Longitudinal copy number, whole exome and targeted deep sequencing of 'good risk' IGHV-mutated CLL patients with progressive disease.

The biological features of IGHV-M chronic lymphocytic leukemia responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in 13 patients presenting with cMBL or Stage A disease and good-risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom 10 have required therapy. Using cytogenetics, fluorescence in situ hybridisation, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing at diagnosis, we identified mutations in established chronic lymphocytic leukemia driver genes in nine patients (69%), non-coding mutations (PAX5 enhancer region) in three patients and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition before therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low-risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good-risk disease.

Evaluation of the Edmonton Functional Assessment Tool (EFAT2) within palliative care: a pilot study.

BACKGROUND: There is increasing need for rehabilitation in both cancer and palliative care. However, there are few validated outcome measures that are suitable for measuring functional performance in this population. The present study evaluated the validity, sensitivity and reliability of the Edmonton Functional Assessment Tool (EFAT2) within a UK palliative care setting. METHODS: Eleven participants aged 65 years and over (mean age 76.5 ± 6.7 years) receiving rehabilitation in a palliative care inpatient setting were studied. Concurrent validity was assessed by comparing EFAT2 with the Barthel Index. Inter-rater reliability of EFAT2 was examined using a sample of four participants recruited from a cancer care ward. RESULTS: A significant negative correlation was observed between the Barthel Index and EFAT2 (r = -0.765, p = 0.01) and both measures were found to be sensitive as determined by Cohen's effect size (EFAT2 = 0.60, Barthel Index = 0.72). High inter-rater reliability was noted for EFAT2 (ICC3, 1 = 0.85) and the agreement between scores was confirmed by Bland-Altman analysis. CONCLUSIONS: EFAT2 showed concurrent validity with the Barthel Index when used to assess the effects of rehabilitation on participants with advanced cancer. The tool was sensitive to change and was found to be reliable when used by different raters. The findings indicate that EFAT2 might be an appropriate outcome measure to use within the palliative care setting. However, the feasibility of using EFAT2 needs to be explored and larger studies are required to confirm its reliability.

Randomized controlled trial of patient-controlled sedation for colonoscopy: Entonox vs modified patient-maintained target-controlled propofol.

AIM: Propofol sedation is often associated with deep sedation and decreased manoeuvrability. Patient-maintained sedation has been used in such patients with minimal side-effects. We aimed to compare novel modified patient-maintained target-controlled infusion (TCI) of propofol with patient-controlled Entonox inhalation for colonoscopy in terms of analgesic efficacy (primary outcome), depth of sedation, manoeuvrability and patient and endoscopist satisfaction (secondary outcomes). METHOD: One hundred patients undergoing elective colonoscopy were randomized to receive either TCI propofol or Entonox. Patients in the propofol group were administered propofol initially to achieve a target concentration of 1.2 µg/ml and then allowed to self-administer a bolus of propofol (200 µg/kg/ml) using a patient-controlled analgesia pump with a handset. Entonox group patients inhaled the gas through a mouthpiece until caecum was reached and then as required. Sedation was initially given by an anaesthetist to achieve a score of 4 (Modified Observer's Assessment of Alertness and Sedation Scale), and colonoscopy was then started. Patients completed an anxiety score (Hospital Anxiety and Depression questionnaire), a baseline letter cancellation test and a pain score on a 100-mm visual analogue scale before and after the procedure. All patients completed a satisfaction survey at discharge and 24 h postprocedure. RESULTS: The median dose of propofol was 174 mg, and the median number of propofol boluses was four. There was no difference between the two groups in terms of pain recorded (95% confidence interval of the difference -0.809, 5.02) and patient/endoscopist satisfaction. There was no difference between the two groups in either depth of sedation or manoeuvrability. CONCLUSION: Both Entonox and the modified TCI propofol provide equally effective sedation and pain relief, simultaneously allowing patients to be easily manoeuvred during the procedures.

13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.

Historically, genes targeted by recurrent chromosomal deletions have been identified within the smallest genomic region shared in all patients, the minimally deleted region (MDR). However, deletions this small do not occur in all patients and are a simplification of the impact larger heterogeneous deletions have during carcinogenesis. We use the example of 13q14 deletions in chronic lymphocytic leukemia to show that genes outside MDRs are associated with disease progression. Genomic profiling of 224 patients identified 205 copy number alterations on chromosome 13 in 132 cases. Deletions including DLEU2 were heterogeneous (845 Kb-96.2 Mb) and identified two breakpoint cluster regions within short interspersed nuclear elements proximal to DLEU2 and within long interspersed nuclear elements/L1 repeats distal to GUCY1B2. After defining a deletion class on the basis of size and location, we show that (a) at diagnosis, larger deletions (class II) were associated with a significantly increased risk of disease progression (odds ratio=12.3; P=0.005), (b) in progressive patients, class II deletions were enriched (P=0.02) and (c) this association was independent of IgVH mutational status, ZAP70 expression and ATM/TP53 deletion. Deletion of a 1 Mb gene cluster (48.2-49.2 Mb), including SETDB2, PHF11 and RCBTB1, was significantly associated (P<0.01) with disease progression. Here, we show that the deletion of genes outside MDRs can influence clinical outcome.

TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of 268 mutations.

The TP53 mutation profile in chronic lymphocytic leukemia (CLL) and the correlation of TP53 mutations with allele status or associated molecular genetics are currently unknown. We performed a large mutation analysis of TP53 at four centers and characterized the pattern of TP53 mutations in CLL. We report on 268 mutations in 254 patients with CLL. Missense mutations appeared in 74% of cases compared with deletions and insertions (20%), nonsense (4%) and splice site (2%) mutations. The majority (243 of 268) of mutations were located in the DNA-binding domain. Transitions were found in 131 of 268 mutations, with only 41 occurring at methylated CpG sites (15%), suggesting that transitions at CpGs are uncommon. The codons most frequently mutated were at positions 175, 179, 248 and 273; in addition, we detected a common 2-nt deletion in the codon 209. Most mutations (199 of 259) were accompanied by deletion of the other allele (17p-). Interestingly, trisomy 12 (without 17p-) was only found in one of 60 cases with TP53 mutation (without 17p-) compared with 60 of 16 in the cohort without mutation (P=0.006). The mutational profile was not different in the cohorts with and without previous therapy, suggesting that the mechanism underlying the development of mutations may be similar, independent of treatment.

Artificial neural networks to predict presence of significant pathology in patients presenting to routine colorectal clinics.

AIM: Artificial neural networks (ANNs) are computer programs used to identify complex relations within data. Routine predictions of presence of colorectal pathology based on population statistics have little meaning for individual patient. This results in large number of unnecessary lower gastrointestinal endoscopies (LGEs - colonoscopies and flexible sigmoidoscopies). We aimed to develop a neural network algorithm that can accurately predict presence of significant pathology in patients attending routine outpatient clinics for gastrointestinal symptoms. METHOD: Ethics approval was obtained and the study was monitored according to International Committee on Harmonisation - Good Clinical Practice (ICH-GCP) standards. Three-hundred patients undergoing LGE prospectively completed a specifically developed questionnaire, which included 40 variables based on clinical symptoms, signs, past- and family history. Complete data sets of 100 patients were used to train the ANN; the remaining data was used for internal validation. The primary output used was positive finding on LGE, including polyps, cancer, diverticular disease or colitis. For external validation, the ANN was applied to data from 50 patients in primary care and also compared with the predictions of four clinicians. RESULTS: Clear correlation between actual data value and ANN predictions were found (r = 0.931; P = 0.0001). The predictive accuracy of ANN was 95% in training group and 90% (95% CI 84-96) in the internal validation set and this was significantly higher than the clinical accuracy (75%). ANN also showed high accuracy in the external validation group (89%). CONCLUSION: Artificial neural networks offer the possibility of personal prediction of outcome for individual patients presenting in clinics with colorectal symptoms, making it possible to make more appropriate requests for lower gastrointestinal endoscopy.

Patient satisfaction with lower gastrointestinal endoscopy: doctors, nurse and nonmedical endoscopists.

AIM: Assessment of patient satisfaction with lower gastrointestinal endoscopy (LGE) comprising colonoscopy and flexible sigmoidoscopy is gaining increasing importance. We have now trained non healthcare professionals such as nonmedical endoscopists (NMEs) to perform LGE to overcome shortage of trained endoscopists. The aim of this study was to prospectively determine patient satisfaction, factors affecting satisfaction with LGE and to compare with nurses, NME and medical endoscopists, in terms of patient satisfaction. METHOD: Consecutive patients undergoing LGE answered specially developed patient satisfaction questionnaire at discharge and 24 h thereafter. This questionnaire was a modification of m-Group Health Association of America questionnaire. Construct and face validity of questionnaire were tested by an expert group. Demographic and clinical data was prospectively collected. Multivariate regression analysis was performed to determine factors influencing patient satisfaction. RESULTS: Some 503 patients were surveyed after LGE. Examinations were performed by nurse (n = 105), doctor (n = 191), or NMEs (n = 155). There were no differences between three groups in terms of completion rates/complications. No differences were detected between endoscopists in patient rating for overall satisfaction (P = 0.6), technical skills (P = 0.58), communication skills (P = 0.61) or interpersonal skills (0.59). Multivariate regression analysis showed that higher preprocedure anxiety, history of pelvic operations/hysterectomy and higher pain scores were associated with adverse patient satisfaction and preprocedure anxiety, history of hysterectomy and female gender were associated with higher pain scores. CONCLUSION: This study has shown that there are no differences in patient satisfaction with LGE performed by nurse, doctor or NME. The most important factor affecting patient satisfaction is degree of discomfort/pain experienced by patient.

Randomized clinical trial of Entonox versus midazolam-fentanyl sedation for colonoscopy.

BACKGROUND: Intravenous sedation for colonoscopy is associated with cardiorespiratory complications and delayed recovery. The aim of this randomized clinical trial was to compare the efficacy of Entonox (50 per cent nitrous oxide and 50 per cent oxygen) and intravenous sedation using midazolam-fentanyl for colonoscopy. METHODS: Some 131 patients undergoing elective colonoscopy were included. Patients completed a Hospital Anxiety and Depression questionnaire, letter cancellation tests and pain scores on a 100-mm visual analogue scale before, immediately after the procedure and at discharge. They also completed a satisfaction survey at discharge and 24 h after the procedure. RESULTS: Sixty-five patients were randomized to receive Entonox and 66 to midazolam-fentanyl. Completion rates were similar (94 versus 92 per cent respectively; P = 0.513). Patients receiving Entonox had a shorter time to discharge. They reported significantly less pain (mean score 16.7 versus 40.1; P < 0.001), and showed better recovery of psychomotor function immediately after the procedure and at discharge. Patient satisfaction was higher among patients who received Entonox (median score 96 versus 89; P = 0.001). CONCLUSION: Entonox provides better pain relief and faster recovery than midazolam-fentanyl and so is more effective for colonoscopy.

Results of the United Kingdoms first pilot study for nonmedical endoscopy practitioners.

OBJECTIVE: Spencer and Ready published the first description of nonmedically qualified endoscopists in 1977 [Dis Colon Rectum 1977; 20: 94]. Since then there has been an explosion of interest in this concept. Duthie et al. [Gut 1998; 43: 711] first described Nurse Endoscopy (NE) in the UK in 1998. In recent years, endoscopy services in England have been subject to review as a consequence of perceived inadequacies in service provision and delivery. METHOD: Increasing pressure on endoscopic services led the Modernisation Agency to commission a pilot study evaluating nontraditional individuals to practice flexible sigmoidoscopy (FS). A science graduate, basic grade nurse and nonclinical member of staff were successfully trained. As a result, a second pilot scheme was introduced, whereby nine nonmedically qualified individuals received education and training in FS. A curriculum was designed, developed and implemented to create an endoscopic practitioner (EP) who was both safe and competent to undertake FS. The student backgrounds included administration, health-care assistants, phlebotomist, junior doctor assistant and clinical physiologist. RESULTS: In the context of academic achievement, prescribed learning outcomes were achieved at a rate of 93% (101 from 108) of success in first attempt at assessment. In terms of practical achievement, each student demonstrated competency in FS. CONCLUSION: The curriculum designed provides a suitable framework to develop individuals with the necessary knowledge, skills and attitudes required for safe, competent practice within FS. This indicates that the curriculum provides a broad range of learning, teaching and assessment strategies befitting to a range of individuals.

Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells.

Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). MicroRNA (miRNA) expression analysis using Ambion (Austin, TX, USA) arrays showed that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Furthermore, nine miRNAs were overexpressed and one underexpressed in integrated HPV-16 cell lines compared to the HPV-negative CaCx cell line C-33A. Based on microarray and/or quantitative real-time PCR and northern blot analyses, microRNA-218 (miR-218) was specifically underexpressed in HPV-positive cell lines, cervical lesions and cancer tissues containing HPV-16 DNA compared to both C-33A and the normal cervix. Expression of the E6 oncogene of high-risk HPV-16, but not that of low-risk HPV-6, reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16-positive cell line increased miR-218 expression. We also demonstrate that the epithelial cell-specific marker LAMB3 is a target of miR-218. We also show that LAMB3 expression is increased in the presence of the HPV-16 E6 oncogene and this effect is mediated through miR-218. These findings may contribute to a better understanding of the molecular mechanisms involved in cervical carcinogenesis.

A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation.

The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies.

P2X7 polymorphism and chronic lymphocytic leukaemia: lack of correlation with incidence, survival and abnormalities of chromosome 12.

The P2X7 receptor, a plasma membrane ATP-gated ion channel that plays a role in lymphocyte apoptosis, has been suggested as an important contributory factor to the pathogenesis of chronic lymphocytic leukaemia (CLL). The P2X7 gene resides on chromosome 12 and is polymorphic in the population at large (1513A/C) with the A and C alleles encoding fully active and nonfunctional proteins, respectively. We have evaluated the significance of this polymorphism by genotyping 144 patients with CLL and 348 healthy controls using a tetraprimer ARMS assay. We found no significant difference in allele frequency between patients and controls. Although patients with the C allele (A/C heterozygotes or C/C homozygotes) had a marginally shorter survival than those who were homozygous for the A allele, this difference was not significant for either the patient group considered as a whole or for IgVH-mutated/unmutated subsets. Finally, no association was found between trisomy 12 and P2X7 genotype. We conclude that the influence, if any, of P2X7 genotype on susceptibility to CLL or clinical outcome is small.

Lymphoid neoplasms.

The majority of B cell lymphomas, but only a minority of T cell lymphomas, are characterized by recurring chromosome translocations. Many involve the immunoglobulin or T cell receptor loci with various partner chromosomes and lead to abnormal proto-oncogene expression. Other recurring translocations result in the production of a novel fusion protein. The detection of translocations is of particular value in diagnosis and in the detection of minimal residual disease. Aneuploidy and deletion of specific chromosome regions are common secondary chromosomal events which are rarely specific to a particular type of lymphoma but provide valuable prognostic information. Analysis by G banding, 24-colour FISH and CGH provides global genomic information; however, more specifically directed investigations utilizing locus-specific FISH probes, PCR techniques or monoclonal antibodies may be more appropriate to answer particular questions regarding diagnosis and prognosis. The known molecular consequences of abnormalities and the appropriate methods of detection are discussed for each subtype of lymphoma.