Utility of next-generation sequencing technologies for the efficient genetic resolution of haematological disorders.

Next-generation sequencing (NGS) has now evolved to be a relatively affordable and efficient means of detecting genetic mutations. Whole genome sequencing (WGS) or whole exome sequencing (WES) offers the opportunity for rapid diagnosis in many paediatric haematological conditions, where phenotypes are variable and either a large number of genes are involved, or the genes are large making sanger sequencing expensive and labour-intensive. NGS offers the potential for gene discovery in patients who do not have mutations in currently known genes. This report shows how WES was used in the diagnosis of six paediatric haematology cases. In four cases (Diamond-Blackfan anaemia, congenital neutropenia (n = 2), and Fanconi anaemia), the diagnosis was suspected based on classical phenotype, and NGS confirmed those suspicions. Mutations in RPS19, ELANE and FANCD2 were found. The final two cases (MYH9 associated macrothrombocytopenia associated with multiple congenital anomalies; atypical juvenile myelomonocytic leukaemia associated with a KRAS mutation) highlight the utility of NGS where the diagnosis is less certain, or where there is an unusual phenotype. We discuss the advantages and limitations of NGS in the setting of these cases, and in haematological conditions more broadly, and discuss where NGS is most efficiently used.

The wide-ranging clinical implications of the short telomere syndromes.

There is an increasing number of inherited disorders in which excessive telomere shortening underlies the molecular defect, with dyskeratosis congenita (DC) being the archetypal short telomere syndrome. DC is classically described as a mucocutaneous triad of oral leukoplakia, nail dystrophy and abnormal skin pigmentation. However, excessive telomere shortening can affect almost any organ system, so the clinical manifestations are protean, including developmental delay, cerebellar hypoplasia, exudative retinopathy, aplastic anaemia, acute myeloid leukaemia, idiopathic pulmonary fibrosis, idiopathic hepatic cirrhosis, head and neck cancer and dental abnormalities, and may be multi-systemic. Undiagnosed patients may be seen by essentially any medical subspecialist. Correct diagnosis is important to ensure appropriate management, and for initiating investigations to identify affected family members. Treatment is often supportive, with transplantation offering cure for pulmonary fibrosis or bone marrow failure. Higher rates of mortality and morbidity with transplantation often require regimen alterations, underscoring the need for correct diagnosis. Short telomeres result from mutations in genes essential for telomere maintenance (e.g. genes encoding subunits of the telomerase enzyme complex). Disease severity reflects not only the severity of the defect, but also the inheritance of short telomeres, giving rise to incomplete penetrance and genetic anticipation. Attendees of the inaugural Australian Short Telomere Syndrome Conference were updated on the current scientific and clinical understanding of these disorders, and discussed the best approach for management of these patients in the Australian context. This review will include recommendations from the conference and aims to increase awareness of short telomere disorders.

Reduced incidence of second solid tumors in survivors of childhood Hodgkin's lymphoma treated without radiation therapy.

BACKGROUND: We assessed the risk of developing second malignancies in children treated for Hodgkin's lymphoma (HL), the majority of whom received chemotherapy only. PATIENTS AND METHODS: The development of second malignancies in children with HL, treated between 1960 and 1999, was assessed. Results were obtained by both chart review and linkage with a centralized cancer registry. Tumor incidence was compared for patients treated with and without radiotherapy (RT) and with the general population. Risk factors for developing second tumors were assessed by multivariate analysis. RESULTS: Of 142 childhood HL patients, 63 had received RT and 79 had not. Overall survival was similar for both groups. Fourteen patients developed second solid tumors, 12 who had received RT and 2 treated with chemotherapy only (P <0.001), with a 30-year cumulative incidence of 24.7% [95% confidence interval (CI) 7.27-47.4] and 5.8% (95% CI 0-58.9), respectively (P = 0.01). The standardized incidence ratio for second solid tumors was 236 (95% CI 112.2-359.0) versus 43.6 (95% CI 0-103.9), respectively. Multivariate analysis showed treatment with RT was the only significant risks factor for developing second solid tumors. CONCLUSIONS: Children with HL without RT have a substantially lower incidence of second tumors than those treated with RT.

Microscopic characterisation of photodetectors used in the hadron calorimeter of the Compact Muon Solenoid experiment.

The Hybrid Photodetector (HPD) is a hybrid unit with a single accelerating gap between a common photocathode and an array of PIN diodes. Customised HPDs with 19 channels were used to detect scintillation light from hadron calorimeter in the Compact Muon Solenoid (CMS) experiment. In this paper, we present results on radiation damage studies carried out on the used HPDs in the outer hadron (HO) and the end-cap hadron (HE) calorimeter of the CMS experiment operating at CERN. The calorimeter is made of alternating layers of scintillating tiles and metals, such as brass or iron. The scintillating light was transmitted to the HPDs by means of optical fibres. Due to excessive exposure to scintillation light and ionising radiation during data taking at the Large Hadron Collider, the performance of the HPDs was expected to degrade significantly in the HE detector. Independent studies on radiation damage of these used photosensors were important to assess the degradation in the performance of the calorimeter. Microscopic scans of relative photon detection efficiencies for two HPDs (one each from HO and HE detector) were made using micron resolution optical scanner. The scanner was specially designed and built for microscopic characterisation of photosensors. Imprints of each fibre (∼1 mm in diameter) on the photocathode with varying damage within the same pixel of the HPD were observed. The localised damage of the photocathode was determined to vary with the amount of scintillation (or calibration) light transmitted by optical fibres to the HPD.

Risk factors for symptomatic venous thromboembolism during therapy for childhood acute lymphoblastic leukemia.

BACKGROUND: Symptomatic venous thromboembolism (VTE) is an unpredictable and life-threatening toxicity, which occurs early in childhood acute lymphoblastic leukemia (ALL) therapy. Approximately 5% of children will experience VTE which is treated with anticoagulation. Asparaginase and corticosteroids are etiologic factors for VTE, however other clinical factors may modify this risk. PROCEDURE: We sought to i) assess published pre-treatment VTE risk factors ii) identify early clinical factors that were associated with VTE and iii) determine whether single nucleotide polymorphisms (SNPs) associated with VTE in non-cancer patients contributed to VTE in children with ALL. We performed a detailed, retrospective analysis of 1021 ALL patients treated between 1998 and 2013. Individual patient records were reviewed to ascertain VTE incidence and document treatment-related clinical variables. RESULTS: The incidence of VTE was 5.1%. Extremes of weight at diagnosis (<5th or >95th centile) was an independent risk factor in multivariable analysis, when added to published risk factors of age ≥10 years and mediastinal mass. When factors during induction/consolidation were considered separately: bacteremia, elevated serum gamma-glutamyl transferase and bilirubin were associated with VTE occurrence. None of the SNPs associated with VTE in non-cancer populations were significantly associated with VTE in our cohort. CONCLUSION: We found two known risk factors (age ≥ 10 years and mediastinal mass) in a large cohort of children treated for ALL and identified other factors associated with VTE such as weight extremes at diagnosis, bacteremia, and abnormal liver function which warrant further study. These VTE risk factors may form the basis of future thromboprophylaxis trials.

Carcinogen binding to various types of dietary fiber.

The percent of the carcinogen 1,2-dimethylhydrazine (DMH) bound to a variety of fibers, such as wheat bran, corn bran, citrus pulp, citrus pectin, and alfalfa, was examined at pH values ranging from 1 to 12. The percent of DMH bound to wheat bran increased from 4% at PH 1 to 55% at pH 2 to 77% at pH 12. A sharp rise in carcinogen binding to corn bran occurred between pH 5% of the DMH was bound and pH 8 where 51% of the DMH was bound. The percent of DMH bound to dehydrated citrus pulp also increased as the pH increased with 10% binding observed at pH 1 and with 57% binding observed at pH 12. Between pH 2 and pH 7, the percent of DMH bound to pectin decreased from 60 to 11%. As the pH became more basic, the percent of DMH bound to pectin increased to 42% at pH 12. The sharpest rise in the percent of DMH bound to alfalfa meal occurred between pH 10.5 and pH 12.0. Results from this experiment showed that the affinity to various types of dietary fibers for the colon carcinogen DMH was differentially affected by pH. These results suggested that the protective effect of certain types of dietary fiber against chemically induced colon cancer my in part be attributed to enhanced carcinogen binding by dietary fiber in the colon.

Multiple myeloma causes clonal T-cell immunosenescence: identification of potential novel targets for promoting tumour immunity and implications for checkpoint blockade.

Tumour-induced dysfunction of cytotoxic T cells in patients with multiple myeloma (MM) may contribute to immune escape and be responsible for the lack of therapeutic efficacy of immune checkpoint blockade. We therefore investigated dysfunctional clonal T cells in MM and demonstrated immunosenescence but not exhaustion as a predominant feature. T-cell clones were detected in 75% of MM patients and their prognostic significance was revalidated in a new post-immunomodulatory drug cohort. The cells exhibited a senescent secretory effector phenotype: KLRG-1+/CD57+/CD160+/CD28-. Normal-for-age telomere lengths indicate that senescence is telomere independent and potentially reversible. p38-mitogen-activated protein kinase, p16 and p21 signalling pathways known to induce senescence were not elevated. Telomerase activity was found to be elevated and this may explain how normal telomere lengths are maintained in senescent cells. T-cell receptor signalling checkpoints were normal but elevated SMAD levels associated with T-cell inactivation were detected and may provide a potential target for the reversal of clonal T-cell dysfunction in MM. Low programmed death 1 and cytotoxic T-lymphocyte-associated antigen 4 expression detected on T-cell clones infers that these cells are not exhausted but suggests that there would be a suboptimal response to immune checkpoint blockade in MM. Our data suggest that other immunostimulatory strategies are required in MM.

Search for a 33.9 MeV/c(2) neutral particle in pion decay

The E815 (NuTeV) neutrino experiment has performed a search for a 33. 9 MeV/c(2) weakly interacting neutral particle produced in pion decay. Such a particle may be responsible for an anomaly in the timing distribution of neutrino interactions in the KARMEN experiment. E815 has searched for this particle's decays in an instrumented decay region; no evidence for this particle was found. The search is sensitive to pion branching ratios as low as 10(-13).

Infectious complications in breast cancer patients undergoing peripheral blood stem cell transplantation: a single center retrospective analysis towards outpatient strategy.

Infectious complications were retrospectively analyzed in 129 transplants, performed in 90 patients, to identify characteristics that qualify breast cancer patients for outpatient-based PBSCT. Thirty-one cases (24%) did not develop fever. Of the remaining 98 cases, 84.7% developed fever during severe neutropenia. On univariate analysis, disease stages II-III, first PBSCT, mucositis grades II-IV and the use of two alkylators were associated with a higher risk of fever development. The latter two factors also affected fever occurrence on multivariate analysis. A longer median time to fever onset was observed in patients conditioned with single as compared to double alkylating agent-containing regimens (respectively 8th vs 6th day, P < 0.00001). As compared with metastatic breast cancer (MBC), high risk breast cancer showed a 2.3-fold increased risk of developing early fever during neutropenia (CI 2.3-3.8), remaining the only variable still significant on multivariate analysis (P = 0.0039). Combination antibiotic therapy was equivalent to single agent therapy. Patients suffering from microbiologically documented fever were at higher risk of undergoing second-line antibiotic therapy. In conclusion, MBC patients treated with a conditioning regimen containing only one alkylating agent and adequate prophylaxis for mucositis may qualify for outpatient-based PBSCT on the basis of a lower risk of infection.

Effect of fat and microflora on hepatic, small intestinal and colonic HMG CoA reductase, cytochrome P450 and cytochrome B5.

High levels of dietary fat caused a significant reduction in HMG CoA reductase activity in the liver of germ-free rats whereas significantly elevated small intestinal enzyme activity was observed. Dietary fat had no significant effect on HMG CoA reductase activity in any tissue studied in the conventional rat. No significant change in colonic HMG CoA reductase activity was observed between any of the experimental groups. Rats fed a high-fat diet tended to exhibit higher cytochrome P450 levels in all tissues studied, regardless of the presence of intestinal microflora.

Use of the internet as a resource for consumer health information: results of the second osteopathic survey of health care in America (OSTEOSURV-II).

BACKGROUND: The Internet offers consumers unparalleled opportunities to acquire health information. The emergence of the Internet, rather than more-traditional sources, for obtaining health information is worthy of ongoing surveillance, including identification of the factors associated with using the Internet for this purpose. OBJECTIVES: To measure the prevalence of Internet use as a mechanism for obtaining health information in the United States; to compare such Internet use with newspapers or magazines, radio, and television; and to identify sociodemographic factors associated with using the Internet for acquiring health information. METHODS: Data were acquired from the Second Osteopathic Survey of Health Care in America (OSTEOSURV-II), a national telephone survey using random-digit dialing within the United States during 2000. The target population consisted of adult, noninstitutionalized, household members. As part of the survey, data were collected on: facility with the Internet, sources of health information, and sociodemographic characteristics. Multivariate analysis was used to identify factors associated with acquiring health information on the Internet. RESULTS: A total of 499 (64% response rate) respondents participated in the survey. With the exception of an overrepresentation of women (66%), respondents were generally similar to national referents. Fifty percent of respondents either strongly agreed or agreed that they felt comfortable using the Internet as a health information resource. The prevalence rates of using the health information sources were: newspapers or magazines, 69%; radio, 30%; television, 56%; and the Internet, 32%. After adjusting for potential confounders, older respondents were more likely than younger respondents to use newspapers or magazines and television to acquire health information, but less likely to use the Internet. Higher education was associated with greater use of newspapers or magazines and the Internet as health information sources. Internet use was lower in rural than urban or suburban areas. CONCLUSIONS: The Internet has already surpassed radio as a source of health information but still lags substantially behind print media and television. Significant barriers to acquiring health information on the Internet remain among persons 60 years of age or older, those with 12 or fewer years of education, and those residing in rural areas. Stronger efforts are needed to ensure access to and facility with the Internet among all segments of the population. This includes user-friendly access for older persons with visual or other functional impairments, providing low-literacy Web sites, and expanding Internet infrastructure to reach all areas of the United States.

Factors associated with intended use of a Web site among family practice patients.

BACKGROUND: The World Wide Web has become a widely utilized source of health information. Although the frequency of health related queries is impressive, the demographics associated with patients making queries has not been clearly delineated. OBJECTIVE: This study s objective was to determine health related Internet usage patterns of family medicine patients. METHODS: Internet use among 824 eligible patients 18 years or older attending seven university based family practice clinics during a two week period in November of 1999 was studied. The survey instrument included 10 items and was designed to collect data in less than five minutes using a paper and pencil format. Statistical significance associated with intended Web site use was computed using a multiple logistic regression model. RESULTS: A response rate of 72.2% was observed with 63.1% being females and 36.9% being males. The mean and median age were 44.0 and 45.7 years, respectively. A steady decline in intended Web site use was observed with advancing age with significant differences observed above 65 years (OR = 0.30; 95% CI = 0.14 - 0.64; p<.002). Other significant findings associated with intended use of a Web site by clinic based patients included having a home computer (OR = 1.99; 95%, CI = 1.05 3.76; p<0.03) and having Internet access at home (OR=5.6, 95%, CI = 2.83-11.18; p<.001). A lack of association between intended Web site use and health insurance status was observed. CONCLUSIONS: Results from this study suggest that Web sites are not likely to be alternative sources of health information for the uninsured or elderly in the near future.