Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.

Background

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.

Materials and methods

We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual γH2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.

Results

Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of γH2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.

Conclusions

The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.

The effectiveness of radiotherapy treatment could be significantly improved if tumour cells could be rendered more sensitive to ionizing radiation without altering the sensitivity of normal tissues. However many of the key, therapeutically exploitable mechanisms that determine intrinsic tumour radiosensitivity are largely unknown. We have conducted a siRNA screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumour radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumour cells (SQ20B) and irradiated normal tissue cells (MRC5). Using γH2AX foci at 24 hours after ionising radiation we identified several genes such as BRCA2, Lig IV and XRCC5, whose knockdown is known to cause increased cell radiosensitivity thereby validating the primary screening endpoint. In addition we identified POLQ (DNA polymerase theta) as a potential tumour-specific target. Subsequent investigations demonstrated that POLQ knockdown resulted in radiosensitisation of a panel of tumour cell lines from different primary sites, whilst having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumour specific radiosensitisation.

Depletion of POLQ (DNA polymerase theta) has recently been shown to render tumour cells more sensitive to radiotherapy whilst having little or no effect on normal tissues. This finding led us to investigate whether tumours that overexpress POLQ are associated with an adverse outcome. We therefore correlated the clinical outcomes of two retrospective series of patients with early breast cancer with the expression levels of POLQ, as determined by microarray gene expression analysis. We found that a significant number of tumours overexpressed POLQ and that overexpression was correlated with ER negative disease (p=0.047) and high tumour grade (p=0.004), both of which are associated with poor clinical outcomes. POLQ overexpression was associated with poor relapse free survival rates on both univariate (HR 5.80; 95% CI, 2.220 to 15.159; p<0.001) and multivariate analysis (HR 8.086; 95% CI 2.340 to 27.948 p=0.001). Analysis of other published clinical series confirmed that POLQ overexpression is associated with adverse clinical outcomes. The poor prognosis associated with POLQ is independent of other clinical or pathological features. The mechanism that causes this adverse outcome remains to be elucidated but may in part arise from resistance to adjuvant treatment. These findings, combined with the limited normal tissue expression of POLQ, make it a very appealing target for possible clinical exploitation.

Depletion of POLQ (DNA polymerase theta) has recently been shown to render tumour cells more sensitive to radiotherapy whilst having little or no effect on normal tissues. This finding led us to investigate whether tumours that overexpress POLQ are associated with an adverse outcome. We therefore correlated the clinical outcomes of two retrospective series of patients with early breast cancer with the expression levels of POLQ, as determined by microarray gene expression analysis. We found that a significant number of tumours overexpressed POLQ and that overexpression was correlated with ER negative disease (p=0.047) and high tumour grade (p=0.004), both of which are associated with poor clinical outcomes. POLQ overexpression was associated with poor relapse free survival rates on both univariate (HR 5.80; 95% CI, 2.220 to 15.159; p<0.001) and multivariate analysis (HR 8.086; 95% CI 2.340 to 27.948 p=0.001). Analysis of other published clinical series confirmed that POLQ overexpression is associated with adverse clinical outcomes. The poor prognosis associated with POLQ is independent of other clinical or pathological features. The mechanism that causes this adverse outcome remains to be elucidated but may in part arise from resistance to adjuvant treatment. These findings, combined with the limited normal tissue expression of POLQ, make it a very appealing target for possible clinical exploitation.

Objective

To describe the characteristics of sudden arrhythmic death syndrome (SADS) and compare its incidence with official national mortality statistics for unascertained deaths.

Design and setting

Sudden unexplained deaths were prospectively surveyed through 117 coroners' jurisdictions in England. Consecutive cases meeting the following criteria were included: white Caucasian, aged 4–64 years, no history of cardiac disease, last seen alive within 12 h of death, normal coroner's autopsy, cardiac pathologist's confirmation of a normal heart and negative toxicology.

Main outcome measures

The estimated mortality from SADS was calculated and the official mortality statistics for unascertained causes of deaths in 4–64‐year‐olds was identified for the same time period.

Results

115 coroner's cases were reported and 56 (49%) SADS victims were identified: mean age 32 years, range 7–64 years and 35 (63%) male. 7 of 39 cases (18%) had a family history of other premature sudden deaths (<45). The estimated mortality from SADS was 0.16/100 000 per annum (95% CI 0.12 to 0.21), compared with an official mortality of 0.10/100 000 per annum for International Classification of Diseases 798.1 (sudden death, cause unknown—instantaneous death) or 1.34/100 000 per annum for unascertained causes of death.

Conclusions

Deaths from SADS occur predominantly in young males. When compared with official mortality, the incidence of SADS may be up to eight times higher than estimated: more than 500 potential SADS cases per annum in England. Families with SADS carry genetic cardiac disease, placing them at risk of further sudden deaths. SADS should therefore be a certifiable cause of death prompting specialised cardiological evaluation of families.

Many inhibitors of the EGFR-RAS-PI3 kinase-AKT signaling pathway are in clinical use or under development for cancer therapy. Here we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3 kinase or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor, but did respond with vascular alterations to RAS or PI3 Kinase inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3 kinase inhibition with decreased tumor hypoxia, increased vascular flow and morphological alterations of their vessels including increased vascular maturity and acquisition of pericyte markers. These changes are similar to the vascular normalization that has been described after anti-angiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetically determined heart muscle disorder presenting clinically with even lethal ventricular arrhythmias, particularly in the young and athletes. It is reported familial with recessive and most commonly dominant inheritance. Disease‐causing genes are increasingly recognised among desmosomal proteins plakoglobin, desmoplakin, plakophilin2, and desmoglein2 displaying phenotypic heterogeneity. Mutations in the plakoglobin and desmoplakin genes have been identified to underlie recessive ARVC associated with woolly hair and palmoplantar keratoderma (Naxos disease), while mutations in plakophilin2, desmoglein2 as well as desmoplakin have been identified to underlie the dominant non‐syndromic form. Preliminary genotype–phenotype assessment indicates that mutations affecting the outer dense plaque of desmosome (desmoglein2, plakoglobin, plakophilin2 and the N‐terminal of desmoplakin) result in ARVC with the ordinary described phenotype. However, mutations at the inner dense plaque, particularly affecting the desmin‐binding site of desmoplakin, may result in ARVC with predominantly left ventricular involvement and clinical overlapping with dilated cardiomyopathy. The interesting finding of abnormal distribution of plakoglobin, independently of the primarily affected protein, might suggest a common pathway for plakoglobin in ARVC pathogenesis.

Objective

To determine the range of survival rates of patients with hypertrophic cardiomyopathy (HCM) by comparing and contrasting the natural history of a cohort of patients seen between 1988 and 2002 with that of other published series.

Methods

956 adult (⩾ 16 years old) patients with HCM (572 men, mean (SD) age 42 (15) years, range 16–88) were evaluated by ECG, Holter, exercise testing, and echocardiography. Patient characteristics and survival data were compared with those in natural history studies from referral and non‐referral centres published between 1960 and January 2003.

Results

The duration of follow up was 69 (45) months. 120 (12.6%) patients died or underwent cardiac transplantation. Sudden cardiac death (n  =  48) was the most common mode of death. The annual rate of sudden death or implantable cardioverter‐defibrillator discharge was 1.02 (95% confidence interval (CI) 0.76 to 1.26). Annual rates for heart failure death or transplantation and stroke related death were 0.55% (95% CI 0.37% to 0.78%) and 0.07% (95% CI 0.02% to 0.19%), respectively. When studies published within the last 10 years of the study period were compared with earlier reports, the size of individual study cohorts was larger (309 (240.6) v 136.5 (98.8), p  =  0.058) and the proportion with severe functional limitation NYHA class III/IV lower (12.4% v 24.8%, p < 0.0001), and fewer patients underwent septal myotomy‐myectomy (5.2% v 18.7%, p < 0.0001). Published sudden death rates over the last 10 years were lower than previously published figures (median 1.0% (range 0.1–1.7) v 2.0% (0–3.5)).

Conclusion

Published survival rates in HCM cohorts have improved progressively over the past 40 years. In the modern era the prevalence of disease related complications is similar in all reporting centres.

Objective

To measure coronary flow reserve (CFR), an index of microvascular function, in Anderson‐Fabry disease (AFD) at baseline and after enzyme replacement therapy (ERT).

Methods and results

Mean (SD) myocardial blood flow (MBF) at rest and during hyperaemia (adenosine 140 μg/kg/min) was measured in 10 male, non‐smoking patients (53.8 (10.9) years, cholesterol 5.5 (1.3) mmol/l) and in 24 age matched male, non‐smoking controls (52.0 (7.6) years, cholesterol 4.5 (0.6) mmol/l) by positron emission tomography (PET). Resting and hyperaemic MBF and CFR (hyperaemic/resting MBF) were reduced in patients compared with controls (0.99 (0.17) v 1.17 (0.25) ml/g/min, p < 0.05; 1.37 (0.32) v 3.44 (0.78) ml/g/min, p < 0.0001; and 1.41 (0.39) v 3.03 (0.85), p < 0.0001, respectively). This coronary microvascular dysfunction was independent of cholesterol concentrations. PET was repeated in five patients after 10.1 (2.3) months of ERT; resting and hyperaemic MBF and CFR were unchanged after ERT (0.99 (0.16) v 0.99 (0.16) ml/g/min; 1.56 (0.29) v 1.71 (0.3) ml/g/min; and 1.6 (0.37) v 1.74 (0.28), respectively; all not significant).

Conclusions

The results of the present study show that patients with AFD have very abnormal coronary microvascular function. These preliminary data suggest that ERT has no effect on coronary microvascular dysfunction. Further work is necessary to determine whether treatment at an earlier stage in the course of the disease may improve coronary microvascular function in patients with AFD.

Objective: To examine the influence of genotype on late gadolinium enhancement (LGE) and the potential of cardiovascular magnetic resonance (CMR) to detect preclinical hypertrophic cardiomyopathy.

Design: Prospective, blinded cohort study of myocardial LGE in a genetically homogeneous population.

Patients: 30 patients with disease causing mutations in the recognised hypertrophic cardiomyopathy gene for cardiac troponin I (TNNI3): 15 with echocardiographically determined left ventricular hypertrophy (LVH+) and 15 without (LVH−).

Main outcome measures: CMR measures of regional left ventricular function, wall thickness, and mass, and the extent and distribution of LGE.

Results: LGE was found in 12 (80%) LVH+ patients but with variable extent (mean 15%, range 3–48%). LGE was also found in two (13%) LVH− patients but the extent was limited (3.6%) and both patients were found to have an abnormal ECG and regional hypertrophy by cine CMR. The extent of LGE was positively associated with clinical markers of sudden death risk (21% with ⩾ 2 risk factors v 7% with ⩽ 1 risk factor, p  =  0.02) and left ventricular mass (r  =  0.56, p < 0.001) and was inversely associated with ejection fraction (r  =  −0.58, p < 0.001). Segmental analysis showed that as regional wall thickness increased, LGE was more prevalent (p < 0.0001) and more extensive (r  =  0.98, p  =  0.001).

Conclusion: In patients with disease causing mutations in TNNI3, focal fibrosis was not detected by LGE CMR before LVH and ECG abnormalities were present. Once LVH is present, LGE is common and the extent correlates with adverse clinical parameters. This suggests that focal fibrosis is closely linked to disease development.

Objectives: To determine the frequency of systolic impairment (SI) and its impact on the natural history of hypertrophic cardiomyopathy (HCM).

Methods: 1080 patients (mean (SD) age 43 (15) years, 660 men) with HCM were evaluated. Initial assessment included history, examination, 48 hour Holter monitoring, cardiopulmonary exercise testing, and echocardiography; SI was defined as a fractional shortening (FS) ⩽ 25%. Survival data were collected at clinic visits or by direct communication with patients and their general practitioners. The results of serial echocardiography in 462 patients with normal FS at presentation are also reported.

Results: 26 (2.4%) patients (49 (14) years, 18 men) had SI at the initial visit. During follow up (58 (49) months), nine (34.6%) died or underwent cardiac transplantation compared with 108 (10.2%) patients with normal FS (p  =  0.01). Five year survival from death (any cause) or transplantation was 90.1% (95% confidence interval (CI) 87.8 to 92.4) in patients with normal systolic function versus 52.4% (95% CI 25.2 to 79.6, p < 0.0001) in patients with SI. In patients who underwent serial echocardiography, 22 (4.8%, aged 41 (15) years) developed SI over 66 (40) months; the annual incidence of SI was 0.87% (95% CI 0.54 to 1.31). On initial evaluation patients who developed SI had a higher frequency of syncope (67 (15.2%) v 10 (45.5%) of those who did not develop SI, p  =  0.001), non-sustained ventricular tachycardia (91 (20.6%) v 11 (50%), p  =  0.002), and an abnormal blood pressure response on exercise (131 (29.7%) v 15 (68.2%), p  =  0.001). Patients with SI had greater wall thinning (p  =  0.001), left ventricular cavity enlargement (p < 0.0005), and deterioration in New York Heart Association functional class (p  =  0.001) during follow up. Thirteen (59.1%) patients who progressed to SI died or underwent transplantation compared with 38 (8.6%) patients who maintained normal systolic function.

Conclusions: SI is an infrequent complication of HCM but, when present, is associated with a poor prognosis.

Objectives: To define physiological upper limits of left ventricular (LV) cavity size in trained adolescent athletes.

Design: Cross sectional echocardiographic study.

Setting: British national sports training grounds and Olympic Medical Institute.

Subjects: 900 elite adolescent athletes (77% boys) aged 15.7 (1.2) years participating in ball, racket, and endurance sports and 250 healthy controls matched for age, sex, and size.

Main outcome measures: LV end diastolic cavity size.

Results: Compared with controls, athletes had a larger LV cavity (50.8 (3.7) v 47.9 (3.5) mm), a difference of 6%. The LV cavity was > 54 mm in 18% athletes, whereas none of the controls had an LV cavity > 54 mm. The LV cavity exceeded predicted sizes in 117 (13%) athletes. Among the athletes with LV dilatation, 78% were boys, LV size ranged from 52–60 mm, and left atrial diameter and LV wall thickness were enlarged. Systolic and diastolic function were normal. None of the athletes in the study had an LV cavity size > 60 mm. LV cavity size correlated with age, sex, heart rate, and body surface area.

Conclusion: Highly trained junior athletes usually have only modest increases in LV cavity size. A proportion of trained adolescent athletes have LV cavity size exceeding predicted values but, in absolute terms, LV cavity rarely exceeds 60 mm as in patients with dilated cardiomyopathy. In highly trained adolescent athletes with an LV cavity size > 60 mm and any impairment of systolic or diastolic function, the diagnosis of dilated cardiomyopathy should be considered.

Objective: To investigate the role of cardiovascular magnetic resonance (CMR) in a series of patients with ECG repolarisation changes and normal echocardiography.

Patients and design: 10 patients with anterolateral T wave inversion for which there was no obvious pathological cause who had normal routine echocardiography without contrast for the exclusion of hypertrophic cardiomyopathy (HCM) also had CMR that was diagnostic of apical HCM.

Results: Apical HCM detected by CMR could be morphologically severe with wall thickness up to 28 mm, or mild. The extent of repolarisation abnormalities did not correlate to the morphological severity.

Conclusions: In patients with unexplained repolarisation abnormalities, a normal routine echocardiogram without contrast does not exclude apical HCM. Further imaging with CMR or contrast echocardiography may be required. The reliance on routine echocardiography to exclude apical HCM may have led to underreporting of this condition.

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor α was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

Objectives: To determine whether pregnancy is well tolerated in hypertrophic cardiomyopathy.

Setting: Referral clinic.

Design: The study cohort comprised 127 consecutively referred women with hypertrophic cardiomyopathy. Forty (31.5%) underwent clinical evaluation before pregnancy. The remaining 87 (68.5%) were referred after their first pregnancy. All underwent history, examination, electrocardiography, and echocardiography. Pregnancy related symptoms and complications were determined by questionnaire and review of medical and obstetric records where available.

Results: There were 271 pregnancies in total. Thirty six (28.3%) women reported cardiac symptoms in pregnancy. Over 90% of these women had been symptomatic before pregnancy. Symptoms deteriorated during pregnancy in fewer than 10%. Of the 36 women with symptoms during pregnancy, 30 had further pregnancies. Symptoms reoccurred in 18 (60%); symptomatic deterioration was not reported. Heart failure occurred postnatally in two women (1.6%). No complications were reported in 19 (15%) women who underwent general anaesthesia and in 22 (17.4%) women who received epidural anaesthesia, three of whom had a significant left ventricular outflow tract gradient at diagnosis after pregnancy. Three unexplained intrauterine deaths occurred in women taking cardiac medication throughout pregnancy. No echocardiographic or clinical feature was a useful indicator of pregnancy related complications.

Conclusions: Most women with hypertrophic cardiomyopathy tolerate pregnancy well. However, rare complications can occur and therefore planned delivery and fetal monitoring are still required for some patients.

The majority of sudden deaths in young athletes occur in the context of underlying inherited or genetic cardiac disorders. The evaluation of every athlete regarding underlying cardiac disease is impractical and therefore needs to be targeted at those who are at a higher risk. A practical approach would be to channel efforts towards athletes with cardiac symptoms, those with a family history of inherited cardiac disease, and those with a family history of premature sudden death. There are potential pitfalls in the evaluation of young athletes using non-invasive tests when making the distinction between physiological adaptations to exercise and cardiac pathology. Physicians evaluating young athletes need to be aware of the spectrum of physiological adaptations and to be familiar with conditions responsible for sudden death in this population.