Liquid biopsy - Performance of the PAXgene® Blood ccfDNA Tubes for the isolation and characterization of cell-free plasma DNA from tumor patients.

BACKGROUND: In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumor patients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications. METHODS: We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined. RESULTS: While the 7day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples. CONCLUSION: The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well.

Factors Associated with Worse Lung Function in Cystic Fibrosis Patients with Persistent Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an important pathogen in cystic fibrosis (CF). However, it is not clear which factors are associated with worse lung function in patients with persistent S. aureus airway cultures. Our main hypothesis was that patients with high S. aureus density in their respiratory specimens would more likely experience worsening of their lung disease than patients with low bacterial loads. METHODS: Therefore, we conducted an observational prospective longitudinal multi-center study and assessed the association between lung function and S. aureus bacterial density in respiratory samples, co-infection with other CF-pathogens, nasal S. aureus carriage, clinical status, antibiotic therapy, IL-6- and IgG-levels against S. aureus virulence factors. RESULTS: 195 patients from 17 centers were followed; each patient had an average of 7 visits. Data were analyzed using descriptive statistics and generalized linear mixed models. Our main hypothesis was only supported for patients providing throat specimens indicating that patients with higher density experienced a steeper lung function decline (p<0.001). Patients with exacerbations (n = 60), S. aureus small-colony variants (SCVs, n = 84) and co-infection with Stenotrophomonas maltophilia (n = 44) had worse lung function (p = 0.0068; p = 0.0011; p = 0.0103). Patients with SCVs were older (p = 0.0066) and more often treated with trimethoprim/sulfamethoxazole (p = 0.0078). IL-6 levels positively correlated with decreased lung function (p<0.001), S. aureus density in sputa (p = 0.0016), SCVs (p = 0.0209), exacerbations (p = 0.0041) and co-infections with S. maltophilia (p = 0.0195) or A. fumigatus (p = 0.0496). CONCLUSIONS: In CF-patients with chronic S. aureus cultures, independent risk factors for worse lung function are high bacterial density in throat cultures, exacerbations, elevated IL-6 levels, presence of S. aureus SCVs and co-infection with S. maltophilia. TRIAL REGISTRATION: ClinicalTrials.gov NCT00669760.

Lactate in cystic fibrosis sputum.

BACKGROUND: Antibiotic therapy is thought to improve lung function in patients with cystic fibrosis (CF) by decreasing neutrophil-derived inflammation. We investigated the origin and clinical significance of lactate in the chronically inflamed CF lung. METHODS: Lactate was measured in sputa of 18 exacerbated and 25 stable CF patients via spectrophotometry and gaschromatography. Lung function was assessed via spirometry. Seven patients with chronic obstructive pulmonary disease (COPD) and three patients with acute lung inflammation served as control groups. Neutrophil and bacterial lactate production was assessed under aerobic and anaerobic conditions. RESULTS: In sputum specimens of patients with respiratory exacerbations lactate concentrations decreased significantly (p<0.005) from 3.4±2.3mmol/L to 1.4±1.4mmol/L after 2-3 weeks of intravenous antibiotics. Successful treatment was reflected in 16 patients (88.9%) by FVC increase associated with lactate decrease (p<0.05). In every single sputum lactate was detectable (3.0±3.1mmol/L, range 0.2-14.1mmol/L). Lactate was lower (1.6±0.8mmol/L) in sputa from seven COPD patients, and it was below the detection limit in three patients with acute lung inflammation. Neutrophil lactate production accumulated up to 10.5mmol/L after 4 days, whereas bacterial lactate production did not appear to contribute substantially to sputum lactate concentrations. CONCLUSIONS: Successful antibiotic therapy is reflected by a decrease in lactate concentrations. Neutrophils are the most likely source for lactate in sputum of CF patients. Therefore lactate may be used to monitor responses to antibiotic therapy as an adjunct to lung function measurements.

Musculoskeletal manifestations and rheumatic symptoms in patients with cystic fibrosis (CF) no observations of CF-specific arthropathy.

OBJECTIVE: To determine the character and frequency of musculoskeletal manifestations and rheumatic symptoms in patients with cystic fibrosis (CF). METHODS: Rheumatic symptoms and signs of 70 patients with CF (age 6 to 61 yrs) were determined by interview and clinical assessment. Age and sex-matched healthy volunteers served as a control group. In CF patients, laboratory measures and bone mineral density (BMD) were investigated. The data were correlated with the CF phenotype [Shwachman Score (ShS), Chrispin-Norman Score (ChNS), and pulmonary function tests (PFT)]. RESULTS: The prevalence of joint pain in the CF patients was 12.9%, with a mean duration of 7 days. Swollen joints were found in 4 patients. None fulfilled the criteria for rheumatoid arthritis or connective tissue disease. Adult CF patients complained more often about noninflammatory back pain and myalgia, and demonstrated reduced spine mobility and impaired everyday life functions compared with the controls. Symptomatic CF patients had elevated erythrocyte sedimentation rate and C-reactive protein levels and performed worse on the ShS, ChNS, and PFT than asymptomatic patients. Antibodies against exotoxin A of Pseudomonas aeruginosa and recombinant Aspergillus fumigatus allergen f4 were found more frequently in CF patients with arthralgia. BMD was decreased in adult patients with more severe CF. CONCLUSION: In CF patients, the prevalence of rheumatic symptoms increases with age and CF severity. Our data suggest an association of infections with P. aeruginosa and A. fumigatus with the occurrence of rheumatic symptoms. However, no association of CF with definite inflammatory joint or connective tissue diseases was observed, and no CF-specific pattern of musculoskeletal symptoms was seen.

[Extranodal non-Hodgkin's lymphoma of MALT-type stage I. A case report]. / Extranodales Marginalzonen-B-Zell-Lymphom (MALT-Lymphom) der Konjunktiven und des Larynx Kasuistik und Literaturübersicht.

BACKGROUND: Extranodal non-Hodgkin's lymphoma are mostly localized in the gastrointestinal tract. A small number of these lymphoma arise from specialized lymphoid cells, the so-called mucosa-associated lymphoid tissue (MALT). We describe one case with a metachronous occurrence of a MALT-type lymphoma of the conjunctiva of both eyelids and supraglottic larynx. PATIENT AND METHOD: A 56-year old woman was first treated in August 1990 for a low-grade B-cell lymphoma in the conjunctiva of the left eyelid. 42 months later an extranodal B-cell lymphoma, located in the conjunctiva of the right eyelid, was found. After a father period of 48 months a MALT-type lymphoma arose in the supraglottic larynx. The findings of staging examinations were normal. The final diagnosis was low-grade B-cell lymphoma of the MALT-type, limited to the conjunctival eyelids and supraglottic larynx, with the clinical staging of IE A. Treatment consisted of a LASER-resection followed by a locoregional radiotherapy, with the dose of 40 Gy. RESULTS: Currently no evidence of disease (NED) at all sites can be proven. CONCLUSIONS: Extranodal, primary low grade B-cell MALT-type lymphoma are rare. In the literature only few cases of each larynx- and conjunctival eyelid involvement described. The radiotherapy has been reported to achieve a long time of relapse-free interval. The present case demonstrates, that even the recurrence of an extranodal, primary low-grade B-cell MALT-type lymphoma responds well to local radiotherapy and can also have a long period of no evidence of disease.