Application of the Lugano Classification for Initial Evaluation, Staging, and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The PRoLoG Consensus Initiative (Part 1-Clinical).

Our objective was to provide consensus recommendations from a consortium of academic and industry experts in the field of lymphoma and imaging for consistent application of the Lugano classification. Methods: Consensus was obtained through a series of meetings from July 2019 until September 2021 sponsored by the Pharma Imaging Network for Therapeutics and Diagnostics (PINTaD) as part of the PINTaD Response Criteria in Lymphoma Working Group (PRoLoG) consensus initiative. Results: Consensus recommendations clarified technical considerations for PET/CT and diagnostic CT from the Lugano classification, including updating the FDG avidity of different lymphoma entities, clarifying the response nomenclature, and refining lesion classification and scoring, especially with regard to scores 4 and 5 and the X category of the 5-point scale. Combination of metabolic and anatomic responses is clarified, as well as response assessment in cases of discordant or missing evaluations. Use of clinical data in the classification, especially the requirement for bone marrow assessment, is further updated on the basis of lymphoma entities. Clarification is provided with regard to spleen and liver measurements and evaluation, as well as nodal response. Conclusion: Consensus recommendations are made to comprehensively address areas of inconsistency and ambiguity in the classification encountered during response evaluation by end users, and such guidance should be used as a companion to the 2014 Lugano classification.

Application of the Lugano Classification for Initial Evaluation, Staging, and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The PRoLoG Consensus Initiative (Part 2-Technical).

The aim of this initiative was to provide consensus recommendations from a consortium of academic and industry experts in the field of lymphoma and imaging for the consistent application of imaging assessment with the Lugano classification. Methods: Consensus was obtained through a series of meetings from July 2019 to October 2021 sponsored by the PINTaD (Pharma Imaging Network for Therapeutics and Diagnostics) as part of the ProLoG (PINTaD RespOnse criteria in Lymphoma wOrking Group) consensus initiative. Results: Consensus recommendations encompass all technical imaging aspects of the Lugano classification. Some technical considerations for PET/CT and diagnostic CT are clarified with regards to required imaging series and scan visits, as well as acquisition and reconstruction of PET images and influence of lesion size and background activity. Recommendations are given on the role of imaging and clinical reviewers as well as on training and monitoring. Finally, an example template of an imaging case report form is provided to support efficient collection of data with Lugano Classification. Conclusion: Consensus recommendations are made to comprehensively address technical and imaging areas of inconsistency and ambiguity in the classification encountered by end users. Such guidance should be used to support standardized acquisition and evaluation with the Lugano 2014.

Radiologists and Clinical Trials: Part 1 The Truth About Reader Disagreements.

The debate over human visual perception and how medical images should be interpreted have persisted since X-rays were the only imaging technique available. Concerns over rates of disagreement between expert image readers are associated with much of the clinical research and at times driven by the belief that any image endpoint variability is problematic. The deeper understanding of the reasons, value, and risk of disagreement are somewhat siloed, leading, at times, to costly and risky approaches, especially in clinical trials. Although artificial intelligence promises some relief from mistakes, its routine application for assessing tumors within cancer trials is still an aspiration. Our consortium of international experts in medical imaging for drug development research, the Pharma Imaging Network for Therapeutics and Diagnostics (PINTAD), tapped the collective knowledge of its members to ground expectations, summarize common reasons for reader discordance, identify what factors can be controlled and which actions are likely to be effective in reducing discordance. Reinforced by an exhaustive literature review, our work defines the forces that shape reader variability. This review article aims to produce a singular authoritative resource outlining reader performance's practical realities within cancer trials, whether they occur within a clinical or an independent central review.

Radiologists and Clinical Trials: Part 2: Practical Statistical Methods for Understanding and Monitoring Independent Reader Performance.

Though many clinical trials rely on medical image evaluations for primary or key secondary endpoints, the methods to monitor reader performance are all too often mired in the legacy use of adjudication rates. If misused, this simple metric can be misleading and sometimes entirely contradictory. Furthermore, attempts to overcome the limitations of adjudication rates using de novo or ad hoc methods often ignore well-established research conducted over the last half-century and can lead to inaccurate conclusions or variable interpretations. Underperforming readers can be missed, expert readers retrained, or worse, replaced. This paper aims to standardize reader performance evaluations using proven statistical methods. Additionally, these methods will describe how to discriminate between scenarios of concern and normal medical interpretation variability. Statistical methods are provided for inter-reader and intra-reader variability and bias, including the adjudicator's bias. Finally, we have compiled guidelines for calculating correct sample sizes, considerations for intra-reader memory recall, and applying alternative designs for independent readers.

Directional inconsistency between Response Evaluation Criteria in Solid Tumors (RECIST) time to progression and response speed and depth.

AIM: We seek to characterize how faster tumour shrinkage rate (k) can lead to paradoxically shorter Response Evaluation Criteria in Solid Tumors (RECIST) time to progression ('TTP20' - tumour size exceeding its minimum by 5 mm and 20%) [1] and, therefore, progression-free survival (PFS). Specifically, we investigate under what conditions this paradoxical behaviour occurs, what fraction of patients satisfy these conditions, whether this phenomenon can invert population-level PFS hazard ratio, and consistency of an alternative time-to-event benefit metric with k. METHODS: We use a mathematical model treating tumour burden as decreasing drug-sensitive and increasing drug-resistant cell subpopulations. We fit this model to data from several clinical trials with different indications [2]. We simulated a more effective treatment and recorded whether patients' TTP20 increased or decreased. We performed a study-level analysis to compare the relationship of speed and depth of response with TTP20 for both the administered 'control' and simulated 'more effective' drug. We propose and test an alternative benefit metric: the model-projected time that tumour size reaches 120% of baseline (TTB120). RESULTS: Depending on indication, 3-27% of patients are estimated to have a paradoxically inverse relationship between k and TTP20. Simulated head-to-head studies show that TTP20-based PFS can favour the less effective drug. In contrast, TTB120 always favours the more effective drug. CONCLUSION: We demonstrate the paradoxical behaviour of RECIST TTP20 - as an exemplar of percent-change-from-nadir based cancer progression criterion - both in theory and in observed patient data at the individual and trial level. We propose an alternative tumour size-based criterion (TTB120) that is directionally consistent with tumour shrinkage rate.

Association of farm-related factors with characteristics profiles of extended-spectrum ß-lactamase- / plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolates from German livestock farms.

Resistance to ß-lactam antibiotics, including third-generation cephalosporins, is of major concern for animal and human health. In this study, extended-spectrum ß-lactamase (ESBL) / plasmid-mediated AmpC (pAmpC) ß-lactamase -producing Escherichia coli isolates from German livestock farms were characterised and associations of these isolate characteristics with farm-related factors were investigated across different types of livestock. A total of 469 isolates originating from 150 farms (34 broiler farms, 38 fattening pig farms, 43 dairy cattle farms, 35 beef cattle farms) was included in the analyses. ESBL-gene family, phylogroup and phenotypic antimicrobial susceptibility for several antimicrobial agents were determined. This data was used to define different profiles characterising the isolates. Multivariate analyses using a distance-based non-parametric approach were performed to investigate associations between the profiles of the isolates and farm-related factors (e.g. management, husbandry, and environment of the farms). Co-occurrence of ESBL-gene families were not found in any of the isolates analysed. Sixty-eight percent of the isolates carried blaCTX-M variant genes. The frequency of phylogroups was as follows: A (55%), B1 (35%), D (17%) and B2 (3%). The most frequent phenotypic non-wildtype profile was non-wildtype status of solely cefepime (27%). Profiles of isolates from broilers differed substantially from those of other isolates. Associations between farm-related factors and characteristics profiles differed, depending on the isolate characteristics included in the analyses. Some factors describing the farm environment, like waterfowl in the surrounding of the farm, were associated with all tested profiles. The epidemiological method applied defines distances between isolates on basis of isolate characteristics data and is capable of analysing associations between isolate characteristics and epidemiological factors. As additional data, such as plasmid characteristics, gene type, or sequence information could be included in future studies, the method is suitable to identify points of action to reduce the occurrence of antimicrobial resistant bacteria.

Cefotaxime-resistant E. coli in dairy and beef cattle farms-Joint analyses of two cross-sectional investigations in Germany.

Resistance to third-generation cephalosporins and other beta-lactam antibiotics is of major concern for animal and human health. Knowledge of the prevalence of resistant bacteria in primary production is an important element to estimate transmission along the stages in the food production chain and the exposure of the human population. The primary objective of this study was to determine the prevalence of cefotaxime-resistant commensal E. coli in dairy and beef cattle production units throughout Germany. Secondarily, the association between management factors and the presence of cefotaxime resistance was investigated. In total, 60 beef cattle and 52 dairy cattle production units all over Germany were included. Cefotaxime-resistant E. coli were isolated from at least one sample in 70% (95% CI: 58-83%) of the farms keeping beef cattle and 85% (95% CI: 75-94%) of the farms keeping dairy cattle. The sample prevalence was 35% (161/455; 95% CI: 31-40%) and 48% (156/323; 95% CI: 43-54%), respectively. Most factors associated with resistance to cefotaxime indicate that less intensive production results in a lower number of positive samples. For beef cattle, antimicrobial treatment of the whole animal group was significantly associated with an increased proportion of samples containing cefotaxime resistant E. coli. In addition, our results indicate that better hygiene management could improve the resistance situation on cattle farms.

Transient transformation of the obligate biotrophic rust fungus Uromyces fabae using biolistics.

Obligate biotrophic pathogens like the rust fungi are important plant pathogens causing enormous losses on food, forage and biomass crops. The analysis of the molecular details underlying obligate biotrophic host-parasite interactions is mainly hampered by the fact that no system for transformation is available for most obligate biotrophic organisms. Here we report the transient transformation of Uromyces fabae, an obligate biotrophic rust fungus using a biolistic approach. Biolistic bombardment of U. fabae urediospores was used to deliver different color markers (ß-glucuronidase (GUS), intron green fluorescent protein (iGFP) and red fluorescent protein (DsRed) and/or a selection marker. Endogenous regulatory elements from U. fabae plasma membrane ATPase (Uf-PMA1) were used to drive expression of the transgenes. In addition to the delivery of color markers, an in planta selection procedure using the fungicide Carboxin was established allowing the propagation of transformants. In addition to mere cytoplasmic expression of the color markers, a nuclear localization signal was fused to DsRed (pRV115-NLS) targeting the fluorescent marker protein to the nuclei. A procedure for the genetic modification of U. fabae was established. The method can be easily adapted for use with other obligate biotrophic fungi. This provides the basis for a more in depth analysis of the molecular principles governing the obligate biotrophic lifestyle.

RT real-time PCR-based quantification of Uromyces fabae in planta.

Quantification of obligate biotrophic parasites has been a long-standing problem in plant pathology. Many attempts have been made to determine how much of a pathogen is present in infected plant tissue. Methods of quantification included scoring disease symptoms, microscopic evaluation, determination of specific compounds like Ergosterol, and lately nucleic acid-based technologies. All of these methods have their drawbacks, and even real-time PCR may not be quantitative if for example the organism of interest has specific and differing numbers of nuclei in different infection structures. We applied reverse transcription (RT) real-time PCR to quantify Uromyces fabae within its host plant Vicia faba. We used three different genes, which have been shown to be constitutively expressed. Our analyses show an exponential increase of fungal material between 4 and 9 days post inoculation and thereafter reaching a steady state of around 45% of total RNA. We also used haustorium-specific genes to determine the amount of haustoria present at each time point. These analyses parallel the development of the whole fungus with the exception of the steady-state level, which is only around 5% of the total RNA. This indicates that RT real-time PCR is a suitable method for quantification of obligate biotrophic parasites, and also for the differentiation of developmental stages.

Modulation of spontaneous breathing via limbic/paralimbic-bulbar circuitry: an event-related fMRI study.

It is well established that pacemaker neurons in the brainstem provide automatic control of breathing for metabolic homeostasis and survival. During waking spontaneous breathing, cognitive and emotional demands can modulate the intrinsic brainstem respiratory rhythm. However the neural circuitry mediating this modulation is unknown. Studies of supra-pontine influences on the control of breathing have implicated limbic/paralimbic-bulbar circuitry, but these studies have been limited to either invasive surgical electrophysiological methods or neuroimaging during substantial respiratory provocation. Here we probed the limbic/paralimbic-bulbar circuitry for respiratory-related neural activity during unlabored spontaneous breathing at rest as well as during a challenging cognitive task (sustained random number generation). Functional magnetic resonance imaging (fMRI) with simultaneous physiological monitoring (heart rate, respiratory rate, tidal volume, end-tidal CO(2)) was acquired in 14 healthy subjects during each condition. The cognitive task produced expected increases in breathing rate, while end-tidal CO(2) and heart rate did not significantly differ between conditions. The respiratory cycle served as the input function for breath-by-breath, event-related, voxel-wise, random-effects image analyses in SPM5. Main effects analyses (cognitive task+rest) demonstrated the first evidence of coordinated neural activity associated with spontaneous breathing within the medulla, pons, midbrain, amygdala, anterior cingulate and anterior insular cortices. Between-condition paired t-tests (cognitive task>rest) demonstrated modulation within this network localized to the dorsal anterior cingulate and pontine raphe magnus nucleus. We propose that the identified limbic/paralimbic-bulbar circuitry plays a significant role in cognitive and emotional modulation of spontaneous breathing.

Spatial dynamics of masked picture repetition effects.

The aim of this study was to further elucidate the mechanisms of early and automatic object processing using a masked picture priming paradigm with both identity and exemplar repetition in functional MRI (fMRI). Masked repetition priming has been commonly used with words to isolate automatic, rapidly occurring mechanisms involved in visual word recognition; however, studies using the technique of masked priming with rapid presentation of pictures have been limited. This study demonstrates how the masked priming technique can be used to study early, automatic processing of rapidly presented complex objects. Temporal-occipital regions previously found to be sensitive to repetition priming in both masked word and unmasked picture studies were found to show repetition suppression for the identity primes only. Most notably, when divided into anterior and posterior divisions, the fusiform gyrus showed anatomically specific repetition suppression only in the posterior portion. This finding is comparable to that found in a previous study of masked word priming, and the similarity may suggest an overlap in the early identification processes for visual word form and visual object processing in this region. Finally, masked repetition of different exemplar objects did not result in reliable neural effects, suggesting that the underlying mechanisms of the more semantic-based, different exemplar priming may occur later or require the intervention of conscious processes.

Masked repetition priming and event-related brain potentials: a new approach for tracking the time-course of object perception.

This study reports a new approach to studying the time-course of the perceptual processing of objects by combining for the first time the masked repetition priming technique with the recording of event-related potentials (ERPs). In a semantic categorization task ERPs were recorded to repeated and unrelated target pictures of common objects that were immediately preceded by briefly presented pattern masked prime objects. Three sequential ERP effects were found between 100 and 650 ms post-target onset. These effects included an early posterior positivity/anterior negativity (N/P190) that was suggested to reflect early feature processing in visual cortex. This early effect was followed by an anterior negativity (N300) that was suggested to reflect processing of object-specific representations and finally by a widely distributed negativity (N400) that was argued to reflect more domain general semantic processing.

A small molecule inhibitor for phosphatase and tensin homologue deleted on chromosome 10 (PTEN).

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphoinositide 3-phosphatase, is an important regulator of insulin-dependent signaling. The loss or impairment of PTEN results in an antidiabetic impact, which led to the suggestion that PTEN could be an important target for drugs against type II diabetes. Here we report the design and validation of a small- molecule inhibitor of PTEN. Compared with other cysteine-based phosphatases, PTEN has a much wider active site cleft enabling it to bind the PtdIns(3,4,5)P3 substrate. We have exploited this feature in the design of vanadate scaffolds complexed to a range of different organic ligands, some of which show potent inhibitory activity. A vanadyl complexed to hydroxypicolinic acid was found to be a highly potent and specific inhibitor of PTEN that increases cellular PtdIns(3,4,5)P3 levels, phosphorylation of Akt, and glucose uptake in adipocytes at nanomolar concentrations. The findings presented here demonstrate the applicability of a novel and specific chemical inhibitor against PTEN in research and drug development.

Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation.

The catalytic properties of the type II phosphoinositide 5-phosphatases of Lowe's oculocerebrorenal syndrome, INPP5B, Synaptojanin1, Synaptojanin2 and SKIP were analysed with respect to their substrate specificity and enzymological properties. Our data reveal that all phosphatases have unique substrate specificities as judged by their corresponding KM and VMax values. They also possessed an exclusive sensitivity towards fatty acid composition, head group phosphorylation and micellar presentation. Thus, the biological function of these enzymes will not just be determined by their corresponding regulatory domains, but will be distinctly influenced by their catalytic properties as well. This suggests that the phosphatase domains fulfil a unique catalytic function that cannot be fully compensated by other phosphatases.

Bisperoxovanadium compounds are potent PTEN inhibitors.

The tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) shares homology with protein tyrosine phosphatases (PTPases). Similarly, bisperoxovanadium (bpV) molecules that are well-established PTPase inhibitors were shown to inhibit PTEN, but at up to 100-fold lower concentrations. The preference and potency of the bpVs towards PTEN was validated in vivo as demonstrated by: (i) an increase of Ser473 phosphorylation of protein kinase B (PKB) at similar low nanomolar doses, (ii) the lack of any effect on the PKB phosphorylation in the PTEN negative cell line UM-UC-3, (iii) the ability to rescue Ly294002-induced phosphoinositide 3-kinase inhibition and (iv) a lack of tyrosine phosphorylation at low nanomolar doses.

Insulin-like growth factor II mRNA is expressed in neurones of the brain of the bony fish Oreochromis mossambicus, the tilapia.

The physiological meaning of insulin-like growth factor II (IGF-II) is still enigmatic. IGF-II occurs in the adult mammalian brain where it is expressed in the mesodermal portion of the choroid plexus and the meninges, but results on its presence in cells of neuroepithelial origin are controversial. However, IGF-II mRNA is transiently expressed in neurones during mammalian early development. In bony fish, IGF-II mRNA is also present in the adult brain but nothing is known about its synthesis sites. Thus, the present study using in situ hybridization with digoxigenin-labelled RNA species-specific probes investigates the cellular distribution of IGF-II mRNA in the adult brain of a bony fish, the tilapia (Oreochromis mossambicus). As in mammals, IGF-II mRNA was strongly expressed in the choroid plexus and meninges. Thus, IGF-II synthesis by choroid plexus and meninges seems to have a long evolutionary history and may be common to all vertebrates. However, as shown by the detailed investigation of landmark nuclei and regions, IGF-II mRNA occurred also in numerous neurones at all levels of the tilapia brain. The distinct localization of IGF-II mRNA in neurones might indicate that neuronal IGF-II acts as transmitter or modulator. However, the widespread occurrence of the IGF-II-producing neurones argues against this assumption and most probably suggests that IGF-II plays a role in the differentiation, maintenance and regeneration of neurones. It is further assumed that the sustained neuronal IGF-II expression in the brain of the adult tilapia correlates with continued post-embryonic up to life-long brain growth as has been shown in many teleost fishes.

Thyroid hormone stimulates hepatic IGF-I mRNA expression in a bony fish, the tilapia Oreochromis mossambicus, in vitro and in vivo.

To gain more knowledge about the physiological regulation of hepatic insulin-like growth factor-I (IGF-I) production in bony fish, we examined the potential influence of thyroid hormone (T3, 3,5,3'-triiodothyronine) on the expression of IGF-I in the liver of the tilapia Oreochromis mossambicus, using in vitro and in vivo methods. The in vitro experiments were performed using a recently established primary hepatocyte cell culture and IGF-I expression was determined by means of semiquantitative RT-PCR. T3 (100 nM) significantly enhanced the synthesis rate of IGF-I mRNA in short (>8h) and long (>42h) time courses. The stimulating effect of T3 was detected already after 1h. After 4h, the IGF-I mRNA expression was more than 150% of the starting amount. In long time courses, after 6h the IGF-I mRNA value was about 170% of that in untreated cells and at the end of the experiment, it was still three times higher than in the control. In addition, the increase in IGF-I mRNA expression evoked by T3 (1 nM to 1 microM) was dose-dependent. In the in vivo approach, 10 individuals of tilapia received 4 daily intraperitoneal injections of T3 (6 microg/g body weight). IGF-I mRNA was assessed using dot blot technique with a tilapia specific IGF-I cRNA probe. The T3 treatment led to an increase of the IGF-I mRNA level up to 45% in the liver compared to the untreated animals. In conclusion, our results show that T3 directly stimulates the hepatic production of IGF-I in the tilapia in vitro and in vivo and indicate that in tilapia liver regulatory mechanisms seem to exist, as they are discussed for mammals.