Predictive Factors for Local Control in Primary and Metastatic Lung Tumours after Four to Five Fraction Stereotactic Ablative Body Radiotherapy: A Single Institution's Comprehensive Experience

AIMS: We report the outcomes of a large lung stereotactic ablative body radiotherapy (SABR) programme for primary non-small cell lung cancer (NSCLC) and pulmonary metastases. The primary study aim was to identify factors predictive for local control.

MATERIALS AND METHODS: In total, 311 pulmonary tumours in 254 patients were treated between 2008 and 2011 with SABR using 48-60 Gy in four to five fractions. Local, regional and distant failure data were collected prospectively, whereas other end points were collected retrospectively. Potential clinical and dosimetric predictors of local control were evaluated using univariate and multivariate analyses.

RESULTS: Of the 311 tumours, 240 were NSCLC and 71 were other histologies. The 2 year local control rate was 96% in stage I NSCLC, 76% in colorectal cancer (CRC) metastases and 91% in non-lung/non-CRC metastases. Predictors of better local control on multivariate analysis were non-CRC tumours and a larger proportion of the planning target volume (PTV) receiving ≥100% of the prescribed dose (higher PTV V100). Among the 45 CRC metastases, a higher PTV V100 and previous chemotherapy predicted for better local control.

CONCLUSIONS: Lung SABR of 48-60 Gy/four to five fractions resulted in high local control rates for all tumours except CRC metastases. Covering more of the PTV with the prescription dose (a higher PTV V100) also resulted in superior local control.

The Impact of Colleague Peer Review on the Radiotherapy Treatment Planning Process in the Radical Treatment of Lung Cancer

AIMS: Modern radiotherapy uses techniques to reliably identify tumour and reduce target volume margins. However, this can potentially lead to an increased risk of geographic miss. One source of error is the accuracy of target volume delineation (TVD). Colleague peer review (CPR) of all curative-intent lung cancer plans has been mandatory in our institution since May 2013. At least two clinical oncologists review plans, checking treatment paradigm, TVD, prescription dose tumour and critical organ tolerances. We report the impact of CPR in our institution.

MATERIALS AND METHODS: Radiotherapy treatment plans of all patients receiving radical radiotherapy were presented at weekly CPR meetings after their target volumes were reviewed and signed off by the treating consultant. All cases and any resultant change to TVD (including organs at risk) or treatment intent were recorded in our prospective CPR database. The impact of CPR over a 13 month period from May 2013 to June 2014 is reported.

RESULTS: One hundred and twenty-two patients (63% non-small cell lung carcinoma, 17% small cell lung carcinoma and 20% 'clinical diagnosis') were analysed. On average, 3.2 cases were discussed per meeting (range 1-8). CPR resulted in a change in treatment paradigm in 3% (one patient proceeded to induction chemotherapy, two patients had high-dose palliative radiotherapy). Twenty-one (17%) had a change in TVD and one (1%) patient had a change in dose prescription. In total, 6% of patients had plan adjustment after review of dose volume histogram.

CONCLUSION: The introduction of CPR in our centre has resulted in a change in a component of the treatment plan for 27% of patients receiving curative-intent lung radiotherapy. We recommend CPR as a mandatory quality assurance step in the planning process of all radical lung plans.

Role played by BRCA1 in transcriptional regulation in response to therapy

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor, implicated in the hereditary predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of cellular processes including DNA repair and recombination, cell cycle checkpoint control, chromatin remodelling and ubiquitination. In addition, substantial data now exist to suggest a role for BRCA1 in transcriptional regulation; BRCA1 has been shown to interact with the Pol II holoenzyme complex and to interact with multiple transcription factors, such as p53 and c-Myc. We have previously identified a range of BRCA1 transcriptional targets and have linked these to specific cellular pathways, including cell cycle checkpoint activation and apoptosis. Current research is focused on the transcriptional mechanisms that underpin the association of BRCA1 deficiency with increased sensitivity to DNA damage-based chemotherapy and resistance to spindle poisons.

A modified method for measuring antibiotic use in healthcare settings: implications for antibiotic stewardship and benchmarking

Objectives: To determine whether adjusting the denominator of the common hospital antibiotic use measurement unit (defined daily doses/100 bed-days) by including age-adjusted comorbidity score (100 bed-days/age-adjusted comorbidity score) would result in more accurate and meaningful assessment of hospital antibiotic use. 

Methods: The association between the monthly sum of age-adjusted comorbidity and monthly antibiotic use was measured using time-series analysis (January 2008 to June 2012). For the purposes of conducting internal benchmarking, two antibiotic usage datasets were constructed, i.e. 2004-07 (first study period) and 2008-11 (second study period). Monthly antibiotic use was normalized per 100 bed-days and per 100 bed-days/age-adjusted comorbidity score. 

Results: Results showed that antibiotic use had significant positive relationships with the sum of age-adjusted comorbidity score (P = 0.0004). The results also showed that there was a negative relationship between antibiotic use and (i) alcohol-based hand rub use (P = 0.0370) and (ii) clinical pharmacist activity (P = 0.0031). Normalizing antibiotic use per 100 bed-days contributed to a comparative usage rate of 1.31, i.e. the average antibiotic use during the second period was 31% higher than during the first period. However, normalizing antibiotic use per 100 bed-days per age-adjusted comorbidity score resulted in a comparative usage rate of 0.98, i.e. the average antibiotic use was 2% lower in the second study period. Importantly, the latter comparative usage rate is independent of differences in patient density and case mix characteristics between the two studied populations. 

Conclusions: The proposed modified antibiotic measure provides an innovative approach to compare variations in antibiotic prescribing while taking account of patient case mix effects. 

Parasite neuropeptide biology:Seeding rational drug target selection?

The rationale for identifying drug targets within helminth neuromuscular signalling systems is based on the premise that adequate nerve and muscle function is essential for many of the key behavioural determinants of helminth parasitism, including sensory perception/host location, invasion, locomotion/orientation, attachment, feeding and reproduction. This premise is validated by the tendency of current anthelmintics to act on classical neurotransmitter-gated ion channels present on helminth nerve and/or muscle, yielding therapeutic endpoints associated with paralysis and/or death. Supplementary to classical neurotransmitters, helminth nervous systems are peptide-rich and encompass associated biosynthetic and signal transduction components - putative drug targets that remain to be exploited by anthelmintic chemotherapy. At this time, no neuropeptide system-targeting lead compounds have been reported, and given that our basic knowledge of neuropeptide biology in parasitic helminths remains inadequate, the short-term prospects for such drugs remain poor. Here, we review current knowledge of neuropeptide signalling in Nematoda and Platyhelminthes, and highlight a suite of 19 protein families that yield deleterious phenotypes in helminth reverse genetics screens. We suggest that orthologues of some of these peptidergic signalling components represent appealing therapeutic targets in parasitic helminths.

Extracellular DNA impedes the transport of vancomycin in Staphylococcus epidermidis biofilms pre-exposed to sub-inhibitory concentrations of vancomycin

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular D-Ala-D-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

Adjuvant bisphosphonate treatment in early breast cancer: meta-analyses of individual patient data from randomised trials

BACKGROUND: Bisphosphonates have profound effects on bone physiology, and could modify the process of metastasis. We undertook collaborative meta-analyses to clarify the risks and benefits of adjuvant bisphosphonate treatment in breast cancer.

METHODS: We sought individual patient data from all unconfounded trials in early breast cancer that randomised between bisphosphonate and control. Primary outcomes were recurrence, distant recurrence, and breast cancer mortality. Primary subgroup investigations were site of first distant recurrence (bone or other), menopausal status (postmenopausal [combining natural and artificial] or not), and bisphosphonate class (aminobisphosphonate [eg, zoledronic acid, ibandronate, pamidronate] or other [ie, clodronate]). Intention-to-treat log-rank methods yielded bisphosphonate versus control first-event rate ratios (RRs).

FINDINGS: We received data on 18 766 women (18 206 [97%] in trials of 2-5 years of bisphosphonate) with median follow-up 5·6 woman-years, 3453 first recurrences, and 2106 subsequent deaths. Overall, the reductions in recurrence (RR 0·94, 95% CI 0·87-1·01; 2p=0·08), distant recurrence (0·92, 0·85-0·99; 2p=0·03), and breast cancer mortality (0·91, 0·83-0·99; 2p=0·04) were of only borderline significance, but the reduction in bone recurrence was more definite (0·83, 0·73-0·94; 2p=0·004). Among premenopausal women, treatment had no apparent effect on any outcome, but among 11 767 postmenopausal women it produced highly significant reductions in recurrence (RR 0·86, 95% CI 0·78-0·94; 2p=0·002), distant recurrence (0·82, 0·74-0·92; 2p=0·0003), bone recurrence (0·72, 0·60-0·86; 2p=0·0002), and breast cancer mortality (0·82, 0·73-0·93; 2p=0·002). Even for bone recurrence, however, the heterogeneity of benefit was barely significant by menopausal status (2p=0·06 for trend with menopausal status) or age (2p=0·03), and it was non-significant by bisphosphonate class, treatment schedule, oestrogen receptor status, nodes, tumour grade, or concomitant chemotherapy. No differences were seen in non-breast cancer mortality. Bone fractures were reduced (RR 0·85, 95% CI 0·75-0·97; 2p=0·02).

INTERPRETATION: Adjuvant bisphosphonates reduce the rate of breast cancer recurrence in the bone and improve breast cancer survival, but there is definite benefit only in women who were postmenopausal when treatment began.

FUNDING: Cancer Research UK, Medical Research Council.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

PURPOSE: Some members of a novel series of pyrrolo-1,5-benzoxazepines (PBOXs) are microtubule-targeting agents capable of inducing apoptosis in a variety of human cancerous cells, hence, they are currently being developed as potential anti-cancer agents. The purpose of this study was to first characterise the activities of a novel PBOX analogue, PBOX-16 and then investigate the anti-angiogenic potential of both PBOX-16 and its prototype PBOX-6.

METHODS: The effects of PBOX-6 and -16 on cancerous cells (chronic myeloid leukaemia K562 cells and ovarian carcinoma A2780 cells) and primary cultured human umbilical vein endothelial cells (HUVECs) were examined by assessing cell proliferation, microtubular organisation, DNA analysis of cell cycle progression and caspase-3/7 activity. Their anti-angiogenic properties were then investigated by examining their ability to interfere with HUVEC differentiation into capillary-like structures and vascular endothelial growth factor (VEGF)-stimulated HUVEC migration.

RESULTS: PBOX-6 and -16 inhibited proliferation of K562, A2780 and HUVEC cells in a concentration-dependent manner. PBOX-16, confirmed as a novel depolymerising agent, was approximately tenfold more potent than PBOX-6. Inhibition of cell proliferation was mediated by G(2)/M arrest followed by varying degrees of apoptosis depending on the cell type; endothelial cells underwent less apoptosis than either of the cancer cell lines. In addition to the antitumourigenic properties, we also describe a novel antiangiogenic function for PBOXs: treatment with PBOXs inhibited the spontaneous differentiation of HUVECs into capillary-like structures when grown on a basement membrane matrix preparation (Matrigel™) and also significantly reduced VEGF-stimulated HUVEC migration.

CONCLUSION: Dual targeting of both the tumour cells and the host endothelial cells by PBOX compounds might enhance the anti-cancer efficacy of these drugs.

Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches

In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ-PCR) and 3-colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time-points during treatment (93.38%), and a combined use of both approaches allowed a multi time-point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0.01% were identified by RQ-PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ-PCR and FC approach, the evaluation of 367 follow-up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0.04), >0.01% at the end of induction (P = 0.02), >0.01% at the end of consolidation (P = 0.01), >0.01% prior to the first delayed intensification (P = 0.01), and >0.1% prior to the second delayed intensification and continued maintenance (P = 0.001) were all associated with relapse and, based on early time-points (end of induction and consolidation) a significant log-rank trend (P = 0.0091) was noted between survival curves for patients stratified into high, intermediate and low-risk MRD groups.

A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance, immune response, and development. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in variety of human chemotherapy resistant cancer cell lines, indicating their potential in the treatment of both solid tumors and tumors derived from the hemopoietic system. Pyrrolobenzoxazepine 4-acetoxy-5-(1-naphtyl)naphtho[2,3-b]pyrrolo[1,2-d][1,4]-oxazepine (PBOX-15) has been shown to depolymerize tubulin in vitro and in the MCF7 breast cancer cell line. We hypothesized that this may suggest a role for this compound in modulating integrin-induced T-cell migration, which is largely dependent on the microtubule dynamics. Experiments were performed using human T lymphoma cell line Hut78 and peripheral blood T-lymphocytes isolated from healthy donors. We observed that human T-lymphocytes exposed to PBOX-15 have severely impaired ability to polarize and migrate in response to the triggering stimulus generated via cross-linking of integrin lymphocyte function associated antigen-1 receptor. Here, we show that PBOX-15 can dramatically impair microtubule network via destabilization of tubulin resulting in complete loss of the motile phenotype of T-cells. We demonstrate that PBOX-15 inhibitory mechanisms involve decreased tubulin polymerization and its post-translational modifications. Novel microtubule-targeting effects of PBOX-15 can possibly open new horizons in the treatment of overactive inflammatory conditions as well as cancer and cancer metastatic spreading.

BubR1 is required for a sustained mitotic spindle checkpoint arrest in human cancer cells treated with tubulin-targeting pyrrolo-1,5-benzoxazepines

Intrinsic or acquired resistance to chemotherapy is a major clinical problem that has evoked the need to develop innovative approaches to predict and ultimately reverse drug resistance. A prolonged G(2)M arrest has been associated with apoptotic resistance to various microtubule-targeting agents (MTAs). In this study, we describe the functional significance of the mitotic spindle checkpoint proteins, BubR1 and Bub3, in maintaining a mitotic arrest after microtubule disruption by nocodazole and a novel series of MTAs, the pyrrolo-1,5-benzoxazepines (PBOXs), in human cancer cells. Cells expressing high levels of BubR1 and Bub3 (K562, MDA-MB-231, and HeLa) display a prolonged G(2)M arrest after exposure to MTAs. On the other hand, cells with low endogenous levels of mitotic spindle checkpoint proteins (SK-BR-3 and HL-60) transiently arrest in mitosis and undergo increased apoptosis. The phosphorylation of BubR1 correlated with PBOX-induced G(2)M arrest in four cell lines tested, indicating an active mitotic spindle checkpoint. Gene silencing of BubR1 by small interfering RNA interference reduced PBOX-induced G(2)M arrest without enhancing apoptotic efficacy. Further analysis demonstrated that PBOX-treated BubR1-depleted cells were both mononucleated and multinucleated with a polyploid DNA content, suggesting a requirement for BubR1 in cytokinesis. Taken together, these results suggest that BubR1 contributes to the mitotic checkpoint induced by the PBOXs.

Identification of tubulin as the molecular target of proapoptotic pyrrolo-1,5-benzoxazepines

We have demonstrated previously that certain members of a series of novel pyrrolo-1,5-benzoxazepine (PBOX) compounds potently induce apoptosis in a variety of human chemotherapy-resistant cancer cell lines and in primary ex vivo material derived from cancer patients. A better understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds is essential to their development as antineoplastic therapeutic agents. This study sought to test the hypothesis that proapoptotic PBOX compounds target the microtubules. We show that a representative proapoptotic PBOX compound, PBOX-6, induces apoptosis in both the MCF-7 and K562 cell lines. An accumulation of cells in G2/M precedes apoptosis in response to PBOX-6. PBOX-6 induces prometaphase arrest and causes an accumulation of cyclin B1 levels and activation of cyclin B1/CDK1 kinase in a manner similar to that of two representative antimicrotubule agents, nocodazole and paclitaxel. Indirect immunofluorescence demonstrates that both PBOX-6 and another pro-apoptotic PBOX compound, PBOX-15, cause microtubule depolymerization in MCF-7 cells. They also inhibit the assembly of purified tubulin in vitro, whereas a nonapoptotic PBOX compound (PBOX-21) has no effect on either the cellular microtubule network or on the assembly of purified tubulin. This suggests that the molecular target of the pro-apoptotic PBOX compounds is tubulin. PBOX-6 does not bind to either the vinblastine or the colchicine binding site on tubulin, suggesting that it binds to an as-yet-uncharacterised novel site on tubulin. The ability of PBOX-6 to bind tubulin and cause microtubule depolymerization confirms it as a novel candidate for antineoplastic therapy.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.