Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.

Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8(+) T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8(+) T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK's immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8(+) T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

Population-average mediation analysis for zero-inflated count outcomes.

Mediation analysis is an increasingly popular statistical method for explaining causal pathways to inform intervention. While methods have increased, there is still a dearth of robust mediation methods for count outcomes with excess zeroes. Current mediation methods addressing this issue are computationally intensive, biased, or challenging to interpret. To overcome these limitations, we propose a new mediation methodology for zero-inflated count outcomes using the marginalized zero-inflated Poisson (MZIP) model and the counterfactual approach to mediation. This novel work gives population-average mediation effects whose variance can be estimated rapidly via delta method. This methodology is extended to cases with exposure-mediator interactions. We apply this novel methodology to explore if diabetes diagnosis can explain BMI differences in healthcare utilization and test model performance via simulations comparing the proposed MZIP method to existing zero-inflated and Poisson methods. We find that our proposed method minimizes bias and computation time compared to alternative approaches while allowing for straight-forward interpretations.

Using a prognostic medical device for early identification of pressure ulcers: protocol for study design.

BACKGROUND: An objective, physiological measurement taken using a medical device may reduce the incidence of pressure ulcers through earlier detection of problems signs before visual signs appear. Research in this field is hampered by variations in clinical practice and patient-level confounders. AIM: The authors outline key considerations for designing a protocol for a study to assess the efficacy and safety of a prognostic medical device in reducing pressure ulcer incidence in a hospital, including comparators, randomisation, sample size, ethics and practical issues. METHOD: Key issues relating to methodology and ethics are considered alongside a theoretical protocol, which could support future researchers in wound care trials. RESULTS: A prospective, three-armed, multi-centre, stratified cluster-randomised controlled trial is proposed. The third arm is recommended as it is expected that patients will need to be moved for the medical device to be used and repositioning is a preventive strategy. A minimum of 16 200 patients in 33 wards would needed to be recruited to achieve statistical significance. Ethical considerations in terms of consent or assent need to be considered. CONCLUSION: The hypothetical study designed to evaluate the effectiveness of a diagnostic or prognostic medical device in reducing pressure ulcer incidence in secondary care, while accounting for biases, would require large sample sizes and involves risks of inter-operator and inter-device reliability, heterogeneity of users and the vague clinical interpretation of device results. Robust research in this field has the potential to influence or change policy and practice relating to the prevention of pressure ulcers in secondary care.

Infection after ureteroscopy for ureteric stones: analysis of 71 305 cases in the Hospital Episode Statistics database.

OBJECTIVES: To investigate the burden of infectious complications following ureteroscopy (URS) for ureteric stones on a national level in England using data from the Hospital Episodes Statistics (HES) data warehouse. MATERIALS AND METHODS: A retrospective cohort was identified and followed up in HES during the period April 2013 to March 2020 for all procedure codes relating to ureteroscopic stone treatment (M27.1, M27.2, M27.3). Treatment episodes relating to the first URS ('index ureteroscopy') for each patient were further analysed. All subsequent admissions within 30 days were also captured. The primary outcome was diagnosis of urinary tract infection (UTI; including all codes relating to a UTI/sepsis within the first 30 days of index URS). Secondary outcomes were critical care attendance, attendance at the accident and emergency department (A&E) within 30 days, and mortality. RESULTS: A total of 71 305 index ureteroscopies were eligible for analysis. The median age was 55 years, and 81% of procedures were elective and 45% were undertaken as day-cases. At the time of index URS, 16% of patients had diabetes, 0.5% had coexisting neurological disease and 40% had an existing stent/nephrostomy. Overall, 6.8% of the cohort (n = 4822) had a diagnosis of UTI within 30 days of index URS (3.9% immediately after surgery). A total of 339 patients (0.5%) required an unplanned stay in critical care during their index URS admission; 8833 patients (12%) attended A&E within 30 days. Overall mortality was 0.18% (60 in-hospital, 65 within 30 days); 40 deaths (0.056%) included infection as a contributing cause of death. CONCLUSION: We present the largest series evaluating infectious complications after ureteroscopic stone treatment. The procedure is safe, with low inpatient infective complication and critical care admission rates.

Self-sustaining smouldering combustion for the treatment of industrial oily waste.

Significant onsite handling and offsite management costs are incurred by oilfield operators annually to properly manage hydrocarbon waste streams such as tank bottoms or other oily sludge or oil impacted soil generated during oil and gas production processes. The current study reports for the first-time technical results of a field trial on use of a smouldering combustion technology performed in an active oilfield. Two treatment batches with oily sludges, stabilized through blending with soil, resulted in permanent hydrocarbon removal (98-99.9% reduction) to create treated soil that met standards for reuse as clean backfill onsite. Emissions profile data collected pre- and post-thermal oxidizer indicated effective removal of volatile organic compounds, CO and SO2, but had increased NO and CO2 due to combustion of propane to affect the thermal oxidation. Regulatory, financial, environmental and safety considerations are discussed in context of future full-scale smouldering technology deployment. The technology has the potential to lower overall unit costs for management of hydrocarbon impacted waste and reduce waste sent to landfills, which can benefit more remote sites.

FAK promotes stromal PD-L2 expression associated with poor survival in pancreatic cancer.

BACKGROUND: Pancreatic Cancer is one of the most lethal cancers, with less than 8% of patients surviving 5 years following diagnosis. The last 40 years have seen only small incremental improvements in treatment options, highlighting the continued need to better define the cellular and molecular pathways contributing to therapy response and patient prognosis. METHODS: We combined CRISPR, shRNA and flow cytometry with mechanistic experiments using a KrasG12Dp53R172H mouse model of pancreatic cancer and analysis of publicly available human PDAC transcriptomic datasets. RESULTS: Here, we identify that expression of the immune checkpoint, Programmed Death Ligand 2 (PD-L2), is associated with poor prognosis, tumour grade, clinical stage and molecular subtype in patients with Pancreatic Ductal Adenocarcinoma (PDAC). We further show that PD-L2 is predominantly expressed in the stroma and, using an orthotopic murine model of PDAC, identify cancer cell-intrinsic Focal Adhesion Kinase (FAK) signalling as a regulator of PD-L2 stromal expression. Mechanistically, we find that FAK regulates interleukin-6, which can act in concert with interleukin-4 secreted by CD4 T-cells to drive elevated expression of PD-L2 on tumour-associated macrophages, dendritic cells and endothelial cells. CONCLUSIONS: These findings identify further complex heterocellular signalling networks contributing to FAK-mediated immune suppression in pancreatic cancer.

Reproductive history differs by molecular subtypes of breast cancer among women aged ≤ 50 years in Scotland diagnosed 2009-2016: a cross-sectional study.

BACKGROUND: The aetiology of breast cancers diagnosed ≤ 50 years of age remains unclear. We aimed to compare reproductive risk factors between molecular subtypes of breast cancer, thereby suggesting possible aetiologic clues, using routinely collected cancer registry and maternity data in Scotland. METHODS: We conducted a cross-sectional study of 4108 women aged ≤ 50 years with primary breast cancer diagnosed between 2009 and 2016 linked to maternity data. Molecular subtypes of breast cancer were defined using immunohistochemistry (IHC) tumour markers, oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), and tumour grade. Age-adjusted polytomous logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for the association of number of births, age at first birth and time since last birth with IHC-defined breast cancer subtypes. Luminal A-like was the reference compared to luminal B-like (HER2-), luminal B-like (HER2+), HER2-overexpressed and triple-negative breast cancer (TNBC). RESULTS: Mean (SD) for number of births, age at first birth and time since last birth was 1.4 (1.2) births, 27.2 (6.1) years and 11.0 (6.8) years, respectively. Luminal A-like was the most common subtype (40%), while HER2-overexpressed and TNBC represented 5% and 15% of cases, respectively. Larger numbers of births were recorded among women with HER2-overexpressed and TNBC compared with luminal A-like tumours (> 3 vs 0 births, OR 1.87, 95%CI 1.18-2.96; OR 1.44, 95%CI 1.07-1.94, respectively). Women with their most recent birth > 10 years compared to < 2 years were less likely to have TNBC tumours compared to luminal A-like (OR 0.63, 95%CI 0.41-0.97). We found limited evidence for differences by subtype with age at first birth. CONCLUSION: Number of births and time since last birth differed by molecular subtypes of breast cancer among women aged ≤ 50 years. Analyses using linked routine electronic medical records by molecularly defined tumour pathology data can be used to investigate the aetiology and prognosis of cancer.

Validation of a Standardised Duplex Ultrasound Classification System for the Reporting and Grading of Peripheral Arterial Disease.

OBJECTIVE: Duplex ultrasound (DUS), a non-invasive means of arterial mapping, allows for the reliable diagnosis of peripheral arterial disease (PAD). One of the authors (C.P.O.), developed a standardised DUS based scoring system, devised for rapid detection and reporting of PAD. The purpose of this study was to validate this system, and to determine the diagnostic performance both overall and per disease severity. METHODS: In total, 250 participants were recruited, based on diagnosis of (n = 125) or absence of PAD (n = 125) from general practice registers. Right and left legs per subject were handled as independent readings, determining actual PAD status via ankle brachial pressure index (ABPI) < 0.9, and then further grading disease severity using suggested ABPI ranges. Data were excluded if no corresponding ABPI value was obtained per DUS determination or if the ABPI reading was > 1.4, owing to the risk of false negatives due to incompressible vessels. Diagnostic sensitivity and specificity were obtained overall, and per severity classification. Furthermore, inter-rater agreement between ABPI and DUS determined PAD severity was determined by linear weighted Cohen's kappa. RESULTS: The sensitivity and specificity in the detection of disease overall was 81.0% (95% confidence interval [CI] 73.4 - 87.2) and 86.3% (95% CI 82.3 - 89.8), respectively. From mild to severe PAD, sensitivity increased from 71.1% (95% CI 55.7 - 83.6) to 89.3% (95% CI 71.8 - 97.7). Furthermore, a Cohen's kappa value of 0.63 (95% CI 0.57 - 0.69) was obtained, indicating moderate agreement between the two diagnostic methods. CONCLUSION: The findings of this study validate the diagnostic performance of the standardised DUS scoring system, as well as its capacity to grade severity of disease, offering a potential tool for the identification of PAD in community/research settings following initial screening methods. Confirmatory work could include a comparison of DUS determined disease with gold standard methods of non-invasive angiography, and novel tools such as toe flex near infrared spectroscopy and multisite photoplethysmography.

Coblation intracapsular tonsillectomy: A cohort study of NHS practice in England using Hospital Episode Statistics.

OBJECTIVES: To identify paediatric intracapsular Coblation tonsillectomy procedures from routine administrative data in England, and determine their safety. DESIGN: Retrospective observational cohort study of four ENT centres using routine data from Hospital Episode Statistics (HES). SETTING: Acute NHS trusts in England conducting exclusively intracapsular Coblation tonsillectomy. PARTICIPANTS: Children (≤16 years old) undergoing bilateral intracapsular Coblation tonsillectomy. MAIN OUTCOME MEASURES: Number of procedures, readmissions for pain, readmissions for bleeding and requirement for additional surgery for regrowth. RESULTS: A total of 5525 procedures were identified. The median patient age was 4 (IQR 2-5). In-hospital complications occurred in 1%, with 0.1% returning to theatre for arrest of primary tonsil bleeding. Almost half of the procedures were conducted as a day-case (44%), with only a small proportion staying in hospital more than one night (7%). Within 28 days, 1.2% of patients were readmitted with bleeding, 0.7% with infection and 0.3% with pain; 0.2% of patients required return to theatre for control of secondary haemorrhage. Longitudinal follow-up has found that revision tonsil surgery is 0.3% at 1 year (n = 4498), 1.1% at 2 years (n = 2938), 1.7% at 3 years (n = 1781), 1.9% at 4 years (n = 905) and 2.2% at 5 years (n = 305). CONCLUSIONS: Intracapsular coblation tonsillectomy safety outcomes in this study show primary and secondary bleeding rates and emergency return to theatre rates are lower than all tonsillectomy techniques reported in the National Prospective Tonsillectomy Audit and also lower than previously published Hospital Episode Statistics analysis of tonsillectomy procedures.

Prostatic urethral lift (UroLift): a real-world analysis of outcomes using hospital episodes statistics.

BACKGROUND: To determine real-world outcomes of prostatic urethral lift (UroLift) procedures conducted in hospitals across England. METHODS: A retrospective observational cohort was identified from Hospital Episode Statistics data including men undergoing UroLift in hospitals in England between 2017 and 2020. Procedure uptake, patient demographics, inpatient complications, 30-day accident and emergency re-attendance rate, requirement for further treatment and catheterization were captured. Kaplan-Meier and hazard analysis were used to analyse time to re-treatment. RESULTS: 2942 index UroLift procedures from 80 hospital trusts were analysed; 85.3% conducted as day-case surgery (admitted to hospital for a planned surgical procedure and returning home on the same day). In-hospital complication rate was 3.4%. 93% of men were catheter-free at 30 days. The acute accident and emergency attendance rate within 30 days was 12.0%. Results of Kaplan Meier analysis for subsequent re-treatment (including additional UroLift and endoscopic intervention) at 1 and 2 years were 5.2% [95% CI 4.2 to 6.1] and 11.9% [10.1 to 13.6] respectively. CONCLUSIONS: This real-world analysis of UroLift shows that it can be delivered safely in a day-case setting with minimal morbidity. However, hospital resource usage for catheterization and emergency hospital attendance in the first 30 days was substantial, and 12% required re-treatment at 2 years.

Sleep and neighborhood socioeconomic status: a micro longitudinal study of chronic low-back pain and pain-free individuals.

Individuals with chronic low back pain (cLBP) frequently report sleep disturbances. Living in a neighborhood characterized by low-socioeconomic status (SES) is associated with a variety of negative health outcomes, including poor sleep. Whether low-neighborhood SES exacerbates sleep disturbances of people with cLBP, relative to pain-free individuals, has not previously been observed. This study compared associations between neighborhood-level SES, pain-status (cLBP vs. pain-free), and daily sleep metrics in 117 adults (cLBP = 82, pain-free = 35). Neighborhood-level SES was gathered from Neighborhood Atlas, which provides a composite measurement of overall neighborhood deprivation (e.g. area deprivation index). Individuals completed home sleep monitoring for 7-consecutive days/nights. Neighborhood SES and pain-status were tested as predictors of actigraphic sleep variables (e.g., sleep efficiency). Analyses revealed neighborhood-level SES and neighborhood-level SES*pain-status interaction significantly impacted objective sleep quality. These findings provide initial support for the negative impact of low neighborhood-level SES and chronic pain on sleep quality.

Temporal summation of mechanical pain prospectively predicts movement-evoked pain severity in adults with chronic low back pain.

BACKGROUND: Biopsychosocial factors above and beyond pathoanatomical changes likely contribute to the severity of chronic low back pain. A pro-nociceptive endogenous pain modulatory balance (↓inhibition and ↑facilitation) may be an important contributor to chronic low back pain severity and physical function; however, additional research is needed to address this possibility. The objective of this study was to determine whether quantitative sensory tests of endogenous pain inhibition and facilitation prospectively predict movement-evoked pain and cLBP severity self-reported on a validated questionnaire. METHODS: One hundred thirty-four individuals with chronic low back pain were enrolled in this two-session study. During the first study session, temporal summation of mechanical pain and conditioned pain modulation were assessed at the lumbar spine to determine endogenous pain facilitation and inhibition, respectively. One week later, participants returned for a second study session whereby they reported their pain severity and pain interference using the Brief Pain Inventory-Short Form. Movement-evoked pain and physical function capacity were assessed upon completion of the balance, walking, and transition from sit to stand tests of the Short Physical Performance Battery. RESULTS: Temporal summation of mechanical pain, but not conditioned pain modulation, significantly and prospectively predicted greater movement-evoked pain and poorer physical function on the Short Physical Performance Battery. Neither temporal summation nor conditioned pain modulation were significantly related to self-reported pain severity or pain interference on the Brief Pain Inventory-Short Form. CONCLUSIONS: Findings suggest that a pro-nociceptive pain modulatory balance characterized by enhanced pain facilitation may be an important driver of movement-evoked pain severity and poor physical function in individuals with chronic low back pain.

Paediatric tonsillectomy in England: A cohort study of clinical practice and outcomes using Hospital Episode Statistics data (2008-2019).

OBJECTIVES: To assess the safety of paediatric tonsillectomy procedures conducted in NHS hospitals in England between 2008 and 2019. DESIGN: Retrospective observational cohort study using Hospital Episode Statistics (HES) data. SETTING: Acute NHS trusts in England conducting paediatric tonsillectomy procedures. PARTICIPANTS: Children (≤16 years old) undergoing bilateral tonsillectomy. MAIN OUTCOME MEASURES: Number of tonsillectomies performed per year by procedural method. In-hospital complications including return to theatre for arrest of haemorrhage. Readmission within 28 days, including those for pain, haemorrhage and surgical arrest of haemorrhage. Long-term outcomes: all-cause mortality, revision tonsillectomy. RESULTS: A total of 318 453 paediatric tonsillectomies were performed from 2008 to 2019:278,772 dissection (87.5%) and 39 681 coblation (12.5%). The proportion of tonsillectomy performed using coblation increased from 7% in 2008/9 to 27% in 2018/9. Five patients died in hospital (including 4 due to respiratory complications). In-hospital complications occurred in 4202 children (1.3%), with the most frequent being haemorrhage. Within 28 days of tonsillectomy, 28 170 patients (8.8%) were readmitted and 7 deaths occurred. Readmission rates for haemorrhage and pain have increased since 2008. The proportion of children undergoing revision tonsillectomy procedures within 5 years following coblation tonsillectomy (1.4%) was approximately double that of dissection (0.6%). CONCLUSIONS: Clinical practice of paediatric tonsillectomy has changed in England over the past 11 years. The overall mortality rate associated with the procedure is 0.0037%. Differences in outcomes have been identified for different procedural methods. However, routine administrative data are limited in differentiating procedural detail (eg we are unable to differentiate intra or extra-capsular techniques from current clinical coding of tonsillectomy procedures). Therefore, prospective national data collection or more granular clinical coding is essential to capture relative outcomes of the different tonsillectomy methods and techniques being used in the NHS.

Unlocking the transcriptomic potential of formalin-fixed paraffin embedded clinical tissues: comparison of gene expression profiling approaches.

BACKGROUND: High-throughput transcriptomics has matured into a very well established and widely utilised research tool over the last two decades. Clinical datasets generated on a range of different platforms continue to be deposited in public repositories provide an ever-growing, valuable resource for reanalysis. Cost and tissue availability normally preclude processing samples across multiple technologies, making it challenging to directly evaluate performance and whether data from different platforms can be reliably compared or integrated. METHODS: This study describes our experiences of nine new and established mRNA profiling techniques including Lexogen QuantSeq, Qiagen QiaSeq, BioSpyder TempO-Seq, Ion AmpliSeq, Nanostring, Affymetrix Clariom S or U133A, Illumina BeadChip and RNA-seq of formalin-fixed paraffin embedded (FFPE) and fresh frozen (FF) sequential patient-matched breast tumour samples. RESULTS: The number of genes represented and reliability varied between the platforms, but overall all methods provided data which were largely comparable. Crucially we found that it is possible to integrate data for combined analyses across FFPE/FF and platforms using established batch correction methods as required to increase cohort sizes. However, some platforms appear to be better suited to FFPE samples, particularly archival material. CONCLUSIONS: Overall, we illustrate that technology selection is a balance between required resolution, sample quality, availability and cost.

Perceived Injustice Helps Explain the Association Between Chronic Pain Stigma and Movement-Evoked Pain in Adults with Nonspecific Chronic Low Back Pain.

OBJECTIVE: For most patients with chronic low back pain (cLBP), the cause is "nonspecific," meaning there is no clear association between pain and identifiable pathology of the spine or associated tissues. Laypersons and providers alike are less inclined to help, feel less sympathy, dislike patients more, suspect deception, and attribute lower pain severity to patients whose pain does not have an objective basis in tissue pathology. Because of these stigmatizing responses from others, patients with cLBP may feel that their pain is particularly unjust and unfair. These pain-related injustice perceptions may subsequently contribute to greater cLBP severity. The purpose of this study was to examine whether perceived injustice helps explain the relationship between chronic pain stigma and movement-evoked pain severity among individuals with cLBP. METHODS: Participants included 105 patients with cLBP who completed questionnaires assessing chronic pain stigma and pain-related injustice perception, as well as a short physical performance battery for the assessment of movement-evoked pain and physical function. RESULTS: Findings revealed that perceived injustice significantly mediated the association between chronic pain stigma and cLBP severity (indirect effect = 6.64, 95% confidence interval [CI] = 2.041 to 14.913) and physical function (indirect effect = -0.401, 95% CI = -1.029 to -0.052). Greater chronic pain stigma was associated with greater perceived injustice (P = 0.001), which in turn was associated with greater movement-evoked pain severity (P = 0.003). CONCLUSIONS: These results suggest that perceived injustice may be a means through which chronic pain stigma impacts nonspecific cLBP severity and physical function.

Balloon dilatation for paediatric airway stenosis: Evidence from the UK Airway Intervention Registry.

OBJECTIVES: To assess the safety and efficacy in routine clinical practice of balloon dilatation procedures in the treatment of paediatric airway stenosis. DESIGN: Observational data collection in prospective online research database. SETTING: Acute NHS Trusts with ENT department undertaking complex paediatric airway work. PARTICIPANTS: Children (<18) undergoing balloon dilatation treatment for airway stenosis. MAIN OUTCOME MEASURES: Airway diameter, complications, hospital resource usage. RESULTS: Fifty-nine patients had 133 balloon procedures during 128 visits to 10 hospitals. Sixty-nine (52%) of balloon procedures were conducted with a tracheostomy. Intra-operative Cotton-Myer grade decreased in 43 (57%). The mean pre-balloon subglottic diameter was 4.2 [95% CI: 3.8 to 4.5] mm, and its rate of increase was 0.8 [0.5 to 1.2] mm per year modelled on 30 patients' long-term data. As the primary treatment of stenosis, the procedural success rate of balloon dilatation (n = 52) was 65% (22% with tracheostomy, 88% without tracheostomy), and 71% as an adjunct to open reconstructive surgery (n = 7). In the 64 hospital visits where a balloon procedure was conducted with a tracheostomy in place, only one in-hospital complication (lower respiratory tract infection) occurred. For those without a tracheostomy in place, in-hospital complications occurred in seven of 64 balloon hospital visits, all related to ongoing or worsening stenosis. Six out-of-hospital complications were deemed related to ongoing or worsening stenosis following the procedure, and two complications were a combination of lower respiratory infection and ongoing or worsening stenosis. CONCLUSIONS: Balloon dilation increases the size of the airway intraoperatively and is associated with long-term increase in airway diameter. Safety outcomes mostly relate to ongoing or worsening stenosis and are more common in patients without a tracheostomy.

The Milk Protein Alpha-Casein Suppresses Triple Negative Breast Cancer Stem Cell Activity Via STAT and HIF-1alpha Signalling Pathways in Breast Cancer Cells and Fibroblasts.

Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

HER2 regulates HIF-2α and drives an increased hypoxic response in breast cancer.

BACKGROUND: Tumour hypoxia is a driver of breast cancer progression associated with worse prognosis and more aggressive disease. The cellular response to hypoxia is mediated by the hypoxia-inducible transcription factors HIF-1 and HIF-2, whose transcriptional activity is canonically regulated through their oxygen-labile HIF-α subunits. These are constitutively degraded in the presence of oxygen; however, HIF-1α can be stabilised, even at high oxygen concentrations, through the activation of HER receptor signalling. Despite this, there is still limited understanding on how HER receptor signalling interacts with HIF activity to contribute to breast cancer progression in the context of tumour hypoxia. METHODS: 2D and 3D cell line models were used alongside microarray gene expression analysis and meta-analysis of publicly available gene expression datasets to assess the impact of HER2 overexpression on HIF-1α/HIF-2α regulation and to compare the global transcriptomic response to acute and chronic hypoxia in an isogenic cell line model of HER2 overexpression. RESULTS: HER2 overexpression in MCF7 cells leads to an increase in HIF-2α but not HIF-1α expression in normoxia and an increased upregulation of HIF-2α in hypoxia. Global gene expression analysis showed that HER2 overexpression in these cells promotes an exaggerated transcriptional response to both short-term and long-term hypoxia, with increased expression of numerous hypoxia response genes. HIF-2α expression is frequently higher in HER2-overexpressing tumours and is associated with worse disease-specific survival in HER2-positive breast cancer patients. HER2-overexpressing cell lines demonstrate an increased sensitivity to targeted HIF-2α inhibition through either siRNA or the use of a small molecule inhibitor of HIF-2α translation. CONCLUSIONS: This study suggests an important interplay between HER2 expression and HIF-2α in breast cancer and highlights the potential for HER2 to drive the expression of numerous hypoxia response genes in normoxia and hypoxia. Overall, these findings show the importance of understanding the regulation of HIF activity in a variety of breast cancer subtypes and points to the potential of targeting HIF-2α as a therapy for HER2-positive breast cancer.

On-treatment biomarkers can improve prediction of response to neoadjuvant chemotherapy in breast cancer.

BACKGROUND: Neoadjuvant chemotherapy is increasingly given preoperatively to shrink breast tumours prior to surgery. This approach also provides the opportunity to study the molecular changes associated with treatment and evaluate whether on-treatment sequential samples can improve response and outcome predictions over diagnostic or excision samples alone. METHODS: This study included a total of 97 samples from a cohort of 50 women (aged 29-76, with 46% ER+ and 20% HER2+ tumours) with primary operable breast cancer who had been treated with neoadjuvant chemotherapy. Biopsies were taken at diagnosis, at 2 weeks on-treatment, mid-chemotherapy, and at resection. Fresh frozen samples were sequenced with Ion AmpliSeq Transcriptome yielding expression values for 12,635 genes. Differential expression analysis was performed across 16 patients with a complete pathological response (pCR) and 34 non-pCR patients, and over treatment time to identify significantly differentially expressed genes, pathways, and markers indicative of response status. Prediction accuracy was compared with estimations of established gene signatures, for this dataset and validated using data from the I-SPY 1 Trial. RESULTS: Although changes upon treatment are largely similar between the two cohorts, very few genes were found to be consistently different between responders and non-responders, making the prediction of response difficult. AAGAB was identified as a novel potential on-treatment biomarker for pathological complete response, with an accuracy of 100% in the NEO training dataset and 78% accuracy in the I-SPY 1 testing dataset. AAGAB levels on-treatment were also significantly predictive of outcome (p = 0.048, p = 0.0036) in both cohorts. This single gene on-treatment biomarker had greater predictive accuracy than established prognostic tests, Mammaprint and PAM50 risk of recurrence score, although interestingly, both of these latter tests performed better in the on-treatment rather than the accepted pre-treatment setting. CONCLUSION: Changes in gene expression measured in sequential samples from breast cancer patients receiving neoadjuvant chemotherapy resulted in the identification of a potentially novel on-treatment biomarker and suggest that established prognostic tests may have greater prediction accuracy on than before treatment. These results support the potential use and further evaluation of on-treatment testing in breast cancer to improve the accuracy of tumour response prediction.

Molecular changes during extended neoadjuvant letrozole treatment of breast cancer: distinguishing acquired resistance from dormant tumours.

BACKGROUND: The risk of recurrence for endocrine-treated breast cancer patients persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate; previous efforts have been limited to in-vitro or in-vivo approaches. In this study, sequential tumour samples from patients receiving extended neoadjuvant aromatase inhibitor therapy were characterised as a novel clinical model. METHODS: Consecutive tumour samples from 62 patients undergoing extended (4-45 months) neoadjuvant aromatase inhibitor therapy with letrozole were subjected to transcriptomic and proteomic analysis, representing before (≤ 0), early (13-120 days), and long-term (> 120 days) neoadjuvant aromatase inhibitor therapy with letrozole. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as "dormant", and the remaining 20 patients as "acquired resistant". RESULTS: Changes in gene expression in dormant tumours begin early and become more pronounced at later time points. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to the resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. The DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared with dormant tissues after extended letrozole treatment. CONCLUSIONS: This is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumours continue to change during treatment whereas acquired resistant tumours more closely resemble their diagnostic samples. Global loss of DNA methylation was observed in resistant tumours under extended treatment. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients, supporting a potential role for therapy targeted at these epigenetic alterations in the management of resistance to oestrogen deprivation therapy.