Protocol for the EACH trial: a multicentre phase II study evaluating the safety and antitumour activity of the combination of avelumab, an anti-PD-L1 agent, and cetuximab, as any line treatment for patients with recurrent/metastatic head and neck squamous cell cancer (HNSCC) in the UK.

INTRODUCTION: Head and neck cancer is the eighth most common cancer in the UK. Current standard of care treatment for patients with recurrent/metastatic squamous cell head and neck carcinoma (HNSCC) is platinum-based chemotherapy combined with the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, cetuximab. However, most patients will have poor median overall survival (OS) of 6-9 months despite treatment. HNSCC tumours exhibit an immune landscape poised to respond to immunotherapeutic approaches, with most tumours expressing the immunosuppressive receptor programmed death-ligand 1 (PD-L1). We undertook the current study to determine the safety and efficacy of avelumab, a monoclonal antibody targeting the interaction between PD-L1 and its receptor on cytotoxic T-cells, in combination with cetuximab. METHODS AND ANALYSIS: This is a multi-centre, single-arm dose de-escalation phase II safety and efficacy study of avelumab combined with cetuximab; the study was to progress to a randomised phase II trial, however, the study will now complete after the safety run-in component. Up to 16 participants with histologically/cytologically recurrent/metastatic squamous cell carcinoma (including HNSCC) who have not received cetuximab previously will be recruited. All patients will receive 10 mg/kg avelumab and cetuximab (500, 400 or 300 mg/m2 depending on the cohort open at time of registration) on days 1 and 15 of 4-week cycles for up to 1 year, (avelumab not given cycle 1 day 1). A modified continual reassessment method will be used to determine dose de-escalation. The primary objective is to establish the safety of the combination and to determine the optimum dose of cetuximab. Secondary objectives include assessing evidence of antitumour activity by evaluating response rates and disease control rates at 6 and 12 months as well as progression-free and OS. ETHICS AND DISSEMINATION: Approval granted by City and East REC (18/LO/0021). Findings will be published in peer-reviewed journals and disseminated at conferences. TRIAL REGISTRATION NUMBER: NCT03494322.

Old Age Psychiatry.

OBJECTIVE: Older Chinese people in New Zealand underutilise mental health services. Lack of recognition of mental health issues and awareness of available treatment is a potential barrier to accessing care. This study investigated depression literacy in older Chinese people. METHOD: A convenience sample of 67 older Chinese people were presented a depression vignette and completed a depression literacy questionnaire. RESULTS: There was a good rate (71.6%) of depression recognition, but no participant chose taking medication as the best method of help. There was a notable level of stigma among participants. CONCLUSION: Older Chinese people would benefit from information regarding mental health conditions and their interventions. Strategies to deliver this information and de-stigmatise mental illness in the Chinese community which incorporate cultural values may be beneficial.

PANTHER: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003.

Phase 0 Study of Vandetanib-Eluting Radiopaque Embolics as a Preoperative Embolization Treatment in Patients with Resectable Liver Malignancies.

PURPOSE: To assess the safety and tolerability of a vandetanib-eluting radiopaque embolic (BTG-002814) for transarterial chemoembolization (TACE) in patients with resectable liver malignancies. MATERIALS AND METHODS: The VEROnA clinical trial was a first-in-human, phase 0, single-arm, window-of-opportunity study. Eligible patients were aged ≥18 years and had resectable hepatocellular carcinoma (HCC) (Child-Pugh A) or metastatic colorectal cancer (mCRC). Patients received 1 mL of BTG-002814 transarterially (containing 100 mg of vandetanib) 7-21 days prior to surgery. The primary objectives were to establish the safety and tolerability of BTG-002814 and determine the concentrations of vandetanib and the N-desmethyl vandetanib metabolite in the plasma and resected liver after treatment. Biomarker studies included circulating proangiogenic factors, perfusion computed tomography, and dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Eight patients were enrolled: 2 with HCC and 6 with mCRC. There was 1 grade 3 adverse event (AE) before surgery and 18 after surgery; 6 AEs were deemed to be related to BTG-002814. Surgical resection was not delayed. Vandetanib was present in the plasma of all patients 12 days after treatment, with a mean maximum concentration of 24.3 ng/mL (standard deviation ± 13.94 ng/mL), and in resected liver tissue up to 32 days after treatment (441-404,000 ng/g). The median percentage of tumor necrosis was 92.5% (range, 5%-100%). There were no significant changes in perfusion imaging parameters after TACE. CONCLUSIONS: BTG-002814 has an acceptable safety profile in patients before surgery. The presence of vandetanib in the tumor specimens up to 32 days after treatment suggests sustained anticancer activity, while the low vandetanib levels in the plasma suggest minimal release into the systemic circulation. Further evaluation of this TACE combination is warranted in dose-finding and efficacy studies.

Ketonuria and Seizure Control in the Medium Chain Triglyceride and Classic Ketogenic Diets.

We hypothesized that children receiving medium-chain triglyceride ketogenic diet (MCTKD) experience similar seizure reduction despite lower ketosis compared with classic ketogenic diet (CKD). Children initiating CKD or MCTKD were enrolled in a prospective observational study. Forty-five children completed 6 months of KD (n = 17 MCTKD, n = 28 CKD). The proportion achieving ≥50% seizure reduction was 71% CKD group and 59% MCTKD group; ≥90% reduction was 32% and 36% in CKD and MCTKD groups, respectively. CKD had higher urine ketones (≥8 mmol/L: 79% vs. 36%, p = 0.005). Children receiving MCTKD experience similar seizure control to CKD despite lower urine ketone measures.

Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS: Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS: Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease-negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4%-84.4%) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION: AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL.

Whole-genome sequencing of single circulating tumor cells from neuroendocrine neoplasms.

Single-cell profiling of circulating tumor cells (CTCs) as part of a minimally invasive liquid biopsy presents an opportunity to characterize and monitor tumor heterogeneity and evolution in individual patients. In this study, we aimed to compare single-cell copy number variation (CNV) data with tissue and define the degree of intra- and inter-patient genomic heterogeneity. We performed next-generation sequencing (NGS) whole-genome CNV analysis of 125 single CTCs derived from seven patients with neuroendocrine neoplasms (NEN) alongside matched white blood cells (WBC), formalin-fixed paraffin-embedded (FFPE), and fresh frozen (FF) samples. CTC CNV profiling demonstrated recurrent chromosomal alterations in previously reported NEN copy number hotspots, including the prognostically relevant loss of chromosome 18. Unsupervised hierarchical clustering revealed CTCs with distinct clonal lineages as well as significant intra- and inter-patient genomic heterogeneity, including subclonal alterations not detectable by bulk analysis and previously unreported in NEN. Notably, we also demonstrated the presence of genomically distinct CTCs according to the enrichment strategy utilized (EpCAM-dependent vs size-based). This work has significant implications for the identification of therapeutic targets, tracking of evolutionary change, and the implementation of CTC-biomarkers in cancer.

Neutropenia in Children Treated With Ketogenic Diet Therapy.

OBJECTIVES: The objectives were to investigate the relationship between ketogenic diet therapy and neutropenia in children with epilepsy. METHODS: A retrospective chart review of children who initiated ketogenic diet at the Hospital for Sick Children between January 1, 2000, and May 1, 2018 was performed. Factors associated with the development of neutropenia during ketogenic diet therapy were evaluated and the relationship between development of a significant or suspected infection and neutrophil count was analyzed. RESULTS: One hundred two children met inclusion criteria and were followed on the diet for up to 24 months. Thirteen of 102 (13%) children were neutropenic at diet initiation. In the remaining 89 children, 27 developed neutropenia. Developing neutropenia was significantly associated with the ketogenic diet at 6 (13%), 12 (23%), and 24 (25%) months follow-up. Developing neutropenia was associated with higher urinary ketones (OR = 4.26, 95% CI: 1.27, 14.15) and longer duration of ketogenic diet therapy (OR = 3.29, 95% CI: 1.42, 7.96). There was no significant association between development of a clinically significant infection and neutropenia. CONCLUSION: Ketogenic diet therapy is associated with neutropenia in children with epilepsy, however, it does not have a significant clinical impact. Concern regarding neutropenia should not discourage the use of the ketogenic diet in children.

Prognostic Threshold for Circulating Tumor Cells in Patients With Pancreatic and Midgut Neuroendocrine Tumors.

BACKGROUND: Circulating tumor cells (CTCs) are detectable in patients with neuroendocrine tumors (NETs) and are accurate prognostic markers although the optimum threshold has not been defined. OBJECTIVE: This work aims to define optimal prognostic CTC thresholds in PanNET and midgut NETs. PATIENTS AND METHODS: CellSearch was used to enumerate CTCs in 199 patients with metastatic pancreatic (PanNET) (90) or midgut NETs (109). Patients were followed for progression-free survival (PFS) and overall survival (OS) for a minimum of 3 years or until death. RESULTS: The area under the receiver operating characteristic curve (AUROC) for progression at 12 months in PanNETs and midgut NETs identified the optimal CTC threshold as 1 or greater and 2 or greater, respectively. In multivariate logistic regression analysis, these thresholds were predictive for 12-month progression with an odds ratio (OR) of 6.69 (P < .01) for PanNETs and 5.88 (P < .003) for midgut NETs. The same thresholds were found to be optimal for predicting death at 36 months, with an OR of 2.87 (P < .03) and 5.09 (P < .005) for PanNETs and midgut NETs, respectively. In multivariate Cox hazard regression analysis for PFS in PanNETs, 1 or greater CTC had a hazard ratio (HR) of 2.6 (P < .01), whereas 2 or greater CTCs had an HR of 2.25 (P < .01) in midgut NETs. In multivariate analysis OS in PanNETs, 1 or greater CTCs had an HR of 3.16 (P < .01) and in midgut NETs, 2 or greater CTCs had an HR of 1.73 (P < .06). CONCLUSIONS: The optimal CTC threshold to predict PFS and OS in metastatic PanNETs and midgut NETs is 1 and 2, respectively. These thresholds can be used to stratify patients in clinical practice and clinical trials.

Development of a novel patient focussed symptom severity index for use in assessing and treating inflammatory conditions of the lactating breast: a Delphi study.

AIM: To investigate the content and face validity of a patient-reported outcome measure used by Australian physiotherapists in the assessment of inflammatory conditions of the lactating breast. METHODS: Sixty one experts representing 'women who previously had inflammatory conditions of the lactating breast' (48%), 'clinicians' (38%) and 'academics' (8%) interested in women's health and 7% unidentified participants were invited to complete a three round Delphi study. RESULTS: Ninety five percent of participants agreed that overall, the patient-reported outcome measure was appropriate for use in assessing and treating inflammatory conditions of the lactating breast. The item 'impact' was added to ensure the appropriate assessment of functional aspects of daily life. The item terminology used in the patient-reported outcome measure was simplified to 'pain', 'redness', 'hardness/tightness', 'temperature of affected area', 'sickness/wellness' and 'affected area. A clinician script was developed to ensure the patient-reported outcome measure has utility and consistency regardless of the experience of the women presenting with inflammatory conditions of the lactating breast or the clinician. CONCLUSION: The resultant Breast Inflammatory Symptom Severity Index (BISSI) is a patient-reported outcome measure for use in the diagnosis and monitoring of clinical changes of symptoms associated with inflammatory conditions of the lactating breast including engorgement, blocked ducts and mastitis. It utilizes person-centred language and assesses symptoms considered important to both patient and clinician. The development of the clinician script facilitates utility regardless of the experience of either the woman with the inflammatory condition or the treating clinician.

VEROnA Protocol: A Pilot, Open-Label, Single-Arm, Phase 0, Window-of-Opportunity Study of Vandetanib-Eluting Radiopaque Embolic Beads (BTG-002814) in Patients With Resectable Liver Malignancies.

BACKGROUND: Transarterial chemoembolization (TACE) is the current standard of care for patients with intermediate-stage hepatocellular carcinoma (HCC) and is also a treatment option for patients with liver metastases from colorectal cancer. However, TACE is not a curative treatment, and tumor progression occurs in more than half of the patients treated. Despite advances and technical refinements of TACE, including the introduction of drug-eluting beads-TACE, the clinical efficacy of TACE has not been optimized, and improved arterial therapies are required. OBJECTIVE: The primary objectives of the VEROnA study are to evaluate the safety and tolerability of vandetanib-eluting radiopaque embolic beads (BTG-002814) in patients with resectable liver malignancies and to determine concentrations of vandetanib and the N-desmethyl metabolite in plasma and resected liver following treatment with BTG-002814. METHODS: The VEROnA study is a first-in-human, open-label, single-arm, phase 0, window-of-opportunity study of BTG-002814 (containing 100 mg vandetanib) delivered transarterially, 7 to 21 days before surgery in patients with resectable liver malignancies. Eligible patients have a diagnosis of colorectal liver metastases, or HCC (Childs Pugh A), diagnosed histologically or radiologically, and are candidates for liver surgery. All patients are followed up for 28 days following surgery. Secondary objectives of this study are to evaluate the anatomical distribution of BTG-002814 on noncontrast-enhanced imaging, to evaluate histopathological features in the surgical specimen, and to assess changes in blood flow on dynamic contrast-enhanced magnetic resonance imaging following treatment with BTG-002814. Exploratory objectives of this study are to study blood biomarkers with the potential to identify patients likely to respond to treatment and to correlate the distribution of BTG-002814 on imaging with pathology by 3-dimensional modeling. RESULTS: Enrollment for the study was completed in February 2019. Results of a planned interim analysis were reviewed by a safety committee after the first 3 patients completed follow-up. The recommendation of the committee was to continue the study without any changes to the dose or trial design, as there were no significant unexpected toxicities related to BTG-002814. CONCLUSIONS: The VEROnA study is studying the feasibility of administering BTG-002814 to optimize the use of this novel technology as liver-directed therapy for patients with primary and secondary liver cancer. TRIAL REGISTRATION: ClinicalTrial.gov NCT03291379; https://clinicaltrials.gov/ct2/show/NCT03291379. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/13696.

Circulating tumour cells and their association with bone metastases in patients with neuroendocrine tumours.

BACKGROUND: Bone metastases are associated with a worse outcome in patients with neuroendocrine tumours (NETs). Tumour overexpression of C-X-C chemokine receptor 4 (CXCR4) appears predictive of skeletal involvement. We investigated the role of circulating tumour cells (CTCs) and CXCR4 expression on CTCs as potential predictors of skeleton invasion. METHODS: Blood from patients with metastatic bronchial, midgut or pancreatic NET (pNET) was analysed by CellSearch. CXCR4 immunohistochemistry was performed on matched formalin-fixed paraffin-embedded (FFPE) samples. RESULTS: Two hundred and fifty-four patients were recruited with 121 midgut and 119 pNETs, of which 51 and 36% had detectable CTCs, respectively. Bone metastases were reported in 30% of midgut and 23% of pNET patients and were significantly associated with CTC presence (p = 0.003 and p < 0.0001). In a subgroup of 40 patients, 85% patients with CTCs had CTCs positive for CXCR4 expression. The proportion of CXCR4-positive CTCs in patients with bone metastases was 56% compared to 35% in those without (p = 0.18) it. Staining for CXCR4 on matched FFPE tissue showed a trend towards a correlation with CXCR4 expression on CTCs (p = 0.08). CONCLUSIONS: CTC presence is associated with bone metastases in NETs. CXCR4 may be involved in CTC osteotropism and present a therapeutic target to reduce skeletal morbidity.

Expression of somatostatin receptors 2 and 5 in circulating tumour cells from patients with neuroendocrine tumours.

BACKGROUND: Neuroendocrine tumours (NET) overexpress somatostatin receptors (SSTR) that can be targeted for therapy. Somatostatin receptor expression is routinely measured by molecular imaging but the resolution is insufficient to define heterogeneity. We hypothesised that SSTR expression could be measured on circulating tumour cells (CTCs) and used to investigate heterogeneity of expression and track changes during therapy. METHODS: MCF-7 cells were transfected with SSTR2 or 5 and spiked into donor blood for analysis by CellSearch. Optimum anti-SSTR antibody concentration and exposure time were determined, and flow cytometry was used to evaluate assay sensitivity. For clinical evaluation, blood was analysed by CellSearch, and SSTR2/5 immunohistochemistry was performed on matched tissue samples. RESULTS: Flow cytometry confirmed CellSearch was sensitive and that detection of SSTR was unaffected by the presence of somatostatin analogue up to a concentration of 100 ng ml-l. Thirty-one NET patients were recruited: grade; G1 (29%), G2 (45%), G3 (13%), primary site; midgut (58%), pancreatic (39%). Overall, 87% had SSTR-positive tumours according to somatostatin receptor scintigraphy or 68-Ga-DOTATE PET/CT. Circulating tumour cells were detected in 21 out of 31 patients (68%), of which 33% had evidence of heterogeneous expression of either SSTR2 (n=5) or SSTR5 (n=2). CONCLUSIONS: Somatostatin receptors 2 and 5 are detectable on CTCs from NET patients and may be a useful biomarker for evaluating SSTR-targeted therapies and this is being prospectively evaluated in the Phase IV CALMNET trial (NCT02075606).

GPs' perspectives on managing the NHS Health Check in primary care: a qualitative evaluation of implementation in one area of England.

OBJECTIVES: To evaluate the implementation of the National Health Service (NHS) Health Check programme in one area of England from the perspective of general practitioners (GPs). DESIGN: A qualitative exploratory study was conducted with GPs and other healthcare professionals involved in delivering the NHS Health Check and with patients. This paper reports the experience of GPs and focuses on the management of the Heath Check programme in primary care. SETTING: Primary care surgeries in the Heart of Birmingham region (now under the auspices of the Birmingham Cross City Clinical Commissioning Group) were invited to take part in the larger scale evaluation. This study focuses on a subset of those surgeries whose GPs were willing to participate. PARTICIPANTS: 9 GPs from different practices volunteered. GPs served an ethnically diverse region with areas of socioeconomic deprivation. Ethnicities of participant GPs included South Asian, South Asian British, white, black British and Chinese. METHODS: Individual semistructured interviews were conducted with GPs face to face or via telephone. Thematic analysis was used to analyse verbatim transcripts. RESULTS: Themes were generated which represent GPs' experiences of managing the NHS Health Check: primary care as a commercial enterprise; 'buy in' to concordance in preventive healthcare; following protocol and support provision. These themes represent the key issues raised by GPs. They reveal variability in the implementation of NHS Health Checks. GPs also need support in allocating resources to the Health Check including training on how to conduct checks in a concordant (or collaborative) way. CONCLUSIONS: The variability observed in this small-scale evaluation corroborates existing findings suggesting a need for more standardisation. Further large-scale research is needed to determine how that could be achieved. Work needs to be done to further develop a concordant approach to lifestyle advice which involves tailored individual goal setting rather than a paternalistic advice-giving model.

Enumeration and Molecular Characterisation of Circulating Tumour Cells in Endometrial Cancer.

BACKGROUND: This is a feasibility study to determine whether circulating tumour cells (CTCs) are detectable and suitable for molecular profiling in advanced endometrial cancer (aEC). METHOD: Between October 2012 and February 2014, 30 patients with aEC had baseline and up to 3 follow-up samples. CTCs and stathmin expression were evaluated using the CellSearch platform. Epithelial cell adhesion molecule (EpCAM) and stathmin immunohistochemistry were performed on FFPE tumour tissue. RESULTS: Eighteen from 30 (60%) patients had detectable CTCs during study [1 CTC (n = 7), 2 (n = 4), 3 (n = 1), 4 (n = 2), 7 (n = 1), 8 (n = 1), 22 (n = 1), 172 (n = 1) in 7.5 ml blood]. Ten from 18 patients had between 50 and 100% of detectable CTCs that were stathmin positive. More CTC-positive than CTC-negative patients had non-endometrioid versus endometrioid histology, tumour size ≥5 versus <5 cm, higher-stage disease and worse survival [hazard ratio 3.3, p > 0.05, 95% confidence interval 0.7-16.2]. Twenty-one tumour blocks were tested for EpCAM and stathmin immunohistochemistry (IHC). Stathmin tumour immunostaining scores (TIS) on IHC were higher in CTC-positive patients. CONCLUSION: CTC enumeration and molecular profiling with stathmin on the CellSearch platform is feasible in aEC. Stathmin TIS on IHC, a known prognostic marker in EC, was associated with CTC positivity.

Early Changes in Circulating Tumor Cells Are Associated with Response and Survival Following Treatment of Metastatic Neuroendocrine Neoplasms.

PURPOSE: To investigate posttreatment circulating tumor cell (CTC) counts in patients with neuroendocrine neoplasms (NENs) as a predictive biomarker for disease progression and overall survival (OS). EXPERIMENTAL DESIGN: Patients with metastatic NENs commencing therapy were prospectively recruited (n = 138). Blood samples were obtained for evaluation of CTCs using the CellSearch platform and for chromogranin A (CgA) at baseline, three to five (median, 4.3) weeks and 10 to 15 (median 13.7) weeks after commencing therapy. Radiologic response and OS data were collected. RESULTS: There was a significant association between first posttreatment CTC count and progressive disease (PD; P < 0.001). Only 8% of patients with a favorable "CTC response" (0 CTCs at baseline and 0 at first posttreatment time-point; or ≥50% reduction from baseline) had PD compared with 60% in the unfavorable group (<50% reduction or increase). Changes in CTCs were strongly associated with OS (P < 0.001), the best prognostic group being patients with 0 CTCs before and after therapy; followed by those with ≥50% reduction in CTCs [hazard ratio (HR), 3.31]; with those with a <50% reduction or increase in CTCs (HR, 5.07) having the worst outcome. In multivariate analysis, changes in CTCs had the strongest association with OS (HR, 4.13; P = 0.0002). Changes in CgA were not significantly associated with survival. CONCLUSIONS: Changes in CTCs are associated with response to treatment and OS in metastatic NENs, suggesting CTCs may be useful as surrogate markers to direct clinical decision making.

A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer.

We report here that a tetra-substituted naphthalene-diimide derivative (MM41) has significant in vivo anti-tumour activity against the MIA PaCa-2 pancreatic cancer xenograft model. IV administration with a twice-weekly 15 mg/kg dose produces ca 80% tumour growth decrease in a group of tumour-bearing animals. Two animals survived tumour-free after 279 days. High levels of MM41 are rapidly transported into cell nuclei and were found to accumulate in the tumour. MM41 is a quadruplex-interactive compound which binds strongly to the quadruplexes encoded in the promoter sequences of the BCL-2 and k-RAS genes, both of which are dis-regulated in many human pancreatic cancers. Levels of BCL-2 were reduced by ca 40% in tumours from MM41-treated animals relative to controls, consistent with BCL-2 being a target for MM41. Molecular modelling suggests that MM41 binds to a BCL-2 quadruplex in a manner resembling that previously observed in co-crystal structures with human telomeric quadruplexes. This supports the concept that MM41 (and by implication other quadruplex-targeting small molecules) can bind to quadruplex-forming promoter regions in a number of genes and down-regulate their transcription. We suggest that quadruplexes within those master genes that are up-regulated drivers for particular cancers, may be selective targets for compounds such as MM41.

Can children with hyperlipidemia receive ketogenic diet for medication-resistant epilepsy?

The very-high-fat ketogenic diet can worsen lipid levels in children with pre-existing hyperlipidemia by increasing serum lipoproteins and reducing antiatherogenic high-density lipoproteins. A retrospective chart review of 160 children treated with the ketogenic diet from September 2000 to May 2011 was performed. Twelve children with pre-existing hyperlipidemia were identified. Lipid levels including total cholesterol, low-density lipoprotein, triglycerides, high-density lipoprotein, and total cholesterol/high-density lipoprotein were measured pre-diet and at 3, 6, and 12 months of treatment. During treatment, there was a significant reduction in mean total cholesterol, low-density lipoprotein, and total cholesterol/high-density lipoprotein. Total cholesterol and low-density lipoprotein were normalized in 8 and 7 children at 6 months; and 9 and 9 children at 12 months respectively. At 6 and 12 months, tot cholesterol/HDL ratio was normalized in 5 and 7 children respectively. Diet modifications were made to achieve healthy lipid levels. By extrapolating the data, it suggests lipid levels can be controlled in children and adults with ketogenic diet treatment.

Evidence for different mechanisms of 'unhooking' for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples.

BACKGROUND: DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the 'unhooking' step is common to all ICLs. METHODS: Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. RESULTS: Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses. CONCLUSIONS: These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are 'unhooked' in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.