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.

Late-onset Pompe disease (LOPD) in Belgium: clinical characteristics and outcome measures.

BACKGROUND: Late-onset Pompe disease (LOPD) is a rare, hereditary, progressive disorder that is usually characterized by limb-girdle muscle weakness and/or respiratory insufficiency. LOPD is caused by mutations in the acid alpha-glucosidase (GAA) gene and treated with enzyme replacement therapy (ERT). METHODS: We studied the clinical, brain imaging, and genetic features of the Belgian cohort of late-onset Pompe disease patients (N = 52), and explored the sensitivity of different outcome measures, during a longitudinal period of 7 years (2010-2017), including the activity limitations ActivLim score, 6 min walking distance (6MWD), 10 m walk test (10MWT), MRC sum score, and forced vital capacity (FVC) sitting/supine. RESULTS: In Belgium, we calculated an LOPD prevalence of 3.9 per million. Mean age at onset of 52 LOPD patients was 28.9 years (SD: 15.8 y), ranging from 7 months to 68 years. Seventy-five percent (N = 39) of the patients initially presented with limb-girdle weakness, whereas in 13% (N = 7) respiratory symptoms were the only initial symptom. Non-invasive ventilation (NIV) was started in 37% (N = 19), at a mean age of 49.5 years (SD: 11.9 y), with a mean duration of 15 years (SD: 10.2 y) after symptom onset. Brain imaging revealed abnormalities in 25% (N = 8) of the patients, with the presence of small cerebral aneurysm(s) in two patients and a vertebrobasilar dolichoectasia in another two. Mean diagnostic delay was 12.9 years. All patients were compound heterozygotes with the most prevalent mutation being c.-32-13 T > G in 96%. We identified two novel mutations in GAA: c.1610_1611delA and c.186dup11. For the 6MWD, MRC sum score, FVC sitting and FVC supine, we measured a significant decrease over time (p = 0.0002, p = 0.0001, p = 0.0077, p = 0.0151), which was not revealed with the ActivLim score and 10MWT (p > 0.05). CONCLUSIONS: Awareness on LOPD should even be further increased because of the long diagnostic delay. The 6MWD, but not the ActivLim score, is a sensitive outcome measure to follow up LOPD patients.

Are PROMs sufficient to record late outcome of breast cancer patients treated with radiotherapy? A comparison between patient and clinician reported outcome through an outpatient clinic after 10years of follow up.

AIM: To investigate whether breast cancer patients' visits to an outpatient clinic for late outcome (OCLO) can be replaced by patient reported outcome measures (PROMs), by comparing late toxicity scored at the OCLO with PROMs. METHODS: All breast cancer patients treated in our institute with adjuvant radiotherapy 10-11years ago were invited to visit the OCLO, and for filling out PROM-questionnaires. Concordance rate between PROMs and OCLO-reported outcome and the percentage of patients with ≥2 degrees difference in toxicity level between patient and clinician was assessed. RESULTS: 686 of 1029 patients were still alive. 249 patients visited the OCLO, and 341 patients returned a questionnaire. At a group level, patients reported higher toxicity rates than clinicians. The mean concordance for individual patients was 58% between patient and clinician reported outcome. In 2.8%, the clinician reported ≥2 degrees higher toxicity than the patients did, whereas in 6.8% patients reported ≥2 degrees higher toxicity. CONCLUSION: PROMs do not underestimate late side-effects at a group level. In spite of the low concordance rate, PROMS can be used to identify patients who experience a heavy burden of side-effects, requiring specific attention. Therefore, patients can be spared a visit to the OCLO.

Genetic and epigenetic differences associated with environmental gradients in replicate populations of two salt marsh perennials.

While traits and trait plasticity are partly genetically based, investigating epigenetic mechanisms may provide more nuanced understanding of the mechanisms underlying response to environment. Using AFLP and methylation-sensitive AFLP, we tested the hypothesis that differentiation to habitats along natural salt marsh environmental gradients occurs at epigenetic, but not genetic loci in two salt marsh perennials. We detected significant genetic and epigenetic structure among populations and among subpopulations, but we found multilocus patterns of differentiation to habitat type only in epigenetic variation for both species. In addition, more epigenetic than genetic loci were correlated with habitat in both species. When we analysed genetic and epigenetic variation simultaneously with partial Mantel, we found no correlation between genetic variation and habitat and a significant correlation between epigenetic variation and habitat in Spartina alterniflora. In Borrichia frutescens, we found significant correlations between epigenetic and/or genetic variation and habitat in four of five populations when populations were analysed individually, but there was no significant correlation between genetic or epigenetic variation and habitat when analysed jointly across the five populations. These analyses suggest that epigenetic mechanisms are involved in the response to salt marsh habitats, but also that the relationships among genetic and epigenetic variation and habitat vary by species. Site-specific conditions may also cloud our ability to detect response in replicate populations with similar environmental gradients. Future studies analysing sequence data and the correlation between genetic variation and DNA methylation will be powerful to identify the contributions of genetic and epigenetic response to environmental gradients.

The epigenetic footprint of poleward range-expanding plants in apomictic dandelions.

Epigenetic modifications, such as DNA methylation variation, can generate heritable phenotypic variation independent of the underlying genetic code. However, epigenetic variation in natural plant populations is poorly documented and little understood. Here, we test whether northward range expansion of obligate apomicts of the common dandelion (Taraxacum officinale) is associated with DNA methylation variation. We characterized and compared patterns of genetic and DNA methylation variation in greenhouse-reared offspring of T. officinale that were collected along a latitudinal transect of northward range expansion in Europe. Genetic AFLP and epigenetic MS-AFLP markers revealed high levels of local diversity and modest but significant heritable differentiation between sampling locations and between the southern, central and northern regions of the transect. Patterns of genetic and epigenetic variation were significantly correlated, reflecting the genetic control over epigenetic variation and/or the accumulation of lineage-specific spontaneous epimutations, which may be selectively neutral. In addition, we identified a small component of DNA methylation differentiation along the transect that is independent of genetic variation. This epigenetic differentiation might reflect environment-specific induction or, in case the DNA methylation variation affects relevant traits and fitness, selection of heritable DNA methylation variants. Such generated epigenetic variants might contribute to the adaptive capacity of individual asexual lineages under changing environments. Our results highlight the potential of heritable DNA methylation variation to contribute to population differentiation along ecological gradients. Further studies are needed using higher resolution methods to understand the functional significance of such natural occurring epigenetic differentiation.

Pain catastrophizing influences the use and the effectiveness of distraction in schoolchildren.

Distraction is an intuitive way of coping with pain and is often used in children's pain treatment programs. However, empirical evidence concerning the effectiveness of distraction is equivocal. One potential explanation might be that distraction does not work for everyone in every situation. In the current series of studies, we examined the role of pain catastrophizing as an influencing factor of distraction effectiveness. In the first study, we investigated the use of pain coping strategies (including distraction) in schoolchildren (N = 828, aged 8-18 years) by means of a questionnaire. Results indicated that children with higher levels of pain catastrophizing reported using less distraction strategies in daily life than children with lower levels of pain catastrophizing. In the second study, a subsample (N = 81, aged 9-18 years) performed a painful cold pressor task (CPT) (12 °C). Participants were randomly assigned to a distraction group, in which an attention-demanding tone-detection task was performed during the CPT, or a control group, in which no distraction task was performed. Results showed that participants in the distraction group were engaged in the distraction task, and reported to have paid less attention to pain than participants in the control group. However, distraction was ineffective in reducing cold pressor pain, and even intensified the pain experience in high catastrophizing children. Caution may be warranted in using distraction as a 'one size fits all' method, especially in high catastrophizing children.

Mothers' and fathers' responses to their child's pain moderate the relationship between the child's pain catastrophizing and disability.

Preliminary evidence suggests that pain catastrophizing in children may be important in understanding how parents respond to their child's pain. However, no study has investigated whether parental responses, in turn, moderate the impact of child's catastrophizing upon pain outcomes. The present study was designed to address this, and investigated the association of the child's catastrophizing with different types of parental responses (ie, solicitousness, discouragement and coping promoting responses) and the extent to which parental responses moderate the association between the child's catastrophizing and disability. Participants were 386 school children and their parents. Analyses revealed significant associations between the child's pain catastrophizing and parental responses, but with mothers and fathers evidencing different patterns; ie, higher levels of the child's catastrophizing were significantly associated with lower levels of solicitousness by fathers, and with higher levels of discouragement by mothers. Moderation analyses indicated that father's solicitiousness moderated the association between catastrophizing and disability; the positive association between catastrophizing and the child's disability was further strengthened when fathers reported low levels of solicitousness, but became less pronounced when fathers reported high levels of solicitousness. Findings also revealed a moderating impact of mothers' and fathers' promotion of their child's well behaviour/coping. Specifically, the detrimental impact of child catastrophizing upon disability was less pronounced when parents reported high promotion of their child's well behaviours/coping. The findings of the present study suggest the importance of assessing and targeting parental responses to their child's pain to alter the adverse impact of the child's pain catastrophizing on pain outcomes.

Habitat-specific natural selection at a flowering-time QTL is a main driver of local adaptation in two wild barley populations.

Understanding the genetic basis of local adaptation requires insight in the fitness effects of individual loci under natural field conditions. While rapid progress is made in the search for genes that control differences between plant populations, it is typically unknown whether the genes under study are in fact key targets of habitat-specific natural selection. Using a quantitative trait loci (QTL) approach, we show that a QTL associated with flowering-time variation between two locally adapted wild barley populations is an important determinant of fitness in one, but not in the other population's native habitat. The QTL mapped to the same position as a habitat-specific QTL for field fitness that affected plant reproductive output in only one of the parental habitats, indicating that the genomic region is under differential selection between the native habitats. Consistent with the QTL results, phenotypic selection of flowering time differed between the two environments, whereas other traits (growth rate and seed weight) were under selection but experienced no habitat-specific differential selection. This implies the flowering-time QTL as a driver of adaptive population divergence. Our results from phenotypic selection and QTL analysis are consistent with local adaptation without genetic trade-offs in performance across environments, i.e. without alleles or traits having opposing fitness effects in contrasting environments.

The effects of parental presence upon the facial expression of pain: the moderating role of child pain catastrophizing.

This experiment investigated the effects of child catastrophic thinking and parental presence on the facial expressions of children when experiencing pain. School children experienced pressure pain in either one of two conditions: (1) when observed by a parent (n=53 children and their parent), or (2) when observed by an adult stranger (n=31 children). Analyses revealed that children showed more facial pain expression in the presence of their parent than in the presence of the stranger. This effect was, however, only found for children with infrequent catastrophic thoughts about pain. Children who have frequent catastrophic thoughts expressed high pain regardless of who they believed was observing them. Results are discussed in terms of the social consequences of pain catastrophizing, and the variables contributing to the expression or suppression of pain display in children and its impact upon others.

Using mating designs to uncover QTL and the genetic architecture of complex traits.

Analysis of quantitative trait loci (QTL) affecting complex traits is often pursued in single-cross experiments. For most purposes, including breeding, some assessment is desired of the generalizability of the QTL findings and of the overall genetic architecture of the trait. Single-cross experiments provide a poor basis for these purposes, as comparison across experiments is hampered by segregation of different allelic combinations among different parents and by context-dependent effects of QTL. To overcome this problem, we combined the benefits of QTL analysis (to identify genomic regions affecting trait variation) and classic diallel analysis (to obtain insight into the general inheritance of the trait) by analyzing multiple mapping families that are connected via shared parents. We first provide a theoretical derivation of main (general combining ability (GCA)) and interaction (specific combining ability (SCA)) effects on F(2) family means relative to variance components in a randomly mating reference population. Then, using computer simulations to generate F(2) families derived from 10 inbred parents in different partial-diallel designs, we show that QTL can be detected and that the residual among-family variance can be analyzed. Standard diallel analysis methods are applied in order to reveal the presence and mode of action (in terms of GCA and SCA) of undetected polygenes. Given a fixed experiment size (total number of individuals), we demonstrate that QTL detection and estimation of the genetic architecture of polygenic effects are competing goals, which should be explicitly accounted for in the experimental design. Our approach provides a general strategy for exploring the genetic architecture, as well as the QTL underlying variation in quantitative traits.

Bell's palsy with ipsilateral numbness.

Bell's palsy is an idiopathic facial palsy of the peripheral type. A herpes virus is the most likely mechanism. We report a patient with the often encountered combination of a facial palsy with ipsilateral sensory changes. Magnetic resonance imaging showed had contrast enhancement in the greater petrosal nerve. Viral spread through anatomical connections could be an explanation for the association of facial palsy with numbness.

Phenotypic selection and regulation of reproduction in different environments in wild barley.

Plasticity of the phenotypic architecture of wild barley, Hordeum spontaneum, was studied in response to water and nutrient stress. Direct and indirect selection on several vegetative and reproductive traits was estimated and path analysis used to reveal how regulating pathways via maternal investment differed between environments. Vegetative traits displayed differential regulating effect on fitness across experimental environments: (1) increase in size was selected for under optimal conditions and under water stress, but not under nutrient stress; (2) allocation to root biomass was selected for under optimal conditions, but it had no effect under nutrient stress and was strongly selected against when water was limiting; (3) delayed onset of reproduction was selected under nutrient limitation whereas earlier onset was selected under water stress. The regulating effect of reproductive traits on final reproductive output also differed across treatments, operating either at the 'early' stage of plant development through varying the number of initiated spikelets per spike (no stress and water stress treatment) or at the 'late' developmental stage adjusting the fertile spikelet weight (no stress and nutrient stress treatment). Reproductive output was regulated via seed abortion under no stress and water stress treatments. Although the underlying mechanism of the regulation through abortion has yet to be discovered, the specific mechanism of abortion under water stress appears to be different from that under optimal conditions. Our results demonstrate that not only is the character architecture in wild barley plastic and sensitive to changing availability of water and nutrients, but the regulating mechanism of maternal investment is also environmentally sensitive.

SPTLC1 mutation in twin sisters with hereditary sensory neuropathy type I.

Hereditary sensory neuropathy type I (HSN I) is an autosomal dominant ulceromutilating disorder of the peripheral nervous system characterized by progressive sensory loss. HSN I locus maps to chromosome 9q22.1-22.3 and is caused by mutations in the gene coding for serine palmitoyltransferase long-chain base subunit 1 (SPTLC1). A novel missense mutation in exon 13 of the SPTLC1 gene (c.1160G-->C; p.G387A) in twin sisters with a severe HSN I phenotype is reported.

Differential selection of growth rate-related traits in wild barley, Hordeum spontaneum, in contrasting greenhouse nutrient environments.

Across-species comparisons show that inherent variation in relative growth rate (RGR) and its underlying traits are correlated with habitat productivity. In this study, we test the hypothesis that growth rate-related traits confer differential selective effects in contrasting nutrient environments. We specifically test whether high RGR is targeted by selection in nutrient-rich environments whereas low values of traits that underlie RGR [specific leaf area (SLA), leaf mass fraction and leaf area ratio (LAR)] confer a direct fitness advantage in nutrient-poor environments, resulting in selection of low RGR as a correlated response. We measured RGR, its underlying component traits, and estimated fitness in a range of wild barley (Hordeum spontaneum) accessions grown under high and low nutrient conditions. Selection on component traits differed between the two environments, while total selection of RGR was not significant. Using multiple regression and path analysis to estimate direct fitness effects, a selective advantage of high LAR and SLA was demonstrated only under nutrient-rich conditions. While supporting the view that observed associations between habitat richness and some RGR-component traits reflect adaptation to differing nutrient regimes, our data suggest that direct selection targets component traits rather than RGR itself.

Localization of the gene for the intermediate form of Charcot-Marie-Tooth to chromosome 10q24.1-q25.1.

Intermediate Charcot-Marie-Tooth neuropathy (CMT) is an inherited sensory motor neuropathy characterized by motor median nerve conduction velocities of 25-45 m/s. We performed a genomewide search in an Italian family with autosomal dominant intermediate CMT and mapped the locus on chromosome 10q. Analysis of key recombinants maps the gene for autosomal dominant intermediate CMT to a 10.7-Mb interval on chromosome 10q24.1-q25.1, between simple tandem repeat markers D10S1709 and D10S1795.

Mutations in the transcriptional activator EYA4 cause late-onset deafness at the DFNA10 locus.

We identified Eyes absent 4 (EYA4), a member of the vertebrate Eya family of transcriptional activators, as the causative gene of postlingual, progressive, autosomal dominant hearing loss at the DFNA10 locus. In two unrelated families from Belgium and the USA segregating for deafness at this locus, we found different mutations in EYA4, both of which create premature stop codons. Although EYA proteins interact with members of the SIX and DACH protein families in a conserved network that regulates early embryonic development, this finding shows that EYA4 is also important post-developmentally for continued function of the mature organ of Corti.

Phenotype-genotype correlations in a CMT2B family with refined 3q13-q22 locus.

OBJECTIVE: To perform genotype-phenotype correlation and genetic linkage analysis in a family with axonal Charcot-Marie-Tooth (CMT) syndrome and ulcero-mutilating features. BACKGROUND: CMT2B is a rare disorder belonging to the group of axonal CMT syndromes that is clinically characterized by marked distal muscle weakness and wasting as well as a high frequency of foot ulcers, infections, and amputations. So far only two families with this disorder have been described in which molecular genetic studies have shown evidence of autosomal dominant inheritance with linkage to chromosome 3q13-q22. METHODS: The authors report a large Austrian family presenting with the typical clinical features of CMT2B. Detailed clinical and electrophysiologic data were obtained in 15 at-risk individuals and DNA samples from 19 family members were collected for genetic linkage studies. RESULTS: Eight family members were definitely affected upon clinical and electrophysiologic examination and the majority revealed pronounced distal muscle wasting and weakness as well as prominent sensory abnormalities, which were frequently complicated by infections and amputations. Electrophysiologic studies showed normal or slightly to moderately slowed motor nerve conduction velocities, markedly reduced compound motor action potential amplitudes with chronodispersion, and absent or reduced amplitudes of sensory nerve action potentials. The molecular genetic study demonstrates linkage to chromosome 3q13-q22. Haplotype analysis in affected individuals indicates that the CMT2B locus is located between the flanking markers D3S1589 and D3S1549, representing a region of 10 cM. CONCLUSIONS: This family is the third CMT2B family reported so far and confirms the existence of the CMT2B locus on chromosome 3q13-q22, which is responsible for a clinically and electrophysiologically homogeneous disorder with prominent distal muscle weakness and wasting, and ulcero-mutilating features. Marked sensory disturbances and the high frequency of foot ulcers, infections, and amputations in our patients seem to be typical for CMT2B. Recombination events in affected individuals reduce the CMT2B candidate gene interval considerably from 25 to 10 cM.

Audiometric analysis of a Belgian family linked to the DFNA10 locus.

OBJECTIVE: To report the otologic and audiometric characteristics of a nonsyndromic postlingual sensorineural hearing impairment in a Belgian family linked to DFNA10. STUDY DESIGN: Retrospective study of the otologic and audiometric data of 17 genetically affected persons. SETTING: Tertiary referral center. PATIENTS: All members of a Belgian kindred who carried the haplotype linked to the inherited hearing impairment of DFNA10. INTERVENTIONS: Diagnostic otologic and audiometric analysis. MAIN OUTCOME MEASURES: Pure-tone audiometry. RESULTS: To find the frequencies that were most affected by the genetic defect, the excess hearing loss of the 17 patients was calculated per frequency in comparison with the respective p50 and p95 thresholds of the normal population. CONCLUSIONS: The genetically affected persons of a Belgian family shared a progressive symmetric sensorineural hearing loss that started in the first to fourth decade. Thirty-five percent of the affected family members had tinnitus, and only one patient had very mild vestibular complaints. At onset, hearing losses were mainly situated at the midfrequencies. With increasing age, all frequencies became affected. The hearing loss was initially mild, with a spontaneous evolution to a moderate or severe hearing impairment. The progression of the hearing loss for the pure-tone average (between 0.5 and 4 kHz) was 1.08 dB/year for this family, compared with 0.50 dB/year and 0.35 dB/year at the 95th and 50th percentiles of the normal population, respectively.

Refined localization and two additional linked families for the DFNA10 locus for nonsyndromic hearing impairment.

DFNA10 originally was mapped to the long arm of chromosome 6 in a large American family segregating for autosomal dominant progressive nonsyndromic hearing impairment. By extending this American family, we have reduced the original DFNA10 candidate region from 13 cM to 3.7 cM. We also report a Belgian family with autosomal dominant nonsyndromic hearing impairment linked to DFNA10 and a Norwegian family with the same condition in which linkage is suggestive, although maximum lod scores are only 2.5. The hearing phenotype in all three DFNA10 families is similar, with losses beginning in the middle frequencies and involving the low and high frequencies later in life.