Altered upper limb kinematics in individuals with dyskinetic cerebral palsy in comparison with typically developing peers - A statistical parametric mapping study.

BACKGROUND: Dyskinetic cerebral palsy (DCP) is clinically characterized by involuntary movements and abnormal postures, which can aggravate with activity. While upper limb movement variability is often detected in the clinical picture, it remains unknown how movement patterns of individuals with DCP differ from typically developing (TD) peers. RESEARCH QUESTION: Do individuals with DCP show i) higher time-dependent standard deviations of upper limb joint angles and ii) altered upper limb kinematics in time and/or amplitude during functional upper limb tasks in comparison with TD individuals? METHODS: Three-dimensional upper limb movement patterns were cross-sectionally compared in 50 individuals with and without DCP during three functional tasks: reach forward (RF), reach and grasp vertical (RGV) and reach sideways (RS). Mean and point-wise standard deviations of angular waveform of the upper limb joint angles were compared between groups to evaluate differences in time and/or amplitude using traditional and non-linear registration statistical parametric mapping. RESULTS: Thirty-five extremities from 30 individuals (mean age 17y4m, range 5-25 y; MACS level I(n = 2); II(n = 15); III(n = 16); IV(n = 2)) with DCP and twenty TD individuals (mean age 16y8m, range 8-25 y) were evaluated. The DCP compared to TD group showed higher point-wise standard deviations at the level of all joints, which was time-dependent and varied between tasks. Mean wrist and elbow flexion was higher for the DCP group during RF (0-83 % wrist; 57-100 % elbow), RGV (0-82 % wrist; 12-100 % elbow) and RS (0-43 % wrist; 70-100 % elbow). SIGNIFICANCE: This is the first study exploring the movement patterns of individuals with DCP during reaching using quantitative measures. Analyzing these individual movement patterns by statistical parametric mapping (SPM) allows us to focus on both specific joint or on specific timing during the movement cycle. The individual information that this method yields can guide individual therapy aiming to improve reaching function in different parts of the movement cycle or evaluate intervention effects on upper extremity treatment.

The Effect of Bimanual Intensive Functional Training on Somatosensory Hand Function in Children with Unilateral Spastic Cerebral Palsy: An Observational Study.

(1) Background: Next to motor impairments, children with unilateral spastic cerebral palsy (CP) often experience sensory impairments. Intensive bimanual training is well known for improving motor abilities, though its effect on sensory impairments is less known. (2) Objective: To investigate whether bimanual intensive functional therapy without using enriched sensory materials improves somatosensory hand function. (3) Methods: A total of twenty-four participants with CP (12-17 years of age) received 80-90 h of intensive functional training aimed at improving bimanual performance in daily life. Somatosensory hand function was measured before training, directly after training, and at six months follow-up. Outcome measures were: proprioception, measured by thumb and wrist position tasks and thumb localization tasks; vibration sensation; tactile perception; and stereognosis. (4) Results: Next to improving on their individual treatment goals, after training, participants also showed significant improvements in the perception of thumb and wrist position, vibration sensation, tactile perception, and stereognosis of the more affected hand. Improvements were retained at six months follow-up. Conversely, proprioception measured by the thumb localization tasks did not improve after training. (5) Conclusions: Intensive functional bimanual training without environmental tactile enrichment may improve the somatosensory function of the more affected hand in children with unilateral spastic CP.

Convergent validity of functional communication tools and spoken language comprehension assessment in children with cerebral palsy.

BACKGROUND: The majority of children with cerebral palsy (CP) experience challenges in functional communication from a young age. A pivotal aspect of functional communication is language comprehension. A variety of classification systems and questionnaires are available to classify and describe functional communication skills in children with CP. A better understanding of the convergent validity of (subsections of) these tools, as well as their relationship with spoken language comprehension, will be valuable in both clinical practice and research. AIMS: To investigate the convergent validity of (subsections of) functional communication tools and the relationship with spoken language comprehension in children with CP. METHODS & PROCEDURES: Cross-sectional data on 138 children were subdivided into three developmental stages based on (Dutch) educational phases: ages 18 months-3;11y (n = 59), 4;0-5;11 years (n = 37) and 6;0-8;11 years (n = 42). The following functional communication tools were used to classify and describe functional communication: Communication Function Classification System (CFCS), subscales of the Caregivers Priorities and Child Health Index of Life with Disabilities-Dutch Version (CPCHILD-DV) and the Focus on Communication Under Six-34 (FOCUS-34) questionnaire. Spoken language comprehension was assessed with the Computer-Based instrument for Low motor Language Testing (C-BiLLT). Correlations between the functional communication tools, and with the C-BiLLT, were calculated using Pearson's and Spearman's correlation coefficients. It was hypothesized a priori that correlations of at least 0.60 suggest good convergent validity. OUTCOMES & RESULTS: At all developmental stages, a significant ordered decreasing tendency of communication outcomes was found across CFCS levels; lower CFCS levels were associated with lower scores on the CPCHILD-DV and FOCUS-34, and with a lower level of spoken language comprehension (C-BiLLT). Correlation coefficients of the functional communication tools exceeded 0.60 at all developmental stages. Correlations between C-BiLLT raw scores and the functional communication tools varied between 0.351 and 0.591 at developmental stage 18 months-3;11 years, between 0.781 and 0.897 at developmental stage 4;0-5;11 years, and between 0.635 and 0.659 at developmental stage 6;0-8;11 years. CONCLUSIONS & IMPLICATIONS: The functional communication tools assessed in this study showed convergent validity at all developmental stages. The CFCS, currently most widely used in paediatric rehabilitation, is adequate in the classification of functional communication. However, for more detailed clinical goal setting and evaluation of change in functional communication, the additional use of FOCUS-34 or CPCHILD-DV is recommended. WHAT THIS PAPER ADDS: What is already known on the subject A range of functional communication tools are available that help describe and classify functional communication in children with CP. These include the CFCS, subsections of CPCHILD-DV and FOCUS-34. The CFCS classifies functional communication in daily life with familiar and unfamiliar partners. Specific subsections of the CPCHILD-DV and FOCUS-34 include items that pertain to communicative participation. The innovative C-BiLLT provides a standardized method to assess spoken language comprehension in children with CP and significant motor impairments. What this paper adds to existing knowledge In the present study, convergent validity was confirmed between CFCS and specific subsections of the CPCHILD-DV and FOCUS-34. Correlations between these functional communication tools and the C-BiLLT were moderate to strong. What are the potential or actual clinical implications of this work? For clinical and research purposes (for instance, accurate prescription of augmentative and alternative communication-AAC), healthcare and educational professionals together with parents need to know how functional communication tools converge and how functional communication levels relate to the comprehension of spoken language. The CFCS provides a valid classification of functional communication abilities in children with CP. However, to measure change in functional communication and to evaluate treatment outcomes, use of additional functional communication tools such as the CPCHILD-DV and FOCUS-34 is recommended. When discrepancies are found between communicative abilities and spoken language comprehension, it is strongly recommended that valid tools are used in a more detailed examination of the child's spoken language comprehension skills and functional communication.

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Preterm Brain Injury, Antenatal Triggers, and Therapeutics: Timing Is Key.

With a worldwide incidence of 15 million cases, preterm birth is a major contributor to neonatal mortality and morbidity, and concomitant social and economic burden Preterm infants are predisposed to life-long neurological disorders due to the immaturity of the brain. The risks are inversely proportional to maturity at birth. In the majority of extremely preterm infants (<28 weeks' gestation), perinatal brain injury is associated with exposure to multiple inflammatory perinatal triggers that include antenatal infection (i.e., chorioamnionitis), hypoxia-ischemia, and various postnatal injurious triggers (i.e., oxidative stress, sepsis, mechanical ventilation, hemodynamic instability). These perinatal insults cause a self-perpetuating cascade of peripheral and cerebral inflammation that plays a critical role in the etiology of diffuse white and grey matter injuries that underlies a spectrum of connectivity deficits in survivors from extremely preterm birth. This review focuses on chorioamnionitis and hypoxia-ischemia, which are two important antenatal risk factors for preterm brain injury, and highlights the latest insights on its pathophysiology, potential treatment, and future perspectives to narrow the translational gap between preclinical research and clinical applications.

Cytidine Diphosphate-Ribitol Analysis for Diagnostics and Treatment Monitoring of Cytidine Diphosphate-l-Ribitol Pyrophosphorylase A Muscular Dystrophy.

BACKGROUND: Many muscular dystrophies currently remain untreatable. Recently, dietary ribitol has been suggested as a treatment for cytidine diphosphate (CDP)-l-ribitol pyrophosphorylase A (CRPPA, ISPD), fukutin (FKTN), and fukutin-related protein (FKRP) myopathy, by raising CDP-ribitol concentrations. Thus, to facilitate fast diagnosis, treatment development, and treatment monitoring, sensitive detection of CDP-ribitol is required. METHODS: An LC-MS method was optimized for CDP-ribitol in human and mice cells and tissues. RESULTS: CDP-ribitol, the product of CRPPA, was detected in all major human and mouse tissues. Moreover, CDP-ribitol concentrations were reduced in fibroblasts and skeletal muscle biopsies from patients with CRPPA myopathy, showing that CDP-ribitol could serve as a diagnostic marker to identify patients with CRPPA with severe Walker-Warburg syndrome and mild limb-girdle muscular dystrophy (LGMD) phenotypes. A screen for potentially therapeutic monosaccharides revealed that ribose, in addition to ribitol, restored CDP-ribitol concentrations and the associated O-glycosylation defect of α-dystroglycan. As the effect occurred in a mutation-dependent manner, we established a CDP-ribitol blood test to facilitate diagnosis and predict individualized treatment response. Ex vivo incubation of blood cells with ribose or ribitol restored CDP-ribitol concentrations in a patient with CRPPA LGMD. CONCLUSIONS: Sensitive detection of CDP-ribitol with LC-MS allows fast diagnosis of patients with severe and mild CRPPA myopathy. Ribose offers a readily testable dietary therapy for CRPPA myopathy, with possible applicability for patients with FKRP and FKTN myopathy. Evaluation of CDP-ribitol in blood is a promising tool for the evaluation and monitoring of dietary therapies for CRPPA myopathy in a patient-specific manner.

Global transcriptional analysis identifies a novel role for SOX4 in tumor-induced angiogenesis.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.

Prognostic relevance of tumor-infiltrating lymphocytes and immune checkpoints in pediatric medulloblastoma.

Pediatric medulloblastomas are the most frequently diagnosed embryonal tumors of the central nervous system. Current therapies cause severe neurological and cognitive side effects including secondary malignancies. Cellular immunotherapy might be key to improve survival and to avoid morbidity. Efficient killing of tumor cells using immunotherapy requires to overcome cancer-associated strategies to evade cytotoxic immune responses. Here, we examined the immune response and immune evasion strategies in pediatric medulloblastomas. Cytotoxic T-cells, infiltrating medulloblastomas with variable activation status, showed no correlation with overall survival of the patients. We found limited numbers of PD1+ T-cells and complete absence of PD-L1 on medulloblastomas. Medulloblastomas downregulated immune recognition molecules MHC-I and CD1 d. Intriguingly, expression of granzyme inhibitors SERPINB1 and SERPINB4 was acquired in 23% and 50% of the tumors, respectively. Concluding, pediatric medulloblastomas exploit multiple immune evasion strategies to overcome immune surveillance. Absence of PD-L1 expression in medulloblastoma suggest limited or no added value for immunotherapy with PD1/PD-L1 blockers.

Oncogenic role of cytomegalovirus in medulloblastoma?

Medulloblastoma is the most common solid tumor among children. Current therapeutic strategies for this malignancy include surgical resection, radiation therapy and chemotherapy. However, these treatments are accompanied with serious side effects such as neurological complications and psychosocial problems, due to the severity of treatment on the developing nervous system. To solve this problem, novel therapeutic approaches are currently being investigated. One of them is targeting human cytomegalovirus in medulloblastoma cancer cells. However, this approach is still under debate, since the presence of cytomegalovirus in medulloblastomas remains controversial. In this review, we discuss the current controversies on the role of cytomegalovirus in medulloblastoma oncogenesis and the potential of cytomegalovirus as a novel (immuno)therapeutic target.

The Thompson Encephalopathy Score and Short-Term Outcomes in Asphyxiated Newborns Treated With Therapeutic Hypothermia.

BACKGROUND: The Thompson encephalopathy score is a clinical score to assess newborns suffering from perinatal asphyxia. Previous studies revealed a high sensitivity and specificity of the Thompson encephalopathy score for adverse outcomes (death or severe disability). Because the Thompson encephalopathy score was developed before the use of therapeutic hypothermia, its value was reassessed. OBJECTIVE: The purpose of this study was to assess the association of the Thompson encephalopathy score with adverse short-term outcomes, defined as death before discharge, development of severe epilepsy, or the presence of multiple organ failure in asphyxiated newborns undergoing therapeutic hypothermia. METHODS: The study period ranged from November 2010 to October 2014. A total of 12 tertiary neonatal intensive care units participated. Demographic and clinical data were collected from the "PharmaCool" multicenter study, an observational cohort study analyzing pharmacokinetics of medication during therapeutic hypothermia. With multiple logistic regression analyses the association of the Thompson encephalopathy scores with outcomes was studied. RESULTS: Data of 142 newborns were analyzed (male: 86; female: 56). Median Thompson score was 9 (interquartile range: 8 to 12). Median gestational age was 40 weeks (interquartile range 38 to 41), mean birth weight was 3362 grams (standard deviation: 605). All newborns manifested perinatal asphyxia and underwent therapeutic hypothermia. Death before discharge occurred in 23.9% and severe epilepsy in 21.1% of the cases. In total, 59.2% of the patients had multiple organ failure. The Thompson encephalopathy score was not associated with multiple organ failure, but a Thompson encephalopathy score ≥12 was associated with death before discharge (odds ratio: 3.9; confidence interval: 1.3 to 11.2) and with development of severe epilepsy (odds ratio: 8.4; confidence interval: 2.5 to 27.8). CONCLUSION: The Thompson encephalopathy score is a useful clinical tool, even in cooled asphyxiated newborns. A score ≥12 is associated with adverse outcomes (death before discharge and development of severe epilepsy). The Thompson encephalopathy score is not associated with the development of multiple organ failure.

Pediatric Primitive Neuroectodermal Tumors of the Central Nervous System Differentially Express Granzyme Inhibitors.

BACKGROUND: Central nervous system (CNS) primitive neuroectodermal tumors (PNETs) are malignant primary brain tumors that occur in young infants. Using current standard therapy, up to 80% of the children still dies from recurrent disease. Cellular immunotherapy might be key to improve overall survival. To achieve efficient killing of tumor cells, however, immunotherapy has to overcome cancer-associated strategies to evade the cytotoxic immune response. Whether CNS-PNETs can evade the immune response remains unknown. METHODS: We examined by immunohistochemistry the immune response and immune evasion strategies in pediatric CNS-PNETs. RESULTS: Here, we show that CD4+, CD8+, γδ-T-cells, and Tregs can infiltrate pediatric CNS-PNETs, although the activation status of cytotoxic cells is variable. Pediatric CNS-PNETs evade immune recognition by downregulating cell surface MHC-I and CD1d expression. Intriguingly, expression of SERPINB9, SERPINB1, and SERPINB4 is acquired during tumorigenesis in 29%, 29%, and 57% of the tumors, respectively. CONCLUSION: We show for the first time that brain tumors express direct granzyme inhibitors (serpins) as a potential mechanism to overcome cellular cytotoxicity, which may have consequences for cellular immunotherapy.

Oropharyngeal squamous cell carcinomas differentially express granzyme inhibitors.

OBJECTIVES: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors (SERPINB1, SERPINB4, and SERPINB9) between HPV-positive and HPV-negative tumors and the relation with survival. METHODS: Protein expression of tumor-infiltrating lymphocytes (TILs) (CD3, CD4, and CD8) and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67%) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors. RESULTS: HPV-positive OPSCCs were significantly more heavily infiltrated by TILs (p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival. CONCLUSION: HPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors.

Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer.

PURPOSE: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. PROCEDURES: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS). RESULTS: Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR. CONCLUSIONS: These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer.

The potential of hypoxia markers as target for breast molecular imaging--a systematic review and meta-analysis of human marker expression.

BACKGROUND: Molecular imaging of breast cancer is a promising emerging technology, potentially able to improve clinical care. Valid imaging targets for molecular imaging tracer development are membrane-bound hypoxia-related proteins, expressed when tumor growth outpaces neo-angiogenesis. We performed a systematic literature review and meta-analysis of such hypoxia marker expression rates in human breast cancer to evaluate their potential as clinically relevant molecular imaging targets. METHODS: We searched MEDLINE and EMBASE for articles describing membrane-bound proteins that are related to hypoxia inducible factor 1α (HIF-1α), the key regulator of the hypoxia response. We extracted expression rates of carbonic anhydrase-IX (CAIX), glucose transporter-1 (GLUT1), C-X-C chemokine receptor type-4 (CXCR4), or insulin-like growth factor-1 receptor (IGF1R) in human breast disease, evaluated by immunohistochemistry. We pooled study results using random-effects models and applied meta-regression to identify associations with clinicopathological variables. RESULTS: Of 1,705 identified articles, 117 matched our selection criteria, totaling 30,216 immunohistochemistry results. We found substantial between-study variability in expression rates. Invasive cancer showed pooled expression rates of 35% for CAIX (95% confidence interval (CI): 26-46%), 51% for GLUT1 (CI: 40-61%), 46% for CXCR4 (CI: 33-59%), and 46% for IGF1R (CI: 35-70%). Expression rates increased with tumor grade for GLUT1, CAIX, and CXCR4 (all p < 0.001), but decreased for IGF1R (p < 0.001). GLUT1 showed the highest expression rate in grade III cancers with 58% (45-69%). CXCR4 showed the highest expression rate in small T1 tumors with 48% (CI: 28-69%), but associations with size were only significant for CAIX (p < 0.001; positive association) and IGF1R (p = 0.047; negative association). Although based on few studies, CAIX, GLUT1, and CXCR4 showed profound lower expression rates in normal breast tissue and benign breast disease (p < 0.001), and high rates in carcinoma in situ. Invasive lobular carcinoma consistently showed lower expression rates (p < 0.001). CONCLUSIONS: Our results support the potential of hypoxia-related markers as breast cancer molecular imaging targets. Although specificity is promising, combining targets would be necessary for optimal sensitivity. These data could help guide the choice of imaging targets for tracer development depending on the envisioned clinical application.

Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

Nuclear localization of the transcriptional coactivator YAP is associated with invasive lobular breast cancer.

BACKGROUND: Yes Associated Protein (YAP) has been implicated in the control of organ size by regulating cell proliferation and survival. YAP is a transcriptional coactivator that controls cellular responses through interaction with TEAD transcription factors in the nucleus, while its transcriptional functions are inhibited by phosphorylation-dependent translocation to the cytosol. YAP overexpression has been associated with different types of cancer, such as lung, skin, prostate, ovary and liver cancer. Recently, YAP was linked to E-cadherin-dependent regulation of contact inhibition in breast cancer cells. RESULTS: In this study we examined YAP protein expression and cellular localization in 237 cases of human invasive breast cancer by immunohistochemistry and related its expression to clinicopathological features and E-cadherin expression. We observed that invasive lobular carcinoma is characterized by higher expression levels of both nuclear and cytosolic YAP (p < 0.001). Nuclear YAP expression did not associate with other variables such as lymph node involvement, tumor grade, tumor size, mitotic activity or the molecular sub-types of invasive breast cancer. We observed that high nuclear and cytosolic YAP expression are associated with the E-cadherin deficient breast cancer subtype ILC (p < 0.001) and cell lines derived from human breast cancers and conditional mouse models of human lobular breast cancer. CONCLUSIONS: Since our data indicate that nuclear YAP localization is more common in breast cancers lacking functional adherens junctions, it suggests that YAP-mediated transcription may be involved in the development and progression of invasive lobular breast cancer.

Molecular imaging with a fluorescent antibody targeting carbonic anhydrase IX can successfully detect hypoxic ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast is difficult to remove completely during surgery as it is not palpable and can therefore require re-excision. Real-time visualization of DCIS using near-infrared fluorescent probes could help the surgeon during surgery as well as the pathologist post-operatively to distinguish the tumor from healthy tissue. As hypoxia-induced necrosis is a common phenomenon in DCIS, we investigated the molecular imaging of DCIS using a fluorescent antibody targeting a hypoxia marker, carbonic anhydrase IX (CAIX), in a preclinical mouse model. A monoclonal antibody against human CAIX was fluorescently labeled with the near-infrared dye IRDye800CW and characterized in vitro. An in vivo study was performed in SCID/Beige mice that were orthotopically transplanted with human breast cancer cells mimicking human DCIS (MCF10DCIS) and MCF10DCIS stably expressing CAIX. A clinically approved fluorescence imaging system was used to monitor probe uptake and to determine tumor-to-normal tissue ratios (TNR). Mean in vivo TNR of CAIX-transduced (CAIX+) tumors was 7.5 ± 0.5. Mean in vivo TNR of DCIS tumors with hypoxic areas reached a plateau level at 48 h after injection of 2.1 ± 0.1 (mean ± SEM) compared to 1.7 ± 0.1 in DCIS without hypoxic areas. Mean intra-operative TNR of DCIS tumors with necrotic regions was higher than that of DCIS tumors without necrotic regions 96 h after injection-2.9 ± 0.1 and 1.5 ± 0.1, respectively-while the TNR of CAIX+ tumors was 11.2 ± 1.0. Specific tumor uptake of MabCAIX-IRDye800CW was confirmed by a biodistribution assay, and immunofluorescence imaging on tumor sections showed specific uptake in hypoxic tumor regions, with higher contrast than conventional chromagen-based immunohistochemistry. Molecular fluorescence imaging with MabCAIX-IRDye800CW can be successfully used to detect hypoxic DCIS before and during surgery to facilitate radical resection. Furthermore, it allows for sensitive CAIX-specific immunofluorescence microscopy of tumor sections, thereby introducing the concept of molecular fluorescence pathology.

Loss of p120-catenin induces metastatic progression of breast cancer by inducing anoikis resistance and augmenting growth factor receptor signaling.

Metastatic breast cancer remains the chief cause of cancer-related death among women in the Western world. Although loss of cell-cell adhesion is key to breast cancer progression, little is known about the underlying mechanisms that drive tumor invasion and metastasis. Here, we show that somatic loss of p120-catenin (p120) in a conditional mouse model of noninvasive mammary carcinoma results in formation of stromal-dense tumors that resemble human metaplastic breast cancer and metastasize to lungs and lymph nodes. Loss of p120 in anchorage-dependent breast cancer cell lines strongly promoted anoikis resistance through hypersensitization of growth factor receptor (GFR) signaling. Interestingly, p120 deletion also induced secretion of inflammatory cytokines, a feature that likely underlies the formation of the prometastatic microenvironment in p120-negative mammary carcinomas. Our results establish a preclinical platform to develop tailored intervention regimens that target GFR signals to treat p120-negative metastatic breast cancers.

Analysis of expression of membrane-bound tumor markers in ductal carcinoma in situ of the breast: paving the way for molecular imaging.

PURPOSE: Achieving radicality during breast conserving surgery for pure ductal carcinoma in situ (DCIS) of the breast and invasive cancer surrounded by DCIS is challenging. Molecular imaging holds promise here, when applied as a tool for image-guided surgery of DCIS. METHODS: Tissue microarrays containing 24 pure DCIS and 63 DCIS with adjacent invasive breast cancer cases were stained by immunohistochemistry for a panel of membrane-bound targets. RESULTS: GLUT1 expression was present in 60.9%, IGF1-R in 55.2% HER2 in 28.7%, MET in 18.4%, EGFR in 16.1%, CD44v6 in 69%, carbonic anhydrase XII (CAXII) in 24.1% and Mammaglobin in 14.9% of DCIS cases. No expression differences between pure DCIS and DCIS with adjacent cancer were observed. Further, HER2 and EGFR expression were correlated with high grade DCIS (p=0.001) and CAXII with low grade DCIS (p=0.027). A putative panel containing HER2, EGFR, GLUT1 and IGF1-R had a detection rate of 90.2% for DCIS and 78.3% for adjacent breast cancer. CONCLUSIONS: We found that membrane-bound targets are more frequently expressed in DCIS than in invasive breast cancer, but that single membrane proteins are too infrequently expressed to serve as single imaging targets for the detection of DCIS. However, a panel of markers consisting of IGF1-R, CD44v6, GLUT1, EGFR, and HER2 was found to be positive in 96.3% of DICS based on marker expression in the adjacent invasive breast cancer as described earlier. This implies that detection of DCIS based on marker expression in the adjacent invasive breast cancer during breast conserving surgery should be possible with a panel of molecular imaging tracers targeting CD44v6, GLUT1, HER2, IGF1-R, and EGFR.