Ostrinia furnacalis (Guenée), can develop strong resistance to Cry1Ab, the most widely commercialized Cry toxin for Bt maize worldwide. It is essential to understand the mechanism of resistance for management of this species, but information on the post-transcriptional regulation of Bt resistance in this target insect is limited. In the present study, RNA was extracted from the ACB in various larval stages (1–5 instar) from Cry1Ab-sensitive (ACB-BtS) and -resistant (ACB-AbR) strains, each of which included two biological replicates. Using Illumina sequencing, a total of 23,809,890 high-quality reads were collected from the four ACB libraries. The numbers of known microRNAs (miRNAs) were 302 and 395 for ACB-BtS and 268 and 287 for ACB-AbR. Using Mireap software, we identified 32 and 16 potential novel miRNAs for ACB-BtS and 18 and 22 for ACB-AbR. Among them, 21 known and 1 novel miRNAs had significantly different expression between ACB-BtS and ACB-AbR. Several miRNAs were observed to target potential Bt receptor genes, such as aminopeptidase N and cadherin-like protein. The glycosylphosphatidylinositol-anchor biosynthetic process and ABC transporters pathway were identified through Gene Ontology and KEGG pathway analysis of target genes of the differentially expressed miRNAs.The Asian corn borer (ACB),  Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most destructive corn-stalk-boring pest in Asia, particularly in China and the Philippines. Estimated yield losses from this pest are 10–20% and may exceed 30%; in some cases, entire harvests are lost in an outbreak year13The Asian corn borer (ACB), DrosophilaDrosophila miRNAs have revealed distinct roles in not only many important developmental events in insects but also various conserved mechanisms in animals, such as ageingIt has been reported that the mutation of aminopeptidase N (APN) genesAutographa califormica multiple nucleo-polyhedrosis virus (AcMNPV) in Spodoptera frugiperda (Sf9) cells resulted in a large number of changes in miRNA expression, such as the upregulation miR-184, miR-998 and miR-10 at 24 h post-infection (hpi) and, for some of these miRNAs, downregulation at 72 hpiBombyx mori larvae infected with B. mori cytoplasmic polyhedrosis virus (BmCPV), 58 miRNAs were found to be significantly upregulated or downregulated in the mid-gut compared with non-infected larvae at 72 and 96 hpiWolbachia-infected and uninfected Tetranychus urticae showed that Wolbachia infection significantly regulated miRNAs in both females and males, with an overall suppression of miRNAs in Wolbachia-infected librariesWolbachia-infected and non-infected mosquitoes, with two miRNAs, aae-miR-2940 and -309a, showing higher abundance in infected onesWolbachia-induced cellular miRNA, aae-miR-12, was confirmed as regulating two target genes, DNA replication licensing factor MCM6 and monocarboxylate transporter MCT1, and shown to be important for the maintenance of Wolbachia density in mosquito cellsBacillus thuringiensis (Bt) toxins has not yet been published.Accumulating evidence in recent years suggests that miRNAs have effects on insect-pathogen interactions, although such studies are limited compared with research on insect development. Infection with In the present study, we investigated and characterized the differential expression of miRNAs in the ACB larvae with different susceptibility to the Cry1Ab toxin using deep sequencing technology. The results will help to further understand the role of miRNAs in the ACB resistance to Bt toxins.Four small RNAs libraries, including two biological replicates of ACB-BtS and ACB-AbR, were constructed and Solexa-sequenced. A total of 23,809,890 high-quality reads were collected from the four libraries (Accession No: SRX976786) . The reaDuring alignment and annotation, some small RNA tags may be mapped to more than one category. To have each unique small RNA map to only one annotation, we followed the priority rule of rRNAetc (in which GenBank > Rfam) > known miRNA > repeat > exon > intronWe identified 900 and 802 unique miRNA genes from the 297,610 and 150,645 total miRNAs in the two replicates of ACB-BtS and 816 and 915 unique miRNA genes from the 210,297 and 337,452 total miRNAs in the two replicates of ACB-AbR. However, due to the lack of a complete ACB genome database, the highest percentage of miRNA in the total sRNAs, which was found in ACB-AbR-2, was only 5.78%. A majority of the information remains unexplored in the datasets.http://www.mirbase.org/). If the miRNA sequence with the most reads hit a known miRNA in miRBase, then the name of the known miRNA was assigned to the miRNA and other variants of the present study. In total, 302, 395, 268 and 287 known miRNAs were identified in ACB-BtS-1, ACB-BtS-2, ACB-AbR-1 and ACB-AbR-2, respectively. Among them, 163 miRNAs were commonly expressed in the four libraries  were significantly differentially expressed between the two strains  . Notablyvice versa. The results revealed that the expression of some of the miRNAs was negatively correlated with the relative abundance of their respective transcripts  . Second,Bacillus thuringiensis (Bt) is a major threat to the long-term use of Bt crops and has been studied extensively. However, understanding of the molecular mechanism of resistance of the ACB, the major corn insect pest in China and other countries of East and Southeast Asia, to Cry toxin is relatively poor. Over the last few years, studies have shown that miRNAs play a key role in host-pathogen interactions by regulating the expression of host resistant genes or viral genes to impair viral replication29Insect resistance to Caenorhabditis elegans over two decades agoet al. used Illumina/Solexa deep sequencing technology to identify 168 known miRNAs belonging to 55 families as well as 204 novel miRNAs of fourth-instar larvae of male and female Eupolyphaga sinensis et al. profiled 1,229 miRNAs of the posterior silk gland at the fifth larval instarSince the discovery of the first miRNA from A total of 163 known and 3 novel miRNAs were commonly expressed in the four libraries of the ACB. Few commonly expressed miRNAs candidates, such as miR-let-7, miR-bantam, and miR-14, have been reported to play essential roles in areas such as insect developmentp-values, only 21 known and 1 novel miRNAs were significantly differentially expressed between the two strains. Among them, miR-31 has been characterized as a tumour suppressor miRNA, with its level in cancer cells varying according to the metastatic state of the tumourManduca sexta and was hypothesized to regulate evolutionarily conserved intermediate in Toll pathway (ECSIT), mitogen-activated protein kinase 1 (MEKK1), and Rel family protein 2B (Rel2B), which are involved in M. sexta immunityPlutella xylostella, miR-31 was identified as one of the five most abundant miRNAsAccording to the fold-changes, 555 known miRNAs and 45 novel miRNAs were differentially expressed between ACB-BtS and ACB-AbR. However, considering the The target genes of miRNAs in the ACB were predicted using the transcriptome of the ACB as a reference. miRNAs were found to target several important candidate Bt receptor genes or other genes that are likely to be involved in insecticide resistance in insects: APN, CAD, ALP, ABC transporter family protein, trypsin-like enzyme, chymotrypsin-like enzyme, CarE, and AChE. Different isoforms of APNs and CAD together with ALP have been shown to interact with different types of Cry toxinsp-values >0.1. It has been shown that several miRNAs have multiple target genes and that numerous genes have mutually common targeting miRNAsThe prediction of putative target transcripts of differentially expressed miRNAs between ACB-BtS and ACB-AbR helps reveal the post-transcriptional regulation of gene expression in ACB with resistance to Cry1Ab. We predicted the target genes of 21 significantly differentially expressed miRNAs. A large number of target genes were predicted from the transcriptome data of the ACB. Considering the results of transcriptome differences between ACB-BtS and ACB-AbR, the expression of some miRNAs and their respective targets were negatively correlated. For example, the expression of APN1 (CL3709.Contig1), APN2 (Unigene9047), APN3 (Unigene33230), and APN4 (CL9114.Contig1) was upregulated in ACB-AbRWolbachia-responsive miRNAs in T. urticaeO. furnacalis to Cry1Ab.GO annotation and KEGG pathway analyses provide a better understanding of the cellular components, molecular functions and biological processes of target genesB. mori that was resistant to Cry1Ab toxin also revealed a mutation in a homologous ABC transporterP. xylostella, eight unigenes from ABCC2 were detected in the Cry1Ac-resistant strain, and a majority of them were downregulatedIn addition, 3.41% of the target genes of known differentially expressed miRNAs were mapped to ABC transporters (ko02010) that have been identified as related to Bt resistance44H. virescens population that underwent selection in the laboratory showed a lack of ALPSpodoptera exigua strain resistant to Cry1Ca and a Trichoplusia ni strain resistant to Cry1Ac also showed reduced APN transcriptsHelicoverpa armigera strain with Cry1Ac resistance was found to contain a deletion mutation in APNM. sexta was found to actuate the localization of APN and ALP in lipid raft micro domains where Cry toxin inserts and forms poresIn addition, the GPI-anchor metabolic/biosynthetic process and GPI anchor biosynthetic pathway (ko00563) were both detected in GO annotation and KEGG pathway analysis. GPI-anchored proteins such as APN and ALP have been identified as Cry-toxin-binding receptors in Lepidoptera. An In summary, this is the first report identifying the ACB miRNAs. The study reveals new information on the ACB miRNAs that are associated with Cry1Ab resistance and may be helpful in studying Bt resistance mechanisms in other Lepidoptera insects. Further function experiments are necessary to elucidate the underlying mechanism of Bt resistance in the ACB.The laboratory strain of the ACB was originally collected from a summer corn field in central China. It was maintained at 27±1°C, 70–80% relative humidity (RH) and a 16:8 (L:D) photoperiod at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing. During this period, the strain had no contact with any insecticides. This strain was considered to be a susceptible strain (designated ACB-BtS). Based on the ACB-BtS strain, trypsin-activated Cry1Ab toxin (94% pure protein) was used as a source of Cry1Ab for the selection diet. The selection treatment strain (ACB-AbR) was initially exposed throughout larval development to Cry1Ab in an artificial diet (2.5 ng toxin/g). The toxin concentration was steadily increased in succeeding generations to target 40–70% mortality in the exposed insects. After 51 generations, the ACB-AbR strain was reared on a diet containing 400 ng toxin/g. Previous work found that ACB-AbR developed more than 100-fold resistance to Cry1Ab after 35 generations of selectionTotal RNAs, each from two biological replicates of ACB-BtS and ACB-AbR, were extracted using TRIzol reagent . Separated on 15% denaturing polyacrylamide gel, small RNA fragments in the 18–30 nt range were purified, and then 3′ and 5′ RNA adaptors were ligated to the RNA pool using T4 RNA ligase. These fragments were then used for reverse transcription and subsequent PCR. The final PCR products were purified and subjected to the proprietary Solexa sequencing-by-synthesis method using the Illumina Genome Analyzer  at the Beijing Genomics Institute .The sequences from the HiSeq sequencing were deposited in the NCBI Sequence Read Archive (SRA). The reads under 50 nt sequence length were first subjected to data cleaning, which included the removal of low-quality reads, reads with 5′ primer contaminants, reads without 3′ primers and reads without the insert tag. The length distribution of the remaining sRNA was then summarized. Then, reads with either poly (A) or shorter than 18 nt were removed, and the final clean reads, which were assigned to two groups including the summary of unique reads and total reads, were used for standard bioinformatics analysis. The common and specific reads of the two groups were summarized, including summaries of unique reads and total reads. As data on the ACB genome are not yet available, the clean reads of the ACB-BtS and ACB-AbR libraries were mapped to the ACB transcript database (Accession No: SRP046207) using SOAP software v1.11 to analyse sRNA expression and distribution in the genome. The clean reads were annotated into different categories to discard rRNAs, tRNAs, snRNAs, and snoRNA using Rfam database v10.1. Because there was no information concerning the miRNAs of the ACB in the miRBase v21.0, the remaining sRNAs were aligned to the miRNA precursors/mature miRNAs of all animals in the miRBase to detect the sequence and count of miRNA families (no specific species) in the samples.Ostrinia scapulalis (Accession No: PRJNA192419) were used as a reference for novel miRNA prediction. The prediction software Mireap (http://sourceforge.net/projects/mireap/) was used to predict novel miRNA candidates by exploring the secondary structure, the Dicer cleavage site, and the minimum free energy of the unannotated small RNA reads that could be mapped to the genome.The characteristic hairpin structure of miRNA precursors can be used to predict novel miRNA candidates. Due to the lack of genome information for the ACB, the genome sequences of P-values, and a scatter plot of the fold-change values was generated. Fold-change was calculated as Fold-change = log2(ACB-AbR/ACB-BtS). The P-value was calculated through equation The expression of miRNAs was compared between ACB-BtS and ACB-AbR to identify differentially expressed miRNAs. First, the expression of miRNA in the four libraries was normalized to transcripts per million (TPM). If the normalized expression of the miRNA was 0, it was modified to 0.01 to enable calculation. If the normalized expression of the miRNA was less than 1 in all libraries, it was ignored to compare for low expression. The normalization formula was: Normalized expression = Actual miRNA count/Total count of clean reads × 10x represents ACB-BtS, y represents ACB-AbR, N1 represents the normalized expression of a miRNA in ACB-BtS library, and N2 represents the normalized expression of the same miRNA in ACB-AbR library.where −(CTmiRNA-CTU6RNA). A t-test was used to examine the significance of expression differences between the two samples using SAS v8.0 software.To validate the relative expression levels of the identified miRNAs in our libraries, we selected 8 differentially expressed miRNAs for qRT-PCR. Total RNA was extracted from the same samples used for deep sequencing. The specific forward primers of 8 selected miRNAs were designed according to the sequence of each miRNA itself and are provided in Supplemental File 9. The reverse transcription reaction was performed with the First Strand cDNA Synthesis Kit  according to the manufacturer’s protocol. qRT-PCR was performed with the SYBRGreen PCR kit  on an ABI-7300 machine  with thermal cycling parameters of 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and then 60 °C for 45 s, according to the manufacturer’s protocol. Three biological replicates, each with three technical replicates, were used for qRT-PCR, and U6 snRNA was used as an internal reference. The threshold cycle (CT) was collected from each reaction, and the relative expression level of each miRNA to U6 snRNA was evaluated using the equation 2http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). The rules used for target prediction were based on those suggested by Allen et al. and Schwab et al.53The putative target sites of miRNA candidates were identified by aligning the miRNA sequences with the ACB transcriptome and other published gene data from Lepidoptera insects using the RNAhybrid software ( http://www.geneontology.org/), calculating gene numbers for each term. Then, a hypergenometric test was used to find GO terms in target gene candidates that were significantly enriched compared with the reference gene background. The calculation used for the test was equation 2:To further analyse the function of differentially expressed miRNAs between ACB-BtS and ACB-AbR, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used on predicted target gene candidates of differentially expressed miRNAs. GO is an international standardized classification system for gene function, which supplies a set of controlled vocabulary to comprehensively describe the property of genes and gene products. There are 3 ontologies in GO: molecular function, cellular component and biological process. The GO results were used to reveal the functions significantly related with predicted target gene candidates of the detected miRNAs. First, all target gene candidates were mapped to GO terms in the database .


Ageing entails cognitive and motor decline as well as brain changes such as loss of gray (GM) and white matter (WM) integrity, neurovascular and functional connectivity alterations. Regarding connectivity, reduced resting-state fMRI connectivity between anterior and posterior nodes of the Default Mode Network (DMN) relates to cognitive function and has been postulated to be a hallmark of ageing. However, the relationship between age-related connectivity changes and other neuroimaging-based measures in ageing is fragmentarily investigated. In a sample of 116 healthy elders we aimed to study the relationship between antero-posterior DMN connectivity and measures of WM integrity, GM integrity and cerebral blood flow (CBF), assessed with an arterial spin labeling sequence. First, we replicated previous findings demonstrating DMN connectivity decreases in ageing and an association between antero-posterior DMN connectivity and memory scores. The results showed that the functional connectivity between posterior midline structures and the medial prefrontal cortex was related to measures of WM and GM integrity but not to CBF. Gray and WM correlates of anterio-posterior DMN connectivity included, but were not limited to, DMN areas and cingulum bundle. These results resembled patterns of age-related vulnerability which was studied by comparing the correlates of antero-posterior DMN with age-effect maps. These age-effect maps were obtained after performing an independent analysis with a second sample including both young and old subjects. We argue that antero-posterior connectivity might be a sensitive measure of brain ageing over the brain. By using a comprehensive approach, the results provide valuable knowledge that may shed further light on DMN connectivity dysfunctions in ageing. As demographic changes in developed countries push up the proportion of elderly adults in the population Cohen, , age-relGM atrophy in both cortical and subcortical structures, evident as early as middle age  and reduced Mean Diffusivity (MD) are the most frequently used DTI measures associated with WM integrity, as they are able to provide summarized information on the state of WM. Diffusion Tensor Imaging measures are altered over the lifespan, presenting an inverted U-shape curve and accelerated decreases during senescence  mPFC-PCU connectivity will be reduced in ageing; (2) mPFC-PCU connectivity will correlate with cognitive performance in elders, essentially with memory function; (3) in ageing, mPFC-PCU connectivity will correlate with grey and WM integrity as well as with perfusion measures; these relationships will be found in, but not limited to, DMN areas and cingulum bundle; (4) regions correlating with mPFC-PCU coupling strength will be located in areas of high age-related vulnerability. To test these hypotheses we used two independent samples. One large sample of healthy elders with small age-related variability was used to extract structural, perfusion and cognitive correlates of mPFC-PCU coupling strength, while a second sample including young and old subjects was used for purposes of group (age-related) comparisons.sample 1.One hundred and sixteen healthy old subjects (age = 68.25 (3.053); range: 63–78, 37 males) were included in this study. Mean years of education (YoE) was 11.09 (4.149). Subjects were recruited in retirement homes and centers for the elderly registered with the Institut Català del Envelliment (ICN) in the area of Barcelona. All participants had normal cognitive profile with Mini-mental State Examination test (MMSE) scores ≥25 and performances not below 1.5SD according to normative scores adjusted for age, gender and education in a neuropsychological evaluation that covered the major cognitive domains. Informed consent was obtained from all participants. The study was approved by the Hospital Clinic de Barcelona ethical committee which follows the guidelines of the Declaration of Helsinki. All subjects underwent MRI acquisition (DTI data were available for 100 subjects). This aged sample was used to assess the relationship between rs-fMRI connectivity on the one hand, and cognition, structural and perfusion indices on the other and will be referred as sample 2) consisting of 25  young and 25  old healthy subjects was used for direct age-groups comparison purposes. Magnetic resonance imaging data from this sample was obtained using exactly the same scanner and sequences as in the primary sample. Mean age was 23.08  and 68.92  for the young and old group respectively. Mean YoE was 19.64 (1.77) and 11.36 (4.02) for the young and old group respectively. Neither age, nor gender and YoE differences between the sample 1 and the old group from the sample 2 were found (p > 0.3). However YoE between old and young groups in the sample 2 differed  as young subjects were recruited in an academic environment. When not specified, methods employed with both samples are assumed to be equivalent. Data from sample 2 has been partially published in previous studies of our group  at the Center Diagnostic per la Imatge in the Hospital Clínic of Barcelona. Magnetic resonance imaging acquisition included the following sequences: a high-resolution 3D structural dataset ; a rs-fMRI sequence  that lasted 5 min (150 volumes); a DTI, sequence  and an Pulsed-Arterial Spin labeling (PASL)-MRI perfusion acquisition .Magnetic resonance imaging analysis was performed using tools from FreeSurferResting-state fMRI: Data preprocessing included removal of the first five volumes, motion correction, skull stripping, spatial smoothing , grand mean scaling, high and low pass filtering (0.1–0.1 Hz) and normalization with two-step linear transformations : Structural data were analyzed with FSL-VBM tool .and Dale . ReconstGray-white matter contrast: GWC was estimated with the reconstructed cortical surface by calculating the non-normalized T1-weighted image intensity contrast (100*[white–grey]/0.5*[white+grey]). Values close to 0 indicate less contrast and thus more blurring of the GM/WM boundary. GM was taken at a 0.3 projection fraction from the boundary while WM was assessed 1 mm below the WM surface. Before performing statistical analyses, the resulting GWC was mapped to a common surface and smoothed with a 15 mm FHWM Gaussian kernel. Two subjects were removed due to problems during cortical reconstruction (n = 114).White matter integrity: Tract-Based Spatial Statistics . The model was re-run, but this time aligning subjects to the standard skeleton instead of using the mean derived skeleton which, allowed comparisons of the results between the two samples. The results did not qualitatively change between the two procedures (data not shown).Perfusion imaging analysis: Arterial Spin labeling preprocessing was carried out with FSL-BASIL toolset . When not specified, data is presented as mean (SD), error bars represent standard error of mean (SEM) and coordinates are reported in MNI space. In the sample 1 age, gender, and YoE were used as covariates for all analyses. Cases were excluded pairwise in the different analyses. In the sample 2 only gender was used as covariate. The rationale for not covariating for education is the overrepresentation of YoE effects due to group differences which biased the results. However, the results were not qualitatively modified when YoE was added as an additional covariate (data not shown).The appropriate tests were carried out with SPSS 20.0  software to handle non-neuroimaging data. Significance was set at sample 1, individual psychometric tests were combined based on functional domains into four composite scales that assessed memory, speed processing, working memory and inhibition in accordance with the ageing literature  correlation between preprocessed and regressed rs-fMRI ROIs time series. Next, correlations were transformed to z-scores. For sake of completeness bilateral IPL and bilateral MTG DMN ROIs were also extracted. The Euclidian distance between posterior midline structures and mPFC ROIs from both samples was small  and mPFC-PCU correlation did not differ between old adults from the sample 2 and sample 1 population  to facilitate interpretation.To obtain the DMN we performed an ICA with rs-fMRI preprocessed datasets  comparisons between mPFC-PCU Z-transformed correlations were performed with the sample 2. For the sake of completeness, correlations between other DMN nodes were also performed.Influence of DMN connectivity on cognition: To evaluate the influence of mPFC-PCU connectivity to cognition we performed a multivariate GLM-based analysis in the main sample, where connectivity, age, YoE and gender were introduced in a model predicting the four cognitive factors.p < 0.05). Relationships of CTh and GWC with connectivity were studied using linear modeling as implemented in FreeSurfer. After thresholding results at p < 0.01, FWE correction for multiple comparisons using a Monte Carlo Null-Z simulation  was performed.Relation of CTh, VBM, FA, MD, CBF and CBF-GM maps to DMN connectivity: To assess the relationship of structural and CBF correlates with anterior-posterior DMN connectivity, multiple voxelwise nonparametric analyses were performed. Nonparametric testing with 5000 permutations as implemented in a randomized FSL tool was used for VBM, MD, FA, CBF and CBF-GM measures followed by threshold-free cluster enhancement (TFCE) and familywise error (FWE) multiple comparison correction (sample 2 (group comparisons) and compared age-related patterns in the brain with those limited by the structural correlates of mPFC-PCU connectivity and those limited to DMN areas (and the cingulum bundle). First, we extracted maps of differences between old and young subjects using GLM-based analysis with group and gender as regressors. Further, differences in the age-related effects on whole brain, connectivity correlates and DMN areas were compared using the raw statistics obtained in the group comparison (thus age-effects) and effect size calculations were performed . To do so, we extracted age-related patterns from the As motion correction may affect fMRI correlations , we found reduced DMN connectivity in ageing, specifically between mPFC and PCU areas in which connectivity was clearly lower in the old group 0.17 (0.27) compared with the young group 0.46 (0.21). Inferential comparisons yielded significant differences in mPFC-PCU coupling strength  as a function of age. Complementarily mPFC-MTG  and mPFC-IPL  couplings also differed in both groups. PCU-MTG  and IPL-MTG  also showed significant age-related difference though they did not survive Bonferroni correction (p < 0.05/6 = 0.0083). No age-related differences were observed between IPL and PCU  ROIs , though this last comparison did not survive Bonferroni corrections (p < 0.05/4 = 0.0125). The working memory and the inhibitory factor were unrelated to connectivity. Years of education was associated with all cognitive factors  while age was only significantly associated with the speed processing factor ; this was expected, given the small standard deviation in this variable.mPFC-PCU coupling was able to predict cognitive performance as it was significantly associated with memory factor  correlations were found between mPFC-PCU connectivity and CTh in bilateral parietal and right superior frontal areas.Gray-White Matter contrast: GWC correlated with mPFC-PCU coupling in a widely distributed set of regions including frontal, temporal and parietal lobes bilaterally. In the left hemisphere three clusters were found: one encompassing the middle and inferior temporal gyrus; another extending towards the PCU, isthmus and posterior cingulate; and a third cluster covered lateral prefrontal areas. In the right hemisphere, a large cluster was found covering medial and lateral parietal lobes, the lateral temporal lobe and the prefrontal cortex with peaks of significance located in pars opercularis, supramarginal and PCU regions  did not show relationship with mPFC-PCU DMN connectivity.Cerebral Blood Flow: No relationship was found between perfusion fMRI measures and DMN connectivity. Neither uncorrected (p < 0.01) nor ROI analyses revealed any consistent differences.Gray Matter Cerebral Blood Flow: When corrected for PVE estimates, CBF-GM estimates did not correlate with connectivity measures. No significant results were found at an uncorrected level (p < 0.01) or with ROI analyses.Summarizing, both GM and WM measures were related to mPFC-PCU DMN connectivity. The topological pattern was not limited either to DMN nodes or to the main tract connecting DMN structures but was distributed across the brain in areas typically characterized as highly affected by ageing. Cerebral blood flow measures were unrelated to antero-posterior DMN connectivity while GWC, a measure of GM/WM blurring, was found to be related to connectivity in distributed brain areas. To gain further insight into these results, mPFC-PCU structural correlates were compared to the age-related patterns of decline extracted with the second sample.Group comparisons with sample 2 yielded evident age-related effects in wide brain areas Figure . Voxel-BStructural correlates of mPFC-PCU within the primary old sample were not limited to the DMN  or to the main tract connecting them, but extended beyond those nodes and were distributed along the cortical mantle. Interestingly, this distribution resembled the impact of age on the brain in each specific neuroimaging modality. To quantify this correspondence, mean effects of age were computed for areas related to mPFC-PCU connectivity and compared to both the overall ageing pattern and the effects of ageing in DMN areas through effect size tests.Unequivocally, voxels that exhibited a relationship with mPFC-PCU DMN connectivity also tended to be more susceptible to the effects of age. Effect sizes were medium for VBM and right hemisphere GWC and high for MD and left hemisphere GWC according to criteria described elsewhere Cohen, . That isIn contrast, DMN areas did not show increased susceptibility (compared to whole brain) to age either in VBM, MD or right hemisphere GWC. In left hemisphere GWC, DMN voxels presented small effects size. Finally, effect size analyses comparing age-vulnerability of DMN areas with areas related to mPFC-PCU connectivity showed, in all the modalities, an increased susceptibility to age effects in the latter regions . However, no significant changes were found in the tests after this variable was added in the statistical models. The relationship of mPFC-PCU with memory and with age (group comparisons) remained significant (p < 0.05), while the relationship of mPFC-PCU connectivity with anatomical correlates was still highly significant (p < 0.001) in all significant clusters. While motion correction impacts connectivity, it does not significantly alter our results.No outliers were found. Mean relative movement was negatively associated with mPFC-PCU connectivity in both samples (Main results can be summarized as follows: (1) mPFC-PCU DMN connectivity is highly reduced in ageing; (2) in ageing, this coupling is related to cognition, specifically to memory performance; (3) mPFC-PCU DMN connectivity is related to measures of GM and WM integrity in several brain areas not restricted to DMN; (4) perfusion measures are unrelated to DMN connectivity; (5) mPFC-PCU DMN structural correlates appear to be located in areas of high age vulnerability.sample 2). Group comparisons suggested that the most significant effect of age was present in the coupling between mPFC and posterior midline structures, in agreement with other studies  and, to some extent, are comparable to those found in our study . While age-related mechanisms affecting structural and functional connectivity might be partially related, the results also suggest that other mechanisms are involved in the maintenance of resting-state DMN connectivity besides those assessed with diffusion-based imaging. In development literature the link between functional and structural anteroposterior DMN connectivity seems to emerge in the late childhood  deposition), the presence of the APOE ɛ4 allele or dopamine depletion. Around 20% of older subjects without cognitive decline have significant Aβ deposition, an early AD biomarker (Chételat et al., post hoc tests did not qualitatively change any of the main results reported here. Post hoc testing was used because no standardized procedures exist to deal with this potential confounding. A third limitation refers to the analyses performed to assess age-related decline, since relatively small group comparisons were carried out. Using this methodology instead of cohorts encompassing the entire life-span or longitudinal approaches may have altered age-related patterns. Still, the patterns were topologically similar to those reported previously in the literature and we preferred to exploit the advantages of using a sample acquired in the same scanner machine. Lastly, the exploratory and correlational character of the study limits the explanatory power of the results.Another potential limitation is the effect of motion over rs-fMRI measures, however, This study provides valuable new information of DMN connectivity in healthy ageing. The main finding is that mPFC-PCU DMN connectivity is related to GM and WM indices of integrity, not only in DMN areas but also in areas of high age-related vulnerability. This coupling is affected by ageing and is related to cognitive performance in elders. Elucidating the basis of disconnection in ageing is of the utmost importance for understanding healthy brain function and cognition as well as the development of brain pathology.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hypertension (HT) is the common adverse event associated with vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKI). The present study was performed to identify the predictive factors of TKI-induced HT and to determine the classes of antihypertensive agents (AHTA) that demonstrate optimal efficacy against this type of HT. The charts of 50 cases of patients that had received VEGFR-TKI treatment were retrospectively examined. The association between patient background and TKI-induced HT, and the effect of administering AHTA were analyzed. High systolic blood pressure at baseline was identified to be a predictive factor for HT. In addition, there was no difference observed between calcium channel blockers (CCBs) and angiotensin receptor II blockers (ARBs) as first-line AHTA for the control of HT. The findings of the present study may aid with predicting the onset of TKI-induced HT, as well as for its management via the primary use of either CCBs or ARBs. It had been indicated that antitumor agents do not have a beneficial effect on patient survival in cases of kidney cancer. Only cytokine therapies, including interferon-α and interleukin-2, have been used for patients with metastatic  kidney cancer (MKC). However, these agents demonstrate insufficient efficacy –3. SubseAll studies were performed retrospectively in Kanazawa University  using the charts of patients who were hospitalized at the Department of Urology. Patients with MKC who underwent VEGFR-TKI  therapy were analyzed. The AHTAs that were administered were categorized according to their mechanisms of action. The study was in accordance with the Declaration of Helsinki Guidelines.de novo HT and commencing AHTA administration, and on HT improvement following AHTA administration. The average BP levels at identical times on three consecutive days were calculated and used for analyses; however, single BP measurements were also used if the patient was discharged and became an outpatient.HT was defined as systolic blood pressure (BP) of >140 mmHg, corresponding to Grade 2 of the National Cancer Institute-Common Terminology Criteria for Adverse Events version 4.0 . The pol2 test to identify trends. The probability of administrating AHTA was estimated using the Kaplan-Meier method. In all analyses, P<0.05 was considered to indicate a statistically significant difference.Statistical analyses were performed using commercially available software . Comparisons between two groups were performed by unpaired two-sided t-test, Fisher’s exact test and χIn total, 50 VEGFR-TKI administration events from 41 patients were analyzed and the patient demographic data are shown in Of the 50 cases, 20 had HT subsequent to VEGFR-TKI administration, and their backgrounds were compared with the 30 non-HT cases . The medde novo HT and no AHTA administration prior to initiation of VEGFR-TKI therapy, were analyzed. The first-line AHTA treatment was either calcium channel blockers (CCBs) or angiotensin receptor II blockers (ARBs). There was no significant difference identified between the control rate of CCB and ARB as first-line treatments (3/8 for CCB treatment and 3/5 for ARB, P=0.5921; The variations in efficacy, between AHTA administration in 13 cases of et al (de novo HT cases, was investigated. As expected, two major categories of AHTA were used as first-line treatments for VEGFR-TKI-induced HT, CCB and ARB, and there was no difference in efficacy identified between these two AHTA categories. Although certain review studies proposed the use of AHTA for VEGFR-TKI-induced HT, there is no evidence that the specific usage of AHTA is a requirement for VEGFR-TKI-induced HT (It is important to identify the predictive factors for key AE that are associated with VEGF-TKI to prevent treatment discontinuation, as well as to predict the population that may show a good response to these agents. Furthermore, it may contribute to an improved outcome. In a study using axitinib treatment for Japanese patients with MKC, Tomita et al  indicateet al . A meta-et al . These sduced HT –16. Howeduced HT . A systeduced HT . In addiduced HT ,20. As pduced HT ,11, ARB


Background: There is growing evidence that human immunodeficiency virus (HIV)-infected women might have a different human papillomavirus (HPV) type distribution in cervical dysplasia specimens as compared to the general population. This has implications for primary prevention.Objective: We aimed to obtain preliminary data on the HPV genotypes prevalent in histological samples of HIV-infected women with cervical intraepithelial neoplasia (CIN) 3/CIS of the cervix in Miami, FL, USA.Methods: Retrospective data were collected on HIV-infected women referred to the University of Miami-Jackson Memorial Hospital colposcopy clinic between years 2000 and 2008. The histology slides of CIN 3/CIS biopsies underwent pathological review and sections were cut from these archived specimens for HPV DNA extraction. HPV genotyping was then performed using the GeneSquare™ HPV genotyping assay. We report on our first set of 23 samples.Results: Eight high-risk HPV types were detected. Types in decreasing order of frequency were 16, 35, 45, 52, 59, 31, 58, and 56. Most cases had multiple infections. HPV type 16 was the most common (45%) followed by HPV-35 and -45 with equal frequency (40%). No samples contained HPV-18.Conclusion: Our preliminary results suggest that cervical dysplasia specimens of HIV-infected women more likely (55%) contain non-16 and -18 high-risk HPV types. We show that this held true for histologically confirmed severe dysplasia and carcinoma-in situ. Epidemiological studies guide vaccine development, therefore HPV type prevalence in CIS and invasive cervical cancer among HIV-infected women should be more rigorously explored to ensure that this highly vulnerable population receives appropriate primary prevention. This histological confirmation was not performed in previously reported studies on HPV type prevalence in HIV-infected women  genotypes are the necessary cause of 99.7% of cervical cancer. Seven of them  account for nearly 90% of all cervical cancer cases in the general population worldwide with little regional variation  and quadrivalent 6/11/16/18) vaccines against additional HR-HPV types –11. The /16/18 va/18 and qMeta-analyses have shown that, in the general population, many HR-HPV types are common in low grade cervical dysplasias however, as the grade of dysplasia increases HPV types 16 and 18 predominate. This follows a fairly linear upward trend. Similarly, as the grade of dysplasia increases, the other HR-HPV types become less common following a downward trend Figure . Our aimAfter obtaining IRB consent, retrospective de-identified clinical data were collected on 510 HIV-infected women with abnormal Pap smears seen at the University of Miami-Jackson Memorial Hospital (UM-JMH) colposcopy clinic between 2000 and 2008  by cervical biopsies. Twenty patients had samples that were considered probably too old for successful DNA extraction and were excluded. Samples were limited to one event per patient using the case with the most severe pathology. All slides underwent central Gynecologic Pathology review. There were 24 of 30 remaining histology slides that were confirmed CIN 3 while the others were down-graded to CIN 2. The corresponding paraffin-embedded blocks of the CIN 3 were pulled and two 20-micron sections were cut from each block. DNA was extracted from these sections using the Ambion™ Recover All kit as per manufacturer’s instructions. We were able to successfully extract DNA for HPV genotyping on 23 samples. Clinical data on the final 23 cases were also obtained including patient demographics, cytological/histological results, as well as CD4 counts and HIV-1 viral load. After DNA was extracted from paraffin sections, we performed Nanodrop analysis to check concentration/ratio of DNA. This was followed by analysis of DNA on an Agilent™ Bioanalyzer chip to assess quality. HPV genotyping of these samples was performed using the Genesquare™ kit developed by Kurabo Industries LTD , which tests for 23 HPV types including all high-risk types and some low risk types. This kit uses multiplex PCR assays with HPV type-specific primers. We chose this PCR-sequencing method because it offers sensitive detection of HPV types at a lower cost and with increased processing speed than the standard Roche Linear array. Furthermore, there is no cross-hybridization because HPV type-specific primers are used as compared to the Roche method, which uses the L1 consensus primer (high-homology region). However, the Genesquare assay has not yet been validated in a large trial.Fifty patients (74 events) were diagnosed with CIN 3  was used for statistical analysis. Descriptive statistics such as mean, standard deviation, median, first and third quantiles were reported for patients’ age and some of patients’ clinical characteristics, such as number of HPV infections, viral load, and CD4 count. Frequencies and percentages were calculated for characteristics such as race/ethnicity, grade of dysplasia, treatment, and recurrence of dysplasia. One-sample exact binomial test was used to test the differences in HPV genotype prevalence in cervical lesion of women between HIV-infected women and the general population reported. Type-I error rate is set to 5%.n = 23), 83% of women were African-American or Haitian-American and 50% were age 40 or younger (Table p < 0.05). However, the prevalence of HR-HPV types 16, 31, 56, and 58 were not significantly different than those in cervical lesions of women in the general population.In this sample n = 3, 83% ofIn conclusion, we found that certain HR-HPV types that are considered relatively rare in high-grade cervical lesions of women in the general population were actually more common in high-grade lesions of HIV-infected women at the UM-JMH colposcopy clinic. We also found that the most prevalent HPV types 16, 35, 45) in these high-grade lesions among HIV-infected women at our institution differed from those of other HIV-infected women in other geographic regions . In that same article, the most carcinogenic HPV types concentrated in species alpha-9 and alpha-7 were distinguished by elevated risk of progression given persistence, rather than persistence alone. Alpha-5 and alpha-6 species were also at elevated risk of progression given persistence  they are not able to evade the immune system as efficiently as HPV types 16 and 18 except in HIV-infected individuals who are immunocompromised. To test this hypothesis regarding immunocompromised status and link with HPV type prevalence, a simple genotyping study in immunocompromised women not due to HIV  could be evaluated.In our sample, 95% 19/20) of the HPV types detected belonged to either alpha-9 or alpha-7 species. The alpha species of HPV are those subgroups of the virus that infect the genito-anal epithelium (/20 of thn = 8) contained a single infection. HPV-16 accounted for 75% of these single infections , however, these preliminary results support the feasibility of retrospective HPV genotyping from archival paraffin-embedded blocks in our lab as well as the need for further research in this area. Because developing countries are known to have higher rates of HIV than the developed world and HPV is more aggressive in HIV-infected individuals, the highest potential for global public health impact by a HPV vaccine would be conferred by its effectiveness in the HIV-infected population or those at risk of acquiring the infection. We are now planning to better characterize differences between HIV-infected and HIV-negative women in terms of their HPV type prevalence and their immunity profiles with the goal of guiding the development of both therapeutic and prophylactic vaccines that would be effective not only in the general population but also in the HIV-infected population.In summary, our study design was based on the positive predictive value of CIN 3 lesions in predicting HPV genotypes in invasive cervical cancer from the same population. Strength of our study was the use of histological pathology review for all samples confirming the grade of the lesions. Our findings are consistent with that of others who found rare HR-HPV types to be more common in dysplasia of HIV-infected women. In this pilot study, the sample was small (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Screening, Brief Intervention, and Referral to Treatment (SBIRT) for substance use has received a great deal of empirical support. Despite the evidence for effectiveness of SBIRT and a compelling rationale for its integration into health-care settings, the circumstances under which it is likely to be implemented and sustained remain elusive. In this project, SBIRT was implemented into emergency departments (EDs) and primary care practices (PCPs) within a large, integrated health system in New York serving areas heavily affected by Hurricane Sandy. The SBIRT model was implemented in the following way: 1) Front-line staff  administered a five-item SBIRT prescreen for alcohol, drug, and tobacco use to all patients during triage; 2) If a positive prescreen resulted, a health educator conducted a full screen using the AUDIT and/or DAST-10; and 3) The health educator conducted a brief intervention and referral to addiction treatment as indicated by the full-screen score. To assist in building the SBIRT process into the normal site workflow, prescreening questions were integrated into the electronic medical record (EMR) in a user-friendly way for front-line staff to administer. The EMR automatically scored the prescreen. As of May 2014, SBIRT was implemented in three EDs and two PCPs within the health system, with services starting on 12/1/13. Implementation was evaluated by reviewing EMR data as well as patient data collected at each site. Approximately 13000 patients were prescreened (13% positive). More women than men were prescreened , and there was a significant older population , as well as diversity in ethnicity (24% Latino) and race (19% African American). Almost 900 full screens were conducted (50% positive), which resulted in 284 brief interventions and 149 referrals to brief or formal addiction treatment. Further analysis revealed that multiple substances were an issue for many patients. For example, 20 percent of patients were positive on both the AUDIT and the DAST-10, and 39 percent of patients who full-screened positive for drugs or alcohol also screened positive for past-year tobacco use. Severity of full-screening scores was greater in the ED setting as compared to the PCP setting. Thirty-nine percent of patients who full-screened positive reported marijuana use in the past 30 days, and 14 percent were daily marijuana users. Overall, results suggest that SBIRT was successfully implemented in several busy sites within this large health system.


Vaccination against the oncogenic human papillomavirus (HPV) types 16 and 18 will reduce the prevalence of these types, thereby also reducing cervical cancer risk in unvaccinated women. This (measurable) herd effect will be limited at first, but is expected to increase over time. At a certain herd immunity level, tailoring screening to vaccination status may no longer be worth the additional effort. Moreover, uniform screening may be the only viable option. We therefore investigated at what level of herd immunity it is cost-effective to also reduce screening intensity in unvaccinated women.We used the MISCAN-Cervix model to determine the optimal screening strategy for a pre-vaccination population and for vaccinated women (~80% decreased risk), assuming a willingness-to-pay of €50,000 per quality-adjusted life year gained. We considered HPV testing, cytology testing and co-testing and varied the start age of screening, the screening interval and the number of lifetime screens. We then calculated the incremental cost-effectiveness ratio (ICER) of screening unvaccinated women with the strategy optimized to the pre-vaccination population as compared to with the strategy optimized to vaccinated women, assuming different herd immunity levels.Primary HPV screening with cytology triage was the optimal strategy, with 8 lifetime screens for the pre-vaccination population and 3 for vaccinated women. The ICER of screening unvaccinated women 8 times instead of 3 was €28,085 in the absence of herd immunity. At around 50% herd immunity, the ICER reached €50,000.From a herd immunity level of 50% onwards, screening intensity based on the pre-vaccination risk level becomes cost-ineffective for unvaccinated women. Reducing the screening intensity of uniform screening may then be considered. Infection with the human papillomavirus (HPV) has been identified as a necessary cause for cervical cancer . Both thIn the Netherlands, a catch-up campaign targeted all 13- to 16-year-old girls in 2009. Since 2010, all 12-year-old girls are offered vaccination. The three-dose vaccination coverage has steadily increased from 49% in the 1993 birth cohort to 61% in the 2000 birth cohort , 8. In tIn many developed countries, vaccinated cohorts are approaching the start age of cervical cancer screening. Especially in settings where both vaccinated and unvaccinated women are well represented, it is unclear what screening strategy should be offered. In the youngest vaccinated cohorts (with limited herd immunity), vaccinated women are at much lower risk than unvaccinated women and screening based on vaccination status is likely more cost-effective than current uniform screening –13. HoweAs long as the follow-up of HPV vaccinated women in trials and population-based settings is not long enough to observe  differences in cervical cancer rates between vaccinated and unvaccinated cohorts, countries are reluctant to reduce the screening frequency. In the U.S., the same screening protocol is recommended for both vaccinated and unvaccinated women , 15. EurWhat is merely realized, is that women at reduced risk (due to either vaccination or herd immunity) could also be harmed by too intensive screening. These women will be offered more screening tests than needed, which increases their probability of being referred to the gynecologist in the absence of clinically relevant lesions. Women with abnormal cytology or HPV positive test results commonly experience fear, self-blame, distress and anxiety about cervical cancer, which reduces their quality of life , 18. TheUsing the MISCAN-Cervix model, we determined two optimal screening strategies: one for a pre-vaccination cohort, and one for a vaccinated cohort. To determine the level of herd immunity for which it would be cost-effective to replace the first strategy by the second, both strategies were applied to an unvaccinated cohort, assuming different levels of herd immunity.The MISCAN-Cervix model, which is described in more detail in the model profile , was useA fraction of these women will acquire HPV-infections and/or develop cervical intraepithelial neoplasia (CIN) lesions. If these precursors progress to cervical cancer, women may die from the disease. If the population undergoes screening, the disease can be detected and treated in an earlier stage. As a result, cervical cancer death may be prevented or postponed.The population at risk for cervical cancer was simulated based on demographic and hysterectomy data , 21; morIn the model, disease is subdivided into seven sequential stages: high-risk HPV-infection, three pre-invasive stages , and three invasive stages  stages IA, IB and II+) . Pre-invWe simulated four different screening policies: (A) primary HPV screening with reflex cytology triage and cytology triage after six months (future Dutch screening program), (B) primary cytology with reflex HPV triage, (C) combined primary HPV and cytology (i.e. co-testing) with HPV triage after 12 months, and (D) primary cytology with cytology and HPV triage after six months and cytology triage after 18 months (current Dutch screening program). Policies (A) and (B) were already found to be cost-effective in case of no herd immunity ; policieScreening schedules differed by start age, screening interval and number of screens in a lifetime. Possible start ages were 25, 30, 35, 40 and 45 years. The screening interval varied from 5 to 20 years and the number of lifetime screens ranged from 1 to 12. Because screening women older than 80 years is not likely to be beneficial [As we aimed to optimized screening for women who adhere to screening guidelines, we assumed full attendance in both primary screening and triage testing . The senDetection of pre-invasive lesions and their associated management, including treatment if necessary, were assumed to lead to a 100% cure rate. A woman can, however, acquire new HPV-infections and develop CIN lesions after CIN treatment. For invasive cancer, we determined age-specific and stage-specific survival probabilities based on data from the NCR . Since cThe estimated costs are based on a societal perspective, and are reported in 2013 euros . Screeni42,43, which is 25.3% for HPV-infections without cytological abnormalities [We assumed the efficacy of the bivalent vaccine as observed in the PATRICIA trialmalities , and 35.malities , the promalities , 41. In In the absence of herd immunity, unvaccinated women were assumed to have the cervical cancer risk as is currently observed in the Netherlands . Full heFor a pre-vaccination and a vaccinated cohort, we simulated the screening strategies described earlier and determined their discounted costs and effects as compared to no screening. For both cohorts, the optimal screening strategy was determined as follows. We first excluded all dominated screening strategies, i.e. those strategies that were more costly and less effective than (combinations of) other strategies. We then ranked the efficient strategies based on the number of quality-adjusted life years (QALYs) gained and calculated their incremental cost-effectiveness ratio (ICER), i.e. the additional costs per additional QALY gained compared to the next less effective, efficient strategy. For each cohort, the optimal screening strategy was then defined as the strategy with an ICER just below the willingness-to-pay threshold of €50,000 per QALY gained, which is a commonly used threshold in cost-effectiveness analyses for cervical cancer screening , 44.The two optimal screening strategies were applied to unvaccinated women assuming herd immunity levels of 0%, 25%, 50%, 75% and 100%. For all these levels, the ICER of screening optimized to the pre-vaccination cohort as compared to screening optimized to the vaccinated cohort was calculated. If the ICER reached above €50,000 per QALY gained, screening optimized to the pre-vaccination risk level was no longer considered cost-effective for unvaccinated women.In the sensitivity analyses, we varied the following parameters.First, we used the vaccine efficacy from two randomized efficacy trials in which the quadrivalent vaccine was used   and in wFinally, this indirect approach of combining the type-specific reduction in HPV prevalence with the HPV-type distribution in HPV-infections, CIN lesions and cervical cancer was also used to determine the vaccine efficacy for the quadrivalent vaccine. The assumed vaccine efficacy was 42.6% for HPV-infections, 28.6%, 50.6%, 57.7% for CIN grade I, II and III respectively and 80.2% for cervical cancer.Instead of assuming an equal background risk for vaccinated and unvaccinated women, we included two sensitivity analyses in which the background risk in unvaccinated women was assumed 50% higher and 50% lower than in vaccinated women.For a pre-vaccination cohort, 6-yearly primary HPV screening in the age range 30–72 years is most cost-effective . This coAs compared to screening 3 times, screening 8 times reduces cervical cancer deaths with 161 per 100,000 unvaccinated women in the absence of herd immunity, and with 28 in case of full herd immunity . It therScreening 8 times instead of 3 increases total costs with approximately €10.9 and €11.1 million assuming no and full herd immunity, respectively. Consequently, the ICER of screening 8 times instead of 3 increased from €28,085 per QALY gained in the absence of herd immunity to €35,042, €47,530, €77,541, and €322,234 for 25%, 50%, 75% and 100% herd immunity, respectively. From When vaccine efficacy was calculated indirectly from the FUTURE trial, the optimal screening strategy for vaccinated women involved an additional screening round at age 71 . In all Similar to the base case analysis, the ICER of using the strategy optimized to the pre-vaccination cohort instead of to the vaccinated cohort, increased with increasing level of herd immunity . In sensFor both a pre-vaccination and a vaccinated cohort, primary HPV screening is more cost-effective than primary cytology or co-testing. The optimal number of lifetime screens varied from 8 for the pre-vaccination cohort, to only 3 for the vaccinated cohort. For unvaccinated women, the adverse effects and costs of screening become more important as the herd immunity level increases. Offering these women 8 instead of 3 lifetime screens incrementally required 34 colposcopy referrals per prevented death for 0% herd immunity, which increased to 118 referrals for 100% herd immunity. The ICER of screening 8 times instead of 3 increased from €28,085 per QALY gained in the absence of herd immunity to €322,234 at full herd immunity. Screening optimized to the risk level in vaccinated women becomes more cost-effective than screening optimized to the pre-vaccination risk level when the herd immunity reaches above 50%-55%.To foresee whether and when the herd immunity will reach this level, countries need to monitor the HPV-16/18 prevalence in unvaccinated women, starting with a reliable pre-vaccination baseline measurement. A recent cross-sectional study among women aged 18–24 years in Australia, in whom vaccination coverage was 55%-74% for 1–3 doses , showed We have not incorporated vaccination coverage as a separate parameter in our analyses, the reason for which is as follows. Vaccination coverage plays a role in two ways: first, it determines how many unvaccinated women there are , and second, it is one of the main determinants of herd immunity. Mathematical models have been created to estimate the level of herd immunity given vaccination coverage –53. ThesThe manuscript primarily focused on the effect of decreasing the screening frequency of uniform screening for unvaccinated women. For vaccinated women, this adjustment would be cost-effective by definition. Meanwhile, it is important to point out that the harms of screening the vaccinated 8 times instead of 3 were smaller than the life years gained , meaningWe optimized the screening strategy to the pre-vaccination risk level and to the risk level in vaccinated women. For partly vaccinated cohorts, it could be beneficial to have a screening strategy that is a compromise of these two strategies. In fact, when ignoring the costs and efforts related to restructuring screening guidelines, it would likely be cost-effective to reduce the screening frequency gradually while the herd immunity level increases. Adjusting national screening guidelines every few years is not a very workable solution though. Likewise, it could be cost-effective to tailor screening to vaccination status. Our results have shown that as soon as the herd immunity level reaches 50%, then it is beneficial (in terms of cost-effectiveness) for unvaccinated women to replace screening optimized to the pre-vaccination risk level with screening optimized to the risk level in vaccinated women. If this already happens within a few years, then establishing tailored screening by e.g. developing a vaccination registry that is linked to the screening invitational system, may not be worthwhile. The (lack of) accumulation of herd immunity over time is crucial in deciding whether the establishment of tailored screening would be worth these additional efforts. We performed our analyses under the assumption that it is most realistic that countries will continue screening all women uniformly, and that a once-only adjustment is made as soon as this seems justified for unvaccinated women.Notable limitations are the following. First, we assumed that the efficacy of the vaccine has a lifelong duration. Although until now, HPV vaccination trials have shown a sustained efficacy , 3, it iTo our knowledge, this is the first study evaluating at what herd immunity level a once-only uniform  screening adaptation becomes, considering risks, benefits and costs, an option. Because vaccinated women are approaching the age at which cervical cancer screening starts, the results of this study will be relevant in the near future. It shows, that as long as stepwise adjustment or dichotomized screening based on vaccination status are considered unfeasible, one may wait until the HPV-16/18 prevalence amongst unvaccinated women drops below 50% of the pre-vaccination level, before considering adjusting screening. Meanwhile, also the necessary evidence for a decrease in cervical cancer risk in vaccinated women should become available.S1 Appendix(DOCX)Click here for additional data file.S1 TableASC-US = atypical squamous cells of undetermined significance; CIN = cervical intraepithelial neoplasia; HSIL = high-grade squamous intraepithelial lesion; HPV = human papillomavirus. *Potential false-positive HPV test results were modelled as HPV-infections with a short duration.(DOCX)Click here for additional data file.S2 TableHPV = human papillomavirus; CIN = cervical intraepithelial neoplasia; FIGO = International Federation of Gynecology and Obstetrics.Costs are in 2013 prices. €1.00 .(DOCX)Click here for additional data file.S3 TableQALY = quality-adjusted life year; ICER = incremental cost-effectiveness ratio; HPV = human papillomavirus.(DOCX)Click here for additional data file.S4 TableQALY = quality-adjusted life year; ICER = incremental cost-effectiveness ratio; HPV = human papillomavirus.(DOCX)Click here for additional data file.S5 TableQALY = quality-adjusted life year; ICER = incremental cost-effectiveness ratio; HPV = human papillomavirus.(DOCX)Click here for additional data file.S6 TableQALY = quality-adjusted life year; ICER = incremental cost-effectiveness ratio; HPV = human papillomavirus.(DOCX)Click here for additional data file.S7 TableQALY = quality-adjusted life year; ICER = incremental cost-effectiveness ratio; HPV = human papillomavirus.(DOCX)Click here for additional data file.


Introduction: The integration of diagnostic testing for the presence of a molecular target is of interest to predict successful targeted radionuclide therapy (TRNT). This so-called ‘theranostic’ approach aims to improve personalized treatment based on the molecular characteristics of cancer cells. Moreover, it offers new insights in predicting adverse effects and provides appropriate tools to monitor therapy responses. Recent findings using nanobodies emphasize their potential as theranostic tools in cancer treatment. Nanobodies are recombinant, small antigen-binding fragments that are derived from camelid heavy-chain-only antibodies.Areas covered: We review the current status of theranostic approaches in TRNT, with a focus on antibodies, peptides, scaffold proteins and emerging nanobodies. In recent years, nanobodies have been evaluated intensively for molecular imaging. In addition, novel data on TRNT using radiolabeled nanobodies for carcinomas and multiple myeloma highlight their promising opportunities in cancer treatment.Expert opinion: We trust that radiolabeled nanobodies will have a future potential as theranostic tools in cancer therapy, both for diagnosis as well as for TRNT. The secblastoma . TargetiThe inclusion of a diagnostic radiotracer with identical or similar pharmacokinetic characteristics as the therapeutic compound, followed by a diagnostic scan to reveal the compound accumulation at the cancer lesion is a preferred and effective strategy to guide the TRNT towards a successful outcome. Diagnostic activities would thereby serve as support for dose estimation and impact the rationalization of treatments based on dose–effect relationships. Additionally, these diagnostic scans anticipate on potential adverse effects of the therapy and are helpful to monitor therapy responses in follow-up studies. The strategy whereby related compounds are employed both for diagnosis and therapy is generally referred to as ‘theranostics’ Here, we provide an overview of possible radionuclides used in TRNT, followed by a survey of antibodies and derived fragments or peptides and even engineered non-immunoglobulin protein scaffolds employed to provide the target specificity. Finally, the recent developments with nanobodies in TRNT are put in the spotlights.2. Particles emitted during atomic decay are classified by means of linear energy transfer (LET) radiation. LET corresponds to the energy released over a certain distance. For the same absorbed dose, high LET is more cytotoxic than low LET radiation.So far, TRNT has been predominantly explored using β-emitting radioisotopes, of which the most widely used examples are described in 223Ra-chloride (Xofigo®) is the first α-emitting radioactive therapeutic agent that is approved by the FDA (in May 2013) for the treatment of castration-resistant prostate cancer and symptomatic bone metastases Currently, TRNT using α-emitters receives more attention . The α-pin vitro and in vivo data have shown that Auger electrons can be efficient as well when targeted to the cell membrane Auger-electron emitters produce intermediate LET values of 4 – 26 keV/μm. In general, it is accepted that these electrons need to be delivered close to the cell nucleus, as their path length is very short . However2.1 131I is used well over 50 years as a self-targeting adjuvant treatment together with surgery in thyroid cancer of both follicular and papillary types 123I or a low activity of 131I can be performed. More recently, diagnostic imaging using iodine-124 (124I) and positron emission tomography (PET/CT) is being evaluated to increase the spatial resolution of the scan. After treatment with high activities of 131I , whole body scintigraphy is performed to confirm the uptake in the known lesions and to demonstrate potential additional lesions that were not evident using low-activity diagnostic iodine imaging. In a subset of patients, iodine-avidity of the cancer lesions decreases over time, due to the loss of expression of the sodium iodine symporter. This loss of expression leads to resistance to 131I therapy, thereby considerably deteriorating the patient’s prognosis 3. 3.1 So far, the principle of TRNT has been mainly explored using mAbs as a vehicle to deliver the toxic radiation and is generally referred to as radioimmunotherapy or RIT. The mAbs are large (150 kDa) and complex molecules comprising two identical light chains and two identical heavy chains, held together by disulfide bonds. Consequently, a Y-shaped molecule is formed containing two identical antigen-binding arms (Fabs) and a glycosylated stem region (Fc) separated by a flexible hinge region . The Fc 90Y-labeled ibritumomab tiuxetan  and 131I-labeled tositumomab , both used in the treatment of relapsed or refractory low-grade B-cell non-Hodgkin’s lymphoma. Lymphomas are classified in either Hodgkin’s (15%) or non-Hodgkin’s lymphoma (85%). Due to the high radiosensitivity of lymphomas, only a relatively low absorbed dose is required to obtain an objective response. Dose estimations are frequently done using single-photon emission computed tomography (SPECT) imaging with Indium-111 (111In)-labeled ibritumomab tiuxetan. Anti-CD20 RIT has proven to be successful, with 80% of the patients responding to therapy, and ∼ 30% showing a complete response The landmarks for RIT consist of the FDA approval of radiolabeled anti-CD20 mAbs 90Y-labeled murine HMFG1 (90Y-muHMFG1) plus standard treatment versus standard treatment alone in patients with epithelial ovarian cancer who had attained a complete clinical remission after cytoreductive surgery and platinum-based chemotherapy. The study revealed that a single intraperitoneal administration of 90Y-muHMFG1 to these patients did not extend survival or time to relapse, most likely due to inefficient dose estimations 90Y-labeled ibritumomab tiuxetan. Pre-targeting is another strategy to reduce the unfavorable pharmacokinetics of mAbs. In pre-targeting, an unconjugated bispecific antibody is given in a first phase. When maximum uptake levels in the tumor are reached, in combination with a substantial clearance from non-target tissues, a small radiolabeled agent that is recognized by one arm of the bispecific antibody is given in a second phase. The latter binds to the bispecific antibody localized at the tumor, while unbound fraction is cleared via the kidneys In contrast to RIT of blood-borne cancers, for larger epithelial tumor burden only limited success has been recorded so far. The majority of attempts in solid tumor-RIT have been focused on colorectal, breast, prostate, ovarian and pancreatic cancer, and some cancers of the CNS, of which the most important completed clinical studies are presented in 3.2 H or VL domains (15 kDa), scFv (30 kDa), Fab (50 kDa) and Fab’2 (110 kDa), as shown schematically in in vivo, these fragments regularly show a significant degree of nonspecific accumulation in healthy tissues. As reviewed elsewhere In an attempt to augment tumor penetration and fasten blood and tissue clearance, much effort has been put in the development of mAb-derived fragments In conclusion, RIT beyond the treatment of B-cell non-Hodgkin lymphoma is still mostly under (pre-) clinical investigation. The prolonged blood residence time of mAbs has important implications on the level of toxicity to bone marrow (BM) and other highly perfused organs like spleen and liver, and specific accumulation of radioactivity in tumor tissue is limited. Recent advances in antibody engineering and pre-targeting strategies can at least partially compensate for these issues.4. 90Y/177Lu-octreotide are identified with 111In-octreotide scintigraphy, and more recently using Gallium-68 (68Ga)-octreotide for PET/CT l-lysine could reduce kidney uptake even further 177Lu-octreotate that was confused with cationic amino acids. Serious nephrotoxicity after PRRNT with 177Lu-octreotate was rare (1.3%). Slight renal impairment could be detected in 43% of cases using 99mTc-DTPA clearance assessments. Consequently, researchers concluded that cumulative administered activity of 177Lu-octreotate is not a major determinant of renal impairment An alternative strategy to target tumor-associated antigens focuses on radiolabeling of natural peptide ligands to cancer-associated receptors. Peptides are defined as short chains of maximally 70 amino acids but are most of the time smaller and generally have a molecular weight below 2000Da. Peptide receptor radionuclide therapy (PRRNT) has been established in neuroblastoma patients that suffer from residual or metastatic disease after surgery. Seventy per cent of neuroblastoma tumors express somatostatin receptors on the cell surface, which can be targeted by octreotide analogs. Patients suitable for PRRNT with 5. Recent advances in TRNT have been complemented by the focus on alternative man-made protein formats, referred to as ‘scaffold molecules’. Over 50 different protein scaffolds have been reported such as Affibodies, DARPins, anticalins, knottins, adnectins and monobodies HER2:342)2 has been fused to an albumin-binding domain (ABD), which interacts with serum albumin and therefore prolongs the blood residence time of the construct. Experimental TRNT of micro-xenografted mice with about 20 MBq of 177Lu-DTPA-ABD-(ZHER2:342)2 leads to a complete prevention of tumor formation 177Lu-labeled Affibody molecule without fusion to an ABD and thus without extended blood residence time. In addition, the biodistribution of anti-HER2 Affibody (ZHER2:4)2, labeled with Astatine-211 (211At) using the precursor N-succinimidyl-p-(trimethylstannyl)benzoate, was evaluated in HER2+ SKOV3 tumor xenografted mice upon co-administration with l-lysine and Na-thiocyanate. Here, researchers noted high doses that were delivered to thyroid (17.24 ± 51.38 Gy/MBq), lung (3.78 ± 0.71 Gy/MBq) and spleen (4.09 ± 1.03 Gy/MBq) and only 2.05 ± 0.19 to tumor. In terms of in vivo nonspecific accumulation and stability, the presented method for 211At-labeling might not be ideal for TRNT A protein scaffold that has been evaluated intensively for molecular imaging but also for TRNT is the Affibody molecule. Affibodies (7 kDa) originate from the Z-domain, which is derived from staphylococcal surface protein A 177Lu, via a DOTA chelator, as novel scaffold molecules for TRNT. Biodistribution measurements showed specific tumor uptake, but again with high renal accumulation as the dose-limiting factor Jiang and coworkers In conclusion, only few man-made scaffold proteins have been investigated in depth for TRNT. Some of them, such as Affibodies, show great potential but additional (pre-) clinical work is definitely required.6. 6.1 Camelidae. These species possess besides conventional antibodies, an additional antibody class that is devoid of light chains and that is referred to as heavy-chain-only antibodies (HCAbs) H1, which is responsible for pairing with CL. The single, variable, antigen-binding domain of the HCAbs, which is referred to as VHH, associates to its cognate target via three loops only. Functional, in vivo affinity-matured VHH fragments (13 – 14 kDa), also known as nanobodies or camel single-domain antibody fragments, are easily cloned from immunized camels or llamas and selected by phage display panning techniques. Consequently, nanobodies can be generated against a plethora of markers. Due to their small paratope, nanobodies target cryptic or hidden antigens that are inaccessible to larger antibodies Nanobodies are derived from a unique antibody format that is present in the family of HH fragment has undergone important adaptations in comparison to a VH of a conventional antibody. A larger region around the first antigen-binding loop (CDR1) is hypervariable Hs. In dromedary VHHs, an extra disulfide bridge often stabilizes these enlarged loops H or VL domains from conventional mAbs.The VEscherichia coli and yeast Moreover, nanobodies resist well against chemical or thermal denaturation and might refold reversibly to their native conformation Hs and are considered low immunogenic, as demonstrated in mice HH can still be further ‘humanized’. Vincke and coworkers described a general strategy to produce a humanized nanobody scaffold that manages to accommodate antigen-binding loops of various nanobodies to graft the antigen specificity of the loop donor Nanobodies share a high degree of sequence identity with human VUsually, an intrinsic affinity in the (sub)nanomolar range is observed for nanobodies and the antigen binding site of mAbs to their cognate antigen. However, in contrast to monovalent nanobodies, the affinity of mAbs having two antigen-binding sites per molecule might improve from avidity effects. Due to their single domain character, however, nanobodies are formatted easily into multivalent or multispecific constructs. Bivalent nanobodies  have bee6.2 in vivo diagnostic tracers in cancer is well documented. In addition, nanobodies against vascular cell adhesion molecule-1 The potential of nanobodies as 99mTc-labeled nanobodies were generally lower (ranging between 5 and 10 %IA/g at 1 – 3 h post-injection (p.i.) and gradually decreasing in time) than those obtained with mAbs at peak time points, but a 10-fold higher specific contrast was generated as early as 1 h post-injection. Radiolabeled nanobodies are cleared fast from the blood, yielding biphasic blood curves. Calculated blood half-lives of the initial phase are situated around 1 min and those of the slow phase around 30 min. At 1 h p.i., the percentage of injected activity per total blood volume generally decreases below 0.5. For clinical translation, the development of PET tracers is of interest, because of the superior image resolution and quantitative properties of clinical PET versus SPECT. Due to the short biological half-life of nanobodies, PET radioisotopes like 68Ga and Fluor-18 (18F) are favorable. In this regard, an anti-EGFR nanobody 68Ga. Moreover, the first clinical trial with a 68Ga-labeled nanobody targeting HER2 in breast cancer patients is currently ongoing (EudraCT 2012-001135-31). SPECT/microCT images of xenografted mice injected with 99mTc-labeled anti-HER2 nanobodies and PET/CT images of xenografted rats injected with 68Ga-labeled anti-HER2 nanobodies are shown in In terms of nuclear imaging of cancer, nanobodies have been developed to target a variety of extracellular cancer cell biomarkers, such as CEA et al. reported the generation of an anti-macrophage mannose receptor (MMR) nanobody for non-invasive imaging of tumor-associated macrophages Movahedi 89Zr) for PET imaging of HGF expression Finally, a recent paper reported the development and characterization of nanobodies targeting the hepatocyte growth factor (HGF) in tandem with Zirconium-89 , co-infused with Gelofusin, in HER2+ SKOV3 tumor-xenografted mice. Dosimetry calculations revealed a dose of 0.90 Gy/MBq that was delivered to both tumor and kidneys and extremely low doses (< 0.05 Gy/MBq) to healthy tissues 177Lu-DTPA-Trastuzumab (anti-HER mAb) supplied six times more radiation to the tumor than untagged 177Lu-DTPA-nanobody, but concomitantly also a 155-, 34-, 80-, 26- and 4180-fold higher radioactivity burden to the lung, liver, spleen, bone and blood, respectively. Nanobody-based TRNT in mice-bearing small established HER2pos tumors (tumor volume of 20 – 30 mm3) led to an almost complete blockade of tumor growth and a significant difference in event-free survival between the treated and the control groups. Based on histology analyses of kidney samples, no evidence of renal inflammation, apoptosis or necrosis was observed, as shown in 51Cr) EDTA or 99mTc-DTPA.In a first attempt to assess the potential of radiolabeled nanobodies for targeted cancer therapy, monovalent nanobodies targeting HER2 were labeled with 177Lu using a DOTA-derivative In another study, bivalent constructs of anti-EGFR nanobodies fused to an ABD were generated and radiolabeled with 125/131I, comparing iodination through the conventional IODO-GEN method with labeling via the residualizing agent Nϵ-(3-[131I]iodobenzoyl)-Lys5-Nα-maleimido-Gly1-GEEEK  in vitro and in vivo compared with nanobody, radiolabeled according to the IODO-GEN and IB-Mal-D-GEEEK methods, as highlighted by higher tumor retention in combination with much lower uptake in healthy tissues including kidneys. SGMIB was found superior, underscoring the importance of matching the labeling method with the normal-tissue clearance and tumor catabolism properties of the protein molecular carrier. Tumor-to-kidney ratios for 125I-SGMIB-nanobody (93 kBq) were < 0.5, < 0.5, 1, 2 and 3 at 1, 2, 4, 8 and 24 h p.i. in HER2+ BT474/M1 tumor-xenografted mice 124I, 123I and 131I for PET and SPECT imaging and for TRNT, respectively.Pruszynski and coworkers reported the radio-iodination of a cysteine-tagged anti-HER2 nanobody (5F7GGC) with Taken together, these novel findings highlight the potential of radiolabeled nanobodies in TRNT. The choice of nanobody format, as well as the radiolabel can have a major impact on both tumor uptake and kidney retention, the latter being generally considered the dose-limiting organ.6.5 99mTc, these generated nanobodies were able to monitor disease progression non-invasively by targeting MM cells in minimal residual disease. Moreover, TRNT using a 177Lu-labeled anti-5T2 MM nanobody led to an inhibition of disease progression in treated mice compared to control animals, as illustrated in Multiple myeloma (MM) is characterized by the monoclonal expansion of malignant plasma cells in BM and by production of monoclonal protein (M-protein). M-protein or paraprotein is a mAb that is secreted into the bloodstream thereupon associating with the MM cell surface in a significant fraction of patients 7. The integration of diagnostic testing for the presence of a molecular target is of interest and allows the prediction of successful TRNT. The streamlined identification of nanobodies with excellent specificity against a variety of tumor-associated antigens is well established, allowing the development of radiolabeled vehicles for use in a generic personalized healthcare strategy in oncology. The recent preclinical progress, both in diagnosis and TRNT, confirms their value as theranostic tools in cancer treatment.89Zr, takes several days until the desired contrast is obtained due to the slow blood clearance kinetics. Moreover, the EPR effect often leads to false positive results. Some of the issues with mAb-probes can be overcome by the genetic engineering into smaller mAb fragments. However, compared to nanobodies mAb fragments are frequently characterized by lower stability, slower clearance, suboptimal tumor targeting and/or undesirable uptake in healthy tissues, which often limits their usefulness as generic tracers for accurate diagnostic imaging.Nanobodies, similar to certain peptides and artificial protein scaffolds, exhibit fast diffusion and clearance kinetics in organisms, specific accumulation in target tissue combined with low nonspecific accumulation. Hence, high-contrast SPECT or PET images are generated as fast as 1 – 3 h p.i., thereby providing an early patient diagnosis. Accordingly, diagnostic scans can be generated using short-lived radioisotopes, which seriously reduce the exposure of patients to radiation. Imaging using mAbs, radiolabeled with for example Radiolabeled nanobodies could be used for early diagnosis/identification of a specific tumor-associated biomarker. Early detection of a specific biomarker attributes to the selection of the appropriate treatment strategy per patient. A whole body scan generates much more information on the presence of a certain biomarker, not only in the primary tumor but also in metastasized lesions, compared to blind biopsies, which is the current standard practice in oncology. Consequently, imaging with nanobodies could be developed to act as a valuable decision-making step in cancer therapy.Nanobodies that are labeled with therapeutic radionuclides reach the tumor with high specificity and only extremely low uptake in healthy tissues is recorded in preclinical dosimetry calculations. In RIT settings, mAbs target the tumor with dosimetry values that are about fivefold higher, but at the expense of delivering a much higher dose to blood and highly perfused organs. Therefore, toxicity to these organs limits the actual radioactive dose that is delivered to the patient by mAb carriers. Genetically engineered mAb fragments display faster clearance kinetics, but for reasons stated above, tend to show lower tumor-to-background ratios and a less optimal dosimetry profile than nanobodies.Due to the monovalent character of nanobodies, they possess a lower avidity compared to mAbs and bivalent mAb fragments such as diabodies and minibodies. Bivalent nanobodies could potentially compensate for this. However, it is clear that the bivalent character of nanobodies should not hamper their beneficial pharmacokinetics and targeting capacities. Due to a lack of studies comparing monovalent versus bivalent nanobodies, this claim is yet to be confirmed.124I could serve for PET imaging and dosimetry purposes, whereas 131I could be applied in TRNT, without substituting the actual theranostic agent. However, 18F- and 68Ga-labeled nanobodies have already shown excellent preclinical and early clinical results and these radioisotopes’ half-lives more closely match the biological half-life of nanobodies.Ideally, the diagnostic and therapeutic nanobody probe should consist of the same format. Predictive SPECT of PET scans could then be applied for preliminary dosimetry estimations. PET tracers are preferred as they generate improved resolution, which is especially important for the identification of small lesions. In this regard, nanobodies that are radio-iodinated with et al. Nanobodies recognizing an epitope, non-competitive to that of a targeted unlabeled therapeutic compound, are easily identified from ‘immune’ nanobody libraries. This opens the possibility to use radiolabeled nanobodies as adjuvant therapies in combination with conventional targeted agents. Due to the exceptionally specific targeting capacity of radiolabeled nanobodies, nanobody-based TRNT can act as an add-on therapy to improved outcome of patients that receive mAb immunotherapy. This claim has also been suggested by Sharma Nanobody-TRNT would most likely find its application in the treatment of micrometastatic and minimal residual disease, where the highly specific deposition of radioactivity to tumor cells is of upmost importance. However, the improved tumor penetration of nanobodies compared to mAbs and most of its derived fragments might open up the road to treating solid tumors. Much more research is, however, necessary to underscore the use of nanobody-based TRNT of solid tumors.51Cr-EDTA or 99mTc-DTPA.Kidney retention of radiolabeled nanobodies is a major limiting factor for TRNT. However, recent studies indicate that this characteristic is reduced through adjustments in the nanobody C-terminal sequence and through a co-infusion with Gelofusin and/or positively charged amino acids. Long-term follow-up of potential kidney toxicity is yet to be done as kidneys are generally considered to be late response tissues. In addition, kidney function could be monitored during TRNT using Fusing nanobodies with an ABD increases the blood circulation times of the construct, their absolute uptake values in tumors and tumor-to-kidney ratios. It would also reduce the necessity to perform repeated dosing. However, this strategy comes at the expense of an elevated delivery of radioactivity to blood and highly perfused organs. Further preclinical and clinical assessments should proof the added value of such nanobody constructs relative to monovalent nanobodies.177Lu and 131I. Regarding nanobody-based TRNT, residualizing radionuclides are of highest interest, as receptor-mediated internalization would trap radionuclides inside the target cell and therefore prolong the exposure. Alternatively, non-residualizing halogens like 131I can be retained in target cells using residualizing prosthetic groups.The choice of radioisotope, as well as the method for radiolabeling can have an important effect on the biodistribution, as shown already for both α-emitting radioisotopes are characterized by high LET values. Due to the highly specific targeting capacity of nanobodies, the combination of nanobody and an α-emitter might deliver a highly specific toxic load to residual or micrometastasized cancer cells, while minimizing impairment to healthy cells. Many α-emitters are characterized by a fast decay, which would be beneficial in terms of dosimetry and would match the short biological half-life of nanobodies. The first study using nanobodies radiolabeled with α-emitting radioisotopes yet needs to be performed.H. No immunogenicity in humans has been documented so far, which creates the opportunity of repeated treatment cycles. This is in contrast to murine mAbs, which are frequently used in RIT instead of humanized mAbs because of improved blood clearance and lower radiation toxicity, where repeated dosing enhances the risk of inducing antibody responses.Nanobodies are highly homologous to human Vin vivo stability, their high specificity of targeting and the very low accumulation in healthy tissues compared to most evaluated mAbs and mAb fragments, we claim nanobodies could generate a new theranostic frame for cancer therapy where it could offer improved personalized medicine opportunities.In conclusion, the use of radiolabeled nanobodies as theranostics in cancer is a fast growing concept. Recent preclinical data have highlighted their potential, but their impact in the clinic is yet to be demonstrated. Moreover, additional preclinical work remains to be performed, focusing on the optimal nanobody construct in combination with the evaluation of a variety of radioisotopes and chelator types, and their influence on dosimetry and therapeutic efficacy. It is, however, clear that there is an unmet need to specifically identify and treat minimal residual and micrometastatic disease after the conventional treatment options like surgery, chemotherapy and external beam radiotherapy. Based on their superior Early diagnostic scans using nanobodies would therefore serve as support for dose estimation and impact the rationalization of treatments based on dose-effect relationships. An initial low activity scan could be performed for dosimetry calculations, followed by a high therapeutic dose, calculated from the dosimetry analysis. We believe nanobodies for both diagnostic and therapeutic applications should be applied in the same monovalent format.68Ga-labeled anti-HER2 nanobody as a molecular imaging tracer targeting the breast cancer marker HER2 is being finalized (EudraCT 2012-001135-31) and will generate the first in-human data on tolerability, dosimetry and its capacity to identify HER2 overexpressing lesions.The first clinical trial with a Article highlights.The integration of diagnostic testing for the presence of a molecular target is of interest to predict successful targeted radionuclide therapy (TRNT).camelidae and are being evaluated intensively as diagnostic markers in both mouse models and clinical settings, especially in cancer.Nanobodies are small variable domains of HCAbs from Recent preclinical advances in TRNT using nanobodies point out their potential for therapeutic applications.Nanobodies against all possible cellular targets are easily identified, emphasizing their potential as generic theranostic tools in cancer.This box summarizes key points contained in the article.M D’Huyvetter is funded by the foundation Emmanuel van der Schueren. T Lahoutte is a Senior Clinical Investigator of the Research Foundation Flanders (FWO). The research at ICMI is funded by the Belgian State, Nationaal Kankerplan, Vlaamse liga tegen kanker, Stichting tegen kanker, FWO-Vlaanderen, IWT and Vrije Universiteit Brussel. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.