Model-Based Selection for Proton Therapy in Breast Cancer: Development of the National Indication Protocol for Proton Therapy and First Clinical Experiences.

AIMS: Proton therapy is a radiation technique that yields less dose in normal tissues than photon therapy. In the Netherlands, proton therapy is reimbursed if the reduced dose to normal tissues is predicted to translate into a prespecified reduction in toxicity, based on nationally approved validated models. The aim of this paper is to present the development of a national indication protocol for proton therapy (NIPP) for model-based selection of breast cancer patients and to report on first clinical experiences. MATERIALS AND METHODS: A national proton therapy working group for breast cancer (PWG-BC) screened the literature for prognostic models able to estimate the individual risk of specific radiation-induced side-effects. After critical appraisal and selection of suitable models, a NIPP for breast cancer was written and subjected to comments by all stakeholders. The approved NIPP was subsequently introduced to select breast cancer patients who would benefit most from proton therapy. RESULTS: The model of Darby et al. (N Engl J Med 2013; 368:987-82) was the only model fulfilling the criteria prespecified by the PWG-BC. The model estimates the relative risk of an acute coronary event (ACE) based on the mean heart dose. The absolute lifetime risk of ACE <80 years was calculated by applying this model to the Dutch absolute incidence of ACE for female and male patients, between 40 and 70 years at breast cancer radiotherapy, with/without cardiovascular risk factors. The NIPP was approved for reimbursement in January 2019. Based on a threshold value of a 2% absolute lower risk on ACE for proton therapy compared with photons, 268 breast cancer patients have been treated in the Netherlands with proton therapy between February 2019 and January 2021. CONCLUSION: The NIPP includes a model that allows the estimation of the absolute risk on ACE <80 years based on mean heart dose. In the first 2 years, 268 breast cancer patients have been treated with proton therapy in The Netherlands.

Effect of breathing motion on robustness of proton therapy plans for left-sided breast cancer patients with indication for locoregional irradiation.

PURPOSE: To investigate the dosimetric impact of breathing motion on robustly optimized proton therapy treatment plans for left-sided breast cancer patients with an indication for locoregional irradiation. MATERIALS AND METHODS: Clinical Target Volumes (CTVs) (left-sided breast, level 1 to 4 axillary lymph nodes, interpectoral and internal mammary lymph node regions) and organs at risk were delineated on 4 D-CTs of ten female patients. After treatment planning to a prescribed dose of 40.05 Gy(RBE) in 15 fractions on the time-averaged CT, the dose was calculated on all ten phases of the breathing cycle. Robustness to setup (5 mm) and range errors (3%) was evaluated for those ten phases. Correlations were evaluated between the phases of the breathing cycle and the D98% of the CTV and the Dmean of the heart. RESULTS: Correlations coefficients were between -0.12 and 0.29. At the most extreme values of the 28 robustness scenarios, the clinical goals were met for all but two patients. The mean heart dose was 0.41 Gy(RBE) with a standard deviation of 0.31 Gy(RBE) of proton therapy plans. CONCLUSION: The effect of breathing motion on the robustness of proton therapy treatment plans for this patient group is minor and not of clinical significance. Based on this patient group, a deep-inspiration breath hold seems to be unnecessary to improve robustness for these patients.

Risk communication in a patient decision aid for radiotherapy in breast cancer: How to deal with uncertainty?

BACKGROUND AND AIM: Patient decision aids for oncological treatment options, provide information on the effect on recurrence rates and/or survival benefit, and on side-effects and/or burden of different treatment options. However, often uncertainty exists around the probability estimates for recurrence/survival and side-effects which is too relevant to be ignored. Evidence is lacking on the best way to communicate these uncertainties. The aim of this study is to develop a method to incorporate uncertainties in a patient decision aid for breast cancer patients to support their decision on radiotherapy. METHODS: Firstly, qualitative interviews were held with patients and health care professionals. Secondly, in the development phase, thinking aloud sessions were organized with four patients and 12 health care professionals, individual and group-wise. RESULTS: Consensus was reached on a pictograph illustrating the whole range of uncertainty for local recurrence risks, in combination with textual explanation that a more exact personalized risk would be given by their own physician. The pictograph consisted of 100 female icons in a 10 x 10 array. Icons with a stepwise gradient color indicated the uncertainty margin. The prevalence and severity of possible side-effects were explained using verbal labels. CONCLUSIONS: We developed a novel way of visualizing uncertainties in recurrence rates in a patient decision aid. The effect of this way of communicating risk uncertainty is currently being tested in the BRASA study (NCT03375801).

Introducing the CSP Analyzer: A novel Machine Learning-based application for automated analysis of two-dimensional NMR spectra in NMR fragment-based screening.

NMR-based screening, especially fragment-based drug discovery is a valuable approach in early-stage drug discovery. Monitoring fragment-binding in protein-detected 2D NMR experiments requires analysis of hundreds of spectra to detect chemical shift perturbations (CSPs) in the presence of ligands screened. Computational tools are available that simplify the tracking of CSPs in 2D NMR spectra. However, to the best of our knowledge, an efficient automated tool for the assessment and binning of multiple spectra for ligand binding has not yet been described. We present a novel and fast approach for analysis of multiple 2D HSQC spectra based on machine-learning-driven statistical discrimination. The CSP Analyzer features a C# frontend interfaced to a Python ML classifier. The software allows rapid evaluation of 2D screening data from large number of spectra, reducing user-introduced bias in the evaluation. The CSP Analyzer software package is available on GitHub https://github.com/rubbs14/CSP-Analyzer/releases/tag/v1.0 under the GPL license 3.0 and is free to use for academic and commercial uses.

Do we have to reduce the recall period? Validity of a daily physical activity questionnaire (PAQ24) in young active adults.

BACKGROUND: Combining the strengths of physical activity (PA) diaries and questionnaires may be needed to improve the unsatisfying measurement quality of existing PA questionnaires. This study investigated the construct validity of a short PA questionnaire (Physical Activity Questionnaire for 24 h [PAQ24]) with a recall period of one day. METHODS: In this cross-sectional study, participants completed the PAQ24 on seven consecutive days while wearing an accelerometer (GENEActiv). Thereafter, the Global Physical Activity Questionnaire (GPAQ) was completed. Spearman correlation coefficients and Bland-Altman analysis were used to assess construct validity. RESULTS: Overall, 50 active adults (11 women, mean age = 25.1 ± 2.5) participated. Relative agreements between Total PA of PAQ24 and accelerometer were 0.37 ≤ ρ ≤ 0.72 for each day with satisfying agreement on five out of seven days. Weekly relative agreement for Total PA was moderate (ρ = 0.44). Relative agreements between PAQ24 and GPAQ were ρ = 0.43 for Total PA. Daily and weekly absolute agreements were poor indicated by wide limits of agreement. CONCLUSIONS: In contrast to weekly Total PA, the majority of daily results of the PAQ24 showed satisfying construct validity. A short recall period may improve the measurement quality of PA questionnaires, but measurement errors and the costs of multiple administrations must be considered in future studies.

Poliadenopatías mediastinales y diagnósticos diferenciales en lupus eritematoso sistémico. Reporte de un caso y revisión de la literatura / Mediastinal polyadenopathies and differential diagnoses in systemic lupus erythematosus. Case report and literature review

Paciente varón de 59 años de edad con diagnóstico de lupus eritematoso sistémico y cuadro de consolidación pulmonar asociada a poliadenopatías mediastinales y derrame pleural, con evolución crónica y tórpida. Se discuten diagnósticos diferenciales e implicancia clínica

A 59-year-old male patient with a diagnosis of systemic lupus erythematosus and pulmonary consolidation associated with mediastinal polyadenopathies and pleural effusion, with chronic and torpid evolution. Differential dignoses and clinical implications are discussed

Inhibitors of PEX14 disrupt protein import into glycosomes and kill Trypanosoma parasites.

The parasitic protists of the Trypanosoma genus infect humans and domestic mammals, causing severe mortality and huge economic losses. The most threatening trypanosomiasis is Chagas disease, affecting up to 12 million people in the Americas. We report a way to selectively kill Trypanosoma by blocking glycosomal/peroxisomal import that depends on the PEX14-PEX5 protein-protein interaction. We developed small molecules that efficiently disrupt the PEX14-PEX5 interaction. This results in mislocalization of glycosomal enzymes, causing metabolic catastrophe, and it kills the parasite. High-resolution x-ray structures and nuclear magnetic resonance data enabled the efficient design of inhibitors with trypanocidal activities comparable to approved medications. These results identify PEX14 as an "Achilles' heel" of the Trypanosoma suitable for the development of new therapies against trypanosomiases and provide the structural basis for their development.

The AP-1 transcription factor JunB is essential for multiple myeloma cell proliferation and drug resistance in the bone marrow microenvironment.

Despite therapeutic advances, multiple myeloma (MM) remains an incurable disease, predominantly because of the development of drug resistance. The activator protein-1 (AP-1) transcription factor family has been implicated in a multitude of physiologic processes and tumorigenesis; however, its role in MM is largely unknown. Here we demonstrate specific and rapid induction of the AP-1 family member JunB in MM cells when co-cultured with bone marrow stromal cells. Supporting a functional key role of JunB in MM pathogenesis, knockdown of JUNB significantly inhibited in vitro MM cell proliferation and survival. Consistently, induced silencing of JUNB markedly decreased tumor growth in a murine MM model of the microenvironment. Subsequent gene expression profiling revealed a role for genes associated with apoptosis, DNA replication and metabolism in driving the JunB-mediated phenotype in MM cells. Importantly, knockdown of JUNB restored the response to dexamethasone in dexamethasone-resistant MM cells. Moreover, 4-hydroxytamoxifen-induced activation of a JunB-ER fusion protein protected dexamethasone-sensitive MM cells against dexamethasone- and bortezomib-induced cytotoxicity. In summary, our results demonstrate for the first time a specific role for AP-1/JunB in MM cell proliferation, survival and drug resistance, thereby strongly supporting that this transcription factor is a promising new therapeutic target in MM.

Relative distribution of quadriceps head anatomical cross-sectional areas and volumes--sensitivity to pain and to training intervention.

INTRODUCTION: Quadriceps heads are important in biomechanical stabilization and in the pathogenesis osteoarthritis of the knee. This is the first study to explore the relative distribution of quadriceps head anatomical cross-sectional areas (ACSA) and volumes, and their response to pain and to training intervention. METHODS: The relative proportions of quadriceps heads were determined in 48 Osteoarthritis Initiative participants with unilateral pain (65% women; age 45-78 years). Quadriceps head volumes were also measured in 35 untrained women (45-55 years) before and after 12-week training intervention. Cross-sectional areas of the vastus medialis (VM), inter-medius (VIM), and lateralis (VL), and of the rectus femoris (RF) were determined from axial T1-weighted MR images. RESULTS: The proportion of the VM on the total quadriceps ACSA increased from proximal to distal. The difference in quadriceps ACSA of painful (vs. pain-free) limbs was -5.4% for the VM (p<0.001), -6.8% for the VL (p<0.01), -2.8% for the VIM (p=0.06), and +3.4% for the RF (p=0.67) but the VM/VL ratio was not significantly altered. The muscle volume increase during training intervention was +4.2% (p<0.05) for VM, +1.3% for VL, +2.0% for VIM (p<0.05) and +1.6% for RF. CONCLUSION: The proportion of quadriceps head relative to total muscle ACSA and volume depends on the anatomical level studied. The results suggest that there may be a differential response of the quadriceps heads to pain-induced atrophy and to training-related hypertrophy. Studies in larger samples are needed to ascertain whether the observed differences in response to pain and training are statistically and clinically significant.

[Salter-Harris type IV epipyseal fracture of the lateral malleolus. A rare injury in childhood]. / Salter-Harris-Typ-IV-Epiphysenfraktur am Außenknöchel. Eine seltene Verletzung im Kindesalter.

This is a case presentation of a 9-year-old boy who sustained a rare Salter-Harris type IV distal fibular fracture including an avulsion fracture of the anterior inferior tibiofibular ligament at the fibular attachment. Treatment consisted of open reduction and internal fixation by Kirschner wire and cerclage. Possible posttraumatic growth disturbances and the major implications are highlighted.

The matricellular protein periostin contributes to proper collagen function and is downregulated during skin aging.

BACKGROUND: Periostin is a secreted 90kDa matricellular protein, which is predominantly expressed in collagen-rich tissues. Collagen is the most abundant protein in mammals and has great tensile strength. Recent investigations have shown that periostin influences collagen fibrillogenesis and biomechanical properties of murine connective tissues. OBJECTIVE: We investigated the function of periostin concerning collagen homeostasis during intrinsic and extrinsic skin aging. For this purpose, human skin samples of young and old donors as well as samples of photoaged and sun-protected skin areas were analyzed for periostin expression. Using in vitro models, we determined the cell types responsible for periostin expression and performed functional analyses with periostin knockdown cells. METHODS: TaqMan Real-Time PCR, UV irradiation, knockdown experiments, immunostaining, electron microscopy, collagen degradation assay, collagen crosslink analysis. RESULTS: Periostin expression is highest in the papillary dermis and downregulated during skin aging. Fibroblasts and non-follicular skin derived precursors were identified as main source for periostin expression in human skin. Periostin knockdown in fibroblasts has no effect on collagen expression, but results in an increased fibril diameter and aberrant collagen structure. This leads to an increased susceptibility of collagen toward proteases, whereas recombinant periostin protects collagen fibrils from degradation. CONCLUSION: Our data show that periostin plays an important role for proper collagen assembly and homeostasis. During skin aging periostin expression decreases and contributes to the phenotype of aged skin.

[Clinically symptom-poor isolated rupture of the biceps tendon of the knee close to the attachment. Can an operative approach always be recommended?]. / Die klinisch symptomarme isolierte ansatznahe Ruptur der Bizepssehne am Kniegelenk. Ist ein operatives Vorgehen uneingeschränkt empfehlenswert?

Isolated avulsions involving the tendon of the biceps femoris muscle are rare injuries. Injury patterns are similar to those of posterolateral knee injuries mostly due to hyperextension and external rotation. Functional loss is common regarding painful limited flexion of the knee. In the case described in this article there was complete avulsion of the biceps femoris tendon but low levels of pain and functional loss despite the proximity to the attachment.

Using combination therapy to override stromal-mediated chemoresistance in mutant FLT3-positive AML: synergism between FLT3 inhibitors, dasatinib/multi-targeted inhibitors and JAK inhibitors.

Acute myeloid leukemia (AML) progenitors are frequently characterized by activating mutations in the receptor tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). Protein tyrosine kinases are integral components of signaling cascades that have a role in both FLT3-mediated transformation as well as viability pathways that are advantageous to leukemic cell survival. The bone marrow microenvironment can diminish AML sensitivity to tyrosine kinase inhibitors. We hypothesized that inhibition of protein kinases in addition to FLT3 may be effective in overriding drug resistance in AML. We used a cell-based model mimicking stromal protection as part of an unbiased high-throughput chemical screen to identify kinase inhibitors with the potential to override microenvironment-mediated drug resistance in mutant FLT3-positive AML. Several related multi-targeted kinase inhibitors, including dasatinib, with the capability of reversing microenvironment-induced resistance to FLT3 inhibition were identified and validated. We validated synergy in vitro and demonstrated effective combination potential in vivo. In particular Janus kinase inhibitors were effective in overriding stromal protection and potentiating FLT3 inhibition in primary AML and cell lines. These results hint at a novel concept of using combination therapy to override drug resistance in mutant FLT3-positive AML in the bone marrow niche and suppress or eradicate residual disease.

Side differences of thigh muscle cross-sectional areas and maximal isometric muscle force in bilateral knees with the same radiographic disease stage, but unilateral frequent pain - data from the osteoarthritis initiative.

OBJECTIVE: To determine whether anatomical thigh muscle cross-sectional areas (MCSAs) and strength differ between osteoarthritis (OA) knees with frequent pain compared with contra-lateral knees without pain, and to examine the correlation between MCSAs and strength in painful vs painless knees. METHODS: Forty-eight subjects (31 women; 17 men; age 45-78 years) were drawn from 4,796 Osteoarthritis Initiative (OAI) participants, in whom both knees displayed the same radiographic stage (KLG2 or 3), one with frequent pain (most days of the month within the past 12 months) and the contra-lateral one without pain. Axial MR images were used to determine MCSAs of extensors, flexors and adductors at 35% femoral length (distal to proximal) and in two adjacent 5 mm images. Maximal isometric extensor and flexor forces were used as provided from the OAI database. RESULTS: Painful knees showed 5.2% lower extensor MCSAs (P=0.00003; paired t-test), and 7.8% lower maximal extensor muscle forces (P=0.003) than contra-lateral painless knees. There were no significant differences in flexor forces, or flexor and adductor MCSAs (P>0.39). Correlations between force and MCSAs were similar in painful and painless OA knees (0.44

Characterization of fibrillar collagen types using multi-dimensional multiphoton laser scanning microscopy.

In dermal photodamage the ratio of the collagen types III to I changes. This makes the investigation of the fibrillar collagen type characteristics interesting for skin research. In this study collagen types were characterized using 5-dimensional multiphoton laser scanning microscopy (5D-IVT) that can be applied in vivo. Second harmonic generation (SHG) signals and fluorescence lifetimes of the collagen autofluorescence were analysed. Collagen type I generates a higher SHG intensity and a longer fluorescence lifetime compared to collagen type III. Thus, the SHG intensity decrease found in photodamaged skin might be explained by the increase in collagen type III. Calculating the in vivo relevant increase of collagen type III gives a negligible difference in fluorescence lifetime not qualifying this method for the determination of collagen type changes in dermal photodamage in vivo in human skin. However, for pathologies that exhibit higher differences in collagen types 5D-IVT analysis might be a suitable method.

Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.

The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.

The JAK2V617F oncogene requires expression of inducible phosphofructokinase/fructose-bisphosphatase 3 for cell growth and increased metabolic activity.

Myeloproliferative neoplasms are characterized by overproduction of myeloid lineage cells with frequent acquisition of oncogenic JAK2V617F kinase mutations. The molecular mechanisms that regulate energy requirements in these diseases are poorly understood. Transformed cells tend to rely on fermentation instead of more efficient oxidative phosphorylation for energy production. Our data in JAK2V617F-transformed cells show that growth and metabolic activity were strictly dependent on the presence of glucose. Uptake of glucose and cell surface expression of the glucose transporter Glut1 required the oncogenic tyrosine kinase. Importantly, JAK2V617F as well as active STAT5 increased the expression of the inducible rate-limiting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which controls glycolytic flux through 6-phosphofructo-1-kinase. PFKFB3 was required for JAK2V617F-dependent lactate production, oxidative metabolic activity and glucose uptake. Targeted knockdown of PFKFB3 also limited cell growth under normoxic and hypoxic conditions and blocked in vivo tumor formation in mice. Overall, these data suggest that inducible PFKFB3 is required for increased growth, metabolic activity and is regulated through active JAK2 and STAT5. Novel therapies that specifically block PFKFB3 activity or expression would, therefore, be expected to inhibit JAK2/STAT5-dependent malignancies and related cancers.

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes.

NADPH oxidases regulate cell growth and migration in myeloid cells transformed by oncogenic tyrosine kinases.

Transformation by tyrosine kinase oncogenes (TKOs) in myeloid malignancies, including BCR-ABL in chronic myeloid leukemia, FLT3ITD in acute myeloid leukemia or JAK2V617F in myeloproliferative neoplasms, is associated with increased growth and cytoskeletal abnormalities. Using targeted approaches against components of the superoxide-producing NADPH-oxidases, including NADPH oxidase 2 (NOX2), NOX4 and the common p22(phox) subunit of NOX1-4, myeloid cells were found to display reduced cell growth and spontaneous migration. Consistent with a role of NOXs as regulators of membrane proximal signaling events in nonphagocytic cells, NOX2 and NOX4 were not involved in the excess production of intracellular reactive oxygen species and did not significantly increase oxygen consumption. All NOX family members are controlled in part through levels of the rate-limiting substrate NADPH, which was found to be significantly elevated in TKO-transformed cells. Also, reduced phosphorylation of the actin filament crosslinking protein myristoylated alanine-rich C-kinase substrate (MARCKS) in response to suppression of p22(phox) hints at a novel effector of NOX signaling. MARCKS was also found to be required for increased migration. Overall, these data suggest a model whereby NOX links metabolic NADPH production to cellular events that directly contribute to transformation.

Drug resistance in mutant FLT3-positive AML.

Mutant Fms-Like Tyrosine kinase-3 (FLT3), which is expressed in the leukemic cells of a subpopulation of acute myeloid leukemia (AML) patients, represents an attractive target for the therapy of AML. There are several FLT3 inhibitors presently in clinical trials with sufficient efficacy and toxicity features to warrant further testing in combination with standard therapies. However, the transient and partial responses observed in AML patients treated with FLT3 inhibitors, coupled with the discovery of drug-resistant leukemic blast cells in AML patients, have made resistance to FLT3 inhibitors a growing concern. In this study, we provide an overview of the role of mutant FLT3 in AML, FLT3 inhibitors under clinical and preclinical investigation, mechanisms of resistance to FLT3 inhibitors, and possible therapeutic approaches to overcoming this resistance.