Natural abundance 13C NMR was used to determine relative amounts of fatty acid subclasses present in fibroadipose tissue from the human breast in healthy and cancer patients and in breast carcinoma tissue. Resonances corresponding to the carbon atoms of triacylglycerides were obtained when adipose tissue constituted more than 10% of the carcinoma. Resonances corresponding to phospholipids and proteins were also observed when the percentage of adipose tissue was lower. No significant difference between the levels of unsaturated fatty acids in adipose tissue from cancer and non-cancer patients was found. However, significant differences in the levels of monounsaturated and saturated fatty acids of carcinoma compared to non-cancerous tissue was found, as was a nearly significant difference for the levels of polyunsaturated fatty acids in these two tissue types. These findings suggest an alteration of cellular lipid composition in neoplastic mammary tissue.


There is increasing evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain anti-tumour alkylating agents and that the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide inter-individual variation in the depletion and subsequent regeneration of ATase in human peripheral blood lymphocytes following sequential DTIC (400 mg m-2) and fotemustine (100 mg m-2) treatment, with the nadir ATase activity occurring approximately 4 h after DTIC administration. We have now measured the formation and loss of O6-methyldeoxyguanosine (O6-MedG) in the DNA of peripheral leucocytes of eight patients receiving this treatment regimen. O6-MedG could be detected within 1 h and maximal levels occurred approximately 3-5 h after DTIC administration. Following the first treatment cycle, considerable inter-individual variation was observed in the peak O6-MedG levels, with values ranging from 0.71 to 14.3 mumol of O6-MedG per mol of dG . Inter- and intra-individual variation in the extent of O6-MedG formation was also seen in patients receiving additional treatment cycles. This may be a consequence of inter-patient differences in the capacity for metabolism of DTIC to release a methylating intermediate and could be one of the determinants of clinical response. Both the pretreatment ATase levels and the extent of ATase depletion were inversely correlated with the amount of O6-MedG formed in leucocyte DNA when expressed either as peak levels (r = -0.59 and -0.75 respectively) or as the area under the concentration-time curve (r = -0.72 and -0.73 respectively). One complete and one partial clinical response were seen, and these occurred in the two patients with the highest O6-MedG levels in the peripheral leucocyte DNA, although the true significance of this observation has yet to be established.


Worldwide, cancer in the esophagus ranks among the 10 most common cancers. Alterations of retinoic acid receptors  expression is considered to play an important role in development of squamous-cell carcinoma (SCC), which is the most common esophageal cancer. Alcohol consumption and smoking, which can alter retinoic acid receptor levels, have been identified as key risk factors in the development of carcinoma in the aero-digestive tract. Therefore, the aim of the present study was to evaluate protein levels of retinoic acid receptors  in esophageal SCC and surrounding normal tissue of patients with untreated SCC and controls.All study participants completed a questionnaire concerning smoking and alcohol drinking habits as well as anthropometrical parameters. Protein levels of RARα, β, γ, and RXRβ were determined by Western Blot in normal esophageal tissue and tissue obtained from SCC of 21 patients with newly diagnosed esophageal SCC and normal esophageal tissue of 10 controls.Protein levels of RARγ were significantly lower by ~68% in SCC compared to normal surrounding tissue in patients with SCC that smoked and/or consumed elevated amounts of alcohol. Furthermore, RARα protein levels were significantly lower (~- 45%) in SCC in comparison to normal esophageal mucosa in patients with elevated alcohol intake. When comparing protein levels of retinoic acid receptors between normal tissue of patients with SCC and controls, RARγ protein levels were found to be significantly higher (~2.7-fold) in normal esophageal tissue of SCC patients than in esophageal tissue obtained from controls. No differences were found for RARα, β, and RXRβ protein levels between normal esophageal tissue of patients and that of controls.In conclusion, results of the present study suggest that alterations of retinoic acid receptors protein may contribute in the development of SCC in esophagus and that in some patients life style  may be a critical component in the alteration of retinoic acid receptor levels in esophagus. Worldwide esophageal cancer ranks among the ten most common cancers  and the Alterations of retinoid metabolism and retinoic acid receptor -mediated gene transcription have been proposed to play an important role in the pathogenesis of SCC -10. It iSince little is known about the abundance of RARs and RXRs protein in esophageal SCC in humans, the major focus of the present study was to determine protein levels of RARα, β, γ, and RXRβ in normal, macroscopically unaffected esophageal tissue and SCC of patients with untreated SCC and controls. In addition, alcohol intake and smoking habits of patients and controls were evaluated and related to retinoic acid receptor levels.The study was approved by the Ethics Committee of the Medical Clinic of the University of Cologne, Germany. Informed consent was obtained from all subjects included in the study. A total of 31 subjects, all of whom were undergoing endoscopies for medical reasons, were included in the study. Twenty-one patients had untreated SCC of the esophagus, and ten SCC-free subjects served as controls. Histopathological analysis of SCC was performed by an experienced physician. Tumor staging was based on a graded rating system and varied from G1 (well differentiated) to G3 (poorly differentiated). In controls, the presence or absence of SCC was confirmed macroscopically. All study participants completed a questionnaire concerning smoking and alcohol drinking habits as well as anthropometrical parameters. Using standard pinch forceps, two biopsies were obtained from macroscopically normal esophageal tissue and SCC of subjects with carcinoma and from the mucosa of healthy controls. Biopsies were placed immediately in liquid nitrogen and stored at -80°C until analysis.® West Dura . Blots were photographed  and immunoquantitation was accomplished by densitometric analysis using the software AIDA . To achieve standardization and comparability of blots, total protein derived from human liver was loaded on each gel at three concentrations  and blottet with the samples. To ensure equal loading, all blots were stained with Ponceau red; haptene signals were normalized to β-actin using a commercially available antibody .Total protein was isolated using Trizol reagent . Using SDS-polyacrylamide gel electrophoresis, 20–30 μg of total protein were separated in a 9% polyacrylamide gel (PAGE). Protein was transferred to a nitrocellulose membrane . Membranes were blocked in Tris-buffered saline (TBS) with 5% non-fat milk powder with Tween 20 , rinsed in TBST, and probed with primary antibodies dissolved in TBS followed by an incubation with the secondary antibody. Primary antibodies against RARα, β, γ, and RXRβ were obtained from Alexis , the secondary peroxidase-conjugated antibodies were purchased from Boehringer . Bands were visualized by enhanced chemiluminescence using SuperSignalt test was used to test for significance of differences in expression levels measured in both normal and malignant tissue of patients with SCC. Fisher's exact test was used for comparison of lifestyle data. Statistical comparison of values originating from separate groups was performed with the Mann-Whitney U-test. P < 0.05 was considered to represent a significant difference.Results are presented as mean ± standard error of the mean (SEM), unless otherwise stated. Wilcoxon Subject age ranged from 38–71 years and most were of normal weight Table .Figures To investigate whether smoking and alcohol consumption influences protein levels of esophageal retinoic acid receptors of patients with SCC were grouped by smoking habits (e.g. non-smoker or smoker) regardless of their alcohol consumption. In smokers, RARγ protein levels were found to be significantly lower  in SCC in comparison to unaffected esophageal tissue in smoking patients (n = 10). Interestingly, a similar difference was not found in patients who were non-smokers . Similar, patients were than sub-grouped by alcohol intake regardless of their smoking habits into patients who (a) reported to consume no alcohol (= "none"), (b) who reported to be "moderate" alcohol consumers not exceeding alcohol intake more than once a week, or (c) who stated to consume alcohol more than 4-times a week or more than 30 g raw ethanol/ d  that basal levels of retinoic acid receptor mRNA expression in normal unaffected tissue might differ between patients with SCC and controls, protein levels of RARα, β, γ, and RXRβ were determined in esophageal tissue obtained form controls and compared with protein levels measured in normal esophageal tissue obtained from patients with esophageal SCC. Representative Western blots are depicted in Figure in situ studies a reduced mRNA expression of RARα was found in esophageal SCC compared with normal tissue [In the present study, the hypothesis was tested that altered protein levels of retinoic acid receptors might be involved in the development of esophageal SCC in humans. Indeed, protein levels of RARγ were found to be significantly lower in SCC relative to normal tissue in patients who smoked and consumed alcohol. A similar difference was found for RARα in SCC patients who reported to consume "elevated" amounts of alcohol. However, diminished levels of RARγ and RARα were only found in these sub-populations of patients with SCC in the esophagus, suggesting that underlying mechanism leading to the development of SCC may vary depending on life style of patients. Several groups have previously determined mRNA expression of RARs and RXRs in normal esophageal tissue and SCC. Zhang et al.  and Qiu l tissue . Howeverin vitro and in vivo studies [Due to the retrospective character of the present study it can only be speculated whether the association of cigarette smoking and reduced protein levels of RARγ in SCC is a cause – and effect relationship or not. However, the action of tobacco carcinogens are believed to be mediated through covalent binding to DNA, RNA, or protein, forming DNA, RNA, and protein adducts . Further studies  suggest in vitro and in vivo by chlormethiazol, an inhibitor of cytochrome P4502E1 (CYP2E1). A significant induction of CYP2E1 was found after one week of chronic alcohol ingestion in human subjects [Several mechanism have been proposed to explain how alcohol ingestion may interfere with retinoid metabolism. First, it has been shown that alcohol may act as a competitive inhibitor of oxidation of retinal to retinoic acid in several organs (e.g. colon and esophagus) -23. Secosubjects . Therefosubjects . This, iUntill now, information on protein levels of retinoic acid receptors in normal unaffected esophageal tissue has been limited. Recently, in a study determining the role of RXR mRNA in Barett's esophagus mRNA levels of RXRβ were found to be higher in non-malignant tissue in patients with adenoma than in healthy controls . In the The results of the present study provide further evidence that a diminished abundance of retinoid acid receptors in esophagus is associated with the development of esophageal SCC in humans. Furthermore, in some patients life style  may contribute to the alteration of retinoic acid receptor levels in esophagus. Further studies will be required to elucidate this interaction of alcohol consumption, smoking, and retinoic acid receptor signaling with the development of SCC in the esophagus.The author(s) declare that they have no competing interest.IB has made substantial contributions to the biochemical analysis and the drafting of article. EW has substantially contributed to the acquisition of data, and conception as well as design of the study. EB, AHH, and CB have made substantial contributions to conception and design as well as the interpretation of data. AP has been involved in the design, the drafting of the article, and revised it critically for important intellectual content. All authors have given final approval of the version to be published.


Overexpression of HAI-1 and matriptase together changed the frequency of carcinoma formation to normal. This suggests that the ratio of matriptase to HAI-1 influences the malignant progression. The aim of this study has been to determine the ratio of matriptase and HAI-1 in colorectal cancer tissue (n = 9), severe dysplasia (n = 15), mild/moderate dysplasia (n = 21) and in normal tissue from the same individuals. In addition, corresponding tissue was examined from healthy volunteers (n = 10). Matriptase and HAI-1 mRNA levels were normalized to β-actin.Using quantitative RT-PCR, we have determined the mRNA levels for Matriptase mRNA level was lower in carcinomas compared to normal tissue from healthy individuals (p < 0.01). In accordance with this, the matriptase mRNA level was also lower in adenomas/carcinomas combined as compared to their adjacent normal tissue (p < 0.01). HAI-1 mRNA levels in both normal and affected tissue from individuals with severe dysplasia or carcinomas and in affected tissue with mild/moderate dysplasia were all significantly lower than mRNA levels observed in corresponding tissue from healthy control individuals. HAI-1 mRNA was lower in carcinomas as compared to normal tissue from healthy individuals (p < 0.001). HAI-1 mRNA levels were significantly lower in tissue displaying mild/moderate (p < 0.001) and severe (p < 0.01) dysplasia compared to normal tissue from the same patients. Both adenomas and carcinomas displayed a significantly different matriptase/HAI-1 mRNA ratio than corresponding normal tissue from healthy control individuals (p < 0.05). In addition statistically significant difference (p < 0.001) could be observed between mild/moderate and severe adenomas and their adjacent normal tissue.matriptase/HAI-1 mRNA ratio occurs early during carcinogenesis. Future studies are required to clarify whether the dysregulated matriptase/HAI-1 ratio was causing the malignant progression or is a consequence of the same.Our results show that dysregulation of the  It has e domain ,8. Matrie domain . Matripte domain -14. HAI-elopment .per se does not seem to be important.It has recently been shown that matriptase possesses a strong oncogenic potential since even a modest overexpression in the skin of transgenic mice caused 100% of the mice to develop tumors, 70% of which progressed into carcinomas . IncreasSeveral studies suggest that matriptase is over-expressed in a wide variety of malignant tumors including prostate, ovarian, uterine, colon, epithelial-type mesothelioma and cervical cell carcinoma ,12,18-22Prompted by the finding that a dysregulated ratio of matriptase to HAI-1 in mice results in tumor formation and carcinogenesis, we have analysed the ratio of matriptase/HAI-1 mRNA expression during colorectal cancer carcinogenesis in humans.β-actin, matriptase and HAI-1 and was therefore excluded from the study. Individuals with dysplasia, who developed carcinomas and were treated at the same hospital, were registered.The KAM cohort  is based on the screening group of the Norwegian Colorectal Cancer Prevention study (The NORCCAP study) in the county of Telemark, Norway . The ID 2 before RNA purification. RNA purification included a DNAse treatment. The cDNA synthesis was performed on approximately 200 ng RNA per 10 μl using Gold RT-RCR kit . Quantitative RT-PCR was performed on ABI7500 sequence detection system (Applied Biosystems) in Universal Mastermix (Applied Biosystems) using 160/140 nM probe and 600/300 nM primers for matriptase and HAI-1, respectively. Matriptase: primers were matrip F, 5'GCGCTCCCTGAAGTCCTTT3'; matrip R 5'GTCCTGGGTCCTCTGTACTGTTTT3'; probe, matrip t 5'FAM-TCACCTCAGTGGTGGCTTTCCCCA-BHQ-1-3'. HAI-1: primers were HAI 1 1532F 5'CGCGGCATCTCCAAGAAG3'; HAI 1 1651R 5'GAACACTGCGACAGCCATCTC3'; probe, HAI 1 1572T 5'FAM-AAATCCCCATTCCCAGCACAGGCTC-BHQ-1-3'. The HAI-1 primers detects both HAI-1 and the splice variant HAI-1B. Primers were designed using Primer Express v2.0 Software. Primers were designed within different exons and with probes covering the exon-exon border to prevent amplification of genomic DNA. 18S rRNA and β-actin primers and probes were obtained from Applied Biosystems. Matriptase and HAI-1 primers and probes were obtained from TAC Copenhagen. In a validation experiment using a control sample, a dilution series was produced and assayed for matriptase, HAI-1 and β-actin as described for the comparative Ct method [matriptase and HAI-1 and that the PCR reactions have similar efficiencies provided that a threshold of 0.2 is used for matriptase and β-actin while the threshold was 0.025 for HAI-1. The threshold is a fixed fluorescence signal level above the baseline. The Ct value of a sample is determined as the fractional cycle number when the samples fluorescence signal exceeds the threshold. The threshold is thus assay specific, determined in the validation experiment and depends on the background of the individual assay. This implies that even though the amplification curves for matriptase and HAI-1 were overlapping, indicating that the two genes had the same relative abundance, the normalised HAI-1 mRNA level seemed higher due to the lower threshold used.Total RNA was purified from tissue as recommended by the manufacturers using Qiagen kit . The tissue had been stored in liquid Nt method . When plMatriptase, HAI-1, β-actin and 18S were quantified separately in triplicates. The average standard deviation on triplicates was 6%. The standard deviation on repeated measurements of the same sample (the control) in separate experiments was 8% and 14% for matriptase and HAI-1 respectively, indicating the day-to-day variation of the assay. Independent PCR reactions of the same samples yielded a correlation coefficient of 0.88. Negative controls (where the RNA was not converted into cDNA) and positive controls were included in all sets. Essentially the same results were obtained whether the expression was normalised to β-actin or 18S (correlation coefficient of 0.921).MiniTab Statistical Software, Release 13.1 Xtra (Minitab Inc.) and GraphPad Prism 4 were used for the statistic calculations. The data were not adjusted for sex since the incidence ratio of colorectal cancer between the genders is 1.1 in Norway .matriptase and HAI-1 were measured in colon tissue samples from healthy control individuals (n = 10) and in healthy and affected tissue from individuals with mild/moderate dysplasia (n = 21), with severe dysplasia (n = 15) and with colorectal cancer (n = 9) by real-time RT-PCR. We chose to normalise the mRNA levels of matriptase and HAI-1 to the mRNA level of β-actin. Essentially identical results were obtained when normalized to 18S rRNA . We found that matriptase, HAI-1 and β-actin mRNA's are present with almost the same abundances  was observed  (Table The range and the interquartile range of mRNA expression of 1) Table .HAI-1 displayed a stronger general decrease in mRNA level with increasing tumor grade for both dysplastic and cancerous tissues as well as in normal tissue than matriptase  (Table 1) Table  in all g1) Table . A compaper se but the ratio of matriptase/HAI-1 expression that determines the frequency of carcinoma formation in a mouse model [matriptase/HAI-1 mRNA expression during colorectal carcinogenesis , severe dysplasia (p < 0.05) and in carcinomas (p < 0.05) when compared to corresponding tissue from healthy individuals. When compared to the ratio of matriptase/HAI-1 mRNA in normal tissue from the same individual there was a statistically significant difference for mild/moderate dysplasia (p < 0.001), for severe dysplasia (p < 0.001) and for all adenomas and carcinomas combined (p < 0.001). No statistically significant difference was seen in carcinoma tissue compared to unaffected tissue from the same person  [β-actin or that the translation frequency of the mRNAs is low. Indeed the half life of HAI-1 when recombinantly expressed in MDCK cells is in the range of a 1.5 hour .Our results have shown that the abundance of the protein) . Likewismatriptase/HAI-1 mRNA ratio as compared to the corresponding tissue in healthy control individuals, is an early event in colorectal cancer carcinogenesis that is maintained during all stages of malignant progression. Future studies are required to clarify whether the dysregulated matriptase/HAI-1 ratio was causing the malignant progression or is a consequence of the same.In conclusion, our study shows that dysregulation of the The author(s) declare that they have no competing interests.LV and UV conceived the idea of the study. EHK designed the cohort and collected the samples. MS and CFS extracted the RNA, carried out the cDNA synthesis and performed most of the statistical calculations. UV and LV designed and validated primers and probes. LV carried out the RT-PCR and drafted the manuscript. KA and EDKP helped writing the manuscript. All authors helped interpret the results, writing the manuscript and read and approved the final version.The pre-publication history for this paper can be accessed here:


Prostasin is proteolytically activated by the serine protease, matriptase, which has a very high oncogenic potential. Prostasin is inhibited by protease nexin-1 (PN-1) and the two isoforms encoded by the mRNA splice variants of prostasin and PN-1 in colorectal cancer tissue (n = 116), severe dysplasia (n = 13), mild/moderate dysplasia (n = 93), and in normal tissue from the same individuals. In addition, corresponding tissues were examined from healthy volunteers (n = 23). A part of the cohort was further analysed for the mRNA levels of the two variants of HAI-1, here denoted HAI-1A and HAI-1B. mRNA levels were normalised to β-actin. Immunohistochemical analysis of prostasin and HAI-1 was performed on normal and cancer tissue.Using quantitative RT-PCR, we have determined the mRNA levels for PN-1 was more that two-fold elevated in colorectal cancer tissue as compared to healthy individuals (p < 0.001) and elevated in both mild/moderate dysplasia (p < 0.01), severe dysplasia (p < 0.05) and in colorectal cancer tissue (p < 0.001) as compared to normal tissue from the same individual. The mRNA levels of HAI-1A and HAI-1B mRNAs showed the same patterns of expression. Immunohistochemistry showed that prostasin is located mainly on the apical plasma membrane in normal colorectal tissue. A large variation was found in the degree of polarization of prostasin in colorectal cancer tissue.The mRNA level of prostasin was slightly but significantly decreased in both mild/moderate dysplasia (p < 0.001) and severe dysplasia (p < 0.01) and in carcinomas (p < 0.05) compared to normal tissue from the same individual. The mRNA level of PN-1 is significantly elevated in colorectal cancer tissue. Future studies are required to clarify whether down-regulation of prostasin activity via up regulation of PN-1 is causing the malignant progression or if it is a consequence of it.These results show that the mRNA level of  Extracellular proteases and protease inhibitors are believed to play an important role during carcinogenesis in many different ways such as degrading the extracellular matrix in order to facilitate invasive growth and activating signal molecules. In accordance with this, proteases were mostly thought of as promoters of carcinogenesis. However, clinical trials where cancer patients were treated with broad-range protease inhibitors have shown that proteases can act as tumour suppressors . StudiesHAI-1, here denoted HAI-1A, and HAI-1B [in vitro invasiveness in both prostate and mammary cancer cell lines [prostasin mRNA levels correlates with longer survival for gastric cancer patients [Prostasin is a glycosylphosphatidylinositol (GPI)-anchored trypsin-like protease expressed in most epithelial cells ,6. It isd HAI-1B . HAI-1B d HAI-1B . Prostasd HAI-1B . Matriptd HAI-1B ,15 and bd HAI-1B . The dowd HAI-1B ,17 and id HAI-1B . It has d HAI-1B  and clead HAI-1B  but may d HAI-1B ,12, and d HAI-1B . Using td HAI-1B . Simultad HAI-1B . Althougll lines ,24 and hpatients .HAI-1 mRNA levels are decreased during colorectal cancer carcinogenesis in both normal and affected tissue from individuals with colorectal cancer adenomas and carcinomas as compared to healthy individuals using an assay that does not differentiate between HAI-1A and HAI-1B [Prostasin is expressed in most epithelial cells and could potentially play a role in most types of cancer. Prostasin has been shown to be down-regulated in gastric cancers  and prosd HAI-1B . Likewisd HAI-1B  and in gd HAI-1B .prostasin and its inhibitors PN-1, HAI-1A, and HAI-1B, during colorectal cancer carcinogenesis in humans.In the present study we have investigated the mRNA levels of The KAM cohort  is based on the screening group of the Norwegian Colorectal Cancer Prevention study (the NORCCAP study) in the county of Telemark, Norway . Additio2 until RNA purification. RNA purification included a DNAse treatment. cDNA synthesis was performed on approximately 200 ng RNA per 20 μl using High-Capacity cDNA Archive Kit . Quantitative RT-PCR for prostasin was performed on the ABI7300 sequence detection system (Applied Biosystems) in Universal Mastermix (Applied Biosystems) using 160/100 nM probe and 600/200 nM primers for prostasin and PN-1, respectively. For detection of prostasin mRNA, primers and probes were: F, 5' GGCCTCCACTGCACTGTCA 3'; R,5' AGTTACACGTCTCACGACTGATCAG '3'; probe, 5'-FAM-CAGTGAGCCTCCTGACGCCCAAG-BHQ-3'. For detection of PN-1 mRNA, primer and probes were: F, 5' CCTCCATGTTTCTCATATCTTGCA 3'; R, 5' ACCAGGGAGGCGATGATCT 3'; probe, 5'FAM-AAGCTTCAGCAGCAACAACTGCAATTCTCA-BHQ-3'. Primers were designed within different exons and with probes covering the exon-exon border to prevent amplification of genomic DNA. DNA coding for HAI-1 gives rise to two mRNA's coding for two proteins where the amino acids sequence differs by 16 residues [HAI-1 mRNA using a probe/primer set that detects both HAI-1A and HAI-1B. For this study we have designed new sets of probe/primers specific for either HAI-1A or HAI-1B. Assays for HAI-1A and HAI-1B were performed using 300 nM probe and 100 nM primers. Primers and probes for detection of HAI-1A were: F, 5'AGA TCT GCA AGA GTT TCG TTT ATG G-3'; R, 5'-GGT GCC AGA GCA CAC TGG AT-3'; probe, 5'-FAM-TGT GCA AGG CCC CTC CAT GGA A-BHQ-1-3'. Primers and probes for detection of HAI-1B were: F, 5'-ATC TGC AAG AGT TTC GTT TAT GGA-3'; R, 5'-GGT GCC AGA GCA CAC TGG AT-3'; probe, FAM-CCT TTG AGA GGC AGC TC-MGBNFQ. HAI-1A and HAI-1B probes were obtained from Applied Biosystems. β-actin primers and probes were obtained as a pre-developed assay (cat. no. 4310881E). All other primers and probes were obtained from TAG Copenhagen (Denmark). In a validation experiment using a control sample, a dilution series was produced and assayed for prostasin, PN-1, HAI-1A, HAI-1B and β-actin as described for the comparative Ct method [t values were plotted against log dilution it was shown that the assays are quantitative over a range of 256-fold dilution and that the PCR reactions have similar efficiencies provided that a threshold of 0.02 is used for prostasin and 0.2 is used for PN-1, HAI-1A, HAI-1B, and β-actin. The threshold is a fixed fluorescence signal level above the baseline and the Ct value of a sample is determined as the fractional cycle number where the sample's fluorescence signal exceeds the threshold. Prostasin, PN-1, HAI-1A, HAI-1B, and β-actin were quantified separately in triplicates. The average standard deviation on the triplicates was 14% or less. The standard deviation on repeated measurements of the same sample (the control) in separate experiments was 20%, 11%, 14%, and 14% for prostasin, PN-1, HAI-1A and HAI-1B respectively, indicating the day-to-day variation of the assay. Independent PCR reactions of the same samples yielded a correlation coefficient of 0.85 for prostasin and 0.95 for PN-1. Negative controls (where the RNA was not converted into cDNA) and positive controls were included in all sets.Total RNA was purified from tissue as recommended by the manufacturers using E.Z.N.A. Total RNA Kit II . The tissue samples were frozen in liquid as soon as possible after surgery and were stored in liquid Nresidues . We havet method . When CtMiniTab Statistical Software, Release 13.1 Xtra (Minitab Inc.) and Graph Pad Prism 4 were used for the statistic calculations. The data were not adjusted for gender since the incidence ratio of colorectal cancer between the genders is 1:1 in Norway .Tissue specimens were fixed in 4% formalin, paraffin embedded and processed for routine histology. Tissue sections were cleared with xylene and rehydrated in a graded series of alcohols and boiled in T-EG buffer  for 20 min for antigen retrieval. The sections were blocked for 30 min in PBS containing 3% BSA and 0.1% Triton X-100 and then incubated at 4°C overnight with mouse anti-prostasin antibody  (0.02 μg/μl) and goat anti-hHAI-1 . The slides were washed 3 times for 5 min with 3% BSA in PBS and 0.1% Triton X-100 and incubated at room temperature for 45 min with the secondary antibody Alexa fluor 555 donkey anti-mouse and Alexa fluor 488 donkey anti-goat. Afterwards, tissue sections were washed 3 times for 5 min with 3% BSA in PBS and 0.1% Triton X-100, followed by 4 times in PBS and finally mounted with DAPI H-1500 (Vector Laboratories Inc.). The sections were examined for the localization of prostasin subjected to laser conventional fluorescence microscopy. Sections where the primary antibodies were omitted served as controls.PN-1 were measured in colon tissue samples from healthy individuals (n = 23), individuals with mild/moderate dysplasia (n = 93), individuals with severe dysplasia (n = 13), and individuals with colorectal cancer (n = 116) by real-time RT-PCR. From individuals with dysplasia a sample of histologically normal tissue was used as well. From individuals with colorectal cancer two sample of histologically normal tissue from the surgically removed tissue were used. Ole sample was obtained as far away from the cancer tissue as possible  and another sample obtained immediately adjacent to the cancer tissue . A good correlation between the mRNA and protein levels for both prostasin [PN-1 [prostasin, PN-1, HAI-1A and HAI-1B were normalised to the mRNA level of β-actin.The mRNA levels of prostasin and rostasin  and PN-1in [PN-1  has prevprostasin normalised to β-actin are shown in Figure prostasin mRNA level of healthy individuals was compared to normal or affected tissue from individuals with dysplasia or colorectal cancer no significant difference could be observed . There was also a significant difference when normal and affected tissue from the same individual were compared for individuals with mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01), and for colorectal cancer both compared to the normal distant (p < 0.05) and to the normal adjacent sample (p < 0.01). This shows that the prostasin mRNA level is slightly but significant lower in mild/moderate dysplasia, severe dysplasia and colorectal cancer tissue as compared to normal tissue.The range and the interquartile range of mRNA levels of d Figure . HoweverPN-1 normalised to β-actin are shown in Figure PN-1 mRNA level showed an increase with increasing tumour grade in dysplastic and cancerous tissues whereas the normal tissue was not affected. A significant almost 3-fold increase (p < 0.001) of PN-1 mRNA was found in carcinomas as compared to corresponding tissues in healthy individuals , severe dysplasia (p < 0.05) and in carcinomas both when compared to the normal distant sample and with normal adjacent sample (p < 0.001),  with carcinomas (p < 0.001) using a one-way ANOVA and Tukey's post test. The PN-1 mRNA levels thus seem to be upregulated both at the transition from normal tissue to mild/moderate dysplasia and then again at the transition between dysplasia and cancer tissue whereas normal tissue is not affected.The range and the interquartile range of mRNA levels of ), Table . In addiprostasin and PN-1 mRNA levels with the designated Dukes'stages. Likewise the adenomas were classified as either low-risk (n = 14) or high-risk (n = 91) adenomas using the criteria described in material and methods. No significant difference in the mRNA levels of either prostasin or PN-1 was seen when comparing high-risk and low-risk adenomas (one-way ANOVA and Tukey's post test for paired comparison). When the prostasin mRNA level in normal tissue from either healthy individuals or individuals with dysplasia or colorectal cancer was plotted against age we were unable to detect any age-related change in the prostasin mRNA level  whereas the PN-1 mRNA level showed a slight increase with higher age. We were unable to detect any gender-related differences in the prostasin or PN-1 mRNA expression levels.Colorectal cancer can be staged according to Dukes' stage A-D where D is the most advanced and metastatic stage. Among the carcinomas in this study 23 carcinomas were designated as Dukes'stage A, 47 carcinomas as stage B, 39 carcinomas as stage C and none of the samples were classified as stage D. No tendency and no significant difference (one-way ANOVA and Tukey's post test for paired comparison) were seen when comparing the HAI-1 transcripts differ by the coding region for 16 amino acids. We have previously shown that HAI-1 is significantly down-regulated in both normal and affected tissue from individuals with adenomas and carcinomas [HAI-1A and HAI-1B respectively. mRNA levels for HAI-1A and HAI-1B were determined in colon tissue samples from healthy individuals (n = 14), in healthy and affected tissue from individuals with mild/moderate dysplasia (n = 19) and in healthy and tumour tissue from patients with colorectal cancer (n = 24). Figure HAI-1A, HAI-1B and the radio of HAI-1A/HAI-1B normalised to β-actin. The levels of HAI-1A and HAI-1B mRNA found are virtually identical in all tissues examined with the levels of HAI-1 found previously [HAI-1A/HAI-1B in the different tissues tested. These data suggest that there is no regulation in the splicing of the HAI-1 transcript during colorectal cancer carcinogenesis.It is possible that prostasin activity is regulated by modulation of the ratio between HAI-1A and HAI-1B as it is not clear whether the two inhibitors have the same kinetic properties. The two rcinomas . In the eviously . Both HANormal and cancerous tissues from five colorectal cancer patients were analysed to investigate the sub-cellular localization of prostasin and HAI-1. Formalin-fixed paraffin-embedded tissue sections were immunohistochemically co-stained for prostasin and HAI-I. In normal colorectal tissue, prostasin was detected mainly on the apical plasma membrane – with only minor staining of the basolateral plasma membrane Figure  and 4D. prostasin, PN-1, HAI-1A and HAI-1B during colorectal cancer carcinogenesis. It has previously been shown that over-expression of prostasin in mammary and prostate cancer cells reduces the invasive properties of cancer cells [prostasin mRNA in mild/moderate dysplasia, severe dysplasia and colorectal cancer tissues combined as compared to corresponding normal tissues from healthy individuals. In a previous study a similar minor but significant down-regulation was seen when comparing normal and affected tissue from gastric cancer patients [prostasin accompanying the transition between severe dysplasia and colorectal cancer. However, the mRNA levels of the inhibitor of prostasin, PN-1 increase at both the transition between normal tissue and mild/moderate dysplasia and again at the transition between severe dysplasia and colorectal cancer.In the present study we have determined the mRNA levels of er cells ,24 and ter cells . It coulpatients . Our resFor PN-1 to be a physiologically relevant inhibitor of prostasin it is required that PN-1 and prostasin have physical access to each other. PN-1 belongs to the serpin family of secretory proteins. We have previously shown that five other members of the serpin family are secreted to both the apical and the basolateral side of the polarized epithelial cell line, MDCK, albeit at different ratios . SimilarHAI-1 mRNA is decreased by 3-fold during colorectal cancer carcinogenesis using an assay that does not distinguish between HAI-1A and HAI-1B [HAI-1A and HAI-1B have expression patterns virtually identical and indistinguishable from the expression pattern of HAI-1 previously found [HAI-1A/HAI-1B remains the same in all tissues investigated during colorectal cancer carcinogenesis. Therefore, the splicing of HAI-1 mRNA is not regulated during carcinogenesis. In the present study we found limited co-localization of prostasin and HAI-1 in normal tissue, as HAI-1 is located mainly on the basolateral plasma membrane and prostasin is located on the apical plasma membrane. This probably does not mean that their interactions is limited due to physical separation as we have previously shown that HAI-1 is transcytosed from the basolateral plasma membrane to the apical plasma membrane [Prostasin is inhibited not only by PN-1 but also by HAI-1A and HAI-1B. We have previously shown that the level of d HAI-1B . In the ly found  and thatmembrane . It is tin vivo. Likewise prostasin-HAI-1B complexes have been purified from a cell line [The enzymatic activity of prostasin is influenced both by the levels of PN-1, HAI-1A and HAI-1B and probably also by the expression levels of other as yet undiscovered inhibitors of prostasin. The relative physiological importance of PN-1, HAI-1A and HAI-1B as inhibitors of prostasin is at present unclear. PN-1 is an inhibitor of not only prostasin, but also of uPA, tPA, thrombin, plasmin, trypsin  and matrell line , suggestPN-1, coincides with acquisition of malignant properties, whereas the mRNA level of prostasin is relative stable during colorectal cancer carcinogenesis. However, further investigations are necessary to understand the role of prostasin, PN-1, HAI-1A and HAI-1B for invasive growth and malignant progression.Proteases have for a long time been thought to play an important role in the growth of cells both during tissue remodelling and invasive growth. However, the action of proteases may be regulated not necessarily by the expression of the protease but by regulating the expression of its inhibitors. Our data shows that elevated mRNA levels for prostasins inhibitor, The authors declare that they have no competing interests.LV conceived the idea of the study. EHK designed the cohort and collected the samples. LV and EHK extracted the RNA. LV and JB designed and validated primers and probes and performed the statistical analyses. JS-P performed the immunohistochemistry. IMBL evaluated the tissue sections. JS-P, SF and LV prepared the figures. KMT and EKH administrated the KAM study. LV and JS-P wrote the first draft of the manuscript. All authors helped interpret the results, writing the manuscript and read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/201/prepub


This virus is antigenically and phylogenetically distinct from rodent-borne hantaviruses. Thottapalayam virus (TPMV) has been placed in the genus Hantavirus of the family Bunyaviridae by virtue of its morphologic features and overall genetic similarities to well-characterized rodentborne hantaviruses. This virus has been isolated from the Asian house shrew (Suncus murinus); however, whether TPMV is naturally harbored by an insectivore host or represents spillover from a rodent reservoir host is unknown. Our analysis of published and unpublished data on the experimental host range, genetics, and molecular phylogeny of TPMV supports coevolution of TPMV with its nonrodent reservoir host. Future studies on the epizootiology of TPMV and investigations of new shrewborne hantaviruses will provide additional insights into the evolutionary origin of hantaviruses in their rodent and insectivore reservoir hosts. Such investigations may also provide clues about determinants of hantavirus pathogenicity and virulence. Hantavirus, similar to other members of the family Bunyaviridae, have a negative-sense, single-stranded RNA genome in 3 segments designated large (L), medium (M), and small (S), which encode an RNA-dependent RNA polymerase, envelope glycoproteins  and nucleocapsid (N) protein, respectively (Apodemus agrarius), Dobrava virus (DOBV) with the yellow-necked field mouse (Apodemus flavicollis), Seoul virus (SEOV) with the Norway rat (Rattus norvegicus), Thailand virus (THAIV) with the bandicoot rat (Bandicota indica), Puumala virus (PUUV) with the bank vole , Tula virus (TULV) with the European common vole , Prospect Hill virus (PHV) with the meadow vole (M. pennsylvanicus), and Sin Nombre virus (SNV) with the deer mouse (Peromyscus maniculatus).Viruses in the genus Hylomyscus simus) (Suncus murinus) captured in 1964 during a survey for Japanese encephalitis virus in southern India , by the intracerebral route with 6,000 PFU of TPMV  by using antisera from experimentally infected animals  or have low pathogenic potential (such as TULV). Among the HFRS- and HPS-causing pathogenic hantaviruses, differential use of β-3 integrins as cellular receptors on platelets and endothelial cells may account for vascular leakage and hemorrhage associated with HFRS and HPS , are present in India, the demonstrated seroreactivity to HTNV may represent cross-reactivity with another Apodemus-borne hantavirus. Alternatively, seroreactivity may indicate infection with a nonrodentborne hantavirus, such as TPMV. Because Asian house shrews are peridomestic, frequently living within or in close proximity to human dwellings, TPMV infection may occur in humans.Immmunoglobulin G (IgG) against HTNV has been detected in sera from persons in southern India  serum samples were reactive to TPMV (geometric mean titer 80.6), and 16 (4.4%) samples were reactive to SEOV (geometric mean titer 103.1). Attempts to verify the specificity of this immunoreactivity by PRNT were unsuccessful . More recently, however, evidence suggestive of TPMV infection was found in a Laotian immigrant with a febrile illness by using a Western immunoblot analysis and a newly developed ELISA. This ELISA used a recombinant TPMV N antigen that contained an E5/G6 epitope captured by MAb E5/G6 (Crocidura leucodon) , Eurasian water shrew (Neomys fodiens), and common mole  in the former Soviet Union (Blarina brevicauda) in the United States (Crocidura russula) (Anourosorex squamipes)    was correctly predicted. When this predictive paradigm is applied to insectivores, species of Crocidura and Sorex genera would be expected to serve as reservoir hosts of hantaviruses because of their close phylogenetic proximity to S. murinus (Crocidura theresae) in Guinea . These newly identified shrewborne hantaviruses provide new knowledge about the genetic diversity of hantaviruses as well as possible insights into their evolutionary origin through host-switching events.Another approach to the targeted discovery of new hantaviruses harbored by shrews relies on molecular phylogeny. By constructing phylogenetic trees based on mitochondrial or nuclear gene DNA sequences, existence of hantaviruses in the Korean field mouse  may be too low for virus transmission. However, this probability is true for most zoonotic microbes, which only rarely infect humans. Thus, in the absence of disease outbreaks, zoonotic diseases frequently go unrecognized. In this regard, HPS would have similarly gone undetected had cases not clustered in time and space and had a closely knit group of dedicated and astute healthcare workers not recognized that something unusual was happening. The long-awaited clue of finding IgG against TPMV in a febrile Laotian immigrant (


Therefore, early assessment of treatment response would be beneficial. In this study, we investigated the in vitro with the MTT assay and in vivo on H727 xenograft tumors. The mice were scanned at baseline with 18F-FDG or 18F-FLT and then treated with either placebo or everolimus (5 mg/kg daily) for 10 days. PET/CT scans were repeated at day 1,3 and 10.The effect of everolimus on the human carcinoid cell line H727 was examined in vitro at concentrations above 1 nM. In vivo tumor volumes measured relative to baseline were significantly lower in the everolimus group compared to the control group at day 3 , day 7  and at day 10 . Uptake of 18F-FDG and 18F-FLT showed little differences between control and treatment groups, but individual mean uptake of 18F-FDG at day 3 correlated with tumor growth day 10 , 18F-FLT mean uptake at day 1 correlated with tumor growth day 7  and at day 3 18F-FLT correlated with tumor growth day 7  and day 10 .Everolimus showed significant inhibition of H727 cell proliferation in vitro and in vivo in human xenografts lung carcinoid NETs and especially early 18F-FLT uptake predicted subsequent tumor growth. We suggest that 18F-FLT PET can be used for tailoring therapy for neuroendocrine tumor patients through early identification of responders and non-responders.Everolimus was effective  Neuroendocrine tumors (NETs) are a heterogeneous group of tumors derived from neuroendocrine cells mainly from the gastrointestinal tract and the bronchopulmonary system. NETs have been considered a rare type of cancer but according to epidemiological data, the incidence has increased five-fold from 1.09 to 5.25/100.000 from 1973 to 2004. Since the survival has increased as well, gastrointestinal NET is now the second most prevalent cancer in the gastrointestinal tract The treatment of NETs has for many years been multifaceted including surgery, somatostatin analogs, interferon alfa, chemotherapy, and peptide receptor radionuclide therapy The gold standard to monitor chemotherapy response in solid tumors has for more than a decade been measurements of the tumor size by CT using the RECIST criteria At the same time, evidence gained from several tumor types show that additional information is obtained when Positron Emission Tomography (PET) is used for treatment monitoring compared to CT measurements alone 18F-fluorodeoxyglucose (FDG).Most widely used in cancer imaging and treatment monitoring is PET using FDG uptake reflects cancer cell glycolysis and has been shown to correlate with early treatment responses both in preclinical studies and patient studies although not in all cases 18F-fluorothymidine (FLT) Another hallmark of cancer, proliferation, can be measured using Everolimus is an inhibitor of the mammalian target of rapamycin (mTOR), a downstream component of the PI3K/AKT pathway involved in the regulation of cell proliferation, metabolism and angiogenesis.It is known that the mTOR pathway is dysregulated in some NETs due to aberrant events such as loss or down-regulation of the tumor suppressor gene PTEN or tuberous sclerosis 2 (TSC2). Low TSC2 and PTEN are linked to progression of the cancer In line with this, everolimus has recently been approved by FDA and EMA for the treatment of advanced pancreatic NETs.At the same time, it is becoming evident that subgroups of NET-patients for some reason have a worse prognosis than others. FDG has shown to be highly prognostic for NET-patients across the classification system Since everolimus has shown promising results in some NETs and the antitumor effect is mediated through inhibiting the mTOR complex, it seems likely that tracers detecting glycolysis and proliferation could serve as early predictors of a treatment response or failure.in vitro and in an animal model using FDG and FLT PET. The aim was to evaluate the effect of everolimus in NET and if FDG/FLT PET imaging could be used as an imaging biomarker for early prediction of the treatment response.In the present study, we monitored the response to everolimus treatment of NET Animal care and experimental procedures were approved by the Danish Animal Welfare Council (license no. 2011/561-14).2 at 370C.The human neuroendocrine tumor cell-line, lung bronchus carcinoid, NCI-H727 obtained from ATCC  was used. The cell line was tested to be free of Mycoplasma at Statens Serum Institute, Copenhagen, Denmark. The cells were cultured in RPMI Medium 1640 with GlutaMAX™  containing 10% fetal calf serum  and 1% penicillin-streptomycin  in 5% COH727 cells were plated in flat bottom 96-well plates at 3000 cells/well and incubated overnight. Treatment with everolimus was done in triplicates at escalating concentrations from 1 to 1000 nM for 72 hours. The cell proliferation was monitored with the MTT assay according to manufacturer protocol  and the proliferation was normalized to the untreated cells.6 cells in 100 uL medium mixed with 100 uL Matrixgel™ Basement Membrane Matrix; BD Sciences, San José, CA, USA). The inoculates were injected subcutaneously on the left and right flank during anesthesia with 1∶1 V/V HypnormR /DormicumR  i.p. Tumors were allowed to grow for 2 weeks. Half of the mice were FLT-PET scanned and the other half were FDG-PET scanned. At day 0 “baseline” examinations with FDG or FLT PET-scans as well as CT-scans were performed. Thereafter we randomized the mice to treatment with everolimus (n = 10) or vehicle (n = 10). The mice were treated daily i.p with 0.250 ml solution, either vehicle consisting of 18 ml 5% glucose and 2 ml DMSO, or 10 mg of everolimus  diluted in 18 ml 5% glucose and 2 ml DMSO. Daily dose of everolimus was 5 mg/kg The study design is illustrated in 18F-FDG was acquired from daily productions for clinical use at our facility .18F-FLT was synthesized using 3-N-Boc-1-[5-O--3-O-nosyl-2-deoxy-b-D-lyxofuranosyl]thymine as precursor and synthesized on a GE TracerLab MX Synthesizer as previously described 2 in N2 and fixed on a bed compatible with both the PET and CT scanner. A 15 min PET scan was acquired using a MicroPET Focus 120  Mice were injected i.v. with approximately 10 MBq of FDG or FLT. Before each FDG scan, the mice were fasted overnight. The mice were anaesthetized with 3% sevoflurane  mixed with 35% 0The PET data were arranged into sinograms and subsequently reconstructed with the maximum a posteriori (MAP) reconstruction algorithm after data acquisition. The pixel size was 0.300×0.300×0.796 mm and average resolution was 1.4 mm full-width-at-half-maximum. The images were not corrected for attenuation or scatter Immediately following the microPET scan, a microCT scan was acquired using a MicroCAT II system  inj, where CT is radioactivity in the tissue, W is weight of the animal and Dinj is injected dose. SUVmean gives information of the mean concentration of tracer and SUVmax is a measure of the voxel within the ROI that have the highest tracer concentration. For tumor volume measurements, ROIs were drawn manually by delineation of the tumor boundary by qualitative assessment in several of the coronal planes on the microCT images. Summation of voxels from all the planes generated the tumor volume. Tumor volume can be correctly assessed by this method Fusion of microPET and microCT images was done in the Inveon software . Before fusion, region of interests (ROIs) were drawn on the CT pictures manually by qualitative assessment covering the whole tumors and subsequently tumor volume and tracer uptake, assessed by standardized uptake values (SUV) mean and maximum, were generated by summation of voxels within the tomographic planes. SUV was defined as (CT*W)/Dmean and tumor growth. Calculations were made in SPSS 20 . Data are reported as mean ± SEM and p<0.05 was considered statistically significant.Unpaired students t-test was used for comparison between treatment and control groups. Paired t-test was used for intra-group comparisons. Shapiro Wilk test for normality and Levenes test for equality of variances were used. Bonferroni correction of p-values for multiple comparisons was applied. Linear regression was used for calculation of correlations between early SUVin vitro at concentrations above 1 nM (Everolimus showed significant inhibition of H727 cell proliferation ove 1 nM .3). The relative size of the tumors, given as the percent of baseline, was used for evaluating the tumor growth.Treatment of H727 xenografts with everolimus 5 mg/kg daily for 10 days resulted in decreases in tumor sizes compared to the vehicle treated control group. There were 36 tumors evaluable for growth monitoring (n = 18 in each group). No significant difference between the mean baseline tumor sizes of the groups was found. The tumor size at baseline was 185±14 . Uptake of FDG and FLT were determined relative to baseline at day 1, 3 and 10 after treatment initiation with everolimus .mean or SUVmax FDG uptake was observed between the treatment and control group at any time. At day 10 FDG SUVmean and SUVmax were significantly increased in the control group when compared to baseline  whereas no difference in FDG uptake was observed in the treatment group on day 10 when compared to baseline.By groups, no significant difference in SUVSixteen tumors were evaluated by FLT PET (n = 8 in each treatment group).mean and SUVmax uptake was significantly increased in both the control group  when compared to baseline. The relative FLT uptake in the control group was significantly raised on day 10 compared to baseline  whereas no difference in FLT uptake was observed in the treatment group. At day 10, the relative FLT SUVmean was significantly higher in the control group compared to the everolimus group  (On day 3 relative FLT SUV= 0.010) .mean (day 1 and 3 ) and subsequent tumor growth (day 7 and 10) are shown for both FDG PET and FLT PET in The correlation between day 1 uptake of FLT PET and tumorvolume at day 10 is shown in Tumor growth was measured as the relative increase in tumor size from baseline to either day 7 or day 10.mean uptake day 1 did not correlate with tumor growth neither day 7 nor day 10. FDG SUVmean uptake at day 3 correlated with tumor growth day 10  and showed a trend towards correlation with day 7 .Within the everolimus treated group, FDG SUVmean of FLT within the everolimus treated group day 1 significantly correlated with tumor growth day 7 , and showed a trend toward correlation at day 10 . FLT SUVmean uptake day 3 was significantly correlated with tumor growth day 7  and day 10 .SUVmax showed same trend (data not shown).Correlations for SUVThe major finding of our study was that everolimus inhibited tumor growth of the human lung carcinoid H727 and that FLT PET performed early after treatment initiation could predict later tumor growth.In our study of human carcinoid lung NET xenografts, everolimus significantly inhibited tumor growth by almost 50% compared to placebo treated tumors, indicating an anti proliferative effect of everolimus on H727 NET.In accordance with this, it has been found that mTOR activation is involved in the proliferation of subtypes of NETs in patients. In pancreatic NET cells, autocrine activation of the pathway is mediated through insulin-like growth factor 1 (IGF1) A phase II trial among patients with progressive pancreatic NET disease showed promising results using everolimus or everolimus in combination with octreotide The subsequent phase III randomized double-blind placebo-controlled trial consisting of 410 patients with progressive well-differentiated pancreatic NET, found that the median progression free survival (PFS) was 11 months with everolimus and 4.6 months with placebo Based on these results, US and European health authorities approved the use of everolimus for advanced pancreatic NET.Everolimus has also been evaluated in a large placebo-controlled phase III trial (RADIANT 2) in different types of NETs  associated with the carcinoid syndrome (n = 429). PFS was prolonged by 5.1 months for patients treated with both everolimus and octreotide LAR  compared to patients receiving placebo and octreotide LAR. The trial documented that everolimus can be used in a broad spectrum of NET patients In vitro studies have also shown that everolimus might be effective in the treatment of lung NETs. Accordingly, Zatelli et al. studied the effect of mTOR on bronchial carcinoids in 24 different bronchial carcinoids dispersed in cell cultures. In 15 of the cell cultures, everolimus significantly reduced cell viability, inhibited p706K activity and blocked IGF1 proliferative effects. The responding cell cultures were found to up-regulate mTOR expression indicating that bronchial carcinoids may be a suitable target for mTOR inhibition Although everolimus is effective in many NETs, the effect of this costly therapy is variable on an individual basis. Therefore, early detection of responders versus non-responders would be of great value. Since everolimus works by inhibiting the mTOR complex, an early detection of changes in mTOR pathway effects, like glycolysis and proliferation, might be visualized by FDG and FLT PET. To the best of our knowledge this has never been tested in NETs.2 = 0.63; P = 0.019). Clinically this would translate into that FLT PET early (days) after initiation of everolimus therapy in NET, could be used to identify responders and non-responders rather than waiting 6–8 weeks to see tumor shrinkage according to RECIST. In this way, non-responders could then early after treatment initiation be changed to alternative therapies and spared to undergo non-effective and expensive treatments. In support of this, non-NETs studies also indicate that FDG and FLT can be used to detect effect of everolimus: Accordingly, Nogová et al. used FDG for detecting the response of everolimus treatment in 8 non-small cell lung cancer patients. They scanned the patients at baseline and after 8 and 28 days of therapy and measured FDG by SUVmax and SUVmean In our study of H727 NET we found that especially FLT was effective in early prediction of later tumor growth. Accordingly, FLT at day one, where no effect on tumor growth was present, could predict growth until day 7 (rOthers have described the use of FDG in a human gastric cell line to determine the optimal biological dose of everolimus soon after therapy initiation vivoWei et al. found that both FLT and FDG uptake decreased and reflected sensitivity towards mTOR inhibition in glioblastoma xenografts in Ours findings that early imaging with FLT rather than FDG predicts later tumor volume is supported by other preclinical studies demonstrating that mTOR-inhibition is better predicted by FLT than FDG in lymphoma cell lines The present study is based on a single bronchial carcinoid cell line. Therefore, we cannot prove that the concept of early prediction of everolimus effect on tumor growth using FLT or FDG holds for all NETs. However, we believe the proof of concept data, are important and final testing will anyhow need to be performed in patients.We found that everolimus was effective in vivo in a human xenografts lung carcinoid NET and that FLT PET could effectively be used for early prediction of tumor growth. We suggest that FLT PET may be used for tailoring therapy in NETs with everolimus through early identification of responders and non-responders. In patients with FLT negative tumors FDG may represent an alternative. Whether such an algorithm translates clinically into better outcome remains to be tested.


The aim of the study was to evaluate changes in cell proliferation and glucose uptake by use of 3’-deoxy-3’-[In vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) were studied after treatment with belinostat. Mice were divided in 2 groups receiving either belinostat (40 mg/kg ip twice daily Day 0–4 and 6–10) or vehicle. Baseline [18F]FLT or [18F]FDG scans were made before treatment (Day 0) and repeated at Day 3, 6 and 10. Tracer uptake was quantified using small animal PET/CT.3) at Day 10 relative to baseline which was significantly different (P = 0.011) from the control group 769 ± 74% (926 mm3). [18F]FLT SUVmax increased from baseline to Day 10  in the control group. No increase was observed in the treatment group. [18F]FDG SUVmean was significantly different in the treatment group compared to the control group (P = 0.0023) at Day 10. Within treatment groups [18F]FDG uptake and to a lesser extent [18F]FLT uptake at Day 3 were significantly correlated with tumor growth at Day 10.Tumors in the belinostat group had volumes that were 462 ± 62% (640 mm[18F]FDG uptake early following treatment initiation predicted tumor sizes at Day 10, suggesting that [18F]FDG may be a valuable biomarker for non-invasive assessment of anti-tumor activity of belinostat. Changes in [18F]FDG uptake following anti-cancer treatment have been analyzed in several clinical studies; however, with variable results FLT and [18F]FDG PET can be used as non-invasive imaging biomarkers for monitoring of belinostat treatment. To do so, we analyzed [18F]FLT and [18F]FDG uptake in a human ovary cancer mouse model before and during treatment with belinostat. Tracer uptake was compared with Ki67, TK1 and glucose transporter 1 (GLUT1) gene expression.7 cells in 100 μL medium mixed with 100 μL Matrixgel™ Basement Membrane Matrix  were injected subcutaneously into the left and right flank respectively during anesthesia with 1:1 v/v mixture of Hypnorm®  and Dormicum® . The cell line was tested free of mycoplasma. A2780 was cultured in RPMI  medium 1640 + GlutaMAX  supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin (Invitrogen) in 5% CO2 at 37°C.Animal care and all experimental procedures were performed under the approval of the Danish Animal Welfare Council (2006/561-1124). Female NMRI  nude mice (8 weeks old) were acquired from Taconic Europe  and allowed to acclimatize for one week in the animal facility before any intervention was initiated. The human ovarian carcinoma cell line A2780  was used. For establishment of xenografts, 10[18F]FLT was synthesized using 3-N-Boc-1-[5-O--3-O-nosyl-2-deoxy-β-D-lyxofuranosyl]thymine as precursor on a GE TracerLab MX Synthesizer as previously described . All reaIn vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) was studied at various time points after treatment initiation. When tumor volumes were approximately 100 mm3 mice were divided in 2 groups receiving either belinostat (40 mg/kg ip) or vehicle  twice daily Day 0–4 and Day 6–10. Baseline [18F]FLT or [18F]FDG PET scans were made before treatment and repeated at Day 3, 6 and 10 after treatment initiation. Tumor volume was followed by CT during the experiments [eriments . Tumor v2 in N2 and fixed on a bed in presence of three fiducial markers allowing fusion of PET and CT pictures. A PET scan was acquired using a MicroPET Focus 120  followed by a microCT scan acquired with a MicroCAT® II system  as previously described [The mice were injected i.v. with 9.5 ± 0.2 (mean ± SD) MBq [18F]FLT or 10.0 ± 0.3 (mean ± SD) MBq [18F]FDG. Mice were fasted overnight before each [18F]FDG PET scan . One houescribed . PET datT*W)/Dinj, where CT is tissue radioactivity concentration, W is weight of the animal and Dinj is injected dose. SUVmean was calculated from the mean radioactivity concentration and SUVmax was calculated from the voxel with the highest tracer concentration.PET and microCT images were fused in the Inveon software . Before fusion region of interests (ROIs) were drawn on the CT pictures manually by qualitative assessment covering the whole tumors and subsequently tumor volume and tracer uptake, assessed by standard uptake value (SUV) was generated by summation of voxels within the tomographic planes. SUV was calculated according to the formula . The concentration of the RNA was determined by NanoDrop 1000 . Total RNA (0.3 μg) was reversed transcribed using the Affinityscript™ QPCR cDNA Synthesis kit  according to the manufacturer’s instructions. Samples were cooled down and stored at −20°C until further use.Primers were designed using Beacon Designer . Primer sequences were Ki67-FP: 5’-tcccgcctgttttctttctgac-3’, Ki67-RP: 5’-ctctccaaggatgatgatgctttac-3’, TK1-FP: 5’-gccgatgttctcaggaaaaagc-3’, TK1-RP: 5’-gcgagtgtctttggcatacttg-3’, GLUT1-FP: 5’-catcatcttcatcccggc-3’, GLUT1-RP: 5’-ctcctcgttgcggttgat-3’, GUSB-FP: 5’-tgagcaagactgatacca-3’, GUSB-RP: 5’-gctagaatagatgaccacaa-3’, HPRT-FP: 5’-caaagcctaagatgagagt-3’, HPRT-RP: 5’-gccacagaactagaacat-3’. For each gene the optimal primer concentration was found. All assays were optimized to have efficiencies between 95% and 105%. All samples were run in triplicate using one μl of cDNA. To each sample a no-reverse transcription control (NoRT) was included, and on each plate a no-template control (NTC) was included.Gene expression was quantified on a Mx3000P® real-time PCR system from Stratagene. All gene of interests (GOIs) and reference genes were quantified with Brilliant® SYBR® Green QPCR Master Mix (Stratagene). The following thermal profile was used in all experiments: 10 minutes of denaturation at 95°C followed by 45 cycles of 30 seconds denaturation at 95°C, 1 minute of annealing at 60°C and 1 minute extension at 72°C. A dissociation curve was afterward acquired by denaturation of the products for 1 minute at 95°C followed by a stepwise increase in temperature from 55°C to 95°C with steps of 0.5°C/cycle where the duration of each cycle was 18 seconds.QPCR data were analyzed in the qBase program. The relative quantification of the GOIs was calculated using two reference genes . Data art-test. Paired t-test was used for intra-group comparisons. Bonferroni correction of P-values for multiple comparisons was applied. Correlations between SUVmean or SUVmax and tumor growth were calculated using linear regression. Calculations were made in PASW 18.0 . Data are reported as mean ± SEM unless stated otherwise and p < 0.05 was considered statistically significant.Comparison between the treatment and control group was calculated using unpaired student’s 3) and control group (118 ± 19 mm3) were identical.Tumors in the control group had volumes that were 419 ± 39% at Day 6 and 769 ± 74% at Day 10 relative to baseline. In the belinostat group tumors were 282 ± 30% at Day 6 and 462 ± 62% at Day 10 relative to baseline which were significantly less than the control group both at Day 6 (p = 0.029) and Day 10 (p = 0.011)  in the control group. No increase in [18F]FLT SUVmax was observed in the treatment group at Day 10 Figures , 3.2 = 0.67; p = 0.02). [18F]FLT SUVmean Day 6 was correlated with tumor growth Day 10/baseline in the belinostat group  however not significant  and treatment group . No significant difference in SUVmax between treatment and control group was observed at any time point. [18F]FDG SUVmean was significant different in the treatment compared to the control group (p = 0.0023) at Day 10. Compared to baseline, [18F]FDG SUVmean uptake was increased at Day 6  and Day 10  in the control group. Compared to baseline, [18F]FDG SUVmean uptake was increased at Day 10  in the treatment group , however not significant. [18F]FDG SUVmean Day 6 was significantly correlated with tumor growth Day 10/baseline . [18F]FDG SUVmax Day 3 was significantly correlated with tumor growth Day10/baseline  as were [18F]FDG SUVmax Day 6   and hypoxanthine phosphoribosyltransferase 1 (HPRT). The levels of Ki67, TK1 and GLUT1 were normalized to the geometric mean of these two reference genes. The gene expression was measured at Day 10 in the treatment relative to the control group. Ki67 gene expression was unchanged in the treatment compared to the control group at Day 10. TK1 gene expression was higher in the treatment compared to the control group at Day 10 . GLUT1 gene expression was lower in the treatment group compared to the control group at Day 10  (Figure In this study we found that 18F]FDG uptake following initiation of treatment with the HDAC inhibitor belinostat predicted tumor sizes at the end of treatment in a mouse model of human ovary cancer. We observed minor effects on [18F]FLT uptake following treatment with belinostat. In a previous study lower tumor uptake of [18F]FLT was observed following treatment with the HDAC inhibitor LAQ824 in a human colon carcinoma mouse model . LAQ824 FFDG uptaEven though we did not find a decrease in [18F]FLT uptake in the belinostat group, within the treatment group [18F]FLT SUVmean at Day 3 and 6 was correlated with tumor growth at Day 10. The tumors having the lowest uptake of [18F]FLT at Day 3 and 6 following initiation of treatment with belinostat were those in which the treatment was most effective.Previously we have observed that [18F]FLT uptake was reduced after initiation of effective anti-cancer therapy in the A2780 tumor model ,49. ThusCompared to the [18F]FLT data, we observed a higher influence on [18F]FDG uptake following treatment with belinostat. At Day 10 uptake of [18F]FDG was decreased in the treatment group compared to the control group. The difference at day 10 did only reach significant difference for SUVmean and not for SUVmax. SUVmean is the mean tracer concentration in tumor and SUVmax is a measure of the pixel within the tumor which has the highest tracer concentration. An explanation to the non-significant change in SUVmax despite changes in SUVmean could therefore be because the anti-cancer treatment is less effective and does not inhibit glucose uptake in the most aggressive parts of the tumor and therefore no significant difference between SUVmax for the treatment and control group was observed. Another explanation to the differences could be that the difference for SUVmax did not reach statistical significance due to a type II error because of the limited amount of animals included in the study.The difference in [18F]FDG uptake between the treatment and control group was supported by underlying changes in gene expression of GLUT1. At Day 10 GLUT1 expressions were lower in the treatment compared to the control group. Other HDAC inhibitors likewise decrease GLUT1 gene expression . GlucoseWithin the treatment group the level of [18F]FDG uptake at Day 3 and 6 was correlated with treatment effect at the end of the study. The tumors which had the lowest [18F]FDG uptake at Day 3 and 6 following treatment start were the tumors which responded best to the treatment. Projecting this into a clinical situation will allow identification of the patients responding best to the treatment. Advantage of this information can be taken in two ways. Firstly, selection of which patients to be included in a clinical trial can be determined depending on drug sensitivity determined early in the treatment course. This will make identification of new compounds which are effective in only a small subset of patients easier. Secondly, in clinical practice, treatment modifications in non-responding patients during a treatment course may be undertaken.Some of the main limitations of the present study were the lack of protein expression levels of molecular markers in tumor tissue. It is therefore unknown whether or not the gene expression levels of Ki67, TK1 and GLUT1 reflected the protein levels of the matching proteins. However, in other studies a positive correlation between Ki67 protein and gene expression has been observed ,56. FurtNo regression in tumor volume was observed following treatment with belinostat; however, the tumor growth was lower in the treatment compared to the control group, thus confirming the anti-cancer effect of belinostat. It is known, that the belinostat compound exerts tumor stasis rather than tumor shrinkage -28. IdenIn conclusion, we found that [18F]FDG uptake early following treatment initiation with belinostat predicted tumor sizes at Day 10, suggesting that [18F]FDG PET may be a biomarker for non-invasive assessment of anti-tumor activity of belinostat. The results from this study supports the addition of [18F]FDG PET scans during clinical trials with belinostat where it may also be used for selection of subjects that may enter such studies.18F]fluorothymidine; [18F]FDG: 2-deoxy-2-[18F]fluoro-D-glucose; PET: Positron emission tomography; CT: Computed tomography; TK1: Thymidine kinase 1; HDAC: Histone deacetylase; ROIs: Region of interests; SUV: Standard uptake value; GOI: Gene of interests; TS: Thymidylate synthease[18F]FLT: 3’-deoxy-3’-[The following co-authors have conflict of interests: Peter Buhl Jensen: Ownership Interests and Employment in Topotarget A/S. Maxwell Sehested: Ownership Interests and Employment in Topotarget A/S. Fredrik Björkling: Employment in Topotarget A/S. Kamille Dumong Erichsen: Employment in Topotarget A/S. All other authors have no conflict of interests.Conceived and designed the experiments: MMJ KDE FB PBJ LH MS AK. Performed the experiments: MMJ KDE CBJ JM. Analyzed the data: MMJ KDE AK. Wrote the paper: MMJ AK. Revised and approved the paper: KDE CBJ FB JM PBJ LH MS AK. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/168/prepub


The aim of this study was non-invasively to study effect of APO866 treatment on [18F]FLT and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake.APO866 is a new anti-tumor compound inhibiting nicotinamide phosphoribosyltransferase (NAMPT). APO866 has an anti-tumor effect in several pre-clinical tumor models and is currently in several clinical phase II studies. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation In vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) was studied at various time points after APO866 treatment. Baseline [18F]FLT or [18F]FDG scans were made before treatment and repeated after 24 hours, 48 hours and 7 days. Tumor volume was followed with computed tomography (CT). Tracer uptake was quantified using small animal PET/CT. One hour after iv injection of tracer, static PET scans were performed. Imaging results were compared with Ki67 immunohistochemistry.Tumors treated with APO866 had volumes that were 114% (24 h), 128% (48 h) and 130% (Day 7) relative to baseline volumes at Day 0. In the control group tumor volumes were 118% (24 h), 145% (48 h) and 339% (Day 7) relative to baseline volumes Day 0. Tumor volume between the treatment and control group was significantly different at Day 7 (P = 0.001). Compared to baseline, [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. Compared to baseline, [18F]FDG SUVmax was significantly different at Day 7 (P = 0.005) in the APO866 group.APO866 treatment caused a significant decrease in [18F]FLT uptake 24 and 48 hours after treatment initiation. The early reductions in tumor cell proliferation preceded decrease in tumor volume. The results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies. The use of positron emission tomography (PET) to discriminate between responders and non-responders early following treatment initiation is desirable. The PET technique allows for non-invasive characterization of biological functionality in tumor tissue. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is used to assess cell proliferation in vitro and several pre-clinical human xenograft tumor models APO866 is a new anti-tumor compound which inhibits nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in the biosynthesis of NAD. APO866 inhibits biosynthesis of cellular NAD from niacinamide by inhibiting NAMPT. Reduction in cellular NAD levels leads to ATP depletion and apoptosis APO866 treatment decreases cellular ATP content 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has in several pre-clinical and clinical studies been tested for its ability to assess early effect of chemotherapeutics; however, with variable results. When new anti-cancer drugs are brought into clinical phase II and III studies, effective methods to discriminate between responders and non-responders are of great value. The aim of this study was therefore non-invasively to study the effect of APO866 treatment on [18F]FLT and [18F]FDG uptake and to evaluate if [18F]FLT or [18F]FDG PET could be used to monitor early effects of APO866 treatment.7 cells in 100 µL medium mixed with 100 µL Matrixgel™ Basement Membrane Matrix  were injected subcutaneously into the left and right flank respectively during anesthesia with 1∶1 v/v mixture of Hypnorm®  and Dormicum® . The cell line has been tested free of mycoplasma. Cells were cultured in RPMI  medium 1640+ GlutaMAX  supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin (Invitrogen) in 5% CO2 at 37°C.Animal care and all experimental procedures were performed under the approval of the Danish Animal Welfare Council (2006/561–1124). Female NMRI  nude mice (6–8 weeks old) were acquired from Taconic Europe  and allowed to acclimatize for one week in the animal facility before any intervention was initiated. The human ovarian carcinoma cell line A2780 3. Mice in the treatment groups received APO866 (2).½ receiving either APO866 or vehicle treatment and [18F]FLT or [18F]FDG scans. Treatment was started at day 14–16 after implantation of tumor cells, where tumor volumes were on average 265 mmd APO866  in salin[18F]FLT was synthesized using 3-N-Boc-1-[5-O--3-O-nosyl-2-deoxy-b-D-lyxofuranosyl]thymine as precursor on a GE TracerLab MX Synthesizer as described earlier 2 in N2 and fixed on a bed in presence of three fiducial markers allowing fusion of PET and CT pictures. A 10 min PET scan was acquired using a MicroPET Focus 120 . After data acquisition, PET data were arranged into sinograms and subsequently reconstructed with the maximum a posteriori (MAP) reconstruction algorithm. The pixel size was 0.866×0.866×0.796 mm and in the center field of view the resolution was 1.4 mm full-width-at-half-maximum. The images were not corrected for attenuation or scatter.Mice were injected i.v. with 10.3±1.4 (mean±SD) MBq [18F]FDG or 9.5±0.9 (mean±SD) MBq [18F]FLT. Mice were fasted overnight before each [18F]FDG scan Following the microPET scan, a microCT scan was acquired with a MicroCAT® II system . A 7 minute and 10 seconds CT scan was performed with parameter settings: 360 rotation steps, tube voltage 60 kV, tube current 500 µA, binning 4 and exposure time 310 ms. The pixel size was 0.091×0.091×0.091 mm.T*W)/Dinj, where CT is radioactivity in the tissue, W is weight of the animal and Dinj is injected dose. SUVmean gives information of the mean concentration of tracer and SUVmax is a measure of the voxel within the ROI which have the highest tracer concentration. For tumor volume measurements ROIs were drawn manually by delineation of the tumor boundary by qualitative assessment in several of the coronal planes on the microCT images. Summation of voxels from all the planes generated the tumor volume.PET and microCT images were fused in the Inveon software . Before fusion region of interests (ROIs) were drawn on the CT pictures manually by qualitative assessment covering the whole tumors and subsequently tumor volume and tracer uptake, assessed by standard uptake values (SUV) mean and maximum, were generated by summation of voxels within the tomographic planes. SUV was defined as  was used. The reactions were detected using standard polymer technique with few modifications. Negative controls were performed by omission of primary antibody. Sections were dewaxed in xylene, rehydrated and pre-treated before immunostaining. The slides were incubated with MIB-1 1:100 diluted in Power Block solution  for 30 min at room temperature. The sections were rinsed in TBST (3×5 minutes) and incubated with rabbit anti-FITC  for 20 min at RT. The reactions were detected with Super Sensitive polymer-HRP reagent  and visualised with DAB ImmPACT Substrate chromogen solution (Vector laboratories cat. co. SK-4105) following the manufactures instructions. Finally, slides were counterstained with Mayers haematoxylin.The level of Ki67 present in the samples was measured as mean of three hot spots on one slide from each tumor. Hotspots were defined as areas with the highest Ki67 intensity and were identified by scanning the entire tumor slide in 10×magnification. Ki67 positive cells were defined as cells with positive nucleus staining. The Ki67 staining index was defined as the number of Ki67 positive cells divided by the total number of tumor cells in an area on the 60×magnification. Positive and negative controls from human tissue were placed on each slide for validation of the immunohistochemical procedure.Comparison of tumor volume between the treatment and control group were calculated using an unpaired student’s t-test. For intra-group comparisons of tracer uptake ANOVA with Bonferroni post-hoc test was used. All data were tested to be normal distributed by means of Kolmogorov-Smirnov test. Correlations between SUVmax ratios and tumor growth were calculated using linear regression. Calculations were made in PASW 17.0 . Data are reported as mean±SEM (unless stated otherwise) and P<0.05 was considered statistically significant.2 = 0.98) and caliper   . APO866  = 0.87) .Baseline levels of [18F]FLT uptake measured as SUVmean were 1.26±0.16 (mean±SD) and SUVmax were 2.18±0.20 (mean±SD). Baseline levels of [18F]FDG uptake measured as SUVmean were 0.67±0.14 (mean±SD) and SUVmax were 1.23±0.29 (mean±SD).Uptake of [18F]FLT measured as SUVmean in the APO866 group was 1.28±0.03 at baseline, 0.98±0.03 at 24 h, 0.90±0.03 at 48 h and 0.96±0.07 at Day 7. Compared to baseline [18F]FLT SUVmean was significantly decreased at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. No significant changes in [18F]FLT SUVmean were observed in the control group. [18F]FLT SUVmax uptake in the APO866 group was 2.20±0.03 at baseline, 1.64±0.04 at 24 h, 1.49±0.03 at 48 h and 1.54±0.11 at Day 7. Compared to baseline [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group  and 5. NUptake of [18F]FDG measured as SUVmean in the APO866 group was 0.70±0.04 at baseline, 0.62±0.03 at 24 h, 0.59±0.04 at 48 h and 0.40±0.08 at Day 7. Compared to baseline [18F]FDG SUVmean was significantly decreased at Day 7 (P<0.001) in the APO866 group. No significant changes in [18F]FDG SUVmean were observed in the control group. [18F]FDG SUVmax uptake in the APO866 group was 1.29±0.09 at baseline, 1.11±0.07 at 24 h, 1.08±0.09 at 48 h and 0.77±0.15 at Day 7. Compared to baseline [18F]FDG SUVmax was significantly decreased at Day 7 (P = 0.005) in the APO866 group. In the control group [18F]FDG SUVmax was 1.16±0.07 at baseline, 0.91±0.03 at 24 h (P = 0.007 vs. baseline), 1.12±0.04 at 48 h and 1.47±0.05 at Day 7 (P<0.001 vs. baseline)  and 5.2 = 0.44) and d7/d0  correlated significantly with final tumor growth and [18F]FDG SUVmax ratio 48 h/0 h  and d7/d0  correlated significantly with final tumor growth . Differences in expression of Ki67 protein between the treatment (28.3%) and control group (52.5%) were significant (P = 0.022) (Expression of Ki67 protein measured by immunohistochemistry showed a significant correlation with [18F]FLT SUVmean (r= 0.022) . Some ofIn this study we investigated the effect of APO866 treatment on tumor uptake of [18F]FLT and [18F]FDG by PET in the A2780 ovarian cancer tumor model. By groups, uptake of [18F]FLT decreased after initiation of treatment and was significantly different from baseline already at 24 hours after treatment initiation. By groups, uptake of [18F]FDG did not change on the first 1–2 days following treatment start, however at Day 7 uptake was significantly decreased to 50% of baseline uptake. At Day 7 we observed decreases in both [18F]FLT and [18F]FDG tracer uptake. On the PET images  it was mA measure of the maximal tracer uptake is often used when assessing treatment responses rather than the mean tracer uptake Lower uptake of tracer at Day 7 compared to baseline for both [18F]FLT and [18F]FDG in the treatment groups was most likely not due to partial volume effect because of a decrease in tumor volume as the tumors did not shrink during the treatment. The changes in tracer uptake were therefore assumed to be due to physiologic changes in the tumor tissue which were further confirmed by the difference in the quantity of Ki67 positive cells between the treatment and control groups. Positive correlation between [18F]FLT uptake and the amount of Ki67 positive cells have earlier been reported by others APO866 is a relatively slow acting anti-cancer drug without any immediate cytotoxicity leading to ATP depletion and apoptosis Anti-cancer drugs being cytostatic or having low cytotoxicity often require long treatment time before exerting their effect on tumor volume. The advantage of measuring physiological changes as we do in this study with [18F]FLT and [18F]FDG PET compared to the tumor volume criteria is that we are able to measure a treatment effect earlier than we can measure a change in tumor volume.When the tumors were studied individually, we found a significant positive correlation between the changes in SUVmax for both [18F]FLT and [18F]FDG between baseline and 48 hours and the tumor growth. Accordingly, those tumors which had the greatest decrease in SUVmax values for both [18F]FDG and [18F]FLT 48 hours after treatment initiation were those which had the lowest increase in tumor volume at the end of the experiment. It indicates that we, in addition to finding a decrease in the whole treatment groups, on the individual tumor level already at 48 hours after treatment initiation were able to identify the tumors that responded best to the treatment as it were those that had the greatest change in SUVmax values.In conclusion, we found a decrease in tumor [18F]FLT uptake following treatment with the nicotinamide phosphoribosyltransferase inhibitor APO866. After 7 days of treatment tumor uptake of [18F]FDG was half of baseline uptake. Additionally, we found that the tumors which responded best to the treatment were those that had the greatest change in both [18F]FLT and [18F]FDG at 48 hours after treatment initiation. These results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies.


To design ARC-111 analogues with improved efficiency, we constructed the QSAR of 22 ARC-111 analogues with RPMI8402 tumor cells. First, the optimized support vector regression (SVR) model based on the literature descriptors and the worst descriptor elimination multi-roundly (WDEM) method had similar generalization as the artificial neural network (ANN) model for the test set. Secondly, seven and 11 more effective descriptors out of 2,923 features were selected by the high-dimensional descriptor selection nonlinearly (HDSN) and WDEM method, and the SVR models (SVR3 and SVR4) with these selected descriptors resulted in better evaluation measures and a more precise predictive power for the test set. The interpretability system of better SVR models was further established. Our analysis offers some useful parameters for designing ARC-111 analogues with enhanced antitumor activity. N,N-dimethylaminoethyl)-2,3-methylenedioxy-5H-dibenzo,6 naphthet al. have compared QSAR modeling of antitumor activity of ARC-111 analogues using stepwise multiple linear regression (stepwise MLR), partial least squares (PLS) and artificial neural network (ANN), and the results showed the ANN model was the most powerful for the test  were reserved by 3 rounds of nonlinear selection. Then the two low-dimensional datasets with 9 and 6 descriptors, respectively, were trained by leave-one-out (LOO) cross-validation and modeled in five Kernel functions . The results of the independent test showed (1) the SVR1 model  with all literature features had higher predictive ability than stepwise MLR and PLS; and (2) the SVR2 model (t = 2) with MSE of 0.061, R2 of 0.950 and Rpred2 of 0.918 for the test set had comparable predictive ability with the ANN  model et = 2) and 10-fold cross-validation will be adopted in future feature selecting, and the Radial Basis Function (t = 2) and LOO cross-validation will be adopted in independent tests.The SVR model with 6 descriptors (SVR2) produced better results than the SVR model with all 9 descriptors (SVR1). We noted that the 6 descriptors were obtained with the WDEM from the 9 descriptors. This showed that the WDEM method might be effective to choose relevant descriptors for more accurate prediction of the activities of ARC-111 analogues. This property will be helpful for the modeling with high-dimensional features. Considering nonlinear function, predictive ability and computing time, the Radial Basis Function (50 (expt.) values] was used for modeling. Because the high-dimensional descriptors had more redundant information, we focused on how to select nonlinearly less but more critical descriptors using SVR. We have developed two novel methods that could select important descriptors from thousands of them. By initial coarse screening using the HDSN method to filter out irrelevant features, the data set would switch from high-dimensional into low-dimensional. Then further careful screening using the WDEM method would turn the data set with low-dimensional features into one with only important descriptors. Throughout the process, the descriptors in modeling with higher MSE values were removed gradually and nonlinearly until the model with the lowest MSE value was obtained. Finally, the SVR models for the test set based on the obtained descriptors were developed and evaluated.To improve drug design of ARC-111 analogues, the analysis of high-dimensional descriptors may result in better prediction. Using the software, PCLIENT, 2,923 molecular descriptors were calculated. Then the high-dimensional dataset containing the independent variables  and the dependent variables  values against RPMI8402 tumor cells ranged from 6.071 to 9.523. To obtain statistically robust QSAR models and compare with the results of MLR, PLS, and ANN in , 2 spatial  and 1 physicochemical (MolPol) descriptors as well as different topological parameters  from the literature were adopted to construct QSAR models. The 9 descriptors were obtained by molecule energy optimization using MM2 ChemOffice 2005, and then were calculated by MODEL and ChemOffice 2005 [First, to understand the QSAR reliability of modeling ARC-111 analogue activities using SVR technique, 4 electronic [http://www.vcclab.org/lab/pclient/) and classified under 24 groups  by our HDSN method [To reduce dimensionality and improve model robustness in QSAR analysis, high-dimensional features would be screened coarsely and nonlinearly into low-dimensional features with lower mean squared error  and the squared predictive correlation coefficient (Rpred2) values calculated by the following equations:The selection of descriptors and the optimization of Kernel functions parameters were examined by 10-fold or LOO validation with the minimum yi, ŷi, ȳ, ŷ̄ and n, respectively, represented the experimental values, the predicted values, the mean values of the experimental values, the mean values of the predicted values and the number of compounds of the test set, and ȳtraining was the mean activity value of the training set. Generally, an acceptable QSAR model was considered to have a higher predictive power only having the lower MSE [R2 [Rpred2 (at least >0.6) [Here ower MSE , the hig MSE [R2  and the st >0.6)  for the In our QSAR analysis, the structural information of 34 ARC-111 analogues was described using 2923 molecular descriptors obtained. Two groups of more important descriptors were obtained using two nonlinear descriptor selection methods, and then used to model the activities of these ARC-111 analogues based on SVR. The two SVR models demonstrated consistently better performance than reference models in terms of prediction accuracy for the test data. Our results offer new theoretical tools for drug design and development.