Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

@#Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Epidermal growth factor receptor and anaplastic lymphoma kinase testing and mutation prevalence in patients with advanced non-small cell lung cancer in Switzerland: A comprehensive evaluation of real world practices.

In order to improve outcomes, identification of the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes has become crucial in advanced non-small-cell lung cancer (NSCLC). The aim of the present study is to analyse time trends and frequency of testing, factors affecting testing as well as prevalence of mutations in the Swiss population. We analysed EGFR and ALK testing in a cohort of patients with newly diagnosed metastasised non-squamous NSCLC in the catchment area of the cancer registry Eastern Switzerland in the years 2008-2014. We analysed prevalence of mutations and studied clinicopathological characteristics and survival of tested and non-tested patients and of patients with and without mutations. Among 718 patients identified, 11% (51/447) harboured an EGFR mutation in the exons 18, 19 or 21 and further 12% (31/265) showed a positive test result for ALK rearrangements. In non-smokers the proportions of mutations were 31% and 23% respectively. Testing rates increased over time and reached 79% in 2014. We observed significantly lower testing rates and poorer survival in elderly, patients with limited life expectancy and patients treated at hospitals not involved in clinical research. Outcomes can be further improved in a considerable proportion of patients with advanced non-squamous NSCLC.

Erlotinib has comparable clinical efficacy to chemotherapy in pretreated patients with advanced non-small cell lung cancer (NSCLC): A propensity-adjusted, outcomes research-based study.

OBJECTIVES: Controversy exists about the integration of erlotinib in patients with EGFR wildtype, advanced NSCLC. MATERIALS AND METHODS: We included patients with advanced NSCLC receiving at least two lines of palliative systemic treatment between January 2005 and December 2014 and not harbouring targetable driver mutations. Primary study endpoint was overall survival (OS), secondary endpoint progression-free survival (PFS). We used Kaplan-Meier statistics, multivariate Cox regression and Propensity score or Inverse Probability Weights (IPW) matching to compare clinical outcome between patients receiving erlotinib in second or further line and those receiving chemotherapy only. The study had a power of 90% to detect a survival superiority of 30%. RESULTS: From a total of 827 patients, we excluded 171 patients with potentially curative treatment, 189 receiving treatment outside of our institute, 206 receiving no or only one line of systemic treatment, 6 with ALK translocations and 28 with EGFR mutations. From 227 patients in the final efficacy analysis, 125 patients received erlotinib in second (89 patients), third (28) or further-line (8), and 102 patients received chemotherapy only. Women and never smokers were significantly overrepresented in the erlotinib group. Both OS (hazard ratio (HR)=1.14, 95% CI 0.80-1.63, P=0.448) and PFS (HR=1.20, 95% CI 0.95-1.52, P=0.119) were similar in the erlotinib compared to the chemotherapy group using IPW-adjusted Cox regression analysis treating the use of erlotinib as a time-dependent covariate starting from second-line treatment and stratified for ECOG performance status and treatment line. ECOG performance status was the most powerful covariate to select patients for erlotinib treatment. CONCLUSION: The present study suggests erlotinib to have similar clinical efficacy compared to chemotherapy in patients with pretreated advanced NSCLC and no known molecular targetable alterations.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

Diffuse large B-cell lymphoma in elderly patients: a retrospective analysis.

BACKGROUND: Few data on patterns of care and outcomes are available for elderly patients with diffuse large B-cell lymphoma (DLBCL) outside of clinical trials. METHODS: We identified patients with DLBCL older than 60 years from a regional cancer registry between 2000 and 2010. Based on registry data and chart review, 128 patients from the oncology network of Eastern Switzerland were analysed for patient characteristics, treatment and outcomes of DLBCL. Three age groups were compared: 60-69, 70-79 and over 80 years old. RESULTS: Median age was 73 years (range: 60 to 95 years). 52/121 treated patients received 6 cycles of R-CHOP/CHOP, of those 30 (58%), 18 (35%) and 4 (7%) patients were 60-69 years, 70-79 years or older than 80 years respectively, with a significant difference by age group, p=0.001. Median OS of patients 60-69, 70-79, and 80 years and older receiving 6 cycles of R-CHOP/CHOP were: 54 months, 31 months and 24 months respectively. In comparison, patients receiving other than 6 cycles of R-CHOP/CHOP treatment regimens had a median OS of 22 months, 17 months and 6 months, respectively. In the multivariable analysis other than 6 cycles of R-CHOP/CHOP were significantly associated with poor survival. The risk of dying increased by a mean of 6% for each year of age from age 60 years onwards. CONCLUSION: In conclusion, treatment regimens other than 6 cycles of R-CHOP/CHOP were significant predictors for survival in our oncology network. The possibility of using R-CHOP treatment regimen should be seriously considered in elderly patients with DLBCL.

Treatment of breast cancer in the elderly: a prospective, population-based Swiss study.

OBJECTIVES: The primary objective of this population-based study is to describe the patterns of care of elderly patients with breast cancer (BC), and evaluate potential causative factors for the decrease in BC-specific survival (BCSS) in the elderly. PATIENTS AND METHODS: We included all or representative samples of patients with newly diagnosed BC from seven Swiss cancer registries between 2003 and 2005 (n=4820). Surgical and non-surgical BC treatment was analyzed over 5 age groups (<65, 65 to <70, 70 to <75, 75 to <80 and ≥80years), and the predictive impact of patient age on specific treatments was calculated using multivariate logistic regression analysis. RESULTS: The proportion of locally advanced, metastatic and incompletely staged BC increased with age. The odds ratio for performing breast-conserving surgery (BCS) in stages I-II BC (0.37), sentinel lymph node dissection (SLND) in patients with no palpable adenopathy (0.58), post-BCS radiotherapy (0.04) and adjuvant endocrine treatment (0.23) were all in disfavor of patients ≥80years of age compared to their younger peers. Only 36% of patients ≥80years of age with no palpable adenopathy underwent SLND. In the adjusted model, higher age was a significant risk factor for omitting post-BCS radiotherapy, SLND and adjuvant endocrine treatment. CONCLUSIONS: This study found an increase in incomplete diagnostic assessment, and a substantial underuse of BCS, post-BCS radiotherapy, SLND and adjuvant endocrine treatment in elderly patients with BC. There is a need for improved management of early BC in the elderly even in a system with universal access to health care services.

Management of elderly patients with advanced non-small cell lung cancer: a single-center experience.

BACKGROUND: Optimal management of elderly patients (≥70 years) with non-small cell lung cancer (NSCLC) remains debatable. We compared survival and treatment of advanced NSCLC between elderly and younger patients. METHODS: From the cancer registry, we identified 188 patients treated with chemotherapy for stage IV NSCLC. Patient characteristics, survival, toxicity, chemotherapy regimen and response were compared between age groups (patients 50-69 vs. ≥70 years). RESULTS: There were 96 young and 92 elderly patients. The majority were male (70%) and had adenocarcinoma (53%). More elderly had an ECOG performance status >1 (59 vs. 42%, p = 0.04). Median survival was longer for young patients (11.5 vs. 10.8 months, hazard ratio, HR 1.43, p = 0.04). Patients ≥75 years had a significantly worse outcome compared to the young and patients aged 70-74 years (11.5 vs. 12.8 vs. 7.7 months, HR 1.71, p = 0.01). Hospitalization rate did not differ. Elderly had more hematological toxicities (56 vs. 32%, p = 0.01) and less frequently received first-line platinum combinations (96 vs. 69%, p < 0.001). CONCLUSIONS: Elderly patients had a marginally worse survival compared to young patients. Despite the less frequent use of combination chemotherapy, elderly patients experienced toxicity more often. Survival of those ≥75 years was significantly worse, indicating the urgent need of further research particularly in this age group.

Lung cancer in the Swiss HIV Cohort Study: role of smoking, immunodeficiency and pulmonary infection.

BACKGROUND: Immunodeficiency and AIDS-related pulmonary infections have been suggested as independent causes of lung cancer among HIV-infected persons, in addition to smoking. METHODS: A total of 68 lung cancers were identified in the Swiss HIV Cohort Study (SHCS) or through linkage with Swiss Cancer Registries (1985-2010), and were individually matched to 337 controls by centre, gender, HIV-transmission category, age and calendar period. Odds ratios (ORs) were estimated by conditional logistic regression. RESULTS: Overall, 96.2% of lung cancers and 72.9% of controls were ever smokers, confirming the high prevalence of smoking and its strong association with lung cancer (OR for current vs never=14.4, 95% confidence interval (95% CI): 3.36-62.1). No significant associations were observed between CD4+ cell count and lung cancer, neither when measured within 1 year (OR for <200 vs ≥500=1.21, 95% CI: 0.49-2.96) nor further back in time, before lung cancer diagnosis. Combined antiretroviral therapy was not significantly associated with lung cancer (OR for ever vs never=0.67, 95% CI: 0.29-1.52), and nor was a history of AIDS with (OR=0.49, 95% CI: 0.19-1.28) or without (OR=0.53, 95% CI: 0.24-1.18) pulmonary involvement. CONCLUSION: Lung cancer in the SHCS does not seem to be clearly associated with immunodeficiency or AIDS-related pulmonary disease, but seems to be attributable to heavy smoking.

Surgical treatment of early stage breast cancer in elderly: an international comparison.

Over 40% of breast cancer patients are diagnosed above the age of 65. Treatment of these elderly patients will probably vary over countries. The aim of this study was to make an international comparison (several European countries and the US) of surgical and radiation treatment for elderly women with early stage breast cancer. Survival comparisons were also made. Data were obtained from national or regional population-based registries in the Netherlands, Switzerland, Ireland, Belgium, Germany, and Portugal. For the US patients were selected from the Surveillance, Epidemiology, and End Results (SEER) database. Early stage breast cancer patients aged ≥ 65 diagnosed between 1995 and 2005 were included. An international comparison was made for breast and axillary surgery, radiotherapy after breast conserving surgery (BCS), and relative or cause-specific survival. Overall, 204.885 patients were included. The proportion of patients not receiving any surgery increased with age in many countries; however, differences between countries were large. In most countries more than half of all elderly patients received breast conserving surgery (BCS), with the highest percentage in Switzerland. The proportion of elderly patients that received radiotherapy after BCS decreased with age in all countries. Moreover, in all countries the proportion of patients who do not receive axillary surgery increased with age. No large differences in survival between countries were recorded. International comparisons of surgical treatment for elderly women with early stage breast cancer are scarce. This study showed large international differences in treatment of elderly early stage breast cancer patients, with the most striking result the large proportion of elderly who did not undergo surgery at all. Despite large treatment differences, survival does not seem to be affected in a major way.

Predictors of state-of-the-art management of early breast cancer in Switzerland.

BACKGROUND: The aim of this study was to investigate predictors of state-of-the-art management of early breast cancer in Switzerland. PATIENTS AND METHODS: The study included 3499 women aged 25-79 years diagnosed with invasive breast cancer stages I-IIIA in 2003-2005. Patients were identified through population-based cancer registries and treated in all kinds of settings. Concordance with national and international recommendations was assessed for 10 items covering surgery, radiotherapy, systemic adjuvant therapy and histopathology reporting. We used multivariate logistic regression to identify independent predictors of high (10 points) and low (≤7 points) concordance. RESULTS: In one-third of the patients, management met guidelines in all items, whereas in about one-fifth, three or more items did not comply. Treatment by a surgeon with caseload in the upper tercile and team involved in clinical research were independent predictors of a high score, whereas treatment by a surgeon with a caseload in the lower tercile was associated with a low score. Socioeconomic characteristics such as income and education were not independent predictors, but patient's place of residence and age independently predicted management according to recommendations. CONCLUSION: Specialization and involvement in clinical research seem to be key elements for enhancing the quality of early breast cancer management at population level.

Changing patterns of cancer incidence in the early- and late-HAART periods: the Swiss HIV Cohort Study.

BACKGROUND: The advent of highly active antiretroviral therapy (HAART) in 1996 led to a decrease in the incidence of Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma (NHL), but not of other cancers, among people with HIV or AIDS (PWHA). It also led to marked increases in their life expectancy. METHODS: We conducted a record-linkage study between the Swiss HIV Cohort Study and nine Swiss cantonal cancer registries. In total, 9429 PWHA provided 20,615, 17,690, and 15,410 person-years in the pre-, early-, and late-HAART periods, respectively. Standardised incidence ratios in PWHA vs the general population, as well as age-standardised, and age-specific incidence rates were computed for different periods. RESULTS: Incidence of KS and NHL decreased by several fold between the pre- and early-HAART periods, and additionally declined from the early- to the late-HAART period. Incidence of cancers of the anus, liver, non-melanomatous skin, and Hodgkin's lymphoma increased in the early- compared with the pre-HAART period, but not during the late-HAART period. The incidence of all non-AIDS-defining cancers (NADCs) combined was similar in all periods, and approximately double that in the general population. CONCLUSIONS: Increases in the incidence of selected NADCs after the introduction of HAART were largely accounted for by the ageing of PWHA.

Geographic variation in breast cancer care in Switzerland.

PURPOSE: Regional disparities in breast cancer outcomes have been reported in Switzerland. The purpose of this study is to investigate geographic variation in early diagnosis and management of breast cancer. METHODS: We used data from a representative sample of 4820 women diagnosed with invasive breast cancer between January 1, 2003 and December 31, 2005 identified by seven Swiss population based cancer registries. We collected retrospectively detailed information on mode of detection, tumor characteristics and treatments. Differences across geographic regions were tested for statistical significance using chi-square tests and uni- and multivariate logistic regression. RESULTS: Considerable disparities in early detection and management of early breast cancer were found across regions. In particular, the proportion of early detected cancer varied from 43% in Valais to 27% in St. Gallen-Appenzell. Mastectomy rates varied from 24% in Geneva to 38% in St. Gallen-Appenzell and Grisons-Glarus. Higher reconstruction rates were observed in regions with lower rates of mastectomy. The use of sentinel node procedure in patients with nodal negative disease was high in Geneva and low in Eastern Switzerland. Differences in compliance with recommendations on the use of endocrine therapy and chemotherapy were less pronounced but statistically significant. CONCLUSIONS: This analysis shows considerable geographic variation in breast cancer care in a health system characterized by high expenditures, universal access to services and high decentralization. Further study into the causes and effects of this variation on short- and long term patient outcomes is needed.

Kaposi sarcoma incidence in the Swiss HIV Cohort Study before and after highly active antiretroviral therapy.

Between 1984 and 2006, 12 959 people with HIV/AIDS (PWHA) in the Swiss HIV Cohort Study contributed a total of 73 412 person-years (py) of follow-up, 35 551 of which derived from PWHA treated with highly active antiretroviral therapy (HAART). Five hundred and ninety-seven incident Kaposi sarcoma (KS) cases were identified of whom 52 were among HAART users. Cox regression was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals (CI). Kaposi sarcoma incidence fell abruptly in 1996-1998 to reach a plateau at 1.4 per 1000 py afterwards. Men having sex with men and birth in Africa or the Middle East were associated with KS in both non-users and users of HAART but the risk pattern by CD4 cell count differed. Only very low CD4 cell count (<50 cells microl(-1)) at enrollment or at HAART initiation were significantly associated with KS among HAART users. The HR for KS declined steeply in the first months after HAART initiation and continued to be low 7-10 years afterwards (HR, 0.06; 95% CI, 0.02-0.17). Thirty-three out of 52 (63.5%) KS cases among HAART users arose among PWHA who had stopped treatment or used HAART for less than 6 months.

Invasive meningococcal and pneumococcal disease in Switzerland: cost-utility analysis of different vaccine strategies.

We performed a cost-utility analysis for various vaccination strategies against meningococcal and pneumococcal diseases (MenC or MenC/PCV-9) in Switzerland. The analysis compared the current recommendations of vaccinating only children with medical risks to the introduction of the vaccination with either the MenC or the MenC/PCV-9 vaccine, administered at 12 or 2, 4 and 6 months of age, into the existing immunisation programme. For a birth cohort of 80,000 children and assuming a vaccine coverage of 80%, the introduction of a generalised vaccination would be cost-effective. The strategy using three doses of MenC/PCV-9 would achieve the highest health benefit, with 440 quality-adjusted life years (QALYs) gained at costs of 34,000 per QALY.

Medical-economical aspects of high sensitivity C-reactive protein assay for the prediction of coronary heart disease. An analysis in Germany and Italy.

BACKGROUND: Elevated plasma concentrations of C-reactive protein (CRP) are associated with increased cardiovascular risk. We studied the cost-effectiveness of CRP determination in primary and secondary prevention settings in two European countries: Germany and Italy. METHODS: Using a decision analytic model we evaluated the costs and consequences of testing or not testing using a high sensitivity (hs)-CRP assay. In a primary prevention model we analyzed a hypothetical cohort of 300000 apparently healthy men divided into three age groups (35-44, 45-54 and 55-64 years). Individuals with CRP levels > 3 mg/l were administered either aspirin or statins according to lipid levels. The cohort was followed for 5 years. In the secondary prevention model hs-CRP testing was evaluated in a cohort of 10000 patients with total cholesterol levels < 4.52 mmol/l and a history of myocardial infarction or unstable angina. The two strategies tested were: 1) administer pravastatin only to those with high CRP values, and 2) treat all patients. The analysis was performed from the societal perspective. Event rates were obtained from epidemiological studies and clinical trials. RESULTS: In the primary prevention model, strategies including testing showed, for men aged 45 years and older, cost-effectiveness ratios between each life year saved (LYS) and cost savings in Germany equal to 10217euro and between each LYS and savings in Italy equal to 16950euro In the age group 35-44 years, therapy with aspirin showed cost-effectiveness ratios of 5318euro and 11203euro per LYS in Germany and in Italy respectively. The widespread use of statins showed an unfavorable cost-effectiveness profile: 44630euro per LYS in Germany and 36270euro per LYS in Italy. In the secondary prevention model, hs-testing for CRP can reduce the cost-effectiveness of pravastatin from 16400 to 6830euro per quality adjusted life year gained. Sensitivity analysis performed on the variables test price and costs of cardiovascular events resulted in minimal changes of the cost-effectiveness ratios. CONCLUSIONS: Both in the primary and the secondary prevention settings, hs-testing for CRP can better target individuals at higher risk, thus improving outcomes and resulting in a more cost-effective strategy.

Experimental study on the fibrogenic properties of different types of alumina.

The fibrogenicity of seven alumina samples was tested in rats by intratracheal instillation, and in mice by intraperitoneal injection. Histopathological studies were carried out on all animals. As an indicator for inflammatory reaction, a bronchoalveolar lavage (BAL) was performed in rats. Lactate dehydrogenase activity and protein content of the supernatant as well as the free-cell population of the lungs were studied. None of the five aluminas used for primary aluminum production showed any fibrogenic potential, while the other two, a chemical grade and a laboratory produced sample, induced fibrotic lesions. A correlation between cytological and biochemical parameters studied in BAL and the fibrosis determined by histology could be established for the quartz-treated rats, but not in the alumina-treated animals.