Improving a regional chemotherapy service by learning from deaths occurring within 30 days of treatment with systemic anti-cancer therapy.

Background: Outwith clinical trials, patient outcomes specifically related to SACT (systemic anti-cancer therapy) are not well reported despite a significant proportion of patients receiving active treatment at the end of life. The NCEPOD reviewing deaths within 30 days of SACT found SACT caused or hastened death in 27% of cases.

Method: Across the Northern Ireland cancer network, 95 patients who died within 30 days of SACT for solid tumours were discussed at the Morbidity and Mortality monthly meeting during 2013. Using a structured template, each case was independently reviewed, with particular focus on whether SACT caused or hastened death.

Results: Lung, GI and breast cancers were the most common sites. Performance status was recorded in 92% at time of final SACT cycle (ECOG PS 0-2 89%).

In 57% the cause of death was progressive disease. Other causes included thromboembolism (13%) and infection (5% neutropenic sepsis, 6% non-neutropenic sepsis). In 26% with death from progressive disease, the patient was first cycle of first line treatment for metastatic disease. In the majority discussion regarding treatment aims and risks was documented. Only one patient was receiving SACT with curative intent, who died from appropriately managed neutropenic sepsis.

A definitive decision regarding SACT's role in death was made in 60%: in 49% SACT was deemed non-contributory and in 11% SACT was deemed the cause of death. In 40% SACT did not play a major role, but a definitive negative association could not be made.

Conclusion: Development of a robust review process of 30-day mortality after SACT established a benchmark for SACT delivery for future comparisons and identified areas for SACT service organisation improvement. Moreover it encourages individual practice reflection and highlights the importance of balancing patients' needs and concerns with realistic outcomes and risks, particularly in heavily pre-treated patients or those of poor performance status.

Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial

BACKGROUND: Long-term hormone therapy has been the standard of care for advanced prostate cancer since the 1940s. STAMPEDE is a randomised controlled trial using a multiarm, multistage platform design. It recruits men with high-risk, locally advanced, metastatic or recurrent prostate cancer who are starting first-line long-term hormone therapy. We report primary survival results for three research comparisons testing the addition of zoledronic acid, docetaxel, or their combination to standard of care versus standard of care alone.

METHODS: Standard of care was hormone therapy for at least 2 years; radiotherapy was encouraged for men with N0M0 disease to November, 2011, then mandated; radiotherapy was optional for men with node-positive non-metastatic (N+M0) disease. Stratified randomisation (via minimisation) allocated men 2:1:1:1 to standard of care only (SOC-only; control), standard of care plus zoledronic acid (SOC + ZA), standard of care plus docetaxel (SOC + Doc), or standard of care with both zoledronic acid and docetaxel (SOC + ZA + Doc). Zoledronic acid (4 mg) was given for six 3-weekly cycles, then 4-weekly until 2 years, and docetaxel (75 mg/m(2)) for six 3-weekly cycles with prednisolone 10 mg daily. There was no blinding to treatment allocation. The primary outcome measure was overall survival. Pairwise comparisons of research versus control had 90% power at 2·5% one-sided α for hazard ratio (HR) 0·75, requiring roughly 400 control arm deaths. Statistical analyses were undertaken with standard log-rank-type methods for time-to-event data, with hazard ratios (HRs) and 95% CIs derived from adjusted Cox models. This trial is registered at ClinicalTrials.gov (NCT00268476) and ControlledTrials.com (ISRCTN78818544).

FINDINGS: 2962 men were randomly assigned to four groups between Oct 5, 2005, and March 31, 2013. Median age was 65 years (IQR 60-71). 1817 (61%) men had M+ disease, 448 (15%) had N+/X M0, and 697 (24%) had N0M0. 165 (6%) men were previously treated with local therapy, and median prostate-specific antigen was 65 ng/mL (IQR 23-184). Median follow-up was 43 months (IQR 30-60). There were 415 deaths in the control group (347 [84%] prostate cancer). Median overall survival was 71 months (IQR 32 to not reached) for SOC-only, not reached (32 to not reached) for SOC + ZA (HR 0·94, 95% CI 0·79-1·11; p=0·450), 81 months (41 to not reached) for SOC + Doc (0·78, 0·66-0·93; p=0·006), and 76 months (39 to not reached) for SOC + ZA + Doc (0·82, 0·69-0·97; p=0·022). There was no evidence of heterogeneity in treatment effect (for any of the treatments) across prespecified subsets. Grade 3-5 adverse events were reported for 399 (32%) patients receiving SOC, 197 (32%) receiving SOC + ZA, 288 (52%) receiving SOC + Doc, and 269 (52%) receiving SOC + ZA + Doc.

INTERPRETATION: Zoledronic acid showed no evidence of survival improvement and should not be part of standard of care for this population. Docetaxel chemotherapy, given at the time of long-term hormone therapy initiation, showed evidence of improved survival accompanied by an increase in adverse events. Docetaxel treatment should become part of standard of care for adequately fit men commencing long-term hormone therapy.

FUNDING: Cancer Research UK, Medical Research Council, Novartis, Sanofi-Aventis, Pfizer, Janssen, Astellas, NIHR Clinical Research Network, Swiss Group for Clinical Cancer Research.

Molecular classification of non-invasive breast lesions for personalised therapy and chemoprevention

Breast cancer screening has led to a dramatic increase in the detection of pre-invasive breast lesions. While mastectomy is almost guaranteed to treat the disease, more conservative approaches could be as effective if patients can be stratified based on risk of co-existing or recurrent invasive disease.Here we use a range of biomarkers to interrogate and classify purely non-invasive lesions (PNL) and those with co-existing invasive breast cancer (CEIN). Apart from Ductal Carcinoma In Situ (DCIS), relative homogeneity is observed. DCIS contained a greater spread of molecular subtypes. Interestingly, high expression of p-mTOR was observed in all PNL with lower expression in DCIS and invasive carcinoma while the opposite expression pattern was observed for TOP2A.Comparing PNL with CEIN, we have identified p53 and Ki67 as predictors of CEIN with a combined PPV and NPV of 90.48% and 43.3% respectively. Furthermore, HER2 expression showed the best concordance between DCIS and its invasive counterpart.We propose that these biomarkers can be used to improve the management of patients with pre-invasive breast lesions following further validation and clinical trials. p53 and Ki67 could be used to stratify patients into low and high-risk groups for co-existing disease. Knowledge of expression of more actionable targets such as HER2 or TOP2A can be used to design chemoprevention or neo-adjuvant strategies. Increased knowledge of the molecular profile of pre-invasive lesions can only serve to enhance our understanding of the disease and, in the era of personalised medicine, bring us closer to improving breast cancer care.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Associations between intensive diabetes therapy and NMR-determined lipoprotein subclass profiles in Type 1 diabetes

Our objective is to define differences in circulating lipoprotein subclasses between intensive vs. conventional management of Type 1 diabetes during the randomization phase of the Diabetes Control and Complications Trial (DCCT). Nuclear magnetic resonance-determined lipoprotein subclass profiles (NMR-LSP), which estimate molar subclass concentrations and mean particle diameters, were determined in 1,294 DCCT subjects after a median of five (interquartile range: four, six) years following randomization to intensive or conventional diabetes management. In cross-sectional analyses, we compared standard lipids and NMR-LSP between treatment groups. Standard total-, LDL- and HDL-cholesterol levels were similar between randomization groups, while triglyceride levels were lower in the intensively treated group. NMR-LSP showed that intensive therapy was associated with larger LDL diameter (20.7 vs. 20.6 nm, p=0.01) and lower levels of small LDL (median: 465 vs. 552 nmol/l, p=0.007), total IDL/LDL (mean: 1000 vs. 1053 nmol/l, p=0.01), and small HDL (mean: 17.3 vs. 18.6 μmol/l, p<0.0001), the latter accounting for reduced total HDL (mean: 33.8 vs. 34.8 μmol/l, p=0.01). In conclusion, intensive diabetes therapy was associated with potentially favorable changes in LDL and HDL subclasses in sera. Further research will determine whether these changes contribute to the beneficial effects of intensive diabetes management on vascular complications.

Identification and validation of the dopamine agonist bromocriptine as a novel therapy for high-risk myelodysplastic syndromes and secondary acute myeloid leukemia

Myelodysplastic syndromes (MDS) represent a broad spectrum of diseases characterized by their clinical manifestation as one or more cytopenias, or a reduction in circulating blood cells. MDS is predominantly a disease of the elderly, with a median age in the UK of around 75. Approximately one third of MDS patients will develop secondary acute myeloid leukemia (sAML) that has a very poor prognosis. Unfortunately, most standard cytotoxic agents are often too toxic for older patients. This means there is a pressing unmet need for novel therapies that have fewer side effects to assist this vulnerable group. This challenge was tackled using bioinformatic analysis of available transcriptomic data to establish a gene-based signature of the development and progression of MDS. This signature was then used to identify novel therapeutic compounds via statistically-significant connectivity mapping. This approach suggested re-purposing an existing and widely-prescribed drug, bromocriptine as a novel potential therapy in these disease settings. This drug has shown selectivity for leukemic cells as well as synergy with current therapies.

Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts

BACKGROUND: Cigarette smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease, however, the role of either nicotine or its primary metabolite cotinine in the progression of periodontitis is unclear. This study aimed to investigate the effects of nicotine and cotinine on the attachment and growth of fibroblasts derived from human periodontal ligament (PDL).

METHODS: Primary cultures were prepared from the roots of extracted premolar teeth. Cells were used at both low (P3 to P5) and high (P11 to P13) passage. Cell numbers were determined over 14 days using either the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay or with a Coulter counter. Cultures were exposed to culture medium supplemented with 1) 15% fetal calf serum (FCS) only; 2) 1% FCS only; 3) 1% FCS and nicotine (concentration range 5 ng/ml to 10 mg/ml); or 4) 1% FCS and cotinine (concentration range 0.5 ng/ml to 10 microg/ml).

RESULTS: Nicotine significantly (P <0.05, by ANOVA) inhibits attachment and growth of low passage cells at concentrations >1 mg/ml and high passage PDL fibroblasts at concentrations >0.5 mg/ml. Cotinine, at the highest concentration used (10 microg/ml), appeared to inhibit attachment and growth of both low and high passage fibroblasts but this was not statistically significant (P >0.05, by ANOVA).

CONCLUSIONS: Tobacco products inhibit attachment and growth of human PDL fibroblasts. This may partly explain the role of these substances in the progression of periodontitis.

Nifedipine-induced gingival hyperplasia

This case study reports on the management of a woman suffering from thrombocytopenic purpura who was referred to the periodontal clinic with gingival swelling and bleeding. This condition was related to nifedipine therapy for hypertension.

Prostate cancer radiotherapy: potential applications of metal nanoparticles for imaging and therapy

Prostate cancer (CaP) is the most commonly diagnosed cancer in males. There have been dramatic technical advances in radiotherapy delivery, enabling higher doses of radiotherapy to primary cancer, involved lymph nodes and oligometastases with acceptable normal tissue toxicity. Despite this, many patients relapse following primary radical therapy, and novel treatment approaches are required. Metal nanoparticles are agents that promise to improve diagnostic imaging and image-guided radiotherapy and to selectively enhance radiotherapy effectiveness in CaP. We summarize current radiotherapy treatment approaches for CaP and consider pre-clinical and clinical evidence for metal nanoparticles in this condition.

Prostate cancer (CaP) is the most commonly diagnosed cancer in males and is responsible for more than 10,000 deaths each year in the UK.1 Technical advances in radiotherapy delivery, including image-guided intensity-modulated radiotherapy (IG-IMRT), have enabled the delivery of higher radiation dose to the prostate, which has led to improved biochemical control. Further improvements in cancer imaging during radiotherapy are being developed with the advent of MRI simulators and MRI linear accelerators.2–4

Nanotechnology promises to deliver significant advancements across numerous disciplines.5 The widest scope of applications are from the biomedical field including exogenous gene/drug delivery systems, advanced biosensors, targeted contrast agents for diagnostic applications and as direct therapeutic agents used in combination with existing treatment modalities.6–11 This diversity of application is especially evident within cancer research, with a myriad of experimental anticancer strategies currently under investigation.

This review will focus specifically on the potential of metal-based nanoparticles to augment the efficacy of radiotherapy in CaP, a disease where radiotherapy constitutes a major curative treatment modality.12 Furthermore, we will also address the clinical state of the art for CaP radiotherapy and consider how these treatments could be best combined with nanotherapeutics to improve cancer outcomes.

Reference gene selection in human periodontal ligament cells

Background: Real-time quantitative PCR (qPCR) is a highly sensitive and specific method which is used extensively for determining gene expression profiles in a variety of cell and tissue types. In order to obtain accurate and reliable gene expression quantification, qPCR data are generally normalised against so-called reference or housekeeping genes. Ideally, reference genes should have abundant and stable RNA transcriptomes under the experimental conditions employed. However, reference genes are often selected rather arbitrarily and indeed some have been shown to have variable expression in a variety of in vitro experimental conditions.
Objective: The objective of the current study was to investigate reference gene expression in human periodontal ligament (PDL) cells in response to treatment with lipopolysaccharide (LPS).
Method: Primary human PDL cells were grown in Dulbecco’s Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum, 100UI/ml penicillin and 100µg/ml streptomycin. RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The expression of a total of 19 reference genes was studied in the presence and absence of LPS treatment using the Roche Reference Gene Panel. Data were analysed using NormFinder and Bestkeeper validation programs.
Results: Treatment of human PDL cells with LPS resulted in changes in expression of several commonly used reference genes, including GAPDH. On the other hand the reference genes β-actin, G6PDH and 18S were identified as stable genes following LPS treatment.
Conclusion: Many of the reference genes studied were robust to LPS treatment (up to 100 ng/ml). However several commonly employed reference genes, including GAPDH varied with LPS treatment, suggesting they would not be ideal candidates for normalisation in qPCR gene expression studies.

Neuropeptide Y in GCF in periodontal health and disease

Objectives: To determine whether neuropeptide Y (NPY) is present in gingival crevicular fluid (GCF) in both periodontal health and disease and to study the relationship of NPY with periodontal inflammation. Methods: GCF samples (30 s) were collected from one site with both pocket depth (>4mm) and loss of periodontal attachment (>4mm) in 20 patients with chronic periodontitis (mean age 41.4, SD 9.6 yrs; 10 m, 10 f). GCF was also collected from clinically healthy sites (< 3mm, no bleeding on probing) in 20 subjects with no periodontitis (mean age 37.4, SD 11.7; 10 m, 10 f). GCF was collected using the periopaper strip method, diluted in 500 ul of phosphate-buffered saline and stored at –70°C. Samples were analysed in duplicate for NPY by radioimmunoassay. NPY levels were compared using the Mann-Whitney test. Results: Measurable NPY was present in all the GCF samples collected from healthy subjects. NPY was below the level of detection in 4 (20%) of the diseased subjects. There was considerable variability in the amount of NPY collected from both groups. There were no differences between the levels of NPY measured in males compared with females in either the healthy or diseased groups. Significantly more (P< 0.0001) NPY (pg) was collected from healthy subjects (Median 165, IQR 80; mean 161, SD 64) than diseased subjects (Median 37.5, IQR 56.3; mean 39.8, SD 35.1). There was more variability in the NPY concentration (pg/ul) which was also significantly higher in healthy (Median 575.7, IQR 562.3; mean 645.7, SD 416.7) compared with diseased subjects (Median 43.6, IQR 117.4; mean 96.4, SD 124.5). Conclusions: It is concluded that the levels of NPY in GCF sampled

LL-37 quantification in gingival crevicular fluid (GCF) from periodontitis patients

Background: LL-37, an anti-microbial peptide belonging to the cathelicidin family, derives its name from two N-terminal leucine residues and the 37 amino acids comprising the peptide. LL-37 is the only known cathelicidin to exist in humans. It exhibits both anti-bacterial and immunomodulatory properties. Objectives: In the current study, LL-37 was quantified in GCF from periodontitis patients. Previous studies have relied on qualitative results from Western blotting to detect LL-37 in GCF. This study aims to quantitatively determine LL-37 levels in GCF. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 sites in 12 patients presenting with advanced periodontitis. Plaque samples were analysed by QPCR for the presence or absence of the periodontopathic bacterium Porphyromonas gingivalis (P. gingivalis). The concentrations of LL-37 in patient samples pre- and post-treatment were deduced by indirect enzyme linked immunosorbent assay (ELISA). Results: Concentrations of LL-37 in samples varied between a minimum and maximum of 1 and 40 ng/ml. LL-37 levels were shown, pre-treatment, to be higher in deep pockets (6-9 mm) compared with shallower pockets (3-5 mm) and highest in those sites which were positive for P. gingivalis. Non-surgical therapy resulted in a significant improvement in clinical indices while expression levels of P. gingivalis were reduced. Following treatment, LL-37 levels in GCF decreased from an average of 6.5 ± 1 - 5.8 ± 1.2 ng/ml. The most interesting observation however was the reduction in LL-37 levels, from an average of 7 ± 1.3 – 2.5 ± 1.1 ng/ml in those sites where P. gingivalis infection was eradicated post-treatment. Conclusions: LL-37 levels are increased at sites showing advanced periodontal disease, reduce following treatment and appear to be linked to the presence of P. gingivalis. This study will further our knowledge of host defence in chronic diseases such as periodontitis.

The role of LL-37 in periodontal wound healing

Introduction: As a result of chronic inflammation during periodontal disease the junctional epithelium becomes micro-ulcerated. The inflammatory process is mediated by both bacterial and host cell products. Host defence peptides such as defensins, secretory leucocyte protease inhibitor (SLPI) and the sole human cathelicidin, LL-37, are secreted by both periodontal cells and neutrophils into gingival crevicular fluid (GCF). They have the ability to modulate the immune response in periodontitis and are thought to have a potential role in periodontal wound healing. Objectives: The aims of this study were to determine the role of LL-37 in the production of Interleukin (IL)-8, IL-6, hepatocyte growth factor (HGF) and basic-fibroblast growth factor (bFGF) by gingival fibroblasts. The role of LL-37 in modulating total matrix metalloproteinase (MMP) activity and expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 by gingival fibroblasts was also investigated. Methods: Primary gingival fibroblasts were co-cultured with concentrations of LL-37 (1, 5 and 10µg/ml) for 24 hours and their supernatants tested for levels of IL-8 and IL-6, HGF, bFGF, TIMP-1 and TIMP-2 by ELISA. Rates of MMP turnover in the supernatants were tested by fluorogenic assay using fluorescence resonance energy transfer (FRET) peptide substrates. Cytotoxicity was measured by MTT assay. Statistical significance was measured using the independent t-test and p<0.05 was considered significant. Results: LL-37 significantly upregulated levels of IL-8, IL-6, HGF, bFGF and TIMP-1 (p<0.05) in a dose-dependent fashion. LL-37 significantly decreased the total MMP activity (p<0.05). None of the LL-37 concentrations tested were cytotoxic to gingival fibroblasts. Conclusion: These results indicate that LL-37 is involved in periodontal wound healing. LL-37 increased levels of proinflammatory cytokines and increased levels of growth factors involved in re-epithelialisation. LL-37 has the ability to regulate remodelling of the periodontium by controlling MMP overactivity both directly and by stimulating production of inhibitors by gingival fibroblasts.