1.9 %ID/g at 6 h p.i., with a slight, although not significant, decrease over time to 3.2 ± 0.2 %ID/g at 72 h p.i. For blood, bone and organs, 111In-Fab demonstrated highest activities at the first time point (6 h), declining with time. The kidneys displayed the highest activity (131 ± 47 %ID/g at 6 h), which rapidly decreased to 67 ± 11 %ID/g by 24 h. The second highest normal organ uptake was observed in the liver (13 ± 5.6 %ID/g at 6 h), rapidly decreasing to 5.6 ± 1.4 %ID/g by 24 h.For oderate H14 and hi125I-Fab generally displayed lower uptake values than 111In-Fab in both tumours and normal organs, with a more pronounced clearance and decrease in activity over time , with the activity rapidly decreasing to 1.4 ± 0.2 at 24 h. The stomach and salivary glands displayed high initial values, both rapidly decreasing to below 0.3 %ID/g 48 h p.i. The exception to this trend was uptake in the thyroid, where no decrease in activity was observed  differed significantly between the CD44v6-negative and CD44v6-positive tumours, with the CD44v6-positive A431 tumours displaying more than two times higher uptake than the CD44v6-negative MDA-MB-231 tumours. This is in line with the values obtained at the same time point (24 h p.i.) in the subsequent biodistribution study in this paper, in which high-CD44v6-expressing A431 tumour uptake of 125I-Fab was approximately 1.3 times higher than the uptake in the moderate-CD44v6-expressing H314 tumours. However, at later time points, the difference in tumour uptake between moderate- and high-CD44v6-expressing tumours was diminished for 125I-Fab, whereas 111In-Fab displayed a clear difference in tumour uptake between the moderate- and high-CD44v6-expressing tumours, ranging between 1.5 to 2 times higher for the high-CD44v6-expressing tumours throughout the study. Thus, we conclude that 111In-Fab displayed higher in vivo target sensitivity at the administered dose.111In-Fab also demonstrated a higher tumour uptake than 125I-Fab at all time points, being more than 15 times higher at the last time point (72 h p.i.). The accumulation and slower dissociation of 111In-Fab in tumours were in line with the slower off-rate of 111In-Fab compared to 125I-Fab observed on these tumour cells in vitro shown in a preceding study [in vivo could nevertheless in part be due to some internalization of the 111In-Fab by the tumour cells as the cellular membrane renews itself, even though our in vitro experiments showed no detectable internalization of 111In-Fab into either H314 or A431 tumour cells up to 24 h of incubation at 37°C.ng study  using no111In [124I [111In- or 124I-labelled AbD15179 is indeed feasible.For successful imaging, the contrast between tumour and surrounding tissue is of even higher importance than the total tumour uptake. In the present study, tumour-to-blood ratios were above 1 already at 24 h p.i. for both conjugates and continued to increase throughout the study. This can be compared to previous studies, where successful CD44v6 imaging in tumour-bearing mice using both 111In  and 124IIn [124I  coupled 125I-labelled Fab fragments were cleared from the circulation more rapidly than the 111In-Fab fragments. This is in line with several other studies using 125I- and 111In-labelled molecules, in which the 125I-labelled compounds have generally shown lower uptake and faster clearance from organs. The reason for this has been suggested to be dehalogenation of the radioiodinated protein or exchange of 111In into iron-binding proteins [125I-Fab than for 111In-Fab [In general, proteins . Another11In-Fab , resulti11In-Fab .111In-Fab displayed a considerably higher uptake than 125I-Fab in the liver, spleen and kidneys. These observations are also in agreement with those reported in the literature when 125I and 111In are compared [111In in the liver could be a result of antibody clearance or exchange with iron-binding protein or a mixture of both processes. The accumulation of 111In in the kidneys can probably be attributed to the active filtration of the fragments and probably to the subsequent exchange of 111In into non-binding proteins within this organ [111In and clear it more slowly is a distinct disadvantage of 111In and may limit its use, although this should be balanced by the fact that for 111In-Fab, the magnitude of tumour uptake was several times higher than seen with the radioiodinated fragment. Additionally, several preclinical and clinical experiments have shown that undesirable renal uptake of 111In can be reduced with pretreatment with e.g. cationic amino acids such as lysine [Furthermore, compared . The obsis organ . The pros lysine -44. More125I-Fab or 111In-Fab in any normal tissue. The transferability of the results to targeting HNSCC in humans is still somewhat limited, at least for organs with known CD44v6 expression as the epithelia of the oral cavity [For imaging in head and neck cancer, tumour-to-tissue contrast of muscle, salivary glands, thyroid and, to some extent, bone is important, in addition to the tumour-to-blood ratio. Some normal mouse tissues containing squamous and transitional epithelium, such as tongue and skin, express murine CD44v6. However, we demonstrate in this study that AbD15179 does not cross-react with the murine CD44v6. Thus, studies with fragments cross-reactive with murine CD44v6 would be needed to study the effect of CD44v6 expression in normal tissues in a mouse model. Consequently, assuming that only non-CD44v6-mediated uptake was measured, our data indicate that there was no unspecific accumulation of l cavity . Howeverl cavity ,26,45,46l cavity . Since wl cavity , this isin vivo imaging use are relatively limited. In this study, we used 111In as a model for radiometals, and 125I as a model for radiohalogens. The choice of radionuclide and the labelling method is an important aspect in successful radio-immunotargeting, and additional improvements in tumour-to-blood ratio could probably be achieved by further optimizing or changing labelling methods or radionuclides. For 111In labelling, a CHX-A″-DTPA chelator-to-Fab molar ratio of 5:1 was chosen. It is possible that a different molar ratio could influence targeting properties of the conjugate. However, in a previous study, conjugation of 111In was studied at three different molar ratios  of CHX-A″-DTPA chelate to panitumumab Fab2in vitro and in vivo. The different ratios had minimal effect on the immunoreactivity and specific binding [125I was used as a surrogate for radionuclides more suitable for imaging such as 124I, or 131I for therapy. The thyroid uptake was relatively high for 125I-Fab, most likely due to uptake of the free lipophilic iodine catabolites after proteolytic degradation of the antibody fragment. Likewise, the initially high uptake in salivary glands and stomach indicates iodine catabolites, since the Na+/I− symporter mediates active I− transport also in these tissues [125I antibody conjugates [The isotopes that have physical and nuclear properties suitable for  tissues . Values njugates ,48. By u2 with a size above the renal threshold [Although intact monoclonal antibodies have been considered candidates for radio-immunodiagnostics due to their specificity, the long residence time in the blood, of days to weeks, limits their applicability. Several studies have demonstrated that the size of antibody-based imaging agents is inversely related to their blood clearance . Consequhreshold , the fra2 or IgG. However, in vitro evaluations of our Fab fragment using SPR demonstrated KD in the low nanomolar range, which is considered optimal for imaging [125I-labelled Fab fragments with lower affinity (KD 30 ± 8 nM) derived from the CD44v6-targeting chimeric antibody U36 [125I-U36-Fab fragments demonstrated lower tumour uptake and lower tumour-to-blood ratios compared to the 125I-Fab in the present study, as well as less optimal tumour-to-organ ratios for e.g. salivary glands, tongue, thyroid and bone. The differences may be the result of many different aspects, for example, tumour cell line and size, as well as administered dose and specific activity of the conjugates. However, since AbD15179 was selected from a large number of fragments due to its high affinity and very slow dissociation from tumour cells [125I-Fab compared to those of 125I-U36-Fab may be the desirable kinetic properties of 125I-Fab. Thus, we believe that not only the fully human format but also the suitable affinity of AbD15179 will be an advantage for radio-immunodiagnostics.Another limitation of Fab fragments pertains to the fact that they are monovalent, often resulting in the loss of functional affinity and reduced binding strength compared to e.g. Fab imaging . Studiesbody U36  have preur cells , one hypin vivo properties of the AbD15179 Fab fragment targeted against CD44v6 labelled with 111In or 125I and assessed its utility as a targeting agent for radio-immunotargeting of HNSCC. Our study demonstrated that 111In-Fab displayed a superior tumour-to-blood ratio in high-expressing CD44v6 tumours at all time points compared to 125I-Fab. Furthermore 111In-Fab was able to discriminate between moderate and high CD44v6 expression in vivo. We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies.We have, for the first time, evaluated the 2: fragment antigen binding; HNSCC: head and neck squamous cell carcinoma; HuCAL: human combinatorial antibody library; IgG: immunoglobulin G; ITLC: instant thin-layer chromatography; KD: equilibrium dissociation constant; NAP-5: gel filtration column; PBS: phosphate-buffered saline; PET: positron emission tomography; p.i.: post injection; SCC: squamous cell carcinomas; SPECT: single-photon emission tomography; SPR: surface plasmon resonance; U36: monoclonal antibody U36.BSA: bovine serum albumin; CAT: chloramine T; CD: cluster of differentiation; CD44v: variable CD44; CD44v6: v6 containing CD44; cMAb: chimeric monoclonal antibody; DMEM: Dulbecco's modified eagle medium; Fc: fragment crystallizables; Fab and FabThe authors declare that they have no competing interests.AKH contributed to the conception and design of the study, carried out the experimental studies and wrote the manuscript. DS, JN, KS and MN contributed to the conception and design of the study, carried out the experimental studies and critically contributed to and revised the manuscript. All authors read and approved the final manuscript.AKH is an otolaryngologist and head and neck surgeon, and a PhD student focusing on methods to detect squamous cell carcinoma in the head and neck region. DS has an MSc degree in Biology and is a PhD student focusing on molecular imaging and therapy of cancer. JN has a PhD degree in biotechnology and is currently a postdoctoral fellow working on antibody engineering. KS is an otolaryngologist and head and neck surgeon with a PhD degree in otorhinolaryngology. MN is an associate professor in Biomedical Radiation Sciences Unit, with a PhD degree in otorhinolaryngology.


Nuclear medicine has become a key part of molecular imaging. In the present review article, we focus on the transbilayer phospholipids as exquisite targets for radiolabelled probes in molecular imaging. Asymmetry of phospholipid distribution is a characteristic of mammalian cell membranes. Phosphatidylcholine and sphyngomyelin cholinophospholipids are primarily located within the external leaflet of the cell membrane. Phosphatidylserine and phosphatidylethanolamine aminophospholipids, and also phosphatidylinositol are primarily located within the internal leaflet of the cell membrane. New radiolabelled tracers have been designed in preclinical and clinical research for PET-CT and SPECT-CT molecular imaging of transbilayer phospholipids. Quantification of PI metabolism requires further optimization with a 18F-radiolabelled DAG compound closer to the PI metabolism than 18F-DAG  [In a rat model, turnover . 11C-DAGfarction . After 3odelling . In 13 pocardium . 18F hasC4, C16) . Table 4SM liposomes-encapsulating 67Ga prepared in a lipid phase with cholesterol  enhanced blood circulation and increased 67Ga delivery to the tumour [T/B) and tumour index (TI = T/B × percentage dose per gram) were higher at 24, 48 and 72 h post-injection.SM is a cholinophospholipid located in the outer leaflet of the membrane bilayer . Liposome tumour . Tumour-67Ga in an aqueous phase offer better radiolabelling properties and 67Ga accumulation for tumour imaging [Neutral SM liposomes with encapsulated  imaging . Table 5In nuclear medicine, the transbilayer phospholipid targets are used in preclinical and clinical research for SPECT-CT and PET-CT molecular imaging with radiolabelled probes to assess key biological functions:PS target allows the visualisation, characterisation, and measurement of apoptosis or necrosis, and thrombosis with radiolabelled annexin V and the 99mTc or 18F-labelled C2A domain of synaptotagmin I and the 99mTc-labelled HYNIC-lactadherin; 99mTc-labelled synthetic PSBP-6-SAAC probe has been designed for molecular imaging of cell death. PS targeting probes may allow imaging of tumour endothelium vasculature with 74As-bavituximab. 77/76As-bavituximab may also serve for β- radioimmunotherapy of tumours with PS exposed endothelium tumour vasculature.1. the PE target allows imaging of apoptosis or necrosis with 99mTc-HYNIC-duramycin. It may also allow tumour imaging with radiolabelled ethanolamine and N,N' dimethylethanolamine.2. the PS and PE targets allow imaging of necrosis with the radiolabelled 64Cu-bis-DOTA hypericin.3. the PC target allows assessment of choline metabolism with 11C-choline or 18F-fluorocholine, and also the evaluation of the acetate and acetoacetate metabolism with 11C-acetate or 18F-fluoroacetate and 11C-acetoacetate.4. the PI target allows evaluation of the neuronal and myocardial PI turnover with 11C or 18F-labelled DAG, and also with 11C-inositol.5. the SM target may be used with liposomes encapsulating 67Ga for imaging purposes.6. the 7. Translation of preclinical research to clinical research will be necessary to optimally assess the pharmacokinetics of radiolabelled probes for molecular imaging of transbilayer phospholipids.18F-FCH: 18F-fluorocholine; 18F-FDG: 18F-fluorodeoxyglucose; kDa: kilodalton; HYNIC: hydrazinonicotinamide; i.e.: id est (in example with complete enumeration); e.g.: exampli gratia (for example with incomplete enumeration); ICM: internal leaflet of the cell membrane; M: molar; 10-9 M: nanomolar; MI: molecular imaging; MRI: magnetic resonance imaging; MW: molecular weight; NSCLC: non-small cell lung cancer; PA: phosphatidic acid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PET-CT: positron emission tomography-computed tomography; p.i: post-intra-venous injection; PI: phosphatidylinositol; PIP: phosphatidylinositol 4-phosphate; PIP 2: phosphatidylinositol 4,5-diphosphate; PIP 3: phosphatidylinositol 3,4,5-triphosphate; PS: phosphatidylserine; SAAC: single amino acid chelator; SM: sphyngomyelin; SPECT-CT: single-photon emission computed tomography-computed tomography; TRAIL: tumour necrosis factor-related apoptosis-inducing ligand; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling.AA: amino acids; ATP: adenosine triphosphate; CT: computed tomography; C2A-GST: C2A Synaptotagmin domain I glutathione-S-transferase; CA4P: combretastatin A4 phosphate; DAG: diacylglycerol; DOTA: 1,4,7,10-tetraazacyclododecane-N, N', N″, N″'-tetraacetic acid; ECM: external leaflet of the cell membrane; FITC: fluorescein isothiocyanate; The authors declare that they have no competing interests.TB has made substantial contribution in drafting the manuscript. FP has made substantial contribution in reviewing the manuscript. All authors read and approved the final manuscript.TB is nuclear medicine physician  and Adjunct Professor at The University of Western Ontario in the department of Medical Imaging .FP is imaging program leader (PhD), Assistant Scientific Director at The Lawson Health Research Institute (LHRI), Professor at The University of Western Ontario in the departments of Medical Imaging, Medical BioPhysics and Physics, and Chief Medical Physicist at St. Joseph's Health Care and London Health Sciences Centre .


Positron emission tomography (PET) may be useful for defining the gross tumour volume for radiation treatment planning and for response monitoring of non-small cell lung cancer (NSCLC) patients. The purpose of this study was to compare tumour sizes obtained from CT- and various more commonly available PET-based tumour delineation methods to pathology findings.18F]-fluoro-2-deoxy-D-glucose PET/CT studies from 19 NSCLC patients were used. Several (semi-)automatic PET-based tumour delineation methods and manual CT-based delineation were used to assess the maximum tumour diameter.Retrospective non-respiratory gated whole body [2=0.82). An absolute SUV threshold of 2.5 also showed a good agreement with pathology after the removal of 5 outliers (R2: 0.79), but showed a significant overestimation in the maximum diameter . Adaptive 50%, relative threshold level and gradient-based methods did not show any outliers, provided only small, non-significant differences in maximum tumour diameter , and showed fair correlation (R2>0.62) with pathology. Although adaptive 70% threshold-based methods showed underestimation compared to pathology (36%), it provided the best precision (SD: 14%) together with good correlation (R2=0.81). Good correlation between CT delineation and pathology was observed (R2=0.77). However, CT delineation showed a significant overestimation compared with pathology .50%, adaptive 41% threshold-based and contrast-oriented delineation methods showed good agreement with pathology after removing two outliers  active part of the tumour for radiotherapy and response monitoring purposes. There is an increasing interest in using FDG PET not only to determine FDG uptake but also to determine the location and extent of the metabolic active part of the tumour. PET is being explored as a tool for e.g. the definition of the gross tumour volume (GTV), location of the metabolic active part of the tumour for radiation oncology or monitoring response during chemotherapy[Positron emission tomography (PET) is a functional imaging modality that provides information about metabolism, physiology and molecular biology of tumour tissue. otherapy. For radotherapy. Accuratotherapy.Another important application of FDG PET is its use for the assessment of (early) treatment response and/or as a prognostic factor. So far, these applications have been mainly explored by studying FDG uptake quantitatively by means of standardised uptake values (SUV). Nevertheless, other parameters, such as the metabolic volume or total lesion glycolysis (product of SUV and metabolic volume), may provide additional valuable information both as prognostic value as well as for treatment response monitoring. Recently, metabolic tumour volume or maximum metabolic tumour diameter have been shown to be independent prognostic factors for oesophageal cancer but only when accurate PET-based tumour delineation methods are used. MoreoveSeveral (semi-)automatic PET-based tumour delineation methods have been studied previously-12. It iIncluded in this study were 19 consecutive patients  with histological proven non-small cell lung cancer, who had undergone a diagnostic whole body PET/CT scan and underwent a surgical resection of their primary lung tumour in the period from December 2003 to December 2004. All patients gave written informed consent prior to inclusion and the study was approved by the Medical Ethics Review Board of the Maastricht University Medical Center.-1, range 4.1-12.0 mmol·L-1) and all patients fasted for at least 6 h before scanning. PET data were reconstructed using ordered subsets expectation maximisation with 4 iterations and 18 subsets, followed by 5 mm full width at half maximum (FWHM) Gaussian smoothing, resulting in an image resolution of about 6.5 mm FWHM. All PET images had a matrix size of 128×128×178, corresponding to a pixel size of 5.31×5.31×5.00 mm3. CT data had an image matrix size of 512×512×178, corresponding to a pixel size of 0.98×0.98×5.00 mm3. CT data were used to correct for tissue attenuation. More acquisition and reconstruction details can be found elsewhere[FDG data were acquired using a whole-body PET/CT scanner . FDG was administered as an intravenous bolus (365±62 MBq). For each patient, plasma glucose levels were measured . Directly after surgical resection, the maximum diameter of this tumour was measured by macroscopic examination in three dimensions using a calliper. Shrinkage of the tumour, estimated to be around 10%, was not considered as no preservation, fixation or inflation was applied prior to the diameter measurements. The obtained maximal diameters ranged from 1.5 to 7.0 cm (mean: 4.0±1.8 cm). The primary tumours were located in the superior (n=8), middle (n=1) or inferior (n=10) lobe.Maximum tumour diameters were measured for each primary lung tumour volume. These tumour volumes were obtained using six different types of PET-based (semi-)automatic tumour delineation volume of interest (VOI) methods:50, VOI70)[1 Fixed threshold of 50% and 70% of maximum voxel value .A41, VOIA50, VOIA70). Same as above, except that it adapts the threshold relative to the local average background[2 Adaptive threshold range of 41-70% of maximum voxel value (VOIckground.Schaefer)[3 Contrast-oriented method (VOIchaefer). This meRTL). This method is an iterative method based on a convolution of the point-spread function that takes into account the differences between various sphere sizes and the scanner resolution[4 Background-subtracted relative-threshold level (RTL) method  algorithm is used to grow the seeds in the gradient basins, thereby creating boundaries on the gradient edges. In our study, two different types of gradient basins were used. The first approach, indicated by Grade tumour-12. The 2.5). Normalised (SUV) voxel intensities at a chosen absolute threshold (2.5) are used to delineate tumour[6 Absolute SUV , mean differences were smaller for all PET-based methods (−9.4 to 13%) than for manual CT delineation (15%). VOI50, VOIA41 and VOISchaefer provided estimates of the maximum diameter  that were closest to those obtained from pathology.Figure50 was similar to that obtained from pathology . For CT-based delineation, corresponding differences in median diameter were significantly larger . In addition, a significant overestimation of median diameter compared to pathology was found for SUV2.5 and GradWT1 . Some other PET-based methods  provided results that were not statistically different from pathology (p>0.10). However, median diameters obtained from VOIA50, VOIRTL and GradWT2 were slightly smaller (<4.7 mm differences) than those obtained from pathology. In contrast, only small differences in median diameter obtained from VOIA41 and VOISchaefer  were observed when compared with pathology.An identical trend can be seen in Table2>0.62; slope 0.69-1.16), except for GradWT1 . Although VOIA70 showed good correlation (R2=0.81), comparable with VOI50, VOIA41 and VOISchaefer, it resulted in an underestimation of the maximum diameter (slope 0.69).TableWT1 was found for small tumours (≤2.5 cm diameter). When excluding these small tumours, mean difference (15.7%) and correlation  between diameters obtained with GradWT1 and pathology moderately improved. Manual CT delineation showed a large maximum diameter for two tumours with heterogeneous FDG uptake  showed a large difference in diameter (>485% difference). The same trend was seen in Bland-Altman plots, as shown in FigureWT1, however, one data point from a patient with a small primary tumour (1.8 cm diameter) was not within the limits of agreement.FigureObtaining accurate (metabolic) tumour boundaries may be important for treatment planning in radiotherapy and/or for use as prognostic factor and/or to monitor response during therapy, and may therefore have a direct impact on clinical outcome.50, VOIA41 and VOISchaefer show good agreement with pathology after removing two outliers , and showed fair correlation  with pathology (TablesWT1 showed only moderate agreement with pathology (R2: 0.43) and overestimated the maximum diameter. In addition, a poor agreement obtained with pathology and GradWT1 was found for small tumours (≤2.5 cm diameter). As the sizes of the small tumours are less than three times the full-width-at-half-maximum, the influence of partial volume effects may be relatively high, causing this poor agreement with pathology. By applying modifications to the algorithm (i.e. GradWT2), the method showed more accurate tumour sizes (R2: 0.62). These findings are in line with a previous study that compared volumes derived with a gradient-based method to pathology[2.5 showed a good agreement with pathology after the removal of 5 outliers (R2: 0.79), but showed a significant overestimation in the maximum diameter . Due to the large number of outliers and large overestimation in diameter size, it is not recommended to use this method for radiotherapy planning purposes.Tumour delineation methods are only suited for radiotherapy planning purposes if they correspond well with pathology. The present results indicate that VOIathology. SUV2.5 A70 showed a large systematic underestimation (−36%) of diameter, this method was the most precise  between maximum diameters obtained from this method and pathology was observed (TableA70 may be a good method for response monitoring in which relative changes in (more active part of) metabolic volume are considered.Assessment of change in tumour size is important when monitoring tumour response during therapy. Although VOIy Figure=0.81 bet2=0.87) with pathology than PET-based percentage threshold methods that did not correct for background activity (R2=0.77). This is in contrast to the present study, where CT-based delineation provided a slightly lower correlation of maximum diameter with pathology than VOI50 . In addition, CT-based delineation showed a moderate overestimation of maximum diameter compared with pathology . Despite the good correlation, a drawback of manual CT delineation is the requirement of both a high resolution image (i.e. not a low dose CT) and an experienced observer. Even if delineation is performed by an experienced observer, manual CT delineation suffers from substantial interobserver variation[Wu et al. showed tariation. In addiariation,16. Moreariation.anatomical (CT) and metabolic (PET) volumes. As previously suggested[For the patient shown in Figureuggested, CT-baseSome factors might limit accurate delineation of tumour volumes and corresponding maximum diameters using the commonly available PET-based (semi-)automatic tumour delineation described in this article. First, primary lesions could be surrounded by high uptake regions, e.g. from suspected locoregional metastases, heart and spine. Therefore, application of tumour delineation methods might be more valid for peripheral tumours and less valid for more centrally located tumours, unless a  bounding box around the tumour is used to prevent delineation of surrounding high uptake regions. Second, the metabolic volume of tumours could show heterogeneous tracer uptake that has been shown to have an impact on threshold-based delineation methods. Moreovein-vivo CT imaging and ex-vivo pathology due to the softness of lung tissue[The present study showed some potential methodological limitations that might have influenced the results. First, it should be noted that deformations could occur between g tissue. The metg tissue where inThe maximum diameter derived from CT-based delineation was overestimated compared to pathology, especially at large tumour diameters. PET-based tumour delineation methods provided maximum diameter sizes in closer agreement with pathology. The PET-based 50%, adaptive 41% threshold-based and contrast-oriented (Schaefer) methods seem to be best suited for assessing tumour sizes  of primary lung tumours, as it provides the best correspondence with pathology data. However, these methods could show a potential difficulty when located close to high uptake regions. Despite only a non-significant small underestimation compared to pathology data, PET-based adaptive 50%, relative threshold level and gradient-based methods could distinguish between the tumour and these adjacent high uptake normal tissues, and are therefore recommended for radiotherapy purposes. An adaptive 70% threshold-based method may be best suited for response monitoring, as it provides the best precision and best correlation with pathology derived size without suffering from outliers.The authors declare that they have no competing interests.PC performed data analysis and data interpretation, and is the main author of the manuscript. RB performed a study design, implemented some of the tumor delineation methods, contributed to the intellectual content (supervision) and reviewed and approved the final content of the manuscript. DdR provided PET image data and critically reviewed the manuscript. WvE provided the data and reviewed the manuscript. AvB collected the data and reviewed the manuscript. MY implemented some of the tools to perform tumor delineations and critically reviewed manuscript. OSH reviewed the manuscript and approved its final content. EFIC performed manually CT delineation and reviewed the manuscript. AAL reviewed the manuscript, contributed to the intellectual content (supervision) and approved the final content of the manuscript. FHPvV performed data interpretation, implemented some of the tumor delineation methods, assisted in drafting the manuscript and supervised the project. All authors reviewed the collected data and interpretation, provided feedback for further research during the study and approved the final submitted version of this manuscript.


Desmoplastic small round cell tumour (DSRCT) is an aggressive and a rare neoplasm. We report on a 34-year-old male who had abdominal discomfort with a large intraperitoneal mass. Histological examination of the tumour biopsy revealed sheets of small round cells. The cells were positive with vimentin and desmin  and negative for WT1 and CD 99 with immunohistochemistry. Cytogenetics showed a translocation disrupting the EWSR 1 gene on 22 q 12 consistent with DSRCT. Electron microscopic examination showed sparse cytoplasmic organelles. The patient succumbed 34 months from disease presentation after multiple chemotherapies and thereafter radiotherapy. In summary, our case exemplifies that it is crucial to combine clinical, histological, and molecular aspects in diagnosing DSRCT especially when characteristic dot positivity with desmin is weak along with deficient marking of WT1 and CD99 by immunohistochemistry. Histology was also less clear than published examples of this entity with a poor desmoplastic response. A multidisciplinary approach including early referral to specialised centres is recommended in these cases as tertiary referral centres will be required to substantiate the diagnosis. Desmoplastic small round cell tumour (DSRCT) is classified as a soft tissue sarcoma. It is an aggressive and rare tumour that primarily presents with an abdominal mass with only a few cases having been reported in the literature . PatientThere are no risk factors that have been identified specific to the disease. The tumour appears to arise from the primitive cells of childhood and is considered a childhood cancer. Research has indicated that there is a relationship between desmoplastic small round cell tumour, Wilm's tumour, and Ewing's sarcoma. Some DSRCT can present clinically, simulating lymphoma or other solid tumours. Therefore, careful selection of diagnostic tools should be employed to differentiate between such tumours. DSRCT is usually associated with a unique chromosomal translocation t p13:q12) resulting in an EWS/WT1 transcript :q12 resu which isDue to the rarity of this tumour, many general practitioners and oncologists are not familiar with the (DSRCT) entity. DSRCT in young patients can be confused with other abdominal tumours including lymphoma, rhabdomyosarcoma, neuroblastoma, primitive neuroectodermal tumour, small cell mesothelioma, Ewing's sarcoma, and Wilm's tumour. Therefore, patients with the likelihood of DSRCT should be referred to a sarcoma specialist centre where adequate expertise would be available for the complex diagnostic tests required for confirmation of the entity. There is no standard protocol for the treatment of the disease. However, recent literature and studies have reported that some patients respond to high-dose chemotherapy with other modalities of treatment such as tumour debulking, cytoreductive surgery, and radiation therapy . OverallWe report on a 34-year-old male who presented in January 2008 to the Launceston General Hospital, Tasmania with 6-month history of abdominal discomfort and pain on lying on his stomach when surfing. There was no other significant past medical history other than tonsillectomy. He was a social smoker and occasionally consumed alcohol. He had a major weight loss from July 2007 until January 2008. He did not have night sweats or any change in his appetite. On examination, he was found to have a painful swelling in the upper left quadrant of his abdomen. The hard mass was about 15 cm in diameter, extending down towards the umbilicus.His full blood count was normal. The liver and renal function tests were normal. He was negative for hepatitis B and C viruses as well as for HIV. His serum CA 125 level was slightly high at 65 U/mL  with other tumour markers being within normal range except for serum lactate dehydrogenase (LDH), which showed a mild increase. He had evidence of past infection with Epstein Barr virus. The cerebrospinal fluid examination did not reveal any abnormal lymphoid or malignant cells. On computer tomography (CT) scan, he was found to have a large cystic intraperitoneal mass with a suspicion of involvement of abdominal lymph nodes. The soft tissue density was present in the area of the pancreas, porta hepatis, para-aortic region and with extension to the thorax where rounded densities over the pericardial region were noted.Two CT-guided biopsies were performed in January and in early February 2008. The first biopsy revealed scanty material and showed only soft connective tissue. The second biopsy had a small piece of fibrous tissue infiltrated by sheets of small cells resembling lymphoid cells. Immunohistochemistry with lymphoma panel markers were negative, and the tissue sample was not sufficient for further studies. Subsequently, in February 2008, he underwent open abdominal surgery, and a large mass in relation to the pancreas was excised. The tumour was extending into the lesser sac and into the right subphrenic space. Omentum was also involved. Complete resection was not possible due to its widespread tissue involvement. The resected specimen had three lobulated greyish masses measuring 4 × 3 × 2 cms. The cut surface was soft, greyish, and uniform. Solid and semi-cystic components were present. Frozen sections showed a small blue round cell tumour. The flow cytometry suggested a nonhaematological malignancy.Histologically, the tumour was composed of large sheets of small round cells forming lobulated nodules . The nucImmunohistochemical stains showed a patchy perinuclear staining pattern with desmin, but characteristic dot-positivity was not prominent . There wCytogenetic studies showed disruptions in the Ewing's sarcoma gene (*22p 11 q), Wilm's tumour gene by FISH probe, and RT-PCR studies were repeated at the Sullivan Nicolaides Pathology, Queensland and showed a positive result with the Ewing's sarcoma dual-colour break-apart probe. This finding is consistent with DSRCT. Electron microscopic examination showed a population of primitive cells. The cytoplasmic organelles were sparse with short strips of rough endoplasmic reticulum and occasional mitochondria. The characteristic whorls of intermediate filaments usually present in DSRCT were not identified, and there were no dense core cytoplasmic granules . The ultDue to the negative immunohistochemical markers for WT1 and CD99 with positive findings for desmin and vimentin it was still thought compatible with a DSRCT even though histologically desmoplasia was minimal. The histology and immunohistochemistry were forwarded for review to two specialist centres because of ambiguity with the proposed diagnosis.The most acceptable consensus is that this case represents a small round blue cell tumour without evidence of significant desmoplasia but should nonetheless be classified as a DSRCT. Moreover, the cells were negatively stained with both WT1 and CD 99, which is against DSRCT. However, Lae et al.  reportedIn a histopathological study  of 39 caIn summary, we present a difficult case of a 34-year-old man with an abdominal tumour simulating lymphoma. The morphological pattern and immunohistochemistry favour a diagnosis of DSRCT, but with less desmoplasia than is usual. Cytogenetics performed on fresh tissue with FISH showed a translocation disrupting the EWS gene on 22p 11q, with an EWS-WT1 gene fusion. The repeat FISH probe and RT-PCR studies performed at the Sullivan Nicolaides Pathology showed a positive result with Ewing's sarcoma dual-colour break-apart probe. This finding is consistent with DSRCT. Electron microscopic studies were consistent with an undifferentiated small cell tumour as dense core granules, characteristic of DSRCT, were not present. The patient was referred to the Peter MacCallum Cancer Centre in Melbourne for further management. He was treated initially according to the VAC protocol , thereafFor diagnosis of DSRCT a combination of clinical, histological, and molecular aspects is required especially when usual markers for the entity WT1 and CD99 are negative. As desmoplasia is minimal and characteristic desmin dot positive is scanty in our case we suggest all the above findings indicate a variant of DSRCT. Finally a multidisciplinary approach including early referral to a specialised centre is recommended in such complex cases.


In the Funding section, the grant number from the funder Key Project of Chinese National Programs for Fundamental Research and Development was listed incorrectly. The correct grant number is: 2013CB429906.


Dear Editor,With introduction of concept like nephron-sparing surgery, partial nephrectomy using surgicel as a haemostatic agent is now being recommended as a procedure of choice for an increasing number of pediatric urology disorders. Although surgicel is considered a safe haemostat, it still has been found to be associated with unusual complications.–4 Thus, A 1.2-year-old female baby with features of left hemihypertrophy associated with complex cystic mass in upper pole of left kidney underwent partial nephrectomy. During partial nephrectomy hemostasis was achieved by ligation of vessels and surgicel was applied in multiple layers after application of Glubran 2 tissue glue. In postoperative period, an increase in drainage in the form of yellowish thick fluid approximately 5-10 ml in 24 hours was observed from fourth postoperative day. The biochemical analysis of fluid in form of creatinine and sodium values done twice on the fifth and eighth postoperative day, corresponded to serum values, thus confirming absence of urine leak. The microbiological analysis showed absence of inflammatory pathology. Thus, presence of thick yellowish sterile fluid was attributed to biodegradation of thick layer of surgicel and the correlation of drain output with ultrasonic thickness of surgicel showed progressive decrease in drainage by the end of second week, which coincided with decrease in ultrasonic thickness of surgicel. Thus, the serum values of fluid and the correlation of ultrasonic thickness with drain output provided evidence that excessive use of surgicel may be a contributing factor for prolonged drainage. The patient recovered well and is asymptomatic on follow-up for the last three months.Since the use of oxidized cellulose by Frantz, sugicel has been used successfully as the biodegradable hemostatic agent.2 Althoug113


Fluid responsiveness is defined based on an arbitrary increase of cardiac output (CO) or stroke volume (SV) of 10 to 15%. We hypothesise that the variation of heart efficiency (Eh) and the slope (S) defined by the relative increase of CO over the relative increase of mean filling pressure (Pmsa) can be used as alternative definitions of fluid responsiveness.Patients admitted to the ICU were monitored with a calibrated LiDCOplus  and Navigator  to estimate Pmsa and Eh . A 250 ml fluid challenge was performed over 5 minutes. Categorical data were compared by Pearson chi-square test. Correlation was assessed by Kappa test. The inflexion point of S to define responders was obtained by ROC curve analysis.P <0.001). The best cutoff or the slope was 0.76 . The proportions of responders identified by the ΔEh . See Figure A total of 104 fluid challenges were observed in 40 patients. ROC curve analysis reveals an area under the curve of 0.93 (95% CI = 0.85 to 1, Moderate agreement is observed between new and current definitions of fluid responsiveness.


We present the case of a rare vulva tumour, in a 33 years Cameroonian old woman and managed in Obstetrics and Gynecology Unit of Yaoundé Central Hospital in Cameroon. It was a painless pedunculated vulva tumour which developed over a period of six months. This gigantic rapidly growing tumour, was treated with simple surgical resection. After surgical resection, histology confirmed an angioneurofibroma hamartoma. There has been no recurrence and presently the patient is symptom-free. A hamartoma is a benign lesion composed of various native tissues, but growing in a disorganized manner. It is not considered a malignant growth, as it grows at the same rate as surrounding cells . It may A 33 year old, G3P2012 (last child 2 years of age), consulted in the outpatient department of Obstetrics and Gynecology Unit of Yaounde Central Hospital in Cameroon two years ago, for a growth in her vulva. This started about 6 months before, and was painless and rapidly growing. She did not present any other symptom. She had consulted several tradi-practitioners but there was no regression.On physical examination, her general condition was satisfactory, and she presented a pedunculated tumour in her vulva. The tumour was implanted in her right labia majora, a pedicle measuring 17cm length and 5cm diameter, and a base of 25cm diameter . The resWe thought it was a vulva tumour probably of embryologic origin and to rule out an acrochordon , a lymphadenoma and fibromyoma of the vulva. She was screened HIV negative and other biological tests were normal.The tumour was excised under general anaesthesia, and the patient discharged the following day on sits baths. On gross examination of the tumour, it was a tumour of 25cm diameter, with a depigmented surface and a mucosa. The pedicle was retracted and 10 cm long with a pigmented surface. There were blood vessels in the pedicle. Histology of the tumour reported a vulva hamartoma of the angioneurofibroma type . Two yeaA hamartoma is a benign, focal malformation that resembles a neoplasm in the tissue of its origin. This is not a malignant tumor, and it grows at the same rate as the surrounding tissues. It is composed of tissue elements normally found at that site, but which are growing in a disorganized manner . AlbrechA hamartoma may consist of a single type of cell, a particular type of tissue or a mixture of tissues. They occur in many different parts of the body and are most often asymptomatic. These benign tumor-like tissues over growths may be congenital or develop later in life. Two types of hamartomas are distinguished: infantile and adult types. The infantile type is a true hamartoma, but that the adult type is referred to as a mixed tumor .Hamartomas of the vulvae are rare benign lesions . Very feOur patient being a 33 year old presented an adult mixed type. The tumour showed an area of well-demarcated interwoven collagen bundles arranged around blood vessels and nerve fibres , and theDifferential diagnosis of such a tumor can be made with an acrochordon which is often multiple and appearing as soft, pedunculated, brown, tan, or skin-colored lesions (0.2-1.5 cm in diameter), and can particularly be found in the inguinal folds of obese and/or diabetic patients, or with a lymphangioma which is usually detected early in infancy on the labia minora or majora as an asymptomatic, raised, compressible, doughy mass, sometimes showing multiple clustered, superficial, thin-walled, translucent, and persistent pseudovesicles filled with clear fluid that may progressively grow over time.Treatment of hamartomas consists of surgical removal of the tumor. The patient should be closely followed for recurrences, which may occur if only partial resection of tumor was done . Our patComplications of the tumor includes: pain, infection, social misfits, and a less than 1% risk of cancerisation in the long term . Some coThe interest of this case is to recall the existence of vulva hamartomas. These tumors can be giant but with simple surgical excision as treatment.