David Lawrence

Spatial and temporal diversity in genomic instability processes defines lung cancer evolution

Spatial and temporal dissection of the genomic changes occurring during the evolution of human non–small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis. We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC. Different regions of a human lung tumor harbor different mutations, possibly explaining why the disease is so tough to treat. [Also see Perspective by Govindan] Space, time, and the lung cancer genome Lung cancer poses a formidable challenge to clinical oncologists. It is often detected at a late stage, and most therapies work for only a short time before the tumors resume their relentless growth. Two independent analyses of the human lung cancer genome may help explain why this disease is so resilient (see the Perspective by Govindan). Rather than take a single “snapshot” of the cancer genome, de Bruin et al. and Zhang et al. identified genomic alterations in spatially distinct regions of single lung tumors and used this information to infer the tumors evolutionary history. Each tumor showed tremendous spatial and temporal diversity in its mutational profiles. Thus, the efficacy of drugs may be short-lived because they destroy only a portion of the tumor. Science, this issue p. 251, p. 256; see also p. 169

Remote ischaemic preconditioning reduces myocardial injury in patients undergoing cardiac surgery with cold-blood cardioplegia: a randomised controlled trial

Background: Remote ischaemic preconditioning (RIPC) induced by brief ischaemia and reperfusion of the arm reduces myocardial injury in coronary artery bypass (CABG) surgery patients receiving predominantly cross-clamp fibrillation for myocardial protection. However, cold-blood cardioplegia is the more commonly used method world wide. Objective: To assess whether RIPC is cardioprotective in CABG patients receiving cold-blood cardioplegia. Design: Single-centre, single-blinded, randomised controlled trial. Setting: Tertiary referral hospital in London. Patients: Adults patients (18–80 years) undergoing elective CABG surgery with or without concomitant aortic valve surgery with cold-blood cardioplegia. Patients with diabetes, renal failure (serum creatinine >130 mmol/l), hepatic or pulmonary disease, unstable angina or myocardial infarction within the past 4 weeks were excluded. Interventions: Patients were randomised to receive either RIPC (n = 23) or control (n = 22) after anaesthesia. RIPC comprised three 5 min cycles of right forearm ischaemia, induced by inflating a blood pressure cuff on the upper arm to 200 mm Hg, with an intervening 5 min reperfusion. The control group had a deflated cuff placed on the upper arm for 30 min. Main outcome measures: Serum troponin T was measured preoperatively and at 6, 12, 24, 48 and 72 h after surgery and the area under the curve (AUC at 72 h) calculated. Results: RIPC reduced absolute serum troponin T release by 42.4% (mean (SD) AUC at 72 h: 31.53 (24.04) μg/l.72 h in controls vs 18.16 (6.67) μg/l.72 h in RIPC; 95% CI 2.4 to 24.3; p = 0.019). Conclusions: Remote ischaemic preconditioning induced by brief ischaemia and reperfusion of the arm reduces myocardial injury in CABG surgery patients undergoing cold-blood cardioplegia, making this non-invasive cardioprotective technique widely applicable clinically. Trial registration number: NCT00397163.

PHASE III PILOT STUDY OF DOSE ESCALATION USING CONFORMAL RADIOTHERAPY IN PROSTATE CANCER: PSA CONTROL AND SIDE EFFECTS

Radical radiotherapy is a standard form of management of localised prostate cancer. Conformal treatment planning spares adjacent normal tissues reducing treatment-related side effects and may permit safe dose escalation. We have tested the effects on tumour control and side effects of escalating radiotherapy dose and investigated the appropriate target volume margin. After an initial 3–6 month period of androgen suppression, 126 men were randomised and treated with radiotherapy using a 2 by 2 factorial trial design. The initial radiotherapy tumour target volume included the prostate and base of seminal vesicles (SV) or complete SV depending on SV involvement risk. Treatments were randomised to deliver a dose of 64 Gy with either a 1.0 or 1.5 cm margin around the tumour volume (1.0 and 1.5 cm margin groups) and also to treat either with or without a 10 Gy boost to the prostate alone with no additional margin (64 and 74 Gy groups). Tumour control was monitored by prostate-specific antigen (PSA) testing and clinical examination with additional tests as appropriate. Acute and late side effects of treatment were measured using the Radiation Treatment and Oncology Groups (RTOG) and LENT SOM systems. The results showed that freedom from PSA failure was higher in the 74 Gy group compared to the 64 Gy group, but this did not reach conventional levels of statistical significance with 5-year actuarial control rates of 71% (95% CI 58–81%) in the 74 Gy group vs 59% (95% CI 45–70%) in the 64 Gy group. There were 23 failures in the 74Gy group and 33 in the 64 Gy group (Hazard ratio 0.64, 95% CI 0.38–1.10, P=0.10). No difference in disease control was seen between the 1.0 and 1.5 cm margin groups (5-year actuarial control rates 67%, 95% CI 53–77% vs 63%, 95% CI 50–74%) with 28 events in each group (Hazard ratio 0.97, 95% CI 0.50–1.86, P=0.94). Acute side effects were generally mild and 18 weeks after treatment, only four and five of the 126 men had persistent ⩾Grade 1 bowel or bladder side effects, respectively. Statistically significant increases in acute bladder side effects were seen after treatment in the men receiving 74 Gy (P=0.006), and increases in both acute bowel side effects during treatment (P=0.05) and acute bladder sequelae (P=0.002) were recorded for men in the 1.5 cm margin group. While statistically significant, these differences were of short duration and of doubtful clinical importance. Late bowel side effects (RTOG⩾2) were seen more commonly in the 74 Gy and 1.5 cm margin groups (P=0.02 and P=0.05, respectively) in the first 2 years after randomisation. Similar results were found using the LENT SOM assessments. No significant differences in late bladder side effects were seen between the randomised groups using the RTOG scoring system. Using the LENT SOM instrument, a higher proportion of men treated in the 74 Gy group had Grade ⩾3 urinary frequency at 6 and 12 months. Compared to baseline scores, bladder symptoms improved after 6 months or more follow-up in all groups. Sexual function deteriorated after treatment with the number of men reporting some sexual dysfunction (Grade⩾1) increasing from 38% at baseline to 66% at 6 months and 1 year and 81% by year 5. However, no consistent differences were seen between the randomised groups. In conclusion, dose escalation from 64 to 74 Gy using conformal radiotherapy may improve long-term PSA control, but a treatment margin of 1.5 cm is unnecessary and is associated with increased acute bowel and bladder reactions and more late rectal side effects. Data from this randomised pilot study informed the Data Monitoring Committee of the Medical Research Council RT 01 Trial and the two studies will be combined in subsequent meta-analysis.

Tracking the Evolution of Non–Small-Cell Lung Cancer

BACKGROUND Among patients with non‐small‐cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early‐stage NSCLC. METHODS In this prospective cohort study, we performed multiregion whole‐exome sequencing on 100 early‐stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence‐free survival. RESULTS We observed widespread intratumor heterogeneity for both somatic copy‐number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy‐number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy‐number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10‐4), which remained significant in multivariate analysis. CONCLUSIONS Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601.)

Effect of remote ischaemic preconditioning on clinical outcomes in patients undergoing cardiac bypass surgery: a randomised controlled clinical trial

Objectives Remote ischaemic preconditioning (RIPC), using brief cycles of limb ischaemia/reperfusion, is a non-invasive, low-cost intervention that may reduce perioperative myocardial injury (PMI) in patients undergoing cardiac surgery. We investigated whether RIPC can also improve short-term clinical outcomes. Methods One hundred and eighty patients undergoing elective coronary artery bypass graft (CABG) surgery and/or valve surgery were randomised to receive either RIPC (2–5 min cycles of simultaneous upper arm and thigh cuff inflation/deflation; N=90) or control (uninflated cuffs placed on the upper arm and thigh; N=90). The study primary end point was PMI, measured by 72 h area under the curve (AUC) serum high-sensitive troponin-T (hsTnT); secondary end point included short-term clinical outcomes. Results RIPC reduced PMI magnitude by 26% (−9.303 difference (CI −15.618 to −2.987) 72 h hsTnT-AUC; p=0.003) compared with control. There was also evidence that RIPC reduced the incidence of postoperative atrial fibrillation by 54% (11% RIPC vs 24% control; p=0.031) and decreased the incidence of acute kidney injury by 48% (10.0% RIPC vs 21.0% control; p=0.063), and intensive care unit stay by 1 day (2.0 days RIPC (CI 1.0 to 4.0) vs 3.0 days control (CI 2.0 to 4.5); p=0.043). In a post hoc analysis, we found that control patients administered intravenous glyceryl trinitrate (GTN) intraoperatively sustained 39% less PMI compared with those not receiving GTN, and RIPC did not appear to reduce PMI in patients given GTN. Conclusions RIPC reduced the extent of PMI in patients undergoing CABG and/or valve surgery. RIPC may also have beneficial effects on short-term clinical outcomes, although this will need to be confirmed in future studies. Trial registration number ClinicalTrials.gov ID: NCT00397163.

Lung cancer diagnosis and staging with endobronchial ultrasound-guided transbronchial needle aspiration compared with conventional approaches: an open-label, pragmatic, randomised controlled trial

Summary Background The diagnosis and staging of lung cancer is an important process that identifies treatment options and guides disease prognosis. We aimed to assess endobronchial ultrasound-guided transbronchial needle aspiration as an initial investigation technique for patients with suspected lung cancer. Methods In this open-label, multicentre, pragmatic, randomised controlled trial, we recruited patients who had undergone a CT scan and had suspected stage I to IIIA lung cancer, from six UK centres and randomly assigned them to either endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) or conventional diagnosis and staging (CDS), for further investigation and staging. If a target node could not be accessed by EBUS-TBNA, then endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) was allowed as an alternative procedure. Randomisation was stratified according to the presence of mediastinal lymph nodes measuring 1 cm or more in the short axis and by recruiting centre. We used a telephone randomisation method with permuted blocks of four generated by a computer. Because of the nature of the intervention, masking of participants and consenting investigators was not possible. The primary endpoint was the time-to-treatment decision after completion of the diagnostic and staging investigations and analysis was by intention-to-diagnose. This trial is registered with ClinicalTrials.gov, number NCT00652769. Findings Between June 10, 2008, and July 4, 2011, we randomly allocated 133 patients to treatment: 66 to EBUS-TBNA and 67 to CDS (one later withdrew consent). Two patients from the EBUS-TBNA group underwent EUS-FNA. The median time to treatment decision was shorter with EBUS-TBNA (14 days; 95% CI 14–15) than with CDS (29 days; 23–35) resulting in a hazard ratio of 1·98, (1·39–2·82, p<0·0001). One patient in each group had a pneumothorax from a CT-guided biopsy sample; the patient from the CDS group needed intercostal drainage and was admitted to hospital. Interpretation Transbronchial needle aspiration guided by endobronchial ultrasound should be considered as the initial investigation for patients with suspected lung cancer, because it reduces the time to treatment decision compared with conventional diagnosis and staging techniques. Funding UK Medical Research Council.

Non-randomised phase II trial of hyperbaric oxygen therapy in patients with chronic arm lymphoedema and tissue fibrosis after radiotherapy for early breast cancer

BACKGROUND Radiation-induced arm lymphoedema is a common and distressing complication of curative treatment for early breast cancer. Hyperbaric oxygen (HBO(2)) therapy promotes healing in bone rendered ischaemic by radiotherapy, and may help some soft-tissue injuries too, but is untested in arm lymphoedema. METHODS Twenty-one eligible research volunteers with a minimum 30% increase in arm volume in the years after axillary/supraclavicular radiotherapy (axillary surgery in 18/21 cases) were treated with HBO(2). The volunteers breathed 100% oxygen at 2.4 ATA for 100 min in a multiplace hyperbaric chamber on 30 occasions over a period of 6 weeks. The volume of the ipsilateral limb, measured opto-electronically by a perometer and expressed as a percentage of contralateral limb volume, was selected as the primary endpoint. A secondary endpoint was local lymph drainage expressed as fractional removal rate of radioisotopic tracer, measured using lymphoscintigraphy. RESULTS Three out of 19 evaluable patients experienced >20% reduction in arm volume at 12 months. Six out of 13 evaluable patients experienced a >25% improvement in (99)Tc-nanocolloid clearance rate from the ipsilateral forearm measured by quantitative lymphoscintigraphy at 12 months. Overall, there was a statistically significant, but clinically modest, reduction in ipsilateral arm volume at 12 months follow-up compared with baseline (P = 0.005). The mean percentage reduction in arm volume from baseline at 12 months was 7.51. Moderate or marked lessening of induration in the irradiated breast, pectoral fold and/or supraclavicular fossa was recorded clinically in 8/15 evaluable patients. Twelve out of 19 evaluable patients volunteered that their arms felt softer, and six reported improvements in shoulder mobility at 12 months. No significant improvements were noted in patient self-assessments of quality of life. CONCLUSION Interpretation is limited by the absence of a control group. However, measurement of limb volume by perometry is reportedly reliable, and lymphoscintigraphy is assumed to be operator-independent. Taking all data into account, there is sufficient evidence to justify a double-blind randomised controlled trial of hyperbaric oxygen in this group of patients.

The divergent roles of protein kinase C epsilon and delta in simulated ischaemia–reperfusion injury in human myocardium

Experimental studies suggest that cardioprotection can be achieved through either the activation of PKC-epsilon prior to the index ischaemic episode or the inhibition of PKC-delta at the onset of reperfusion. However, whether these PKC isoforms exert such divergent roles in human myocardium, subjected to simulated ischaemia-reperfusion injury, is unclear. Human atrial trabeculae were isolated from right atrial appendages harvested from patients undergoing elective cardiac surgery. These were subjected to 90 min of hypoxia followed by 120 min of reoxygenation, at the end of which the recovery of baseline contractile function was determined. Atrial trabeculae were randomised to receive various treatment protocols comprising a peptide activator of PKC-epsilon, a peptide inhibitor of PKC-delta and their respective inactive control peptides. Administering the PKC-delta peptide inhibitor at reoxygenation improved the recovery of function at all the concentrations tested (39.3+/-1.4% at 5 nM, 52.4+/-2.9% at 50 nM and 46.8+/-2.9% at 500 nM versus the control group, 27.5+/-1.4%: N > or = 6/group: P<0.02). Preconditioning with the PKC-epsilon peptide activator improved the recovery of function (40.0+/-0.8% at 50 nM and 49.7+/-3.1% at 500 nM versus the control group 27.5+/-1.4%: N > or = 6/group: P<0.02). This cardioprotective effect was comparable to that achieved by a standard hypoxic preconditioning protocol (52.3+/-3.2%). Interestingly, administering the PKC-epsilon activator (500 nM) at the onset of reperfusion also improved the recovery of contractile function (40.7+/-2.1% versus 27.5+/-1.5%: N > or = 6/group: P < 0.05). In human myocardium, cardioprotection can be achieved by either inhibiting PKC-delta or activating PKC-epsilon at the onset of reperfusion. In addition, PKC-epsilon activation offers cardioprotection when administered as a preconditioning strategy.

Assessment of two novel ventilatory surrogates for use in the delivery of gated/tracked radiotherapy for non-small cell lung cancer

BACKGROUND In selected patients with NSCLC the therapeutic index of radical radiotherapy can be improved with gating/tracking technology. Both techniques require real-time information on target location. This is often derived from a surrogate ventilatory signal. We assessed the correlation of two novel surrogate ventilatory signals with a spirometer-derived signal. The novel signals were obtained using the VisionRT stereoscopic camera system. The VisionRT-Tracked-Point (VRT-TP) signal was derived from tracking a point located midway between the umbilicus and xiphisternum. The VisionRT-Surface-Derived-Volume (VRT-SDV) signal was derived from 3D body surface imaging of the torso. Both have potential advantages over the current surrogate signals. METHODS Eleven subjects with NSCLC were recruited. Each was positioned as for radiotherapy treatment, and then instructed to breathe in five different modes: normal, abdominal, thoracic, deep and shallow breathing. Synchronous ventilatory signals were recorded for later analysis. The signals were analysed for correlation across all modes of breathing, and phase shifts. The VRT-SDV was also assessed for its ability to determine the mode of breathing. RESULTS Both novel respiratory signals showed good correlation (r>0.80) with spirometry in 9 of 11 subjects. For all subjects the correlation with spirometry was better for the VRT-SDV signal than for the VRT-TP signal. Only one subject displayed a phase shift between the VisionRT-derived signals and spirometry. The VRT-SDV signal could also differentiate between different modes of breathing. Unlike the spirometer-derived signal, neither VisionRT-derived signal was subject to drift. CONCLUSION Both the VRT-TP and VRT-SDV signals have potential applications in ventilatory-gated and tracked radiotherapy. They can also be used as a signal for sorting 4DCT images, and to drive 4DCT single- and multiple-parameter motion models.

Thoracic empyema: a 12-year study from a UK tertiary cardiothoracic referral centre.

Background Empyema is an increasingly frequent clinical problem worldwide, and has substantial morbidity and mortality. Our objectives were to identify the clinical, surgical and microbiological features, and management outcomes, of empyema. Methods A retrospective observational study over 12 years (1999–2010) was carried out at The Heart Hospital, London, United Kingdom. Patients with empyema were identified by screening the hospital electronic ‘Clinical Data Repository’. Demographics, clinical and microbiological characteristics, underlying risk factors, peri-operative blood tests, treatment and outcomes were identified. Univariable and multivariable statistical analyses were performed. Results Patients (n = 406) were predominantly male (74.1%); median age = 53 years (IQR = 37–69). Most empyema were community-acquired (87.4%) and right-sided (57.4%). Microbiological diagnosis was obtained in 229 (56.4%) patients, and included streptococci (16.3%), staphylococci (15.5%), Gram-negative organisms (8.9%), anaerobes (5.7%), pseudomonads (4.4%) and mycobacteria (9.1%); 8.4% were polymicrobial. Most (68%) cases were managed by open thoracotomy and decortication. Video-assisted thoracoscopic surgery (VATS) reduced hospitalisation from 10 to seven days (P = 0.0005). All-cause complication rate was 25.1%, and 28 day mortality 5.7%. Predictors of early mortality included: older age (P = 0.006), major co-morbidity (P = 0.01), malnutrition (P = 0.001), elevated red cell distribution width (RDW, P<0.001) and serum alkaline phosphatase (P = 0.004), and reduced serum albumin (P = 0.01) and haemoglobin (P = 0.04). Conclusions Empyema remains an important cause of morbidity and hospital admissions. Microbiological diagnosis was only achieved in just over 50% of cases, and tuberculosis is a notable causative organism. Treatment of empyema with VATS may reduce duration of hospital stay. Raised RDW appears to associate with early mortality.