The frequency and clinical significance of centromere enumeration probe 17 alterations in human epidermal growth factor receptor 2 immunohistochemistry-equivocal invasive breast cancer.

BACKGROUND AND AIMS: Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score. METHODS AND RESULTS: A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5-< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007). CONCLUSION: The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT.

Retrospective observational study of HER2 immunohistochemistry in borderline breast cancer patients undergoing neoadjuvant therapy, with an emphasis on Group 2 (HER2/CEP17 ratio ≥2.0, HER2 copy number <4.0 signals/cell) cases.

BACKGROUND: The ASCO/CAP guidance on HER2 testing in breast cancer (BC) has recently changed. Group 2 tumours with immunohistochemistry score 2+ and HER2/CEP17 ratio ≥2.0 and HER2 copy number <4.0 signals/cell were re-classified as HER2 negative. This study aims to examine the response of Group 2 tumours to neoadjuvant chemotherapy (NACT). METHODS: 749 BC cases were identified from 11 institutions. The association between HER2 groups and pathological complete response (pCR) was assessed. RESULTS: 54% of immunohistochemistry HER2 positive (score 3+) BCs showed pCR, compared to 19% of immunohistochemistry 2+ FISH amplified cases. 27% of Group 2 treated with HER2 targeted therapy achieved pCR, compared to 19 and 11% in the combined Groups 1 + 3 and Groups 4 + 5, respectively. No difference in pCR rates was identified between Group 2 and Group 1 or combined Groups 1 + 3. However, Group 2 response rate was higher than Groups 4 + 5 (p = 0.017). CONCLUSION: No difference in pCR was detected in tumours with a HER2/CEP17 ratio ≥2.0 and a HER2 score 2+ by IHC when stratified by HER2 gene copy number. Our data suggest that ASCO/CAP HER2 Group 2 carcinomas should be evaluated further with respect to eligibility for HER2 targeted therapy.

Transcriptome profiles of stem-like cells from primary breast cancers allow identification of ITGA7 as a predictive marker of chemotherapy response.

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

Predictors of pathological complete response to neoadjuvant treatment and changes to post-neoadjuvant HER2 status in HER2-positive invasive breast cancer.

The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.

Digital pathology for primary diagnosis of screen-detected breast lesions - experimental data, validation and experience from four centres.

AIM: The rate of deployment of digital pathology (DP) systems for primary diagnosis in the UK is accelerating. The flexibility and resilience of digital versus standard glass slides could be of great benefit in the NHS breast screening programme (NHSBSP). This study aims to document the safety and benefits of DP for preoperative tissue diagnosis of screen-detected breast lesions. METHODS AND RESULTS: Concordance data for glass and digital slides of the same cases from four sites were subjected to detailed concordance-discordance analysis. A literature review of DP in the primary diagnosis of breast lesions is presented, making this the most comprehensive synthesis of digital breast cancer histopathological diagnostic data to date. Detailed concordance analysis of experimental data from two histopathology departments reveals clinical concordance rates for breast biopsies of 96% (216 of 225) and 99.6% (249 of 250). Data from direct comparison validation studies in two histopathology departments, utilising the protocol recommended by the Royal College of Pathologists, found concordance rates for breast histology cases of 99.4% (180 of 181) and 99.0% (887 of 896). An intraobserver variation study for glass versus digital slides for difficult cases from the NHSBSP yielded a kappa statistic of 0.80, indicating excellent agreement. Discordances encountered in the studies most frequently concerned discrepancies in grading attributable to mitotic count-scoring and identification of weddelite. CONCLUSIONS: The experience of four histopathology laboratories and our review of pre-existing literature suggests that DP is safe for the primary diagnosis of NHSBSP breast histology specimens, and does not increase the risk of misclassification.

Digital pathology for the primary diagnosis of breast histopathological specimens: an innovative validation and concordance study on digital pathology validation and training.

AIM: To train and individually validate a group of breast pathologists in specialty-specific digital primary diagnosis by using a novel protocol endorsed by the Royal College of Pathologists' new guideline for digital pathology. The protocol allows early exposure to live digital reporting, in a risk-mitigated environment, and focuses on patient safety and professional development. METHODS AND RESULTS: Three specialty breast pathologists completed training in the use of a digital microscopy system, and were exposed to a training set of 20 challenging cases, designed to help them identify personal digital diagnostic pitfalls. Following this, the three pathologists viewed a total of 694 live, entire breast cases. All primary diagnoses were made on digital slides, with immediate glass slide review and reconciliation before final case sign-out. There was complete clinical concordance between the glass and digital impression of the case in 98.8% of cases. Only 1.2% of cases had a clinically significant difference in diagnosis/prognosis on glass and digital slide reads. All pathologists elected to continue using the digital microscope as the standard for breast histopathology specimens, with deferral to glass for a limited number of clinical/histological scenarios as a safety net. CONCLUSION: Individual training and validation for digital primary diagnosis allows pathologists to develop competence and confidence in their digital diagnostic skills, and aids safe and responsible transition from the light microscope to the digital microscope.

Effect of exercise on passive myocardial stiffness in mice with diastolic dysfunction.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome, characterized by increased diastolic stiffness and a preserved ejection fraction, with no effective treatment options. Here we studied the therapeutic potential of exercise for improving diastolic function in a mouse model with HFpEF-like symptoms, the TtnΔIAjxn mouse model. TtnΔIAjxn mice have increased diastolic stiffness and reduced exercise tolerance, mimicking aspects of HFpEF observed in patients. We investigated the effect of free-wheel running exercise on diastolic function. Mechanical studies on cardiac muscle strips from the LV free wall revealed that both TtnΔIAjxn and wildtype (WT) exercised mice had a reduction in passive stiffness, relative to sedentary controls. In both genotypes, this reduction is due to an increase in the compliance of titin whereas ECM-based stiffness was unaffected. Phosphorylation of titin's PEVK and N2B spring elements were assayed with phospho-site specific antibodies. Exercised mice had decreased PEVK phosphorylation and increased N2B phosphorylation both of which are predicted to contribute to the increased compliance of titin. Since exercise lowers the heart rate we examined whether reduction in heart rate per se can improve passive stiffness by administering the heart-rate-lowering drug ivabradine. Ivabradine lowered heart rate in our study but it did not affect passive tension, in neither WT nor TtnΔIAjxn mice. We conclude that exercise is beneficial for decreasing passive stiffness and that it involves beneficial alterations in titin phosphorylation.

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction. METHODS: Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene), with dimethyl sulfoxide (DMSO)-injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels. RESULTS: cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. CONCLUSIONS: Inhibition of the RNA binding motif-20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.

Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy.

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Deleting titin's I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function.

Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the thick filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (∼ 40-fold higher) but low, relative to that of the myosin-based thick filament (∼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for thick filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.

Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin.

BACKGROUND: The purpose of this study was to determine whether patients with heart failure and a preserved ejection fraction (HFpEF) have an increase in passive myocardial stiffness and the extent to which discovered changes depend on changes in extracellular matrix fibrillar collagen and cardiomyocyte titin. METHODS AND RESULTS: Seventy patients undergoing coronary artery bypass grafting underwent an echocardiogram, plasma biomarker determination, and intraoperative left ventricular epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no hypertension or diabetes mellitus), hypertension (HTN) without (-) HFpEF (n=31), and HTN with (+) HFpEF (n=22). One or more of the following studies were performed on the biopsies: passive stiffness measurements to determine total, collagen-dependent and titin-dependent stiffness (differential extraction assay), collagen assays (biochemistry or histology), or titin isoform and phosphorylation assays. In comparison with controls, patients with HTN(-)HFpEF had no change in left ventricular end-diastolic pressure, myocardial passive stiffness, collagen, or titin phosphorylation but had an increase in biomarkers of inflammation (C-reactive protein, soluble ST2, tissue inhibitor of metalloproteinase 1). In comparison with both control and HTN(-)HFpEF, patients with HTN(+)HFpEF had increased left ventricular end-diastolic pressure, left atrial volume, N-terminal propeptide of brain natriuretic peptide, total, collagen-dependent, and titin-dependent stiffness, insoluble collagen, increased titin phosphorylation on PEVK S11878(S26), reduced phosphorylation on N2B S4185(S469), and increased biomarkers of inflammation. CONCLUSIONS: Hypertension in the absence of HFpEF did not alter passive myocardial stiffness. Patients with HTN(+)HFpEF had a significant increase in passive myocardial stiffness; collagen-dependent and titin-dependent stiffness were increased. These data suggest that the development of HFpEF depends on changes in both collagen and titin homeostasis.

Expression of phosphorylated eIF4E-binding protein 1, but not of eIF4E itself, predicts survival in male breast cancer.

BACKGROUND: Male breast cancer is rare and treatment is based on data from females. High expression/activity of eukaryotic initiation factor 4E (eIF4E) denotes a poor prognosis in female breast cancer, and the eIF4E pathway has been targeted therapeutically. Eukaryotic initiation factor 4E activity in female breast cancer is deregulated by eIF4E overexpression and by phosphorylation of its binding protein, 4E-BP1, which relieves inhibitory association between eIF4E and 4E-BP1. The relevance of the eIF4E pathway in male breast cancer is unknown. METHODS: We have assessed expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 (p4E-BP1) using immunohistochemistry in a large cohort of male breast cancers (n=337) and have examined correlations with prognostic factors and survival. RESULTS: Neither eIF4E expression nor estimated eIF4E activity were associated with prognosis. However, a highly significant correlation was found between p4E-BP1 expression and disease-free survival (DFS), linking any detectable p4E-BP1 with poor survival (univariate log rank P=0.001; multivariate HR 8.8, P=0.0001). CONCLUSIONS: Our data provide no support for direct therapeutic targeting of eIF4E in male breast cancer, unlike in females. However, as p4E-BP1 gives powerful prognostic insights that are unrelated to eIF4E function, p4E-BP1 may identify male breast cancers potentially suitable for therapies directed at the upstream kinase, mTOR.

Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force.

Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKIIδ and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (∼30%) passive force reduction caused by CaMKIIδ and a much smaller (∼6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKIIδ can alter the passive force of myocytes in the heart (although CaMKIIδ appears to be more potent) during physiological and pathophysiological states.

Adding value to rare tissue samples donated to biobanks: characterisation of breast tissue and primary cell cultures obtained from a female-to-male transgender patient.

Biobanks provide a window of opportunity to store and add value to material from rare cases allowing their future use in biomedical research. One such example is the opportunityto obtain good quality tissue from patients undergoing gender re-assignment. Following patient agreement to donate tissue samples to our biobank we catalogued the histological appearance, defined the expression of the hormone receptors ERα, PR, AR and the proliferation marker Ki67, and generated and characterised primary cell cultures in a female to male (FTM) transgender patient referred to our unit for surgery. Immunohistochemistry was performed for ERα, PR and AR and the proliferation marker Ki67. Hormone receptor expression was confined to epithelial cells lining the breast ducts. Ki67 immunoreactivity was sparse indicating little proliferation of luminal epithelium, consistent with normal mammary gland. Cultures of epithelial cells and fibroblasts were derived from surplus tissue. The latter lacked expression of epithelial markers and hormone receptors but exhibited expression of vimentin. Culture of the former on Matrigel saw an outgrowth of more rounded "epithelial-like" cells. Immunofluoresence characterisation showed a mixed phenotype with expression of vimentin and both myoepithelial and luminal epithelial markers. Sporadic weak ERα expression and moderate PR expression was seen. In summary, as well as routinely collecting tissue and blood samples, we have characterised and stored tissue and cells from a FTM transgender patient, adding value to this resource which,available from the Breast Cancer Campaign Tissue Bank for those interested in further studying the biology of FTM transgender tissue.

PSMD9 expression predicts radiotherapy response in breast cancer.

BACKGROUND: More than 50% of cancer patients are recommended to receive radiotherapy. Recommendations are based mainly on clinical and pathological factors and not intrinsic tumour radio-sensitivity. Use of radiotherapy according to predictive markers would potentially reduce costly over-treatment, and improve the treatment risk-benefit ratio and cancer outcomes. Tumour expression of the 26S proteasome has been reported to predict radiotherapy response: low expression was associated with higher rates of local recurrence after radiotherapy, suggesting that low proteasome expression and activity was associated with radio-resistance. However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers. Our aim was to further analyse the relevance of 26S proteasome expression, focussing specifically on the PSMD9 subunit, in the largest clinical cohort to date, and to investigate the functional role of PSMD9 in radio-sensitivity in breast cancer cell lines. METHODS: We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences. The value of expression as a prognostic or radiotherapy predictive marker was tested using Kaplan-Meier and Cox regression analyses. PSMD9 function was examined in breast cancer cell lines MCF7 and MDA-MB-231 using siRNA knock-downs and colony forming assays after irradiation. RESULTS: Low tumour PSMD9 expression was significantly associated with a reduced incidence of local recurrence in patients receiving adjuvant radiotherapy (univariate log rank p = 0.02; multivariate regression p = 0.009), but not in those treated without radiotherapy, suggesting that low PSMD9 expression was associated with relative tumour radio-sensitivity. In support of this, reduction of PSMD9 expression using siRNA in breast cancer cell lines in vitro sensitized cells to radiotherapy. CONCLUSIONS: We conclude that PSMD9 expression may predict radiotherapy benefit, with low expression indicative of relative radio-sensitivity, the opposite of previous reports relating to 26S proteasome expression. Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

The sodium channel ß1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis.

Voltage-gated Na(+) channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller ß subunits. The ß subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). ß1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. ß1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/ß1 protein were up-regulated in BCa specimens, compared with normal breast tissue. ß1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. ß1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and ß1 over-expressing tumour cells had an elongate morphology. In vitro, ß1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. ß1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na(+) current, thus replicating the mechanism by which ß1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, ß1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for ß1 in tumour growth and metastasis in vivo. We propose that ß1 warrants further study as a potential biomarker and targeting ß1-mediated adhesion interactions may have value as a novel anti-cancer therapy.

Improving ostomates' confidence with a discharge-solidifying agent.

Not every person with a stoma will develop problems or even feel that he or she is unable to manage a stoma independently. However, for the individual who does experience problems and struggles to cope, the stoma nurse is pivotal in providing resources and product experience. He or she can enable the ostomate to adapt to the stoma and maintain their quality of life. This article provides an overview of a solidifying agent and case studies illustrating how it can be used to help patients achieve independence.

Managing high-output stomas: module 3 of 3.

Enterocutaneous fistulae (ECF) and high-output stomas are challenging to manage, owing to the large volume of loss that may result in severe dehydration, electrolyte imbalances, malnutrition and sepsis. It is imperative that this group of patients receive adequate nutrition, as malnutrition and sepsis are the leading cause of death. Treatment is complex and based on various situations, treatment can be medical/conservative management or surgical. Depending on the site of the fistula and the length of residual intestine contributing as the cause of a high output stoma with the nutritional status of the patient, clinicians have to decide whether parenteral nutrition (PN) or enteral nutrition (EN) should be established. As previously discussed in module 1 and 2 (Gabe, 2013; Gabe and Slater, 2013) the theme of nutritional management and appliance/accessory selection to manage patients with ECF and high output stomas was outlined. The aim of providing an understanding of the nutritional needs and the practicalities of maintaining appliance adherence, and in turn, a reduction in the breakdown of the peri-stomal skin was described. Module 3 aims to provide understanding for the reader that may encounter patients undergoing surgery for the management of their ECF or high-output stoma. Lastly it was felt necessary to discuss the subject of intestinal transplantation. This complex surgical option is not available to all patients with intestinal failure and only undertaken at a couple of recognised centres. The process of referring patients that are deemed suitable for intestinal transplantation will be addressed and what the surgery entails with long-term outcomes and the quality of life for the patient.

Concordance of HER2-low scoring in breast carcinoma among expert pathologists in the United Kingdom and the republic of Ireland -on behalf of the UK national coordinating committee for breast pathology.

BACKGROUND: Recent clinical evidence showed that breast cancer with low HER2 expression levels responded to trastuzumab deruxtecan therapy. The HER2-low cancers comprise immunohistochemistry (IHC) score 1+ and 2+ ISH non-amplified tumours, currently classified as HER2 negative. Little data exists on the reproducibility of pathologists reporting of HER2-low cancer. PATIENT AND METHODS: Sixteen expert pathologists of the UK National Coordinating Committee for Breast Pathology scored 50 digitally scanned HER2 IHC slides. The overall level of agreement, Fleiss multiple-rater kappa statistics and Cohen's Kappa were calculated. Cases with low concordance were re-scored by the same pathologists after a washout period. RESULTS: Absolute agreement was achieved in 6% of cases, all of which scored 3+. Poor agreement was found in 5/50 (10%) of cases. This was due to heterogeneous HER2 expression, cytoplasmic staining and low expression spanning the 10% cut-off value. Highest concordance (86%) was achieved when scores were clustered as 0 versus others. Improvement in kappa of overall agreement was achieved when scores 1+ and 2+ were combined. Inter-observer agreement was moderate to substantial in the whole cohort but fair to moderate in the HER2-low group. Similarly, consensus-observer agreement was substantial to almost perfect in the whole cohort and moderate to substantial in the HER2-low group. CONCLUSION: HER2-low breast cancer suffers from lower concordance among expert pathologists. While most cases can reproducibly be classified, a small proportion (10%) remained challenging. Refining the criteria for reporting and consensus scoring will help select appropriate patients for targeted therapy.

Abundant non-canonical dUTP found in primary human macrophages drives its frequent incorporation by HIV-1 reverse transcriptase.

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nm), compared with activated CD4(+) T cells (2-5 µm). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 µm) is ∼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2',3'-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.